Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms

Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10^-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.     

Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.     

Peroxiredoxin I and II in Human Eyes: Cellular Distribution and Association with Pterygium and DNA Damage

Peroxiredoxin I and II are both 2-Cys members of the peroxiredoxin family of antioxidant enzymes and inactivate hydrogen peroxide. On western blotting, both enzymes appeared as 22-kD proteins and were present in the sclera, retina and iris. Immunohistochemistry showed strong cytoplasmic labeling in the basal cells of the corneal epithelial layer and the corneoscleral limbus. The melanocytes within the stroma of the iris and the anterior epithelial cells of the lens also showed strong cytoplasmic labeling. The fibrous structure of the stroma and the posterior surface of the ciliary body were also labeled. There was also strong labeling for both enzymes in the photoreceptors and the inner and outer plexiform layers of the retina. There was increased labeling of peroxiredoxin I and II in pterygium. In normal conjunctiva and cornea, only the basal cell layer showed labeling for peroxiredoxin I and II, whereas, in pterygia, there was strong cytoplasmic labeling in most cells involving the full thickness of the epithelium. Co-localization of the DNA oxidation product 8-hydroxy-2’-deoxyguanosine antibody with the nuclear dye 4’,6’-diamidino-2-phenylindole dihydrochloride indicated that the majority of the oxidative damage was cytoplasmic; this suggested that the mitochondrial DNA was most affected by the UV radiation in this condition.     

Microbial interactions: Population and sporulation studies ofBacillus sphaericus grown in association withErwinia atroseptica

A study was made of a phenomenon, previously reported, in whichBacillus sphaericus failed to sporulate in the usual peptone media, but would sporulate in these media when grown in association withErwinia atroseptica.Pure culture studies withB. sphaericus indicated that the stimulus driving the cells toward further vegetative growth and the resulting failure of the cells to sporulate was associated with the peptide fraction of peptone media; inhibition of sporulation could be reversed by reduction of the peptone level of the medium or by replacement of the peptone with known amino acids, with known amino acids and short-chain peptides, or with complete hydrolysates of casein.Population and sporulation studies were performed onB. sphaericus cultured inE. atroseptica spent medium and on mixed cultures of the two organisms. A variety of population and sporulation responses were obtained through alteration of the chemical and physical nature of the media byE. atroseptica, cultured alone or in mixed culture withB. sphaericus.It is suggested that removal of pro-vegetative peptides byE. atroseptica is responsible for the enhancement of sporulation observed inB. sphaericus in peptone media.From a thesis submitted to the Graduate School of the University of Maryland, by the senior author, in partial fulfillment of the requirements for the Ph. D. degree.     

Thermal imaging of the periorbital regions during the presentation of an auditory startle stimulus

Infrared thermal imaging of the inner canthi of the periorbital regions of the face can potentially serve as an input signal modality for an alternative access system for individuals with conditions that preclude speech or voluntary movement, such as total locked-in syndrome. However, it is unknown if the temperature of these regions is affected by the human startle response, as changes in the facial temperature of the periorbital regions manifested during the startle response could generate false positives in a thermography-based access system. This study presents an examination of the temperature characteristics of the periorbital regions of 11 able-bodied adult participants before and after a 102 dB auditory startle stimulus. The results indicate that the startle response has no substantial effect on the mean temperature of the periorbital regions. This indicates that thermography-based access solutions would be insensitive to startle reactions in their user, an important advantage over other modalities being considered in the context of access solutions for individuals with a severe motor disability.     

A Naturally Occurring Novel Allele of Escherichia coli Outer Membrane Protein A Reduces Sensitivity to Bacteriophage

A novel Escherichia coli outer membrane protein A (OmpA) was discovered through a proteomic investigation of cell surface proteins. DNA polymorphisms were localized to regions encoding the protein's surface-exposed loops which are known phage receptor sites. Bacteriophage sensitivity testing indicated an association between bacteriophage resistance and isolates having the novel ompA allele.     

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.     

Ultrafast Red Light Activation of Synechocystis Phytochrome Cph1 Triggers Major Structural Change to Form the Pfr Signalling-Competent State

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel ‘head-to-head’ arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.     

The Advocacy for Pedestrian Safety Study: Cluster Randomised Trial Evaluating a Political Advocacy Approach to Reduce Pedestrian Injuries in Deprived Communities

Objective

To determine whether advocacy targeted at local politicians leads to action to reduce the risk of pedestrian injury in deprived areas.

Design

Cluster randomised controlled trial.

Setting

239 electoral wards in 57 local authorities in England and Wales.

Participants

617 elected local politicians.

Interventions

Intervention group politicians were provided with tailored information packs, including maps of casualty sites, numbers injured and a synopsis of effective interventions.

Main outcome measures

25–30 months post intervention, primary outcomes included: electoral ward level: percentage of road traffic calmed; proportion with new interventions; school level: percentage with 20 mph zones, Safe Routes to School, pedestrian training or road safety education; politician level: percentage lobbying for safety measures. Secondary outcomes included politicians’ interest and involvement in injury prevention, and facilitators and barriers to implementation.

Results

Primary outcomes did not significantly differ: % difference in traffic calming (0.07, 95%CI: −0.07 to 0.20); proportion of schools with 20 mph zones (RR 1.47, 95%CI: 0.93 to 2.32), Safe Routes to School (RR 1.34, 95%CI: 0.83 to 2.17), pedestrian training (RR 1.23, 95%CI: 0.95 to 1.61) or other safety education (RR 1.16, 95%CI: 0.97 to 1.39). Intervention group politicians reported greater interest in child injury prevention (RR 1.09, 95%CI 1.03 to 1.16), belief in potential to help prevent injuries (RR 1.36, 95%CI 1.16 to 1.61), particularly pedestrian safety (RR 1.55, 95%CI 1.19 to 2.03). 63% of intervention politicians reported supporting new pedestrian safety schemes. The majority found the advocacy information surprising, interesting, effectively presented, and could identify suitable local interventions.

Conclusions

This study demonstrates the feasibility of an innovative approach to translational public health by targeting local politicians in a randomised controlled trial. The intervention package was positively viewed and raised interest but changes in interventions were not statistically significance. Longer term supported advocacy may be needed.

Trial Registration

Current Controlled Trials ISRCTN91381117