Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains

The Type VII protein secretion system, found in Gram‐positive bacteria, secretes small proteins, containing a conserved W‐x‐G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.     

Field of Dreams: Restitution of Pollinator Services in Restored Bird‐Pollinated Plant Populations

Ecosystem functionality is an increasingly important objective of ecological restoration. Despite this, a few studies have rigorously assessed reproductive functionality within restored plant populations, and it is largely assumed that pollinators follow restoration of plant communities—“build it and they will come.” Here, we applied an ecological genetic approach to determine the impact of spatial separation on mating in Banksia menziesii (Proteaceae), a dominant bird‐pollinated species of Banksia woodlands of Western Australia. All plants at three post‐mining restored sites (n = 72 [13 years old], n = 21 [8 years old], and n = 20 [9 years old]), as well as a sample from an adjacent natural reference site (n = 42), were genotyped at nine microsatellite loci. Seed set, mating system parameters, realized pollen dispersal through the assignment of paternity to seed, and avian pollinator species composition, abundance and behavior, were assessed. All patches displayed equivalent heterozygosity (H_e = 0.53–0.59) and very weak genetic divergence (F_ST ≤ 0.01). Seed of plants within restored sites showed complete outcrossing and relatively high seed set, 26% of which were sired by pollen donors located beyond the local patch. Similar abundance and movement of nectar‐feeding birds was observed in restored and natural sites, despite lower bird species diversity in the restored site, where a smaller, less aggressive species was dominant. Our results demonstrate the restitution of wide outcrossing in these restored Banksia patches within an active mine‐site, and suggest that restored bird‐pollinated Banksia populations are resilient to human impacts, due largely to their generalist pollinator requirements and highly‐mobile avian pollinators.     

Concomitant Detection of HER2 Protein and Gene Alterations by Immunohistochemistry (IHC) and Silver Enhanced In Situ Hybridization (SISH) Identifies HER2 Positive Breast Cancer with and without Gene Amplification

Introduction

HER2 status assessment became a mandatory test assay in breast cancer, giving prognostic and predictive information including eligibility for adjuvant anti-HER2 therapy. Precise and reliable assessment of HER2 status is therefore of utmost importance. In this study we analyzed breast cancer samples by a novel technology for concomitant detection of the HER2 protein and gene copy number.

Methods

Tissue microarrays containing 589 invasive breast cancer samples were analyzed with a double immunohistochemistry (IHC) and silver labeled in situ hybridization (SISH) assay simultaneously detecting HER2 protein and gene copy number in the same tumor cells. This bright-field assay was analyzed using scores according to the modified ASCO guidelines and the results were correlated with patient prognosis.

Results

Overall concordance rate between protein expression and the presence of gene amplification was 98%. Fifty-seven of 60 tumors (95%) with IHC score 3+, 6 of 10 tumors with IHC score 2+ (60%) and only 3 of 519 tumors (0.6%) with IHC score 0/1+ were amplified by SISH. Patients with gene amplification despite IHC score 0/1+ had a tendency for worse overall survival (p = 0.088, reaching nearly statistical significance) compared to IHC score 0/1+ without amplification. In contrast, there was no difference in overall survival in IHC score 3+/2+ tumors with and without gene amplification.

Conclusions

The novel double IHC and SISH assay for HER2 is efficient in the identification of breast cancer with discordant HER2 protein and HER2 gene status, especially for the prognostically relevant groups of HER2 protein negative tumors with HER2 amplification and HER2 protein positive tumors without HER2 amplification. Breast cancer without HER2 amplification among IHC score 2+/3+ tumors (10% in our cohort) suggests that other mechanisms than gene amplification contribute to protein overexpression in these cells.     

MR Angiography Follow-Up 10 Years after Cryptogenic Nonperimesencephalic Subarachnoid Hemorrhage

ObjectivesLong-term magnetic resonance angiography (MRA) follow-up studies regarding cryptogenic nonperimesencephalic subarachnoid hemorrhage (nSAH) are scarce. This single-centre study identified all patients with angiographically verified cryptogenic nSAH from 1998 to 2007: The two main objectives were to prospectively assess the incidence of de novo aneurysm with 3.0-MRI years after cryptogenic nSAH in patients without evidence for further hemorrhage, and retrospectively assess patient demographics and outcome.MethodsFrom prospectively maintained report databases all patients with angiographically verified cryptogenic nSAH were identified. 21 of 29 patients received high-resolution 3T-MRI including time-of-flight and contrast-enhanced angiography, 10.2 ± 2.8 years after cryptogenic nSAH. MRA follow-up imaging was compared with initial digital subtraction angiography (DSA) and CT/MRA. Post-hemorrhage images were related to current MRI with reference to persistent lesions resulting from delayed cerebral ischemia (DCI) and post-hemorrhagic siderosis. Patient-based objectives were retrospectively abstracted from clinical databases.Results29 patients were identified with cryptogenic nSAH, 17 (59%) were male. Mean age at time of hemorrhage was 52.9 ± 14.4 years (range 4 – 74 years). 21 persons were available for long-term follow-up. In these, there were 213.5 person years of MRI-follow-up. No de novo aneurysm was detected. Mean modified Rankin Scale (mRS) during discharge was 1.28. Post-hemorrhage radiographic vasospasm was found in three patients (10.3%); DCI-related lesions occurred in one patient (3.4%). Five patients (17.2%) needed temporary external ventricular drainage; long-term CSF shunt dependency was necessary only in one patient (3.4%). Initial DSA retrospectively showed a 2 x 2 mm aneurysm of the right distal ICA in one patient, which remained stable. Post-hemorrhage siderosis was detected 8.1 years after the initial bleeding in one patient (4.8%).ConclusionPatients with cryptogenic nSAH have favourable outcomes and do not exhibit higher risks for de novo aneurysms. Therefore the need for long-term follow up after cryptogenic nSAH is questionable.     

Cost-Effectiveness of Coronary Artery Calcium Testing for Coronary Heart and Cardiovascular Disease Risk Prediction to Guide Statin Allocation: The Multi-Ethnic Study of Atherosclerosis (MESA)

Background

The Multi-Ethnic Study of Atherosclerosis (MESA) showed that the addition of coronary artery calcium (CAC) to traditional risk factors improves risk classification, particularly in intermediate risk asymptomatic patients with LDL cholesterol levels <160 mg/dL. However, the cost-effectiveness of incorporating CAC into treatment decision rules has yet to be clearly delineated.

Objective

To model the cost-effectiveness of CAC for cardiovascular risk stratification in asymptomatic, intermediate risk patients not taking a statin. Treatment based on CAC was compared to (1) treatment of all intermediate-risk patients, and (2) treatment on the basis of United States guidelines.

Methods

We developed a Markov model of first coronary heart disease (CHD) and cardiovascular disease (CVD) events. We modeled statin treatment in intermediate risk patients with CAC≥1 and CAC≥100, with different intensities of statins based on the CAC score. We compared these CAC-based treatment strategies to a “treat all” strategy and to treatment according to the Adult Treatment Panel III (ATP III) guidelines. Clinical and economic outcomes were modeled over both five- and ten-year time horizons. Outcomes consisted of CHD and CVD events and Quality-Adjusted Life Years (QALYs). Sensitivity analyses considered the effect of higher event rates, different CAC and statin costs, indirect costs, and re-scanning patients with incidentalomas.

Results

We project that it is both cost-saving and more effective to scan intermediate-risk patients for CAC and to treat those with CAC≥1, compared to treatment based on established risk-assessment guidelines. Treating patients with CAC≥100 is also preferred to existing guidelines when we account for statin side effects and the disutility of statin use.

Conclusion

Compared to the alternatives we assessed, CAC testing is both effective and cost saving as a risk-stratification tool, particularly if there are adverse effects of long-term statin use. CAC may enable providers to better tailor preventive therapy to patients'' risks of CVD.     

Pili-Induced Clustering of N. gonorrhoeae Bacteria

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.     

Design and characterization of auxotrophy-based amino acid biosensors

Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that - upon deletion - should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used.     

Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC₅₀ = 1.4 μM), suramin sodium salt (IC₅₀ = 3.6 μM), NF 023 hydrate (IC₅₀ = 6.2 μM) and tyrphostin AG 538 (IC₅₀ = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.