Functional Elements of EspF\u3csub\u3eu\u3c/sub\u3e, an Enterohemorrhagic \u3cem\u3eE. coli\u3c/em\u3e Effector that Stimulates Actin Assembly: A Dissertation

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an attaching and effacing pathogen that upon attachment to host cells, induce characteristic attaching and effacing lesions and formation of F-actin rich pedestals beneath sites of bacterial attachment. EHEC harbors a Type III secretion system through which it delivers dozens of effectors into the host cell. The two secreted effectors critical for EHEC-mediated actin pedestal formation are the translocated intimin receptor (Tir) and EspFU. EspFU consists of an N-terminal secretion signal and a C-terminus containing six tandem 47-residue proline-rich repeats, each of which can bind and activate the actin nucleation promoting factor N-WASP. Structural and functional analyses described here have identified the mechanism of N-WASP activation by EspFU and the minimal domains and specific residues required for this activity. While EspFU and Tir are the only bacterial effectors required for F-actin pedestal formation, recruitment of EspFU to Tir is mediated by an unidentified putative host factor. To identify the host factor responsible for linking these two effectors, a combination of in vitro and functional assays were used to identify the host factor, IRTKS and the residues required for these interactions were defined. Further, the presence of at least two 47-residue repeats in all characterized clinical isolates of canonical EHEC strains led us to address the minimal requirements for EspFU functional domains to promote recruitment to Tir and N-WASP activation. Here we show that two proline-rich elements of EspFU are required for recruitment of EspFU by IRTKS to sites of bacterial attachment. Furthermore, once artificially clustered at the membrane, a single N-WASP binding element of EspFU can induce actin pedestal formation

Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome

Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1beta, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1beta. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, and introduction of RNase H, which degrades such hybrids, into infected cells specifically inhibited inflammasome activation. Notably, an E. coli rnhA mutant, which is incapable of producing RNase H and thus harbors increased levels of RNA:DNA hybrid, induced elevated levels of NLRP3-dependent caspase-1 activation and IL-1beta maturation. Collectively, these findings identify RNA:DNA hybrids of bacterial origin as a unique microbial trigger of the NLRP3 inflammasome

The role of individual and social variables in task performance.

This paper reports on a data-based study in which we explored - as part of a larger-scale British-Hungarian research project - the effects of a number of affective and social variables on foreign language (L2) learners’ engagement in oral argumentative tasks. The assumption underlying the investigation was that students’ verbal behaviour in oral task situations is partly determined by a number of non-linguistic and non-cognitive factors whose examination may constitute a potentially fruitful extension of existing task-based research paradigms. The independent variables in the study included various aspects of L2 motivation and several factors characterizing the learner groups the participating students were members of (such as group cohesiveness and intermember relations), as well as the learners’ L2 proficiency and ‘willingness to communicate’ in their L1. The dependent variables involved objective measures of the students’ language output in two oral argumentative tasks (one in the learners’ L1, the other in their L2): the quantity of speech and the number of turns produced by the speakers. The results provide insights into the interrelationship of the multiple variables determining the learners’ task engagement, and suggest a multi-level construct whereby some independent variables only come into force when certain conditions have been met

Optimisation and validation of an integrated magnetic resonance imaging-only radiotherapy planning solution

Background and purpose: Magnetic resonance imaging (MRI)-only treatment planning is gaining in popularity in radiation oncology, with various methods available to generate a synthetic computed tomography (sCT) for this purpose. The aim of this study was to validate a sCT generation software for MRI-only radiotherapy planning of male and female pelvic cancers. The secondary aim of this study was to improve dose agreement by applying a derived relative electron and mass density (RED) curve to the sCT. Method and materials: Computed tomography (CT) and MRI scans of forty patients with pelvic neoplasms were used in the study. Treatment plans were copied from the CT scan to the sCT scan for dose comparison. Dose difference at reference point, 3D gamma comparison and dose volume histogram analysis was used to validate the dose impact of the sCT. The RED values were optimised to improve dose agreement by using a linear plot. Results: The average percentage dose difference at isocentre was 1.2% and the mean 3D gamma comparison with a criteria of 1%/1 mm was 84.0% ± 9.7%. The results indicate an inherent systematic difference in the dosimetry of the sCT plans, deriving from the tissue densities. With the adapted REDmod table, the average percentage dose difference was reduced to −0.1% and the mean 3D gamma analysis improved to 92.9% ± 5.7% at 1%/1 mm. Conclusions: CT generation software is a viable solution for MRI-only radiotherapy planning. The option makes it relatively easy for departments to implement a MRI-only planning workflow for cancers of male and female pelvic anatomy.</p

A multifaceted approach to investigating pre-task planning effects on paired oral test performance

Despite the growing popularity of paired format speaking assessments, the effects of pre-task planning time on performance in these formats are not yet well understood. For example, some studies have revealed the benefits of planning but others have not. Using a multifaceted approach including analysis of the process of speaking performance, the aim of this paper is to investigate the effect of pre-task planning in a paired format. Data were collected from 32 students who carried out two decision-making tasks in pairs, under planned and unplanned conditions. The study used analyses of rating scores, discourse analytic measures, and conversation analysis (CA) of test-taker discourse to gain insight into co-constructing processes. A post-test questionnaire was also administered to understand the participants’ perceptions toward planned and unplanned interactions. The results from rating scores and discourse analytic measures revealed that planning had limited effect on performance, and analysis of the questionnaires did not indicate clear differences between the two conditions. CA, however, identified the possibility of a contrastive mode of discourse under the two planning conditions, raising concerns that planning might actually deprive test-takers of the chance to demonstrate their abilities to interact collaboratively

Sinteza, in vitro antitumorsko ispitivanje i radiosenzitirajuće vrednovanje novih derivata 4-[3-(supstituiranih)tioureido]-N-(kinoksalin-2-il)benzensulfonamida

Sulfonamides and quinoxaline derivatives possess many types of biological activities and have been recently reported to show substantial antitumor activity. This paper reports the synthesis of novel thioureidosulfaquinoxaline derivatives. All the newly synthesized compounds were evaluated for their in vitro anticancer activity against a human liver cell line (HEPG2) and showed higher activity than the reference drug doxorubicin. 4-(3-(4-Ethylbenzoate)thioureido)-N-(quinoxalin-2-yl)benzenesulfonamide (9) (IC50 = 15.6 µmol L1), N-(pyridin-2-yl)-4-(3-(4-(N-quinoxalin-2-yl-sulfamoyl)phenyl)thioureido)benzene-sulfonamide (10) (IC50 = 26.8 µmol L1) and N-(quinoxalin-2-yl)-4-(3-(4-(N-thiazol-2-ylsulfamoyl)phenyl)thioureido)benzenesulfonamide (11) (IC50 = 24.4 µmol L1) were the most potent compared to doxorubicin (IC50 = 71.8 µmol L1). The most potent compounds 9, 10 and 11 were evaluated as radiosensitizing agents by subjecting the compounds to γ-irradiation (8 kGy).Derivati sulfonamida i kinoksalina imaju raznoliko biološko djelovanje, između ostalog i antitumorsko djelovanje. U radu je opisana sinteza novih derivata tioureido sulfakinoksalina. Svim novim spojevima ispitano je antitumorsko djelovanje in vitro na humanoj staničnoj liniji jetre (HEPG 2). Svi ispitani spojevi pokazuju jači učinak nego referentni lijek doksorubicin. Najjači učinak imali su 4-(3-(4-etilbenzoat)tioureido)-N-(kinoksalin-2-il)benzen-sulfonamid (9) (IC50 = 15,6 µmol L1), N-(piridin-2-il)-4-(3-(4-(N-kinoksalin-2-il-sulfamoil)fenil)tioureido)-benzen-sulfonamid (10) (IC50 = 26,8 µmol L1) i N-(kinoksalin-2-il)-4-(3-(4-(N-tiazol-2-ilsulfamoil)fenil)tioureido)benzen-sulfonamid (11) (IC50 = 24,4 µmol L1), dok je IC50 vrijednost bila 71,8 µmol L1. Najaktivniji spojevi 9, 10 i 11 evaluirani su kao radziosenzitirajuća sredstva nakon izlaganja spojeva γ-zračenju (8 kGy)

A novel locus for restless legs syndrome maps to chromosome 19p in an Irish pedigree

Restless legs syndrome (RLS) is a common, sleep-related movement disorder. The symptoms follow a circadian pattern, worsening in the evening or night, leading to sleep disruption and daytime somnolence. Familial forms of RLS have been described and usually display an autosomal dominant pattern of inheritance. To date, linkage analysis has identified nine RLS loci, but no specific causative gene has been reported. Association mapping has highlighted a further four genomic areas of interest. We have conducted a genome-wide linkage analysis in an Irish autosomal dominant RLS pedigree with 11 affected members. Significant linkage was found on chromosome 19p for a series of microsatellite markers, with a maximum two-point LOD score of 3.59 at θ = 0.0 for marker D19S878. Recombination events, identified by haplotype analysis, define a genetic region of 6.57 cM on chromosome 19p13.3, corresponding to an interval of 2.5 Mb. This study provides evidence of a novel RLS locus and provides further evidence that RLS is a genetically heterogenous disorder

Validation of an MRI-only planning workflow for definitive pelvic radiotherapy

Purpose: Previous work on Magnetic Resonance Imaging (MRI) only planning has been applied to limited treatment regions with a focus on male anatomy. This research aimed to validate the use of a hybrid multi-atlas synthetic computed tomography (sCT) generation technique from a MRI, using a female and male atlas, for MRI only radiation therapy treatment planning of rectum, anal canal, cervix and endometrial malignancies. Patients and methods: Forty patients receiving radiation treatment for a range of pelvic malignancies, were separated into male (n = 20) and female (n = 20) cohorts for the creation of gender specific atlases. A multi-atlas local weighted voting method was used to generate a sCT from a T1-weighted VIBE DIXON MRI sequence. The original treatment plans were copied from the CT scan to the corresponding sCT for dosimetric validation. Results: The median percentage dose difference between the treatment plan on the CT and sCT at the ICRU reference point for the male cohort was − 0.4% (IQR of 0 to − 0.6), and − 0.3% (IQR of 0 to − 0.6) for the female cohort. The mean gamma agreement for both cohorts was &gt; 99% for criteria of 3%/2 mm and 2%/2 mm. With dose criteria of 1%/1 mm, the pass rate was higher for the male cohort at 96.3% than the female cohort at 93.4%. MRI to sCT anatomical agreement for bone and body delineated contours was assessed, with a resulting Dice score of 0.91 ± 0.2 (mean ± 1 SD) and 0.97 ± 0.0 for the male cohort respectively; and 0.96 ± 0.0 and 0.98 ± 0.0 for the female cohort respectively. The mean absolute error in Hounsfield units (HUs) within the entire body for the male and female cohorts was 59.1 HU ± 7.2 HU and 53.3 HU ± 8.9 HU respectively. Conclusions: A multi-atlas based method for sCT generation can be applied to a standard T1-weighted MRI sequence for male and female pelvic patients. The implications of this study support MRI only planning being applied more broadly for both male and female pelvic sites. Trial registration This trial was registered in the Australian New Zealand Clinical Trials Registry (ANZCTR) (www.anzctr.org.au) on 04/10/2017. Trial identifier ACTRN12617001406392.</p