The Novel DNA Binding Mechanism of Ridinilazole, a Precision Clostridiodes difficile Antibiotic.

Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.

Engineering human MEK-1 for structural studies: A case study of combinatorial domain hunting.

Structural biology studies typically require large quantities of pure, soluble protein. Currently the most widely-used method for obtaining such protein involves the use of bioinformatics and experimental methods to design constructs of the target, which are cloned and expressed. Recently an alternative approach has emerged, which involves random fragmentation of the gene of interest and screening for well-expressing fragments. Here we describe the application of one such fragmentation method, combinatorial domain hunting (CDH), to a target which historically was difficult to express, human MEK-1. We show how CDH was used to identify a fragment which covers the kinase domain of MEK-1 and which expresses and crystallizes significantly better than designed expression constructs, and we report the crystal structure of this fragment which explains some of its superior properties. Gene fragmentation methods, such as CDH, thus hold great promise for tackling difficult-to-express target proteins.

Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications.

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.

Counting CAG repeats in the Huntington's disease gene by restriction endonuclease EcoP15I cleavage.

Huntington's disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.

Safety and efficiency of perilesional sclerotherapy in leg ulcer patients with postthrombotic syndrome and/or oral anticoagulation with phenprocoumon.

BACKGROUND: The positive effects of perilesional sclerotherapy for venous leg ulcers is well documented. Although many patients with venous leg ulcers require oral anticoagulation or have had a deep vein thrombosis, the effects of these factors on perilesional sclerotherapy are unknown. The aim of this study was to review effects of oral anticoagulation and/or postthrombotic syndrome on perilesional sclerotherapy. PATIENTS AND METHODS: 28 patients with venous leg ulcers were observed. 12/28 had a postthrombotic syndrome, 5/12 were on oral anticoagulants (phenprocoumon with INR 2-3). During each treatment session, 1 ml sclerosing foam (1:5, polidocanol 2 %, method of Tessari) was injected. Treatment was continued until all extrafascial veins in the 15cm surrounding the ulcer were closed. RESULTS: Closure of the perilesional veins was achieved in all patients with 2.5 +/- 1.8 injections. In 10 of 28 patients (35.7 %), just one injection was needed. More injections were needed, both in patients with postthrombotic syndrome (3.3 +/- 2.1 vs. 1.8 +/- 1.3) and on anticoagulation with phenprocoumon (4.2 +/- 1.2 vs. 2.1 +/- 1.7). There were only two complications: an ascending phlebitis up to the accessory saphenous vein and a superficial erosion at an injection site which healed within 1 week. CONCLUSIONS: Perilesional sclerotherapy with foam is a safe and efficient therapy for patients with chronic venous leg ulcers even with postthrombotic syndrome and/ or ongoing anticoagulation.

Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I.

Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site. For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule. DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation. In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures. One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites. The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy. Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes.

A combinatorial method to enable detailed investigation of protein-protein interactions.

BACKGROUND: Successful structural investigations of protein-protein interactions can be facilitated by studying only the core interacting regions of the constituent proteins. However, attempting the discovery of stable core complexes using informed trial-and-error approaches can prove time and resource intensive. METHODS: We describe a valuable extension of combinatorial domain hunting (CDH), a technology for the timely elucidation of soluble protein truncations. The new method, CDH(2), enables empirical discovery of stable protein-protein core complexes. CDH(2) is demonstrated ab initio using a previously well-characterized Hsp90/Cdc37 complex. RESULTS: Without using a priori information, we demonstrate the isolation of stable protein-protein complexes, suitable for further analyses. DISCUSSION: This resource-efficient process can provide protein complexes for screening of compounds designed to modulate protein-protein interactions, thus facilitating novel drug discovery.

Functional characterization and modulation of the DNA cleavage efficiency of type III restriction endonuclease EcoP15I in its interaction with two sites in the DNA target.

EcoP15I is a Type III restriction endonuclease requiring the interaction with two inversely oriented 5'-CAGCAG recognition sites for efficient DNA cleavage. Diverse models have been developed to explain how enzyme complexes bound to both sites move toward each other, DNA translocation, DNA looping and simple diffusion along the DNA. Conflicting data also exist about the impact of cofactor S-adenosyl-L-methionine (AdoMet), the AdoMet analogue sinefungin and the bases flanking the DNA recognition sequence on EcoP15I enzyme activity. To clarify the functional role of these questionable parameters on EcoP15I activity and to optimize the enzymatic reaction, we investigated the influence of cofactors, ionic conditions, bases flanking the recognition sequence and enzyme concentration. We found that AdoMet is not necessary for DNA cleavage. Moreover, the presence of AdoMet dramatically impaired DNA cleavage due to competing DNA methylation. Sinefungin neither had an appreciable effect on DNA cleavage by EcoP15I nor compensated for the second recognition site. Moreover, we discovered that adenine stretches on the 5' or 3' side of CAGCAG led to preferred cleavage of this site. The length of the adenine stretch was pivotal and had to be different on the two sides for most efficient cleavage. In the absence of AdoMet and with enzyme in molar excess over recognition sites, we observed minor cleavage at two communicating DNA sites simultaneously. These results could also be exploited in the high-throughput, quantitative transcriptome analysis method SuperSAGE to optimize the crucial EcoP15I digestion step.

Changes in venous refilling time from childhood to adulthood in subjects with apparently normal veins.

OBJECTIVE: Measuring venous refilling time by photoplethysmography is one of the most commonly used methods in venous diagnostics. Numerous studies have been done in adults to investigate its suitability to assess the overall venous calf pump function and to discriminate healthy subjects from patients with venous disease. The present study investigated any changes in the behavior of venous calf pump function from childhood to adulthood in subjects with healthy veins. METHODS: The study population consisted of 73 healthy (CEAP clinical classes C0 and C1) subjects (52 females, 21 males) who were all of the same age group (10 to 12 years old). It was a representative selection of the participants in a comprehensive longitudinal vein study known as the Bochum study (BO). The data of this study were obtained from (original) pupils of 11 secondary schools in the city of Bochum, Germany at four different periods (BO I 1982-1983, 10 to 12 years; BO II 1986-1987, 14 to 16 years; BO III 1991, 18 to 20 years; and BO IV 2001-2002, 29 to 31 years), 1990 RESULTS: The distribution patterns of venous refilling time showed a successive shift to the right from childhood to adulthood. The distribution peak was in the range of 10.1 to 20 seconds in BO I, broadened to 10.1 to 30 seconds in BO II, and changed to 40.1 to 50 seconds in BO III and BO IV. The median values increased from 24 seconds to more than 45 seconds. When we compared the two subgroups classified as C0 and C1, there was no significant difference in the behavior of their venous refilling time. CONCLUSIONS: To our knowledge, this is the first time the physiologic changes of venous calf pump function have been documented in a longitudinal study in young volunteers with healthy veins. The median venous refilling time successively lengthened, corresponding to a maturing of the venous calf pump function during adolescence and then stayed on a stable level. Therefore, measurements of venous calf pump function are not a means for assessing malfunction of the venous system during childhood and adolescence.

[Trichoteiromania]. / Trichoteiromanie.

BACKGROUND: The physical influences leading to traumatic hair injury may be the result of cosmetic treatments, may be accidental or self-inflicted. The most frequent cause of self-inflected hair loss is trichotillomania, in which the hair is plucked, while trichotemnomania, in which the hair is deliberately cut, is less frequent. Freyschmidt-Paul et al. proposed the term trichoteiromania for yet another type of artificial hair loss, which results from perpetual rubbing of the scalp with fracturing of the hair shafts. PATIENTS AND METHODS: Four patients with trichoteiromania are further characterized on the basis of clinical, morphological and psychopathological criteria. RESULTS: In contrast to trichotillomania, trichoteiromania has no diagnostic histopathological features and has a normal trichogram. Traumatic changes to the hair shaft are more conspicuous, with splitting at the ends of the hairs, giving the impression of white tips. The underlying mental disorder varis among the patients, though scalp dysaesthesia, not explained through any specific dermatological disorder, is a common denominator in all cases. CONCLUSIONS: While trichotillomania is considered to be an obsessive-compulsive disorder, the underlying mental disorder in trichoteiromania represents a more heterogeneous group. Cooperation with the psychiatrist is indicated, as much as the management and prognosis of trichoteiromania will depend on recognition of the underlying mental disorder and its specific psychotherapeutic and pharmacological treatment.

[Extracorporeal photopheresis in progressive systemic sclerosis: discrimination of responders and non-responders]. / Die Extrakorporale Photopherese bei Progressiver Systemischer Sklerodermie: Unterscheidung von Respondern und Non-respondern.

BACKGROUND: The benefit of extracorporeal photopheresis (ExP) in progressive systemic sclerosis (PSS) is controversial. There is limited experience with the long-term use of ExP in PSS. The purpose of the present study was to distinguish between responders and non-responders by using ExP in PSS and to evaluate activation markers for PSS. PATIENTS AND METHODS: 20 subjects with PSS were treated for 12 months with ExP (interval: 1x/month) as an immunomodulating monotherapy (11/20) or combination therapy (9/20). The course of PSS was assessed by both specially designed clinical score and serological parameters (CRP, ANA, beta-galactosidase, P-III-P, CD4/CD8-ratio, TNF-alpha, 11-2-R, 11-6). RESULTS: After 12 cycles of ExP, 30% of the subjects showed a partial remission and 25%, stable disease (55% responders) while 45% had a progression (non-responders). Although there was no correlation between the clinical course and the serological parameters, an increase of beta-galactosidase during therapy marked a progression of PSS in non-responders. Responders with a short PSS-course before ExP, moderate ANA titres, normal TNF-alpha levels and lack of Scl-70 had a good prognosis. CONCLUSIONS: About the half of the subjects with PSS profited by the long-term use of ExP. Thereby the mild immunomodulating effect of ExP seems to be insufficient to control markedly progressive courses of PSS.

Gene expression analysis of plant host-pathogen interactions by SuperSAGE.

The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure "SuperSAGE." By applying SuperSAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a "nonmodel" organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up- or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.