Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue

<p>Abstract</p> <p>Background</p> <p>The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes <it>C. elegans nhr-67, Drosophila tailless </it>and <it>dissatisfaction</it>, and vertebrate Tlx (NR2E2, NR2E4, NR2E1), and the NR2E3 subclass, which includes <it>C. elegans fax-1 </it>and vertebrate PNR (NR2E5, NR2E3). PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members.</p> <p>Results</p> <p>We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind <it>in vivo</it>.</p> <p>Conclusion</p> <p>These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1 and NR2E3 subclasses. For the NR2E1 protein NHR-67, Asp-19 permits binding to AAGTCA half-sites, while Asn-19 permits binding to AGGTCA half-sites. The apparent conservation of DNA-binding properties between vertebrate and nematode NR2E receptors allows for the possibility of evolutionarily-conserved regulatory patterns.</p

A biomechanical model of the effect of subtalar arthroereisis on the adult flexible flat foot.

OBJECTIVE: The hypothesis tested was that the increased load on the medial arch in the adult flat foot can be reduced through a 6 mm subtalar arthroereisis. DESIGN: A three-dimensional multisegment biomechanical model was used in conjunction with experimental data and data from the literature. BACKGROUND: Biomechanical models have been used to study the plantar fascia, medial arch height, subtalar motion, medial displacement calcaneal osteotomy and distribution of forces in the foot. METHODS: Responses of a normal foot, a flat foot, and a flat foot with a subtalar arthroereisis to an applied load of 683 N were analyzed and the distribution of support among the metatarsal heads and the moment about various joints were computed. RESULTS: The flattened foot results in an increase in the load on the head of the first metatarsal from 10% to 24% of the body weight, and an increase in the moment about the talo-navicular joint from 3.4 to 11.9 Nm. Insertion of a 6 mm cylinder into the sinus tarsi, subtalar arthroereisis, results in a shift of the load back toward the lateral column, decreasing the load on the first metatarsal to 6% of the body weight and decreasing the moment about the talo-navicular joint to 6.0 Nm. CONCLUSIONS: Our analysis indicates that a 6 mm subtalar arthroereisis in an adult flat foot model decreases the load on the medial arch

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-2

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>irs, respectively). Competition experiments with DR2A, DR3A, and DR1A are shown at 10-fold and 100-fold molar excess (wedges) or 10-fold molar excess only (DR3A). We observed additional shifted species in this experiment, however these bands could not be competed with equivalent unlabeled oligonucleotides

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-3

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>smid derived from pLacZi that contained a single DR1A binding site upstream of the gene. Each strain included either no activator or a fusion construct containing a nematode nuclear receptor DBD fused to the yeast GAL4 activation domain. B. β-galactosidase activity for yeast containing DR1G binding sites. Error bars show standard deviations. Asterisks indicate results that are significantly different than no activator control by student's t-test (p < 0.05). The difference between the FAX-1 and FAX-1 N19D mutant activators on DR1A sites is also statistically significant. We performed equivalent experiments using strains containing negative control DRNC sites and activator constructs. These strains did not show β-gal activity relative to controls that had no activator (data not shown). We performed equivalent experiments using strains containing MON1 sites and HRWS sites (both AAGTCA monomers) and a FAX-1 DBD activator, which also did not show β-gal activity relative to controls (data not shown)

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-0

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>-1 antiserum [31]. B. EMSA of FAX-1 protein binding to DR1A sequences (AAGTCA DR1) and failing to bind to DRNC sequences (AATTCA repeats). Binding could be competed with 10-fold and 100-fold molar excess of unlabeled competitor DR1G and DR1A DNA, but not DRNC DNA (wedges). Similar results (not shown) were obtained using radioactively-labeled DR1G DNA (AGGTCA DR1). C. and D. EMSA of FAX-1 protein binding to DR1T sequences (ATGTCA DR1) and DR1C sequences (ACGTCA DR1), respectively

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue-1

<p><b>Copyright information:</b></p><p>Taken from "Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of is partially mediated via a subclass-specific P-box residue"</p><p>http://www.biomedcentral.com/1471-2199/9/2</p><p>BMC Molecular Biology 2008;9():2-2.</p><p>Published online 7 Jan 2008</p><p>PMCID:PMC2225407.</p><p></p>MON1 and DR1A DNA sequences did not reduce non-specific background bands. Similar results (not shown) were obtained with MON2 sequences (single AAGTCA site in different position). B. EMSA of FAX-1 protein binding to HRSW sequences (AAGTCA strong binding site followed by AATTCA weak binding site). Binding could be competed with 10-fold and 100-fold molar excess of unlabeled competitor HRSW DNA, but not DRNC DNA (wedges). Although we obtained strong shifted bands, the proportion of labeled DNA shifted was considerably less than that observed with DR1A sequences (Table 1). We obtained similar results (not shown) with HRWS DNA (AATTCA weak site followed by AAGTCA strong site)

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31â127 anaesthetic procedures in 30â874 children with a mean age of 6·35 years (SD 4·50) were included. The incidence of perioperative severe critical events was 5·2% (95% CI 5·0â5·5) with an incidence of respiratory critical events of 3·1% (2·9â3·3). Cardiovascular instability occurred in 1·9% (1·7â2·1), with an immediate poor outcome in 5·4% (3·7â7·5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10â000. This was independent of type of anaesthesia. Age (relative risk 0·88, 95% CI 0·86â0·90; p<0·0001), medical history, and physical condition (1·60, 1·40â1·82; p<0·0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0·99, 0·981â0·997; p<0·0048 for respiratory critical events, and 0·98, 0·97â0·99; p=0·0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia. Funding European Society of Anaesthesiology

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6.35 years (SD 4.50) were included. The incidence of perioperative severe critical events was 5.2% (95% CI 5.0-5.5) with an incidence of respiratory critical events of 3.1% (2.9-3.3). Cardiovascular instability occurred in 1.9% (1.7-2.1), with an immediate poor outcome in 5.4% (3.7-7.5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0.88, 95% CI 0.86-0.90; p<0.0001), medical history, and physical condition (1.60, 1.40-1.82; p<0.0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0.99, 0.981-0.997; p<0.0048 for respiratory critical events, and 0.98, 0.97-0.99; p=0.0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia