Delineation of the Ancestral Tus-Dependent Replication Fork Trap.

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a 'locked' Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for 'back-up' Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

Dermatologic features of Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is characterized by distinctive facial and skeletal features, developmental delay, cognitive impairment, and behavioral abnormalities, including self-injurious behaviors. We aimed to investigate whether cutaneous features are common in SMS. We performed a complete skin examination in 20 young SMS patients. Skin features secondary to self-injurious behavior, such as bites, abrasions, dystrophic scars, limited spots of hyperkeratosis, anomalies of the nails, and whitlows, were found in the majority of patients. Acral pachydermia and fissured plantar keratoderma were common. Xerosis was constant and associated with extensive keratosis pilaris in the majority of patients. Dermatofibromas were frequent in older patients. The hair was dense and shiny, with an unusual hairline. Eyelash trichomegaly and heavy brows were common, as well as folliculitis on the back. The skin features of SMS have rarely been reported in the literature. Some of these are the consequence of neurobehavioral features, but some cutaneous features and abnormalities of appendages have not been reported in other related syndromes. Skin manifestations of SMS are varied, sometimes induced by self-injurious behavior and sometimes more specific. It remains to be determined whether the combination of the two kinds of signs could contribute to early diagnosis of the syndrome.

Application of the pulsed light technology to mycotoxin degradation and inactivation.

The persistence of mycotoxins and their metabolites in agricultural products is a major safety concern because of their high resistance to all kinds of decontamination techniques. In this study, we evaluated the effectiveness of the pulsed light technology for the degradation of mycotoxins. We report that eight flashes of pulsed light destroyed of 84.5 ± 1.9, 72.5 ± 1.1, 92.7 ± 0.8 and 98.1 ± 0.2% of zearalenone, deoxynivalenol, aflatoxin B1 and ochratoxin in solution. The degradation of the molecules was monitored by HPLC and LC-MS/MS analysis. We estimated the potential toxicity of zearalenone and deoxynivelenol after exposure to a pulsed light treatment using the Caenorhabditis elegans survival tests. The genotoxicity of aflatoxin B1 was also investigated using a complete Ames test. The results show that the treatment of zearalenone and deoxynivelenol by single or multiple flashes of pulsed light is associated with a stagnation or marginal decrease of the toxicity of the mycotoxins and that treatment of aflatoxin B1 by pulsed light can completely eliminate the mutagenic potential of this mycotoxin. This work provides the first demonstration of a nonthermal technology allowing mycotoxin destruction and inactivation of their mutagenic activity.

A polyplex qPCR-based binding assay for protein-DNA interactions.

The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.

Modulation of TCR signalling components occurs prior to positive selection and lineage commitment in iNKT cells.

iNKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. Their unique functional characteristics are induced by a transcriptional program initiated by positive selection mediated by CD1d expressed by CD4+CD8+ (double positive, DP) thymocytes. Here, using a novel Vα14 TCR transgenic strain bearing greatly expanded numbers of CD24hiCD44loNKT cells, we examined transcriptional events in four immature thymic iNKT cell subsets. A transcriptional regulatory network approach identified transcriptional changes in proximal components of the TCR signalling cascade in DP NKT cells. Subsequently, positive and negative selection, and lineage commitment, occurred at the transition from DP NKT to CD4 NKT. Thus, this study introduces previously unrecognised steps in early NKT cell development, and separates the events associated with modulation of the T cell signalling cascade prior to changes associated with positive selection and lineage commitment.

Site-specific covalent attachment of DNA to proteins using a photoactivatable Tus-Ter complex.

Investigations into the photocrosslinking kinetics of the protein Tus with various bromodeoxyuridine-substituted Ter DNA variants highlight the potential use of this complex as a photoactivatable connector between proteins of interest and specific DNA sequences.

Selective protein unfolding: a universal mechanism of action for the development of irreversible inhibitors.

High-throughput differential scanning fluorimetry of GFP-tagged proteins (HT-DSF-GTP) was applied for the identification of novel enzyme inhibitors acting by a mechanism termed: selective protein unfolding (SPU). Four different protein targets were interrogated with the same library to identify target-selective hits. Several hits selectively destabilized bacterial biotin protein ligase. Structure-activity relationship data confirmed a structure-dependent mechanism of protein unfolding. Simvastatin and altenusin were confirmed to irreversibly inactivate biotin protein ligase. The principle of SPU combined with HT-DSF-GTP affords an invaluable and innovative workflow for the identification of new inhibitors with potential applications as antimicrobials and other biocides.

Clinical phenotype of scabies by age.

OBJECTIVE: Scabies has a clinical presentation that seems to vary according to age. We conducted a prospective study with the goal of delineating the clinical presentation of the disease into 3 groups of age: infants, <2 years; children, 2 to 15 years; and adults, >15 years. METHODS: This trial was a prospective, multicenter observational study in consecutive patients with a confirmed diagnosis of scabies who were seen in 13 French Departments of Dermatology and Pediatric Dermatology between April 2010 and April 2011. A standardized questionnaire was completed for each patient. To identify factors associated with patient age, comparisons between the 3 age groups were conducted by using univariate and multivariate multinomial logistic regression analyses. RESULTS: A total of 323 individuals were included; the gender ratio (female:male) was 1.2:1. In univariate analysis, infants were more likely to have facial involvement. In multivariate logistic regression, relapse was more frequent in children (odds ratio [OR]: 2.45 [95% confidence interval (CI):1.23-4.88]) and infants (OR: 3.26 [95% CI: 1.38-7.71]). In addition, family members with itch (OR: 2.47 [95% CI: 1.04-5.89]), plantar (OR: 20.57 [95% CI: 7.22-58.60]), and scalp (OR: 16.94 [95% CI: 3.70-77.51]) involvement were also found to be independently associated with the age group <2 years. CONCLUSIONS: There is a specific clinical presentation of scabies in infants and children. Taking into account these specificities may be helpful for the early diagnosis and the identification of cases to prevent the propagation of the disease.

Dissecting the salt dependence of the Tus-Ter protein-DNA complexes by high-throughput differential scanning fluorimetry of a GFP-tagged Tus.

The analysis of the salt dependence of protein-DNA complexes provides useful information about the non-specific electrostatic and sequence-specific parameters driving complex formation and stability. The differential scanning fluorimetry of GFP-tagged protein (DSF-GTP) assay has been geared with an automatic Tm peak recognition system and was applied for the high-throughput (HT) determination of salt-induced effects on the GFP-tagged DNA replication protein Tus in complex with various Ter and Ter-lock sequences. The system was designed to generate two-dimensional heat map profiles of Tus-GFP protein stability allowing for a comparative study of the effect of eight increasing salt concentrations on ten different Ter DNA species at once. The data obtained with the new HT DSF-GTP allowed precise dissection of the non-specific electrostatic and sequence-specific parameters driving Tus-Ter and Tus-Ter-lock complex formation and stability. The major factor increasing the thermal resistance of Tus-Ter-lock complexes in high-salt is the formation of the TT-lock, e.g. a 10-fold higher Kspe was obtained for Tus-GFP:Ter-lockB than for Tus-GFP:TerB. It is anticipated that the system can be easily adapted for the study of other protein-DNA complexes.

Differential Tus-Ter binding and lock formation: implications for DNA replication termination in Escherichia coli.

In E. coli, DNA replication termination occurs at Ter sites and is mediated by Tus. Two clusters of five Ter sites are located on each side of the terminus region and constrain replication forks in a polar manner. The polarity is due to the formation of the Tus-Ter-lock intermediate. Recently, it has been shown that DnaB helicase which unwinds DNA at the replication fork is preferentially stopped at the non-permissive face of a Tus-Ter complex without formation of the Tus-Ter-lock and that fork pausing efficiency is sequence dependent, raising two essential questions: Does the affinity of Tus for the different Ter sites correlate with fork pausing efficiency? Is formation of the Tus-Ter-lock the key factor in fork pausing? The combined use of surface plasmon resonance and GFP-Basta showed that Tus binds strongly to TerA-E and G, moderately to TerH-J and weakly to TerF. Out of these ten Ter sites only two, TerF and H, were not able to form significant Tus-Ter-locks. Finally, Tus's resistance to dissociation from Ter sites and the strength of the Tus-Ter-locks correlate with the differences in fork pausing efficiency observed for the different Ter sites by Duggin and Bell (2009).

Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system.

Information about the stability of proteins is paramount to determine their optimal storage or reaction conditions. It is also essential to determine protein stability in high-throughput when screening for new or improved functions of proteins obtained from large mutant libraries. In drug discovery programs, monitoring of ligand-induced stabilization effects can be used to identify lead compounds in high-throughput. These studies require expensive biophysical instrumentation and large quantities of purified proteins. To address these issues, we developed a new method, using GFP as a reporter system to quantify the stability of a protein and its ligand-associated stabilization effects that requires neither special equipment nor extensive purification steps. Here, GFP is fused to a protein of interest (POI) through a linker and is used as a reporter system for protein unfolding and aggregation. The three POIs used in this study include the Ter-binding protein Tus, glycerol kinase and chloramphenicol acetyl transferase. The fluorescent fusion protein is subjected to irreversible thermal denaturation leading to formation of aggregates, which are eliminated by a centrifugation step. The residual fluorescence of the soluble fraction can be directly related to the stability of the POI and can be quantitatively monitored using a fluorescence plate reader. The GFP-based stability assay (GFP-Basta) was able to identify stabilizing compounds and afforded a new quantitative method for the screening and ranking of ligands for three different proteins. These applications are particularly useful for drug discovery, directed evolution, structural and functional genomics.