Multiple Degrees of Freedom in the Fish Skull and Their Relation to Hydraulic Transport of Prey in Channel Catfish.

Fish perform many complex manipulation behaviors without hands or flexible muscular tongues, instead relying on more than 20 movable skeletal elements in their highly kinetic skulls. How fish use their skulls to accomplish these behaviors, however, remains unclear. Most previous mechanical models have represented the fish skull using one or more planar four-bar linkages, which have just a single degree of freedom (DoF). In contrast, truncated-cone hydrodynamic models have assumed up to five DoFs. In this study, we introduce and validate a 3D mechanical linkage model of a fish skull that incorporates the pectoral girdle and mandibular and hyoid arches. We validate this model using an in vivo motion dataset of suction feeding in channel catfish and then use this model to quantify the DoFs in the fish skull, to categorize the motion patterns of the cranial linkage during feeding, and to evaluate the association between these patterns and food motion. We find that the channel catfish skull functions as a 17-link, five-loop parallel mechanism. Despite having 19 potential DoFs, we find that seven DoFs are sufficient to describe most of the motion of the cranial linkage, consistent with the fish skull functioning as a multi-DoF, manipulation system. Channel catfish use this linkage to generate three different motion patterns (rostrocaudal wave, caudorostral wave, and compressive wave), each with its own associated food velocity profile. These results suggest that biomechanical manipulation systems must have a minimum number of DoFs to effectively control objects, whether in water or air.

An XROMM Study of Food Transport and Swallowing in Channel Catfish.

Most predatory ray-finned fishes swallow their food whole, which can pose a significant challenge, given that prey items can be half as large as the predators themselves. How do fish transport captured food from the mouth to the stomach? Prior work indicates that, in general, fish use the pharyngeal jaws to manipulate food into the esophagus, where peristalsis is thought to take over. We used X-Ray Reconstruction of Moving Morphology to track prey transport in channel catfish (Ictalurus punctatus). By reconstructing the 3D motions of both the food and the catfish, we were able to track how the catfish move food through the head and into the stomach. Food enters the oral cavity at high velocities as a continuation of suction and stops in the approximate location of the branchial basket before moving in a much slower, more complex path toward the esophagus. This slow phase coincides with little motion in the head and no substantial mouth opening or hyoid depression. Once the prey is in the esophagus, however, its transport is surprisingly tightly correlated with gulping motions (hyoid depression, girdle retraction, hypaxial shortening, and mouth opening) of the head. Although the transport mechanism itself remains unknown, to our knowledge, this is the first description of synchrony between cranial expansion and esophageal transport in a fish. Our results provide direct evidence of prey transport within the esophagus and suggest that peristalsis may not be the sole mechanism of esophageal transport in catfish.

Thrombin inhibitor design.

Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.

Non-peptide fibrinogen receptor antagonists. 7. Design and synthesis of a potent, orally active fibrinogen receptor antagonist.

The design, synthesis, and pharmacological evaluation of L-734,217, a potent, low-molecular weight, orally active fibrinogen receptor antagonist, is reported. A strategy for producing low-molecular weight inhibitors from the peptide c-[(Ac)CRGDC] A, previously reported from these laboratories, is outlined. This strategy combines a retrodesign analysis of the conformationally defined cyclic peptide A with stereochemical information present in the arginine-glycine-aspartic acid (RGD) tripeptide sequence, culminating with the discovery of L-734,217. L-734,217 inhibited the aggregation of human, dog, and chimpanzee platelets at concentrations below 100 nM and was found to be > 15000-fold less effective at inhibiting the attachment of human umbilical vein endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting the aggregation of platelets. L-734,217 showed significant ex vivo antiplatelet activity following oral administration in dogs and chimpanzees at doses of 1.0 and 2.0 mg/kg, respectively, and has been selected as a clinical candidate for development as an antithrombotic agent.

Design of novel, potent, noncovalent inhibitors of thrombin with nonbasic P-1 substructures: rapid structure-activity studies by solid-phase synthesis.

Study of surface representations of the inhibitor-bound thrombin P-1 pocket revealed a lipophilic recess in this pocket which is not occupied by any known inhibitor. Solid-phase synthesis was used to generate benzylamides of D-diphenylAlaPro by aminolysis of Boc dipeptide Kaiser resin. The resulting amides inhibited thrombin in the range IC50 = 3-13,000 nM, and the structure-activity relationships and molecular modeling suggest a unique fit of the benzyl side chain into P-1 with the meta substituent occupying the recess.

Discovery of a novel, selective, and orally bioavailable class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position.

A novel class of thrombin inhibitors incorporating aminopyridyl moieties at the P1 position has been discovered. Four of these thrombin inhibitors (13b,c,e and 14d) showed nanomolar potency (Ki 0.8-12 nM), 300-1500-fold selectivity for thrombin compared with trypsin, and good oral bioavailability (F = 40-76%) in rats or dogs. The neutral P1 was expected to increase metabolic stability and oral absorption. Identification of this novel aminopyridyl group at P1 was a key step in our search for a clinical candidate.

Design and synthesis of a series of potent and orally bioavailable noncovalent thrombin inhibitors that utilize nonbasic groups in the P1 position.

As part of an ongoing effort to prepare therapeutically useful orally active thrombin inhibitors, we have synthesized a series of compounds that utilize nonbasic groups in the P1 position. The work is based on our previously reported lead structure, compound 1, which was discovered via a resin-based approach to varying P1. By minimizing the size and lipophilicity of the P3 group and by incorporating hydrogen-bonding groups on the N-terminus or on the 2-position of the P1 aromatic ring, we have prepared a number of derivatives in this series that exhibit subnanomolar enzyme potency combined with good in vivo antithrombotic and bioavailability profiles. The oxyacetic amide compound 14b exhibited the best overall profile of in vitro and in vivo activity, and crystallographic studies indicate a unique mode of binding in the thrombin active site.

Discovery and development of the novel potent orally active thrombin inhibitor N-(9-hydroxy-9-fluorenecarboxy)prolyl trans-4-aminocyclohexylmethyl amide (L-372,460): coapplication of structure-based design and rapid multiple analogue synthesis on solid support.

Early studies in these laboratories of peptidomimetic structures containing a basic P1 moiety led to the highly potent and selective thrombin inhibitors 2 (Ki = 5.0 nM) and 3 (Ki = 0.1 nM). However, neither attains significant blood levels upon oral administration to rats and dogs. With the aim of improving pharmacokinetic properties via a more diverse database, we devised a resin-based route for the synthesis of analogues of these structures in which the P3 residue is replaced with a range of lipophilic carboxylic amides. Assembly proceeds from the common P2-P1 template 7 linked via an acid-labile carbamate to a polystyrene support. Application of the methodology in a repetitive fashion afforded several interesting analogues out of a collection of some 200 compounds. Among the most potent of the group, N-(9-hydroxy-9-fluorenecarboxy)-prolyl trans-4-aminocyclohexylmethyl amide (L-372,460 8, Ki = 1.5 nM), in addition to being fully efficacious in a rat model of arterial thrombosis at an infusion rate of 10 micrograms/kg/min, exhibits oral bioavailability of 74% in dogs, and oral bioavailability of 39% in monkeys with a serum half-life of just under 4 h. On the basis of its favorable biological properties, inhibitor 8 has been subject to further evaluation as a possible treatment for thrombogenic disorders.

Synthesis of a series of potent and orally bioavailable thrombin inhibitors that utilize 3,3-disubstituted propionic acid derivatives in the P3 position.

As part of an effort to prepare efficacious and orally bioavailable analogs of the previously reported thrombin inhibitors 1a, b, we have synthesized a series of compounds that utilize 3,3-disubstituted propionic acid derivatives as P3 ligands. By removing the N-terminal amino group, the general oral bioavailability of this class of compounds was enhanced without excessively increasing the lipophilicity of the compounds. The overall properties of the molecules could be drastically altered depending on the nature of the groups substituted onto the 3-position of the P3 propionic acid moiety. A number of the compounds exhibited good oral bioavailability in rats and dogs, and numerous compounds were efficacious in a rat FeCl3-induced model of arterial thrombosis. Compound 7, the 3,3-diphenylpropionic acid derivative, showed the best overall profile of in vivo and in vitro activity. Molecular modeling studies suggest that these compounds bind in the thrombin active site in a manner essentially identical to that previously reported for compound 1a.

Efficacious, orally bioavailable thrombin inhibitors based on 3-aminopyridinone or 3-aminopyrazinone acetamide peptidomimetic templates.

We have addressed the key deficiency of noncovalent pyridinone acetamide thrombin inhibitor L-374,087 (1), namely, its modest half-lives in animals, by making a chemically stable 3-alkylaminopyrazinone bioisostere for its 3-sulfonylaminopyridinone core. Compound 3 (L-375,378), the closest aminopyrazinone analogue of 1, has comparable selectivity and slightly decreased efficacy but significantly improved pharmacokinetics in rats, dogs, and monkeys to 1. We have developed an efficient and versatile synthesis of 3, and this compound has been chosen for further preclinical and clinical development.

Chevalier Jackson lecture. Esophagology: an update.

Because of the great interest in the problem of gastroesophageal reflux, much emphasis has been placed on factors which control the competence of the lower esophageal sphincter (LES). Much study has been devoted to the effect of hormones and drugs on the LES. Of the various diagnostic methods available, 24-hour pH testing seems to offer the most information regarding reflux and its complications. Medical therapy should be given a careful trial before surgical procedures for reflux are considered. Diagnostic esophagoscopy is usually performed with fiberoptic instruments. Open-tube esophagoscopes are still preferable for most types of endoscopy is usually performed with fiberoptic instruments. Open-tube esophagoscopes are still preferable for most types of endoscopic therapy. However, flexible fiberscopes may be used for injection of esophageal varices and for laser coagulation of bleeding lesions or even tumors. Guide wires may be introduced through flexible scopes to aid in the dilation of esophageal strictures and also to aid in the placement of prosthetic tubes in patients with obstructing cancers. The performance of esophagoscopy by practitioners of several disciplines has resulted in fragmentation of the specialty. Ideally, all esophagoscopists should be competent with both open-tube and fiberoptic scopes and should be familiar with all of the newer knowledge of the physiology and pharmacology of the esophagus and its sphincters.

Designing serological surveillance programmes to document freedom from disease with special reference to exotic viral diseases of pigs in Denmark.

Surveillance programmes based on laboratory screening tests are increasingly used to document freedom from disease in order to facilitate trade. The following aspects must be considered when designing such programmes: diseases to be selected; epidemiology of the diseases; unit of analysis (animal or herd); target age group (or target farm type); test characteristics and sample size. Issues related to these aspects are discussed and illustrated using the example of serological surveillance for exotic viral diseases in the pig population of Denmark. Sampling designs based on individual animal samples are compared with herd-based sampling (two-stage sampling). While the latter is likely to require a larger sample size, the increased level of information and the reliability of the results obtained are considered to be worth the expense. Issues related to the development of international standards for declaring freedom from disease are discussed. The authors conclude that international standards are desirable, providing that these standards represent scientifically valid principles.

Evaluation of the antibacterial residue surveillance programme in Danish pigs using Bayesian methods.

Residues of pharmacological active substances or their metabolites might be found in food products from food-producing animals. Maximum Residue Limits for pharmacological active substances in foodstuffs of animal origin are established to assure high food safety standards. Each year, more than 20,000 samples are analysed for the presence of antibacterial residues in Danish pigs. This corresponds to 0.1% of the size of the slaughter pig population and more than 1% of the sows slaughtered. In this study, a Bayesian model was used to evaluate the Danish surveillance system accuracy and to investigate the impact of a potential risk-based sampling approach to the residue surveillance programme in Danish slaughter pigs. Danish surveillance data from 2005 to 2009 and limited knowledge about true prevalence and test sensitivity and specificity were included in the model. According to the model, the true antibacterial residue prevalence in Danish pigs is very low in both sows (∼0.20%) and slaughter pigs (∼0.01%). Despite data constraints, the results suggest that the current screening test used in Denmark presents high sensitivity (85-99%) and very high specificity (>99%) for the most relevant antibacterial classes used in Danish pigs. If high-risk slaughter pigs could be identified by taking into account antibacterial use or meat inspection risk factors, a potential risk-based sampling approach to antibacterial residue surveillance in slaughter pigs would allow reducing the sample size substantially, while increasing or maintaining the probability of detection. Hence, the antibacterial residue surveillance programme in Danish pigs would be more cost-effective than today.