Sacral Neuromodulation in Patients With Low Anterior Resection Syndrome: The SANLARS Randomized Clinical Trial.

BACKGROUND: Sacral neuromodulation might be effective to palliate low anterior resection syndrome after rectal cancer surgery, but robust evidence is not available. OBJECTIVE: To assess the impact of sacral neuromodulation on low anterior resection syndrome symptoms as measured by validated scores and bowel diaries. DESIGN: Randomized, double-blind, 2-phased, controlled, multicenter crossover trial (NCT02517853). SETTINGS: Three tertiary hospitals. PATIENTS: Patients with major low anterior resection syndrome 12 months after transit reconstruction after rectal resection who had failed conservative treatment. INTERVENTIONS: Patients underwent an advanced test phase by stimulation for 3 weeks and received the pulse generator implant if a 50% reduction in low anterior resection syndrome score was achieved. These patients entered the randomized phase in which the generator was left active or inactive for 4 weeks. After a 2-week washout, the sequence was changed. After the crossover, all generators were left activated. MAIN OUTCOME MEASURES: The primary outcome was low anterior resection syndrome score reduction. Secondary outcomes included continence and bowel symptoms. RESULTS: After testing, 35 of 46 patients (78%) had a 50% or greater reduction in low anterior resection syndrome score. During the crossover phase, all patients showed a reduction in scores and improved symptoms, with better performance if the generator was active. At 6- and 12-month follow-up, the mean reduction in low anterior resection syndrome score was -6.2 (95% CI -8.97 to -3.43; p < 0.001) and -6.97 (95% CI -9.74 to -4.2; p < 0.001), with St. Mark's continence score -7.57 (95% CI -9.19 to -5.95, p < 0.001) and -8.29 (95% CI -9.91 to -6.66; p < 0.001). Urgency, bowel emptiness sensation, and clustering episodes decreased in association with quality-of-life improvement at 6- and 12-month follow-up. LIMITATIONS: The decrease in low anterior resection syndrome score with neuromodulation was underestimated because of an unspecific measuring instrument. There was a possible carryover effect in sham stimulation sequence. CONCLUSIONS: Neuromodulation provides symptoms and quality-of-life amelioration, supporting its use in low anterior resection syndrome. See Video Abstract . NEUROMODULACIN SACRA EN PACIENTES CON SNDROME DE RESECCIN ANTERIOR BAJA ENSAYO CLNICO ALEATORIZADO SANLARS: ANTECEDENTES:La neuromodulación sacra podría ser eficaz para paliar el síndrome de resección anterior baja después de la cirugía de cáncer de recto, pero no hay pruebas sólidas disponibles.OBJETIVO:Evaluar el impacto de la neuromodulación sacra en los síntomas del síndrome de resección anterior baja, medido mediante puntuaciones validadas y diarios intestinales.DISEÑO:Ensayo cruzado multicéntrico, controlado, aleatorizado, doble ciego, de dos fases (NCT02517853).LUGARES:Tres hospitales terciarios.PACIENTES:Pacientes con puntuación de resección anterior baja importante, 12 meses después de la reconstrucción del tránsito después de la resección rectal en quienes había fracasado el tratamiento conservador.INTERVENCIONES:Los pacientes se sometieron a una fase de prueba avanzada mediante estimulación durante tres semanas y se les implantó el generador de impulsos si se lograba una reducción del 50% en la puntuación del síndrome de resección anterior baja, ingresando a la fase aleatorizada en la que el generador se dejaba activo o inactivo durante cuatro semanas. Después de observar por 2 semanas, se cambió la secuencia. Después del cruce, todos los generadores quedaron activados.PRINCIPALES MEDIDAS DE RESULTADO:El resultado primario fue la reducción de la puntuación del síndrome de resección anterior baja. Los resultados secundarios incluyeron continencia y síntomas intestinales.RESULTADOS:Después de las pruebas, 35 de 46 pacientes (78%) tuvieron una reducción ≥50% en la puntuación del síndrome de resección anterior baja. Durante el cruce, todos los pacientes mostraron una reducción en las puntuaciones y una mejora de los síntomas, con un mejor rendimiento si el generador estaba activo. A los 6 y 12 meses de seguimiento, la reducción media en la puntuación del síndrome de resección anterior baja fue -6,2 (-8,97; -3,43; p < 0,001) y -6,97 (-9,74; -4,2; p < 0,001), con Puntuación de continencia de St. Mark's -7,57 (-9,19; -5,95, p < 0,001) y -8,29 (-9,91; -6,66; p < 0,001). La urgencia, la sensación de vacío intestinal y los episodios de agrupamiento disminuyeron en asociación con una mejora en la calidad de vida a los 6 y 12 meses de seguimiento.LIMITACIONES:La disminución en la puntuación del síndrome de resección anterior baja con neuromodulación se subestimó debido a un instrumento de medición no específico. Posible efecto de arrastre en la secuencia de estimulación simulada.CONCLUSIONES:La neuromodulación mejora los síntomas y la calidad de vida, lo que respalda su uso en el síndrome de resección anterior baja. (Traducción-Dr. Mauricio Santamaria ).

Leveraging a nationwide infection surveillance program to implement a colorectal surgical site infection reduction bundle: a pragmatic, prospective, and multicenter cohort study.

BACKGROUND: Bundled interventions usually reduce surgical site infection (SSI) when implemented at single hospitals, but the feasibility of their implementation at the nationwide level and their clinical results are not well established. MATERIALS AND METHODS: Pragmatic interventional study to analyze the implementation and outcomes of a colorectal surgery care bundle within a nationwide quality improvement program. The bundle consisted of antibiotic prophylaxis, oral antibiotic prophylaxis (OAP), mechanical bowel preparation, laparoscopy, normothermia, and a wound retractor. Control group (CG) and Intervention group (IG) were compared. Overall SSI, superficial (S-SSI), deep (D-SSI), and organ/space (O/S-SSI) rates were analyzed. Secondary endpoints included microbiology, 30-day mortality, and length of hospital stay. RESULTS: A total of 37 849 procedures were included, 19 655 in the CG and 18 194 in the IG. In all, 5462 SSIs (14.43%) were detected: 1767 S-SSI (4.67%), 847 D-SSI (2.24%), and 2838 O/S-SSI (7.5%). Overall SSI fell from 18.38% (CG) to 10.17% (IG), odds ratio (OR) of 0.503 [0.473-0.524]. O/S-SSI rates were 9.15% (CG) and 5.72% (IG), OR of 0.602 [0.556-0.652]. The overall SSI rate was 16.71% when no measure was applied and 6.23% when all six were used. Bundle implementation reduced the probability of overall SSI (OR: 0.331; CI 95 : 0.242-0.453), and also O/S-SSI rate (OR: 0.643; CI 95 : 0.416-0.919). In the univariate analysis, all measures except normothermia were associated with a reduction in overall SSI, while only laparoscopy, OAP, and mechanical bowel preparation were related to a decrease in O/S-SSI. Laparoscopy, wound retractor, and OAP decreased overall SSI and O/S-SSI in the multivariate analysis. CONCLUSIONS: In this cohort study, the application of a specific care bundle within a nationwide nosocomial infection surveillance system proved feasible and resulted in a significant reduction in overall and O/S-SSI rates in the elective colon and rectal surgery. The OR for SSI fell between 1.5 and 3 times after the implementation of the bundle.

An interventional nationwide surveillance program lowers postoperative infection rates in elective colorectal surgery. A cohort study (2008-2019).

BACKGROUND: Colorectal surgery is associated with the highest rate of surgical site infection (SSI). This study analyses the effectiveness of an interventional surveillance program on SSI rates after elective colorectal surgery. MATERIAL AND METHODS: Cohort study showing temporal trends of SSI rates and Standardized Infection Ratio (SIR) in elective colorectal surgery over a 12-year period. Prospectively collected data of a national SSI surveillance program was analysed and the effect of specific interventions was evaluated. Patient and procedure characteristics, as well as SIR and SSI rates were stratified by risk categories and type of SSI analysed using stepwise multivariate logistic regression models. RESULTS: In a cohort of 42,330 operations, overall cumulative SSI incidence was 16.31%, and organ-space SSI (O/S-SSI) was 8.59%. There was a 61.63% relative decrease in SSI rates (rho = -0.95804). The intervention which achieved the greatest SSI reduction was a bundle of 6 measures. SSI in pre-bundle period was 19.73% vs. 11.10% in post-bundle period (OR 1.969; IC 95% 1.860-2.085; p < 0.0001). O/S-SSI were 9.08% vs. 6.06%, respectively (OR 1.547; IC 95% 1.433-1.670; p < 0.0001). Median length of stay was 7 days, with a significant decrease over the studied period (rho = -0.98414). Mortality of the series was 1.08%, ranging from 0.35% to 2.0%, but a highly significant decrease was observed (rho = -0.67133). CONCLUSIONS: Detailed analysis of risk factors and postoperative infection in colorectal surgery allows strategies for reducing SSI incidence to be designed. An interventional surveillance program has been effective in decreasing SIR and SSI rates.

Preoperative oral antibiotics and surgical-site infections in colon surgery (ORALEV): a multicentre, single-blind, pragmatic, randomised controlled trial.

BACKGROUND: Previous studies have found that mechanical bowel preparation with oral antibiotics can reduce the incidence of surgical-site infections, but no randomised controlled trial has assessed oral antibiotics alone without mechanical bowel preparation. The aim of this study was to determine whether prophylaxis with oral antibiotics the day before elective colon surgery affects the incidence of postoperative surgical-site infections. METHODS: In this multicentre, pragmatic, randomised controlled trial (ORALEV), patients undergoing colon surgery were recruited from five major hospitals in Spain and 47 colorectal surgeons at these hospitals participated. Patients were eligible for inclusion if they were diagnosed with neoplasia or diverticular disease and if a partial colon resection or total colectomy was indicated. Participants were randomly assigned (1:1) using online randomisation tables to either administration of oral antibiotics the day before surgery (experimental group) or no administration of oral antibiotics before surgery (control group). For the experimental group, ciprofloxacin 750 mg was given every 12 h (two doses at 1200 h and 0000 h) and metronidazole 250 mg every 8 h (three doses at 1200 h, 1800 h, and 0000 h) the day before surgery. All patients were given intravenous cefuroxime 1·5 g and metronidazole 1 g at the time of anaesthetic induction. The primary outcome was incidence of surgical-site infections. Patients were followed up for 1 month after surgery and all postsurgical complications were registered. This study was registered with EudraCT, 2014-002345-21, and ClinicalTrials.gov, NCT02505581, and is closed to accrual. FINDINGS: Between May 2, 2015, and April 15, 2017, we assessed 582 patients for eligibility, of whom 565 were eligible and randomly assigned to receive either no oral antibiotics (n=282) or oral antibiotics (n=282) before surgery. 13 participants in the control group and 16 in the experimental group were subsequently excluded; 269 participants in the control group and 267 in the experimental group received their assigned intervention. The incidence of surgical-site infections in the control group (30 [11%] of 269) was significantly higher than in the experimental group (13 [5%] of 267; χ2 test p=0·013). Oral antibiotics were associated with a significant reduction in the risk of surgical-site infections compared with no oral antibiotics (odds ratio 0·41, 95% CI 0·20-0·80; p=0·008). More complications (including surgical-site infections) were observed in the control group than in the experimental group (76 [28%] vs 51 [19%]; p=0·017), although there was no difference in severity as assessed by Clavien-Dindo score. No differences were noted between groups in terms of local complications, surgical complications, or medical complications that were not related to septic complications. INTERPRETATION: The administration of oral antibiotics as prophylaxis the day before colon surgery significantly reduces the incidence of surgical-site infections without mechanical bowel preparation and should be routinely adopted before elective colon surgery. FUNDING: Fundación Asociación Española de Coloproctología.

Identification of potential aryl hydrocarbon receptor ligands by virtual screening of industrial chemicals.

We have developed a virtual screening procedure to identify potential ligands to the aryl hydrocarbon receptor (AhR) among a set of industrial chemicals. AhR is a key target for dioxin-like compounds, which is related to these compounds' potential to induce cancer and a wide range of endocrine and immune system-related effects. The virtual screening procedure included an initial filtration aiming at identifying chemicals with structural similarities to 66 known AhR binders, followed by 3 enrichment methods run in parallel. These include two ligand-based methods (structural fingerprints and nearest neighbor analysis) and one structure-based method using an AhR homology model. A set of 6445 commonly used industrial chemicals was processed, and each step identified unique potential ligands. Seven compounds were identified by all three enrichment methods, and these compounds included known activators and suppressors of AhR. Only approximately 0.7% (41 compounds) of the studied industrial compounds was identified as potential AhR ligands and among these, 28 compounds have to our knowledge not been tested for AhR-mediated effects or have been screened with low purity. We suggest assessment of AhR-related activities of these compounds and in particular 2-chlorotrityl chloride, 3-p-hydroxyanilino-carbazole, and 3-(2-chloro-4-nitrophenyl)-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2(3H)-one.

Outcomes of GatekeeperTM prosthesis implantation for the treatment of fecal incontinence: a multicenter observational study.

BACKGROUND: The implantation of Gatekeeper ™ (GK) represents a new option for the treatment of fecal incontinence (FI). The aim of this study was to analyze the postoperative morbidity associated with GK and to determine its clinical efficacy after at least 1 year of follow-up. METHODS: This was a multicenter, retrospective and longitudinal study of patients with FI who were treated with GK at our institutions between January 2010 and December 2015. Patients with FI without sphincter lesions or with sphincter injuries < 120° and with low anterior resection syndrome were included. Postoperative complications, long-term adverse effects and migration were recorded. FI severity was assessed using the Vaizey score. Patients were classified as responders or non-responders according to the improvement of the Vaizey score (≥ than 50 and < 50%, respectively) during the first 6 months after implantation. RESULTS: Forty-nine consecutive patients treated with GK between 2010 and 2015 were included (11 males and 38 females, mean age 63.3 years, SD 13.5). No postoperative and long-term complications were observed. Prosthesis migration was observed in 51% of patients. Twenty-three patients (48%) were classified as responders and 25 (52%) as non-responders. The mean Vaizey score at baseline, 6, 12 months and last visit post-surgery in the responder group was, respectively, 13.3 (SD 3.8), 4.3 (SD 2.1), 4.2 (SD 3.6) and 5.7 (SD 5.3). Significant differences were observed between the mean baseline Vaizey score and the mean 6, 12 and last follow-up Vaizey score values (p < 0.001). In long-term follow-up (2.7 years (SD 1.1)), responders maintained an improvement of more than 50% of the baseline Vaizey score. In the non-responder group the mean number of migrated prostheses was higher than in the responder group (2.4 SD 2.5 vs. 1.0 SD 1.6; p = 0.040). CONCLUSIONS: GK is a safe and effective procedure in more than 50% of the patients for at least 1 year after the implantation.

Preventing Parastomal Hernia Using a Modified Sugarbaker Technique With Composite Mesh During Laparoscopic Abdominoperineal Resection: A Randomized Controlled Trial.

OBJECTIVE: The aim of this study was to assess the reduction in the incidence of parastomal hernia (PH) after placement of prophylactic synthetic mesh using a modified Sugarbaker technique when a permanent end-colostomy is needed. SUMMARY OF BACKGROUND DATA: Prevention of PH formation is crucial given the high prevalence of PH and difficulties in the surgical repair of PH. METHODS: A randomized, prospective, double-blind, and controlled trial. Rectal cancer patients undergoing laparoscopic abdominoperineal resection with permanent colostomy were randomized (1 : 1) to the mesh and nonmesh arms. In the mesh group, a large-pore lightweight composite mesh was placed in the intraperitoneal/onlay fashion using a modified Sugarbaker technique. PH was detected by computed tomography (CT) after a minimum follow-up of 12 months. Analysis was per-protocol. RESULTS: The mesh group included 24 patients and the control group 28. Preoperative data, surgical time, and postoperative morbidity were similar. The median follow-up was 26 months. After CT examination, 6 of 24 PHs (25%) were observed in the mesh group compared with 18 of 28 (64.3%) in the nonmesh group (odds ratio 0.39, 95% confidence interval 0.18-0.82; P = 0.005). The Kaplan-Meier curves showed significant differences in favor of the mesh group (long-rank = 4.21, P = 0.04). The number needed to treat was 2.5, which confirmed the effectiveness of the intervention. CONCLUSIONS: Placement of a prosthetic mesh by the laparoscopic approach following the modified Sugarbaker technique is safe and effective in the prevention of PH, reducing significantly the incidence of PH.

Tumor neuroendocrino en divertículo de Meckel: un hallazgo inusual / Neuroendocrine tumor in Meckel's diverticulum: an unusual finding

No disponible

An Aryl Hydrocarbon Receptor from the Salamander Ambystoma mexicanum Exhibits Low Sensitivity to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

Structural features of the aryl hydrocarbon receptor (AHR) can underlie species- and population-specific differences in its affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). These differences often explain variations in TCDD toxicity. Frogs are relatively insensitive to dioxin, and Xenopus AHRs bind TCDD with low affinity. Weak TCDD binding results from the combination of three residues in the ligand-binding domain: A354 and A370, and N325. Here we sought to determine whether this mechanism of weak TCDD binding is shared by other amphibian AHRs. We isolated an AHR cDNA from the Mexican axolotl (Ambystoma mexicanum). The encoded polypeptide contains identical residues at positions that confer low TCDD affinity to X. laevis AHRs (A364, A380, and N335), and homology modeling predicts they protrude into the binding cavity. Axolotl AHR bound one-tenth the TCDD of mouse AHR in velocity sedimentation analysis, and in transactivation assays, the EC50 for TCDD was 23 nM, similar to X. laevis AHR1ß (27 nM) and greater than AHR containing the mouse ligand-binding domain (0.08 nM). Sequence, modeled structure, and function indicate that axolotl AHR binds TCDD weakly, predicting that A. mexicanum lacks sensitivity toTCDD toxicity. We hypothesize that this characteristic of axolotl and Xenopus AHRs arose in a common ancestor of the Caudata and Anura.

Factores predictivos de infección de herida en cirugía colorrectal. Estudio observacional multicéntrico de casos y controles / Predictors of wound infection in elective colorectal surgery. Multicenter observational case-control study

INTRODUCCIÓN: El objetivo de este estudio es evaluar la utilidad del material de sutura Vicryl Plus® en reducir la tasa de infección de herida postoperatoria en cirugía colorrectal electiva. MÉTODO: Estudio de casos y controles prospectivo multicéntrico sobre 480 pacientes intervenidos de cirugía colorrectal electiva entre el 2006 y 2007, divididos en 2 grupos de igual tamaño muestral, sometidos a cierre de pared abdominal mediante uso de Vicryl Plus® (grupo 1) y PDS II® (grupo 2). En el estudio participaron 5 centros hospitalarios del Estado Español. La infección de herida fue clasificada en superficial y profunda. Fueron incluidos todos los pacientes diagnosticados de infección de herida durante la estancia hospitalaria y hasta 30 días después del alta. Para el estudio estadístico fueron utilizados el test del Chi-cuadrado y el exacto de Fisher para el análisis bivariante y el modelo de regresión logística para el análisis multivariante. RESULTADOS: La tasa de infección de herida observada fue significativamente inferior en el grupo 1: 14,6 frente al 29,2 del grupo 2. Según el estudio multivariante, el riesgo de infección de herida es superior en los pacientes con neoplasia, enfermedad pulmonar, anemia, tiempo operatorio superior a 2 h, falta de segunda dosis profiláctica intraoperatoria y cierre de laparotomía con sutura de PDS II®. CONCLUSIONES: El uso de material de sutura recubierto de triclosan puede ser una herramienta profiláctica eficaz para disminuir la tasa de infección de herida operatoria en los pacientes intervenidos mediante cirugía colorrectal electiva

INTRODUCTION: The aim of this study is to evaluate the usefulness of Vicryl Plus® suture in reducing the rate of postoperative wound infection in elective colorectal surgery. METHODS: A prospective case-control multicenter study with 480 patients undergoing elective colorectal surgery was performed between 2006 and 2007. Patients were divided in 2 groups of equal sample size: group 1, closure of the abdominal wall using Vicryl Plus® and group 2 where PDS II® was used. The study involved 5 hospitals in the Spanish State. Wound infection was classified into superficial and deep. All patients diagnosed of wound infection during the hospital stay and up to 30 days after discharge were studied. For the statistical analysis Chi-square test and Fisher exact were used for bivariate analysis and logistic regression model for multivariate analysis. Results Wound infection rates were significantly lower in group 1: 14.6 vs 29.2. Multivariate analysis showed that risk of wound infection was higher in patients with cancer, lung disease, anemia, operative time greater than 2 h, lack of second dose intra-operative prophylactic antibiotic and laparotomy closure with PDS suture II®. CONCLUSIONS: The use of suture coated with triclosan can be an effective prophylactic tool in reducing wound infection rate in patients undergoing elective colorectal surgery

Design of gliadin peptide analogues with low affinity for the celiac disease associated HLA-DQ2 protein.

The HLA-DQ2.5 receptors bind gluten-derived peptides and present them to the T cells in the intestinal mucosa thus inducing the development of immune responses typical of the celiac disease. On the basis of the X-ray structure of the domain of HLA-DQ2.5 bound to the DQ2.5-glia-α1a epitope, fifteen peptides were designed with the aim of lowering the epitope binding affinity, thus reducing the autoimmune response. Hydroxylation of Pro residues was proposed as a suitable functionalization given that both enzymatic and chemical synthetic methods are available. Then, a computational study on the effects of Pro hydroxylation on HLA-DQ2.5 binding was performed by molecular docking. A docking protocol able to reproduce the binding geometry of the known crystallographic complex was set up and applied to the designed DQ2.5-glia-α1a analogues. Among them, the one including four di-hydroxylated Pro residues was predicted to lower the binding affinity to the greatest extent. Therefore, the same functionalization was also computationally tested for other celiac disease relevant epitopes, DQ2.5-glia-α1b and DQ2.5-glia-α2, and their ability for inhibiting the binding to HLA-DQ2.5 was confirmed. On this basis, these hydroxylated peptides are expected to significantly affect the gluten activity involved in celiac disease and, after experimental validation, a synthetic method will be developed for introducing this gluten modification directly in flour. The proposed approach is a promising tool to study the binding of other gliadin and glutenin derived T-cell epitopes as well as their variants.

Predictors of wound infection in elective colorectal surgery. Multicenter observational case-control study.

INTRODUCTION: The aim of this study is to evaluate the usefulness of Vicryl Plus(®) suture in reducing the rate of postoperative wound infection in elective colorectal surgery. METHODS: A prospective case-control multicenter study with 480 patients undergoing elective colorectal surgery was performed between 2006 and 2007. Patients were divided in 2 groups of equal sample size: group 1, closure of the abdominal wall using Vicryl Plus(®) and group 2 where PDS II(®) was used. The study involved 5 hospitals in the Spanish State. Wound infection was classified into superficial and deep. All patients diagnosed of wound infection during the hospital stay and up to 30 days after discharge were studied. For the statistical analysis Chi-square test and Fisher exact were used for bivariate analysis and logistic regression model for multivariate analysis. RESULTS: Wound infection rates were significantly lower in group 1: 14.6 vs. 29.2. Multivariate analysis showed that risk of wound infection was higher in patients with cancer, lung disease, anemia, operative time greater than 2 h, lack of second dose intra-operative prophylactic antibiotic and laparotomy closure with PDS suture II(®). CONCLUSIONS: The use of suture coated with triclosan can be an effective prophylactic tool in reducing wound infection rate in patients undergoing elective colorectal surgery.

Ginsenosides are novel naturally-occurring aryl hydrocarbon receptor ligands.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals. In this study, we examined the ability of a series of ginsenosides extracted from ginseng, a traditional Chinese medicine, to bind to and activate/inhibit the AHR and AHR signal transduction. Utilizing a combination of ligand and DNA binding assays, molecular docking and reporter gene analysis, we demonstrated the ability of selected ginsenosides to directly bind to and activate the guinea pig cytosolic AHR, and to stimulate/inhibit AHR-dependent luciferase gene expression in a recombinant guinea pig cell line. Comparative studies revealed significant species differences in the ability of ginsenosides to stimulate AHR-dependent gene expression in guinea pig, rat, mouse and human cell lines. Not only did selected ginsenosides preferentially activate the AHR from one species and not others, mouse cell line was also significantly less responsive to these chemicals than rat and guinea pig cell lines, but the endogenous gene CYP1A1 could still be inducted in mouse cell line. Overall, the ability of these compounds to stimulate AHR signal transduction demonstrated that these ginsenosides are a new class of naturally occurring AHR agonists.

Artificial neural networks for efficient clustering of conformational ensembles and their potential for medicinal chemistry.

The biological function of proteins is strictly related to their molecular flexibility and dynamics: enzymatic activity, protein-protein interactions, ligand binding and allosteric regulation are important mechanisms involving protein motions. Computational approaches, such as Molecular Dynamics (MD) simulations, are now routinely used to study the intrinsic dynamics of target proteins as well as to complement molecular docking approaches. These methods have also successfully supported the process of rational design and discovery of new drugs. Identification of functionally relevant conformations is a key step in these studies. This is generally done by cluster analysis of the ensemble of structures in the MD trajectory. Recently Artificial Neural Network (ANN) approaches, in particular methods based on Self-Organising Maps (SOMs), have been reported performing more accurately and providing more consistent results than traditional clustering algorithms in various data-mining problems. In the specific case of conformational analysis, SOMs have been successfully used to compare multiple ensembles of protein conformations demonstrating a potential in efficiently detecting the dynamic signatures central to biological function. Moreover, examples of the use of SOMs to address problems relevant to other stages of the drug-design process, including clustering of docking poses, have been reported. In this contribution we review recent applications of ANN algorithms in analysing conformational and structural ensembles and we discuss their potential in computer-based approaches for medicinal chemistry.

Specific ligand binding domain residues confer low dioxin responsiveness to AHR1ß of Xenopus laevis.

The aryl hydrocarbon receptor (AHR) is a Per-ARNT-Sim (PAS) family protein that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vertebrates. Frogs are remarkably insensitive to TCDD, and AHRs from Xenopus laevis bind TCDD with low affinity. We sought to identify structural features of X. laevis AHR1ß associated with low TCDD sensitivity. Substitution of the entire ligand binding domain (LBD) with the corresponding sequence from mouse AHR(b-1) dramatically increased TCDD responsiveness in transactivation assays. To identify the amino acid residues responsible, we constructed a comparative model of the AHR1ß LBD using homologous domains of PAS proteins HIF2α and ARNT. The model revealed an internal cavity with dimensions similar to those of the putative binding cavity of mouse AHR(b-1), suggesting the importance of side chain interactions over cavity size. Of residues with side chains clearly pointing into the cavity, only two differed from the mouse sequence. When A354, located within a conserved ß-strand, was changed to serine, the corresponding mouse residue, the EC50 for TCDD decreased more than 15-fold. When N325 was changed to serine, the EC50 decreased 3-fold. When the mutations were combined, the EC50 decreased from 18.6 to 0.8 nM, the value nearly matching the TCDD sensitivity of mouse AHR. Velocity sedimentation analysis confirmed that mutant frog AHRs exhibited correspondingly increased levels of TCDD binding. We also assayed mutant AHRs for responsiveness to a candidate endogenous ligand, 6-formylindolo[3,2-b]carbazole (FICZ). Mutations that increased sensitivity to TCDD also increased sensitivity to FICZ. This comparative study represents a novel approach to discerning fundamental information about the structure of AHR and its interactions with biologically important agonists.