Conformation and Dynamics of Human Urotensin II and Urotensin Related Peptide in Aqueous Solution.

Conformation and dynamics of the vasoconstrictive peptides human urotensin II (UII) and urotensin related peptide (URP) have been investigated by both unrestrained and enhanced-sampling molecular-dynamics (MD) simulations and NMR spectroscopy. These peptides are natural ligands of the G-protein coupled urotensin II receptor (UTR) and have been linked to mammalian pathophysiology. UII and URP cannot be characterized by a single structure but exist as an equilibrium of two main classes of ring conformations, open and folded, with rapidly interchanging subtypes. The open states are characterized by turns of various types centered at K8Y9 or F6W7 predominantly with no or only sparsely populated transannular hydrogen bonds. The folded conformations show multiple turns stabilized by highly populated transannular hydrogen bonds comprising centers F6W7K8 or W7K8Y9. Some of these conformations have not been characterized previously. The equilibrium populations that are experimentally difficult to access were estimated by replica-exchange MD simulations and validated by comparison of experimental NMR data with chemical shifts calculated with density-functional theory. UII exhibits approximately 72% open:28% folded conformations in aqueous solution. URP shows very similar ring conformations as UII but differs in an open:folded equilibrium shifted further toward open conformations (86:14) possibly arising from the absence of folded N-terminal tail-ring interaction. The results suggest that the different biological effects of UII and URP are not caused by differences in ring conformations but rather by different interactions with UTR.

Can Simulations and Modeling Decipher NMR Data for Conformational Equilibria? Arginine-Vasopressin.

Arginine vasopressin (AVP) has been suggested by molecular-dynamics (MD) simulations to exist as a mixture of conformations in solution. The (1)H and (13)C NMR chemical shifts of AVP in solution have been calculated for this conformational ensemble of ring conformations (identified from a 23 µs molecular-dynamics simulation). The relative free energies of these conformations were calculated using classical metadynamics simulations in explicit water. Chemical shifts for representative conformations were calculated using density-functional theory. Comparison with experiment and analysis of the results suggests that the (1)H chemical shifts are most useful for assigning equilibrium concentrations of the conformations in this case. (13)C chemical shifts distinguish less clearly between conformations, and the distances calculated from the nuclear Overhauser effect do not allow the conformations to be assigned clearly. The (1)H chemical shifts can be reproduced with a standard error of less than 0.24 ppm (<2.2 ppm for (13)C). The combined experimental and theoretical results suggest that AVP exists in an equilibrium of approximately 70% saddlelike and 30% clinched open conformations. Both newly introduced statistical metrics designed to judge the significance of the results and Smith and Goodman's DP4 probabilities are presented.

A Three-Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors.

Molecular-dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V2 -receptor (V2 R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V2 R and its V1a R-analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three-site mechanism separates agonists from antagonists and explains subtype selectivity.

Anticoagulant rodenticide toxicity in six dogs presenting for ocular disease.

OBJECTIVE: To describe cases of suspected anticoagulant rodenticide toxicity manifesting with predominantly ocular signs. MATERIALS AND METHODS: Six canine cases that presented to veterinary referral hospitals for ocular abnormalities and were diagnosed with suspected or confirmed anticoagulant rodenticide ingestion were reviewed for commonalities in presentation and outcome. RESULTS: Five dogs had unilateral ocular signs and one dog had bilateral manifestations. Signs included subconjunctival hemorrhage, exophthalmos, and commonly orbital pain without other significant physical examination findings. Prothrombin time was measured in 5 of 6 dogs and was prolonged in all. Partial thromboplastin time was measured in 4 of 6 dogs and was prolonged in all. Complete blood cell count and serum chemistry profiles demonstrated mild, if any, abnormalities. Five dogs had known anticoagulant rodenticide exposure, and rodenticide ingestion was suspected in 1 additional case based on clinical signs, clinical pathologic abnormalities, and response to treatment. Five of 6 cases were hospitalized overnight for plasma transfusions along with oral or injectable vitamin K1 , and all dogs were treated with oral vitamin K1 for 30 days. All dogs experienced complete resolution of clinical signs within 6 weeks of initiating treatment. CONCLUSIONS: Anticoagulant rodenticide toxicity can present with predominantly ocular manifestations. Rodenticide ingestion should be considered in dogs with unilateral or bilateral subconjunctival hemorrhage, exophthalmos, and orbital pain.

In Silico Adoption of an Orphan Nuclear Receptor NR4A1.

A 4.1 µs molecular dynamics simulation of the NR4A1 (hNur77) apo-protein has been undertaken and a previously undetected druggable pocket has become apparent that is located remotely from the 'traditional' nuclear receptor ligand-binding site. A NR4A1/bis-indole ligand complex at this novel site has been found to be stable over 1 µs of simulation and to result in an interesting conformational transmission to a remote loop that has the capacity to communicate with a NBRE within a RXR-α/NR4A1 heterodimer. Several features of the simulations undertaken indicate how NR4A1 can be affected by alternate-site modulators.

A pulse-dose topical 1% 5-fluorouracil treatment regimen in a young dog with corneal squamous cell carcinoma.

PURPOSE: To describe the use of a pulse-dose topical 5-fluorouracil (5-FU) treatment regimen in a Pug dog with corneal squamous cell carcinoma (SCC). METHODS: A 1-year-old, spayed female Pug was evaluated for a corneal perforation of the right eye, which was surgically stabilized with a conjunctival pedicle graft. At the time of medial canthoplasty 7 weeks later, two areas of gray-white discoloration had developed medial and lateral to the graft. Biopsy samples were obtained via superficial keratectomy while under general anesthesia. RESULTS: Definitive diagnosis of corneal SCC was made through histopathological examination of the surgical biopsies. Thoracic radiography and submandibular lymph node cytology revealed no evidence of metastatic neoplasia. Following healing of the corneal biopsy sites, topical 1% 5-FU ointment was applied four times daily for four consecutive days once a month, for six treatment cycles. Twenty-three months after diagnosis, the patient remains visual and comfortable with no evidence of SCC recurrence. Long-term therapy with once daily topical 1% cyclosporine solution was used to manage corneal pigmentation bilaterally. CONCLUSIONS: The pulse-therapy 1% 5-FU protocol was a successful, convenient, and cost-effective adjunctive treatment with few adverse effects.

Effect of topical ophthalmic dorzolamide(2%)-timolol(0.5%) solution and ointment on intraocular pressure in normal horses.

OBJECTIVE: To compare the effect of commercially available solution and compounded ointment formulations of dorzolamide(2%)-timolol(0.5%) on intraocular pressure (IOP) of normal horses. ANIMALS: Eighteen clinically normal horses. PROCEDURES: A randomized, masked prospective design was used with horses divided into two equal groups. One eye of each horse was selected for topical ophthalmic treatment with either 0.2 mL of dorzolamide(2%)-timolol(0.5%) solution or 0.2 g of dorzolamide(2%)-timolol(0.5%) ointment every 12 h for 5 days. The contralateral eye of horses in both groups was untreated. Rebound tonometry was performed every 6 h starting 2 days prior to and ending 2 days after the treatment period. RESULTS: The mean IOP reduction in eyes treated with the solution or ointment formulations was 13%. Untreated eyes in both groups experienced a lesser but still statistically significant reduction in IOP. The IOP values did not return to baseline within 48 h of the last treatment. CONCLUSIONS AND CLINICAL RELEVANCE: The commercially available solution and compounded ointment formulations of ophthalmic dorzolamide(2%)-timolol(0.5%) had similar effects on IOP in normal horses. Persistent IOP reduction following cessation of treatment may indicate prolonged drug effect or acclimation of horses to tonometry.

Conformation and dynamics of 8-Arg-vasopressin in solution.

Arginine-vasopressin was subjected to a long (11 µs) molecular dynamics simulation in aqueous solution. Analysis of the results by DASH and principal components analyses revealed four main ring conformations that move essentially independently of the faster-moving tail region. Two of these conformations (labeled "saddle") feature well-defined ß-turns in the ring and conserved transannular hydrogen bonds, whereas the other two ("open") feature neither. The conformations have been identified and defined and are all of sufficient stability to be considered candidates for biological conformations in their cognate receptors.

A solid-phase extraction method for rapidly determining the adsorption coefficient of pharmaceuticals in sewage sludge.

The partitioning of pharmaceuticals in the environment can be assessed by measuring their adsorption coefficients (Kd) between aqueous and solid phases. Measuring this coefficient in sewage sludge gives an indication of their partitioning behaviour in a wastewater treatment plant and hence contributes to an understanding of their subsequent fate. The regulatory approved method for measuring Kd in sewage sludge is the US Environmental Protection Agency's Office of Prevention, Pesticides and Toxic Substances (OPPTS) guideline 835.1110, which is labour intensive and time consuming. We describe an alternative method for measuring the Kd of pharmaceuticals in sewage sludge using a modified solid-phase extraction (SPE) technique. SPE cartridges were packed at different sludge/PTFE ratios (0.4, 6.0, 24.0 and 40.0% w/w sludge) and eluted with phosphate buffer at pH 7.4. The approach was tested initially using three pharmaceuticals (clofibric acid, diclofenac and oxytetracycline) that covered a range of Kd values. Subsequently, the sorption behaviour of ten further pharmaceuticals with varying physico-chemical properties was evaluated. Results from the SPE method were comparable to those of the OPPTS test, with a correlation coefficient of 0.93 between the two approaches. SPE cartridges packed with sludge and PTFE were stable for up to one year; use within one month reduced variability in measurements (to a maximum of 0.6 log units). The SPE method is low-cost, easy to use and enables the rapid measurement of Kd values for a large number of chemicals. It can be used as an alternative to the more laborious full OPPTS test in environmental fate studies and risk assessments.

APTE: identification of indirect read-out A-DNA promoter elements in genomes.

BACKGROUND: Transcriptional regulation is normally based on the recognition by a transcription factor of a defined base sequence in a process of direct read-out. However, the nucleic acid secondary and tertiary structure can also act as a recognition site for the transcription factor in a process known as indirect read-out, although this is much less understood. We have previously identified such a transcriptional control mechanism in early Xenopus development where the interaction of the transcription factor ilf3 and the gata2 promoter requires the presence of both an unusual A-form DNA structure and a CCAAT sequence. Rapid identification of such promoters elsewhere in the Xenopus and other genomes would provide insight into a less studied area of gene regulation, although currently there are few tools to analyse genomes in such ways. RESULTS: In this paper we report the implementation of a novel bioinformatics approach that has identified 86 such putative promoters in the Xenopus genome. We have shown that five of these sites are A-form in solution, bind to transcription factors and fully validated one of these newly identified promoters as interacting with the ilf3 containing complex CBTF. This interaction regulates the transcription of a previously uncharacterised downstream gene that is active in early development. CONCLUSIONS: A Perl program (APTE) has located a number of potential A-form DNA promotor elements in the Xenopus genome, five of these putative targets have been experimentally validated as A-form and as targets for specific DNA binding proteins; one has also been shown to interact with the A-form binding transcription factor ilf3. APTE is available from http://www.port.ac.uk/research/cmd/software/ under the terms of the GNU General Public License.

Sharpening the toolbox of computational chemistry: a new approximation of critical f-values for multiple linear regression.

Multiple linear regression is a major tool in computational chemistry. Although it has been used for more than 30 years, it has only recently been noted within the cheminformatics community that the standard F-values used to assess the significance of the resulting models are inappropriate in situations where the variables included in a model are chosen from a large pool of descriptors, due to an effect known in the statistical literature as selection bias. We have used Monte Carlo simulations to estimate the critical F-values for many combinations of sample size (n), model size (p), and descriptor pool size (k), using stepwise regression, one of the methods most commonly used to derive linear models from large sets of molecular descriptors. The values of n, p, and k represent cases appropriate to contemporary cheminformatics data sets. A formula for general n, p, and k values has been developed from the numerical estimates that approximates the critical stepwise F-values at 90%, 95%, and 99% significance levels. This approximation reproduces both the original simulated values and an interpolation test set (within the range of the training values) with an R2 value greater than 0.995. For an extrapolation test set of cases outside the range of the training set, the approximation produced an R2 above 0.93.

The extraction of information and knowledge from trained neural networks.

In the past, neural networks were viewed as classification and regression systems whose internal representations were incomprehensible. It is now becoming apparent that algorithms can be designed that extract comprehensible representations from trained neural networks, enabling them to be used for data mining and knowledge discovery, that is, the discovery and explanation of previously unknown relationships present in data. This chapter reviews existing algorithms for extracting comprehensible representations from neural networks and outlines research to generalize and extend the capabilities of one of these algorithms, TREPAN. This algorithm has been generalized for application to bioinformatics data sets, including the prediction of splice junctions in human DNA sequences, and cheminformatics. The results generated on these data sets are compared with those generated by a conventional data mining technique (C5) and appropriate conclusions are drawn.

Pattern recognition based on color-coded quantum mechanical surfaces for molecular alignment.

A pattern recognition algorithm for the alignment of drug-like molecules has been implemented. The method is based on the calculation of quantum mechanical derived local properties defined on a molecular surface. This approach has been shown to be very useful in attempting to derive generalized, non-atom based representations of molecular structure. The visualization of these surfaces is described together with details of the methodology developed for their use in molecular overlay and similarity calculations. In addition, this paper also introduces an additional local property, the local curvature (C (L)), which can be used together with the quantum mechanical properties to describe the local shape. The method is exemplified using some problems representing common tasks encountered in molecular similarity.

Seropositivity rates for Bartonella henselae, Toxocara canis and Toxoplasma gondii in New Zealand blood donors.

BACKGROUND: Bartonella henselae, Toxocara canis and Toxoplasma gondii organisms can all cause vision-threatening posterior segment disease. Diagnosis relies heavily on clinical findings, but detection of antibodies in the patient's serum may be helpful in some cases. The significance of a positive serology result depends on the seroprevalence in the general population. Seroprevalence rates for these organisms vary widely around the world and this study aimed to determine the rates for a New Zealand population. METHODS: One hundred and forty healthy blood donors from the Waikato region in the North Island of New Zealand had their serum tested using an indirect latex agglutination test for detection of T. gondii antibodies, an indirect immunofluorescent assay for B. henselae antibodies and an enzyme-linked immunosorbent assay test for T. canis antibodies. RESULTS: Seropositivity rates for T. canis, T. gondii and B. henselae in this New Zealand population were 0.7 +/- 1.65%, 42.9 +/- 8.12% and 5 +/- 3.61%, respectively. CONCLUSION: Seropositivity rates for T. gondii and B. henselae in New Zealand are similar to rates reported in some other countries; however, T. canis seroprevalence may be lower than reported elsewhere in the world.

Specific occupant and collision characteristics are associated with motor vehicle collision-related blunt cerebrovascular artery injury.

BACKGROUND: Blunt cerebrovascular artery injury (BCI) remains difficult to diagnose but is recognized with increasing frequency after motor vehicle collisions (MVCs). Failure to detect this injury in a timely fashion can be devastating. Criteria that can be used to heighten the suspicion of this injury have been suggested; however, more encompassing screening has been recommended. To address this need, we sought to describe occupant, vehicle, and collision characteristics among MVC occupants who sustained a BCI. METHODS: All cases of BCI identified in the National Automotive Sampling System Crashworthiness Data System, a national probability sample of passenger vehicles involved in police-reported tow-away MVCs, between 1993 and 2001 were selected. Information on occupant (e.g., demographics, seating position, and restraint use), collision (e.g., collision type and severity), and vehicle characteristics were obtained and summarized using descriptive statistics. RESULTS: Nine-hundred forty individuals with BCI were identified in the Crashworthiness Data System data files. Over half were belted (57.4%) and 82.3% had airbag deployment; 16.2% were partially or completely ejected from the vehicle. Head and thoracic injuries were common (44.4% and 40.8%, respectively); 27.8% sustained a cervical spine fracture and 21.0% sustained a soft-tissue injury to the neck. The mean Injury Severity Score was 33.6. The case fatality rate was 44.5%. The majority of BCI occupants were drivers (76.0%). Among belted occupants, the lap/shoulder was the most commonly attributed as the injury source (61.4%). Among unbelted occupants, frequent injury sources included air bags (15.0%), windshield (13.7%), and other interior objects. With respect to collision characteristics, the average change in velocity (Delta V) was 43.3 km/h. The majority of collisions were frontal (76.2%). CONCLUSION: This study indicates that BCI is both a rare and lethal injury typified by specific occupant and collision characteristics. These characteristics provide insight as to the cause of this injury that may aid in the evaluation and management of the blunt trauma patient at risk for BCI.

Early results of lower extremity infrageniculate revascularization with a new polytetrafluoroethylene graft.

When an autologous vein is not available for lower extremity revascularization, prosthetic grafts are often required. However, prosthetic bypass grafts have limited patency for infrageniculate reconstruction. To potentially improve patency, a new geometric modification of the polytetrafluoroethylene (PTFE) graft, Distaflo (Impra, Tempe, AZ), has been developed for lower extremity bypass. We reviewed our early experience with the Distaflo graft in patients who required infrageniculate bypass for lower extremity ischemia when no suitable autologous saphenous vein was available. All patients were maintained on warfarin anticoagulation postoperatively. All grafts were followed at 6- to 12-week intervals with duplex ultrasound evaluation. Patient characteristics, operative procedures, and graft surveillance information were maintained on a computerized registry. Thirty-two patients with limb-threatening ischemia underwent 35 infrageniculate reconstructions with a Distaflo graft between February 26, 1999, and August 24, 2000. Thirty-two of 35 bypasses were performed on extremities that had previously undergone a surgical procedure. Forty-eight previous revascularization procedures were done on these 25 extremities. Thirty grafts were constructed to the tibial outflow sites, whereas the remaining five grafts were placed to the below-knee popliteal artery. One patient died on the second postoperative day secondary to unrelated causes, and only one graft (3%) failed during the same hospitalization. Fifteen of 35 grafts (43%) remained patent 1 to 30 months later. Four patent grafts (6%) were ligated between 2 and 14 months for infectious indications. When considering the 20 failed grafts, 9 patients underwent major amputation, 5 patients remain with chronically ischemic limbs, and 6 patients underwent additional bypass grafts. Twenty-three patients (72%) maintained limb salvage. The Distaflo PTFE graft achieves promising early patency for complex infrageniculate revascularization and may be used as an alternative conduit in patients with critical limb ischemia who do not have an adequate vein for lower extremity revascularization.

Abdominal aortic aneurysms in "high-risk" surgical patients: comparison of open and endovascular repair.

OBJECTIVE: To evaluate the early results of endovascular grafting for high-risk surgical candidates in the treatment of abdominal aortic aneurysms (AAA). SUMMARY BACKGROUND DATA: Since the approval of endoluminal grafts for treatment of AAA, endovascular repair of AAA (EVAR) has expanded to include patients originally considered too ill for open AAA repair. However, some concern has been expressed regarding technical failure and the durability of endovascular grafts. METHODS: The University of Alabama at Birmingham (UAB) Computerized Vascular Registry identified all patients who underwent abdominal aneurysm repair between January 1, 2000, and June 12, 2002. Patients were stratified by type of repair (open AAA vs. EVAR) and were classified as low risk or high risk. Patients with at least one of the following classifications were classified as high risk: age more than 80 years, chronic renal failure (creatinine > 2.0), compromised cardiac function (diminished ventricular function or severe coronary artery disease), poor pulmonary function, reoperative aortic procedure, a "hostile" abdomen, or an emergency operation. Death, systemic complications, and length of stay were tabulated for each group. RESULTS: During this 28-month period, 404 patients underwent AAA repair at UAB. Eighteen patients (4.5%) died within 30 days of their repair or during the same hospitalization. Two hundred seventeen patients (53%) were classified as high risk. Two hundred fifty-nine patients (64%) underwent EVAR repair, and 130 (50%) of these were considered high-risk patients (including four emergency procedures). One hundred forty-five patients (36%) underwent open AAA repair, including 15 emergency operations. All deaths occurred in the high-risk group: 12 (8.3%) died after open AAA repair and 6 (2.3%) died after EVAR repair. Postoperative length of stay was shorter for EVAR repair compared to open AAA. CONCLUSIONS: High-risk and low-risk patients can undergo EVAR repair with a lower rate of short-term systemic complications and a shorter length of stay compared to open AAA. Despite concern regarding the durability of EVAR, high-risk patients should be evaluated for EVAR repair before committing to open AAA repair.

A consensus neural network-based technique for discriminating soluble and poorly soluble compounds.

BCUT [Burden, CAS, and University of Texas] descriptors, defined as eigenvalues of modified connectivity matrices, have traditionally been applied to drug design tasks such as defining receptor relevant subspaces to assist in compound selections. In this paper we present studies of consensus neural networks trained on BCUTs to discriminate compounds with poor aqueous solubility from those with reasonable solubility. This level was set at 0.1 mg/mL on advice from drug formulation and drug discovery scientists. By applying strict criteria to the insolubility predictions, approximately 95% of compounds are classified correctly. For compounds whose predictions have a lower level of confidence, further parameters are examined in order to flag those considered to possess unsuitable biopharmaceutical and physicochemical properties. This approach is not designed to be applied in isolation but is intended to be used as a filter in the selection of screening candidates, compound purchases, and the application of synthetic priorities to combinatorial libraries.

Determining patient preference for treatment of extracranial carotid artery stenosis: carotid angioplasty and stenting versus carotid endarterectomy.

Revascularization of extracranial carotid artery stenosis (ECAS) continues to be the subject of spirited academic debate. Conflicting studies in the literature have fostered uncertainty among patients choosing between CEA and CAS. We obtained preference-based utilities from prospective patients being evaluated for ECAS and incorporated them into a decision analytic model. Patients being evaluated for ECAS in an outpatient setting were interviewed prior to their initial visit with a vascular surgeon. Patient preference data were elicited using probability trade-off (PTO) assessment and time trade-off (TTO) method. Decision analysis was performed to compare CEA with CAS. Morbidity and mortality rates were obtained from recent literature reports from the same institution. Our results showed that when patients are informed, they prefer and will more often choose CEA over CAS for revascularization of ECAS. Among patients preferring CAS, they expect no more than a 46% increase in the rate of stroke and/or death. Future clinical studies on true stroke rates for CAS will be required to further refine this analysis.

Use of hybrid aortic stent grafts for endovascular repair of abdominal aortic aneurysms: indications and outcomes.

BACKGROUND: Only two aortic stent grafts (Ancure-Guidant, Menlo Park, CA, and AneuRx-Medtronic, Sunnyvale, CA) have been FDA-approved for endovascular aortic aneurysm repair (EndoAAA). These grafts differ significantly in construction and clinical advantage, and combining components of these grafts (hybrid graft) is occasionally necessary. The role and outcome of hybrid aortic stent grafts is unknown. METHODS: All EndoAAA procedures during an 18-month period (10/99-4/01) were reviewed using the hospital record and a computer registry. Endografts were classified as hybrid if components from more than one type of stent graft were used or standard if constructed from only one stent graft type. Hybrid grafts were further classified as "anticipated" or "unanticpated." Outcomes were compared between hybrid and standard grafts using Fisher's exact test. RESULTS: One hundred forty-five EndoAAA repairs were performed (AneuRx, 67; Ancure, 70; and custom-made, 8) of which 14 (9.6%) were hybrid grafts. The majority of hybrid grafts (11) were constructed by adding AneuRx aortic or iliac cuffs to Ancure grafts. In most cases, the need for a hybrid graft was unanticipated (10) and related to an intraoperative proximal type I endoleak (7). Conversion to open operation was avoided in six patients by constructing hybrid grafts. When anticipated (4), hybrid grafts were constructed to treat complex iliac aneurysms. Outcomes in all categories were similar (P > 0.05) for hybrid vs standard grafts: technical success (93 vs 99%), conversion toopen AAA (7.1 vs 2.3%), vascular complications (7.1 vs 7.6%), systemic complications (21 vs 11%), endoleak (15 vs 14%), and rupture (0 vs 0%). CONCLUSIONS: The short-term safety and effectiveness of hybrid grafts are similar to those of standard grafts. Combining graft components to create hybrid grafts may increase the ability to treat complex iliac aneurysmal disease and may reduce conversions to open AAA repair. Thus, access to multiple graft types may be an important factor in the success and safety of EndoAAA repair.