Purification and Characterization of Biofilm-Associated EPS Exopolysaccharides from ESKAPE Organisms and Other Pathogens

In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kDa) exopolysaccharides from biofilms of eight human pathogens, including species of Staphylcococcus, Klebsiella, Acinetobacter, Pseudomonas, and a toxigenic strain of Escherichia coli O157:H7. Glycosyl composition analysis indicated a high total mannose content across all strains with P. aeruginosa and A. baumannii exopolysaccharides comprised of 80–90% mannose, K. pneumoniae and S. epidermidis strains containing 40–50% mannose, and E. coli with ∼10% mannose. Galactose and glucose were also present in all eight strains, usually as the second and third most abundant carbohydrates. N-acetyl-glucosamine and galacturonic acid were found in 6 of 8 strains, while arabinose, fucose, rhamnose, and xylose were found in 5 of 8 strains. For linkage analysis, 33 distinct residue-linkage combinations were detected with the most abundant being mannose-linked moieties, in line with the composition analysis. The exopolysaccharides of two P. aeruginosa strains analyzed were consistent with the Psl carbohydrate, but not Pel or alginate. The S. epidermidis strain had a composition rich in mannose and glucose, which is consistent with the previously described slime associated antigen (SAA) and the extracellular slime substance (ESS), respectively, but no polysaccharide intracellular adhesion (PIA) was detected. The high molecular weight exopolysaccharides from E. coli, K. pneumoniae, and A. baumannii appear to be novel, based on composition and/or ratio analysis of carbohydrates.     

Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.     

A Genome-Wide Scan for Breast Cancer Risk Haplotypes among African American Women

Genome-wide association studies (GWAS) simultaneously investigating hundreds of thousands of single nucleotide polymorphisms (SNP) have become a powerful tool in the investigation of new disease susceptibility loci. Haplotypes are sometimes thought to be superior to SNPs and are promising in genetic association analyses. The application of genome-wide haplotype analysis, however, is hindered by the complexity of haplotypes themselves and sophistication in computation. We systematically analyzed the haplotype effects for breast cancer risk among 5,761 African American women (3,016 cases and 2,745 controls) using a sliding window approach on the genome-wide scale. Three regions on chromosomes 1, 4 and 18 exhibited moderate haplotype effects. Furthermore, among 21 breast cancer susceptibility loci previously established in European populations, 10p15 and 14q24 are likely to harbor novel haplotype effects. We also proposed a heuristic of determining the significance level and the effective number of independent tests by the permutation analysis on chromosome 22 data. It suggests that the effective number was approximately half of the total (7,794 out of 15,645), thus the half number could serve as a quick reference to evaluating genome-wide significance if a similar sliding window approach of haplotype analysis is adopted in similar populations using similar genotype density.     

Root Exudation of Phytochemicals in Arabidopsis Follows Specific Patterns That Are Developmentally Programmed and Correlate with Soil Microbial Functions

Plant roots constantly secrete compounds into the soil to interact with neighboring organisms presumably to gain certain functional advantages at different stages of development. Accordingly, it has been hypothesized that the phytochemical composition present in the root exudates changes over the course of the lifespan of a plant. Here, root exudates of in vitro grown Arabidopsis plants were collected at different developmental stages and analyzed using GC-MS. Principle component analysis revealed that the composition of root exudates varied at each developmental stage. Cumulative secretion levels of sugars and sugar alcohols were higher in early time points and decreased through development. In contrast, the cumulative secretion levels of amino acids and phenolics increased over time. The expression in roots of genes involved in biosynthesis and transportation of compounds represented in the root exudates were consistent with patterns of root exudation. Correlation analyses were performed of the in vitro root exudation patterns with the functional capacity of the rhizosphere microbiome to metabolize these compounds at different developmental stages of Arabidopsis grown in natural soils. Pyrosequencing of rhizosphere mRNA revealed strong correlations (p<0.05) between microbial functional genes involved in the metabolism of carbohydrates, amino acids and secondary metabolites with the corresponding compounds released by the roots at particular stages of plant development. In summary, our results suggest that the root exudation process of phytochemicals follows a developmental pattern that is genetically programmed.     

Intracranial Injection of AAV Expressing NEP but Not IDE Reduces Amyloid Pathology in APP+PS1 Transgenic Mice

The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer’s disease pathology. Several proteases, including Neprilysin (NEP), endothelin converting enzyme (ECE), and insulin degrading enzyme (IDE), have been shown to cleave β-amyloid peptides (Aβ). We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n) or IDE (IDE-n), or engineered secreted forms of NEP (NEP-s) or IDE (IDE-s). In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD.     

A Randomized Controlled Pilot Study of Home-Based Step Training in Older People Using Videogame Technology

Background

Stepping impairments are associated with physical and cognitive decline in older adults and increased fall risk. Exercise interventions can reduce fall risk, but adherence is often low. A new exergame involving step training may provide an enjoyable exercise alternative for preventing falls in older people.

Purpose

To assess the feasibility and safety of unsupervised, home-based step pad training and determine the effectiveness of this intervention on stepping performance and associated fall risk in older people.

Design

Single-blinded two-arm randomized controlled trial comparing step pad training with control (no-intervention).

Setting/Participants

Thirty-seven older adults residing in independent-living units of a retirement village in Sydney, Australia.

Intervention

Intervention group (IG) participants were provided with a computerized step pad system connected to their TVs and played a step game as often as they liked (with a recommended dose of 2–3 sessions per week for 15–20 minutes each) for eight weeks. In addition, IG participants were asked to complete a choice stepping reaction time (CSRT) task once each week.

Main Outcome Measures

CSRT, the Physiological Profile Assessment (PPA), neuropsychological and functional mobility measures were assessed at baseline and eight week follow-up.

Results

Thirty-two participants completed the study (86.5%). IG participants played a median 2.75 sessions/week and no adverse events were reported. Compared to the control group, the IG significantly improved their CSRT (F_31,1 = 18.203, p<.001), PPA composite scores (F_31,1 = 12.706, p = 0.001), as well as the postural sway (F_31,1 = 4.226, p = 0.049) and contrast sensitivity (F_31,1 = 4.415, p = 0.044) PPA sub-component scores. In addition, the IG improved significantly in their dual-task ability as assessed by a timed up and go test/verbal fluency task (F_31,1 = 4.226, p = 0.049).

Conclusions

Step pad training can be safely undertaken at home to improve physical and cognitive parameters of fall risk in older people without major cognitive and physical impairments.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12611001081909.     

Assessing Insecticide Hazard to Bumble Bees Foraging on Flowering Weeds in Treated Lawns

Maintaining bee-friendly habitats in cities and suburbs can help conserve the vital pollination services of declining bee populations. Despite label precautions not to apply them to blooming plants, neonicotinoids and other residual systemic insecticides may be applied for preventive control of lawn insect pests when spring-flowering weeds are present. Dietary exposure to neonicotinoids adversely affects bees, but the extent of hazard from field usage is controversial. We exposed colonies of the bumble bee Bombus impatiens to turf with blooming white clover that had been treated with clothianidin, a neonicotinoid, or with chlorantraniliprole, the first anthranilic diamide labeled for use on lawns. The sprays were applied at label rate and lightly irrigated. After residues had dried, colonies were confined to forage for six days, and then moved to a non-treated rural site to openly forage and develop. Colonies exposed to clothianidin-treated weedy turf had delayed weight gain and produced no new queens whereas those exposed to chlorantraniliprole-treated plots developed normally compared with controls. Neither bumble bees nor honey bees avoided foraging on treated white clover in open plots. Nectar from clover blooms directly contaminated by spray residues contained 171±44 ppb clothianidin. Notably, neither insecticide adversely impacted bee colonies confined on the treated turf after it had been mown to remove clover blooms present at the time of treatment, and new blooms had formed. Our results validate EPA label precautionary statements not to apply neonicotinoids to blooming nectar-producing plants if bees may visit the treatment area. Whatever systemic hazard through lawn weeds they may pose appears transitory, however, and direct hazard can be mitigated by adhering to label precautions, or if blooms inadvertently are contaminated, by mowing to remove them. Chlorantraniliprole usage on lawns appears non-hazardous to bumble bees.     

Enamel Matrix Derivative Inhibits Adipocyte Differentiation of 3T3-L1 Cells via Activation of TGF-βRI Kinase Activity

Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-β can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-β in vitro. Herein, we examined whether TGF-β signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-βRI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-βRI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro.     

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10^−/− mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10^−/− mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.