Decreased plakophilin-1 expression promotes increased motility in head and neck squamous cell carcinoma cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

The toxin/immunity network of Burkholderia pseudomallei contact-dependent growth inhibition (CDI) systems

Burkholderia pseudomallei is a category B pathogen and the causative agent of melioidosis – a serious infectious disease that is typically acquired directly from environmental reservoirs. Nearly all B. pseudomallei strains sequenced to date (> 85 isolates) contain gene clusters that are related to the contact‐dependent growth inhibition (CDI) systems of γ‐proteobacteria. CDI systems from Escherichia coli and Dickeya dadantii play significant roles in bacterial competition, suggesting these systems may also contribute to the competitive fitness of B. pseudomallei. Here, we identify 10 distinct CDI systems in B. pseudomallei based on polymorphisms within the cdiA‐CT/cdiI coding regions, which are predicted to encode CdiA‐CT/CdiI toxin/immunity protein pairs. Biochemical analysis of three B. pseudomallei CdiA‐CTs revealed that each protein possesses a distinct tRNase activity capable of inhibiting cell growth. These toxin activities are blocked by cognate CdiI immunity proteins, which specifically bind the CdiA‐CT and protect cells from growth inhibition. Using Burkholderia thailandensis E264 as a model, we show that a CDI system from B. pseudomallei 1026b mediates CDI and is capable of delivering CdiA‐CT toxins derived from other B. pseudomallei strains. These results demonstrate that Burkholderia species contain functional CDI systems, which may confer a competitive advantage to these bacteria.     

Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease

A comprehensive, unbiased inventory of synuclein forms present in Lewy bodies from patients with dementia with Lewy bodies was carried out using two-dimensional immunoblot analysis, novel sandwich enzyme-linked immunosorbent assays with modification-specific synuclein antibodies, and mass spectroscopy. The predominant modification of alpha-synuclein in Lewy bodies is a single phosphorylation at Ser-129. In addition, there is a set of characteristic modifications that are present to a lesser extent, including ubiquitination at Lys residues 12, 21, and 23 and specific truncations at Asp-115, Asp-119, Asn-122, Tyr-133, and Asp-135. No other modifications are detectable by tandem mass spectrometry mapping, except for a ubiquitous N-terminal acetylation. Small amounts of Ser-129 phosphorylated and Asp-119-truncated alpha-synuclein are present in the soluble fraction of both normal and disease brains, suggesting that these Lewy body-associated forms are produced during normal metabolism of alpha-synuclein. In contrast, ubiquitination is only detected in Lewy bodies and is primarily present on phosphorylated synuclein; it therefore likely occurs after phosphorylated synuclein has deposited into Lewy bodies. This invariant pattern of specific phosphorylation, truncation, and ubiquitination is also present in the detergent-insoluble fraction of brain from patients with familial Parkinson's disease (synuclein A53T mutation) as well as multiple system atrophy, suggesting a common pathogenic pathway for both genetic and sporadic Lewy body diseases. These observations are most consistent with a model in which preferential accumulation of normally produced Ser-129 phosphorylated alpha-synuclein is the key event responsible for the formation of Lewy bodies in various Lewy body diseases.     

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.     

A genetic similarity rule determines arthropod community structure

We define a genetic similarity rule that predicts how genetic variation in a dominant plant affects the structure of an arthropod community. This rule applies to hybridizing cottonwood species where plant genetic variation determines plant-animal interactions and structures a dependent community of leaf-modifying arthropods. Because the associated arthropod community is expected to respond to important plant traits, we also tested whether plant chemical composition is one potential intermediate link between plant genes and arthropod community composition. Two lines of evidence support our genetic similarity rule. First, in a common garden experiment we found that trees with similar genetic compositions had similar chemical compositions and similar arthropod compositions. Second, in a wild population, we found a similar relationship between genetic similarity in cottonwoods and the dependent arthropod community. Field data demonstrate that the relationship between genes and arthropods was also significant when the hybrids were analysed alone, i.e. the pattern is not dependent upon the inclusion of both parental species. Because plant-animal interactions and natural hybridization are common to diverse plant taxa, we suggest that a genetic similarity rule is potentially applicable, and may be extended, to other systems and ecological processes. For example, plants with similar genetic compositions may exhibit similar litter decomposition rates. A corollary to this genetic similarity rule predicts that in systems with low plant genetic variability, the environment will be a stronger factor structuring the dependent community. Our findings argue that the genetic composition of a dominant plant can structure higher order ecological processes, thus placing community and ecosystem ecology within a genetic and evolutionary framework. A genetic similarity rule also has important conservation implications because the loss of genetic diversity in one species, especially dominant or keystone species that define many communities, may cascade to negatively affect the rest of the dependent community.     

Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

Background

Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota.

Methodology/Principal Findings

We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients'' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds—approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics.

Conclusions/Significance

The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure—reducing some bacteria while selecting for others.     

Planning for exercises of chemical, biological, radiological, and nuclear (CBRN) forensic capabilities

A forensic capability to help identify perpetrators and exclude innocent people should be an integral part of a strategy against terrorist attacks. Exercises have been conducted to increase our preparedness and response capabilities to chemical, biological, radiological, and nuclear (CBRN) terrorist attacks. However, incorporating forensic components into these exercises has been deficient. CBRN investigations rely on forensic results, so the need to integrate a forensic component and forensics experts into comprehensive exercises is paramount. This article provides guidance for planning and executing exercises at local, state, federal, and international levels that test the effectiveness of forensic capabilities for CBRN threats. The guidelines presented here apply both to situations where forensics is only a component of a more general exercise and where forensics is the primary focus of the exercise.     

Technology mediator: a new role for the reference librarian?

The Arizona Health Sciences Library has collaborated with clinical faculty to develop a federated search engine that is useful for meeting real-time clinical information needs. This article proposes a technology mediation role for the reference librarian that was inspired by the project, and describes the collaborative model used for developing technology-mediated services for targeted users.