Epidemiology and control of an outbreak of foot-and-mouth disease in the Republic of Ireland in 2001.

An outbreak of foot-and-mouth disease was confirmed in a flock of sheep on a farm in the Cooley peninsula, County Louth, on March 22, 2001. The virus was similar to other viruses of the serotype O PanAsian strain and virtually indistinguishable from other isolates from Northern Ireland and Great Britain. The epidemiological evidence suggested that infected sheep brought from Great Britain on February 19, 2001, were the source of the infection. The disease was eradicated by epidemiological investigation, serological testing and extensive culling.

Control of foot and mouth disease: lessons from the experience of Ireland.

The authors outline the measures applied in Ireland to prevent the introduction of foot and mouth disease (FMD) from the United Kingdom, France and The Netherlands, to stamp out the disease in Ireland following confirmation of an outbreak on 22 March 2001 and to conduct serological surveillance in order to prove freedom from the disease. Successful control was the result of prompt action and extensive culling in the area around the infected premises. This was achieved by the State Veterinary Service operating with the assistance of the personnel and equipment resources of many Government departments, private industry and private veterinary practitioners, with the co-operation of the farming community and general public. In order to ensure effective use of these resources, good systems of communication and information technology are vital, as are the existence of detailed contingency plans and trained staff.

Modeling of deoxy- and dideoxyaldohexopyranosyl ring puckering with MM3(92).

Extensive variations of the ring structures of three deoxyaldohexopyranoses, L-fucose, D-quinovose, and L-rhamnose, and four dideoxyaldohexopyranoses, D-digitoxose, abequose, paratose, and tyvelose, were studied by energy minimization with the molecular mechanics algorithm MM3(92). Chair conformers, 4C(1) in D-quinovose and the equivalent 1C(4) in L-fucose and L-rhamnose, overwhelmingly dominate in the three deoxyhexoses; in the D-dideoxyhexoses, 4C(1) is again dominant, but with increased amounts of 1C(4) forms in the alpha anomers of the three 3,6-dideoxyhexoses, abequose, paratose, and tyvelose and in both alpha and beta anomers of the 2,6-dideoxyhexose D-digitoxose. In general, modeled proton-proton coupling constants agreed well with experimental values. Computed anomeric ratios strongly favor the beta configuration except for D-digitoxose, which is almost equally divided between alpha and beta configurations, and L-rhamnose, where the beta configuration is somewhat favored. MM3(92) appears to overstate the prevalence of the equatorial beta anomer in all three deoxyhexoses, as earlier found with fully oxygenated aldohexopyranoses.

Measuring service-specific performance and educational value within a general surgery residency: the power of a prospective, anonymous, Web-based rotation evaluation system in the optimization of resident satisfaction.

BACKGROUND: We used a Web-based evaluation system to institute specific changes to various clinical teaching services in our integrated residency in an effort to optimize the overall quality of the educational experience and measured the resident satisfaction in these rotations. METHODS: Residents rated 8 categories of experience on a scale of 1 to 5 (maximum summation score, 40 points). Data were analyzed by t-test for equality of means. A probability value of less than.05 was considered significant. RESULTS: Compliance with completion of the evaluations was 100%. The Chronbach's alpha reliability coefficient of the tool was 0.826. Tukey's estimate of power to achieve additivity was 1.5. Six under-performing services were re-engineered with prominent effects on 7 postgraduate year (PGY) rotations. On 2 general surgery services at 1 hospital, the workload was redistributed, and a dedicated team teaching time was instituted (PGY-3 [a]: before, 22 points/after, 31 points; P =.003; PGY-3 [b]: before, 25 points/after, 31 points; P =.004; PGY-1: before, 24 points/after, 29 points; P =.07). A general surgery service at another hospital redistributed coverage of the attending surgeons to create a nonteaching service (PGY-1: before, 22 points/-after, 27 points; P =.01). The transplantation service (PGY-3) was examined, and the role of the point was redefined (before, 24 points/after, 31 points; P =.01). One vascular service (PGY-2) redistributed cases and workload (before, 27 points/after, 22 points; P =.07). The vascular PGY-2 position was eliminated and replaced by a mid-level practitioner. The cardiothoracic service (PGY-1) rotation was converted into a preceptorship (before, 23 points/after, 30 points; P =.015). CONCLUSIONS: A web-based clinical rotation evaluation provides a means for the assessment of the impact of programmatic changes while preserving resident anonymity and maintaining accountability.

Polysaccharide recognition by surfactant protein D: novel interactions of a C-type lectin with nonterminal glucosyl residues.

Surfactant protein D (SP-D), a C-type lectin, is an important pulmonary host defense molecule. Carbohydrate binding is critical to its host defense properties, but the precise polysaccharide structures recognized by the protein are unknown. SP-D binding to Aspergillus fumigatus is strongly inhibited by a soluble beta-(1-->6)-linked but not by a soluble beta-(1-->3)-linked glucosyl homopolysaccharide (pustulan and laminarin, respectively), suggesting that SP-D recognizes only certain polysaccharide configurations, likely through differential binding to nonterminal glucosyl residues. In this study we have computationally docked alpha/beta-D-glucopyranose and alpha/beta-(1-->2)-, alpha/beta-(1-->3)-, alpha/beta-(1-->4)-, and alpha/beta-(1-->6)-linked glucosyl trisaccharides into the SP-D carbohydrate recognition domain. As with the mannose-binding proteins, we found significant hydrogen bonding between the protein and the vicinal, equatorial OH groups at the 3 and 4 positions on the sugar ring. Our docking studies predict that alpha/beta-(1-->2)-, alpha-(1-->4)-, and alpha/beta-(1-->6)-linked but not alpha/beta-(1-->3)-linked glucosyl trisaccharides can be bound by their internal glucosyl residues and that binding also occurs through interactions of the protein with the 2- and 3-equatorial OH groups on the glucosyl ring. By using various soluble glucosyl homopolysaccharides as inhibitors of SP-D carbohydrate binding, we confirmed the interactions predicted by our modeling studies. Given the sequence and structural similarity between SP-D and other C-type lectins, many of the predicted interactions should be applicable to this protein family.

Beyond requirements: residency management through the Internet.

HYPOTHESIS: An Internet application could collect information to satisfy documentation required by the Residency Review Committee. Beyond replacing a difficult and inefficient paper system, it would collect, process, and distribute information to administration, faculty, and residents. DESIGN: Descriptive study. SETTING: An integrated residency of 18 services at a university teaching hospital with 4 affiliated institutions. PARTICIPANTS: Residency administrators, faculty, and residents. INTERVENTIONS: The application included a procedure recorder, resident evaluation of faculty and rotations, goals and objectives (stratified by service and resident level), and matching faculty evaluation of residents with these goals as competencies. Policies, schedules, research opportunities, clinical site information, and curriculum support were created. MAIN OUTCOME MEASURES: Degree of compliance with Residency Review Committee standards, number of deficiencies corrected, and quantity and quality of information available to administration, faculty, and residents. RESULTS: The Internet system increased resident compliance for faculty and rotation evaluations from 20% and 34%, respectively, to 100%, which was maintained for 22 months. These evaluations can be displayed individually, in summary grids, and as postgraduate year-specific averages. Faculty evaluations of residents can be reviewed throughout the system. The defined category report for procedures, which had deficiencies in the preceding 6 years, had none for the last 2 years. The Internet application provides Accreditation Council for Graduate Medical Education-validated operative logs to regulatory agencies. CONCLUSIONS: A Web-based system can satisfy requirements and provide processed data that are of better quality and more complete than our paper system. We are now able to use scarce time and personnel to nurture developing surgical residents instead of shuffling paper.

Genetic typing of ruminant pestivirus strains from Northern Ireland and the Republic of Ireland.

A study was performed to investigate the genotypes and sub-groups of pestiviruses present in ruminants in Ireland. These comprised one ovine and eighteen bovine pestiviruses from Northern Ireland and six bovine pestiviruses from the Republic of Ireland. A 288 base pair (bp) portion of the 5'-non coding region (5'-NCR) from each of 25 pestiviruses collected over a period of 31 years was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) and the product directly sequenced. From each pestivirus, nucleotide sequences corresponding to bases 130 to 374 of the 5'-NCR of NADL were aligned and compared with each other and with the corresponding sequences of a number of reference, field or vaccinal strains of BVDV types I and II, border disease virus and classical swine fever virus. All of the 25 sequenced pestiviruses were found to be BVDV type Ia. These were closely related to the constituent viruses of the 2 inactivated vaccines currently licensed for use in Northern Ireland and to recent bovine isolates from England.

Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability.

Three single-residue mutations, Asp71-->Asn, Gln409-->Pro and Gly447-->Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28- Val29-Ser30-->Asn-Pro-Pro (23-30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301-->Ala-G ly-Ala (297-301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Delta439-441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N:-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O:-glycosylation in the already highly O:-glycosylated belt region that extends around the globular catalytic domain. The 23-30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297-301 replacement mutant GA was made to understand the function of the random coil region between alpha-helices 9 and 10. Delta439-441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23-30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4 degrees C increase in operating temperature at constant thermostability.

Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site.

The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.

Characterization of kinetics and thermostability of Acremonium strictum glucooligosaccharide oxidase.

The kinetic and thermostability properties of a glucooligosaccharide oxidase from Acremonium strictum were determined. This enzyme produces only maltobionic acid from maltose. It is most active at pH 9 to 10.5, and is most stable at pH 6.5. Values of both K(M) and V(max) on maltose are highest at pH 10. The highest values of K(M) and V(max) occur with glucose, maltopentaose, and maltoheptaose, whereas the lowest values of K(M) are with maltotriose and of V(max) are with maltohexaose. Values of K(M) with any substrate and at any pH are always substantially above 1 mM. Activation energies for catalysis and thermoinactivation are 23 kJ/mol and 421 kJ/mol, respectively. The N-terminal sequence is not homologous with any other oxidase, but has some homology with other proteins having different functions. These unusual properties suggest that glucooligosaccharides may not be the primary substrates of this enzyme.

Automated docking of maltose, 2-deoxymaltose, and maltotetraose into the soybean beta-amylase active site.

In this study, products and substrates were docked into the active site of beta-amylase using the simulated annealing algorithm AutoDock. Lowest-energy conformers reproduced known crystallographic atom positions within 0.4 to 0.8 A rmsd. Docking studies were carried out with both open and closed configurations of the beta-amylase mobile flap, a loop comprising residues 96 to 103. Ligands with two rings docked within the cleft near the active site when the flap was open, but those with four rings did not. The flap must be closed for alpha-maltotetraose to adopt a conformation allowing it to dock near the crystallographically determined subsites. The closed flap is necessary for productive but not for nonproductive binding, and therefore it plays a essential role in catalysis. The gain in total binding energy upon closing of the flap for alpha-maltose docked to subsites -2, -1 and +1, +2 is about 22 kcal/mol, indicating more favorable interactions are possible with the flap closed. Larger intermolecular interaction energies are observed for two alpha-maltose molecules docked to subsites -2, -1 and +1, +2 than for one alpha-maltotetraose molecule docked from subsites -2 to +2, suggesting that it is only upon cleavage of the alpha-1,4 linkage that optimal closed-flap binding can occur with the crytallographically determined enzyme structure.

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20-->Cys/Ala27-->Cys, Ser30-->Pro, Gly137-->Ala, 311-4 loop, Ser411-->Ala and Ser436-->Pro.

Six previously constructed and nine newly constructed Aspergillus awamori glucoamylases with multiple mutations made by combining existing single mutations were tested for their ability to produce glucose from maltodextrins. Multiple mutations have cumulative effects on glucose yield, specific activity and thermostability. No general correlation between glucose yield and thermostability was observed, although mutations that presumably impede unfolding at high temperatures uniformly increase thermostability and generally increase glucose yield. Peak glucose yields decrease with increasing temperature. The best combination of high glucose yield, high specific activity and high thermostability occurs in Asn20-->Cys/Ala27-->Cys/Ser30-->Pro/Gly137-->Ala glucoamylase.

Mutations to alter Aspergillus awamori glucoamylase selectivity. III. Asn20-->Cys/Ala27-->Cys, Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Lys108-->Met, Gly137-->Ala, 311-314 Loop, Tyr312-->Trp and Ser436-->Pro.

Mutations Asn20-->Cys/Ala27-->Cys (SS), Ala27-->Pro, Ser30-->Pro, Lys108-->Arg, Gly137-->Ala, Tyr312-->Trp and Ser436-->Pro in Aspergillus awamori glucoamylase, along with a mutation inserting a seven-residue loop between Tyr311 and Gly314 (311-314 Loop), were made to increase glucose yield from maltodextrin hydrolysis. No active Lys108-->Met glucoamylase was found in the supernatant after being expressed from yeast. Lys108-->Arg, 311-314 Loop and Tyr312-->Trp glucoamylases have lower activities than wild-type glucoamylase; other GAs have the same or higher activities. SS and 311-314 Loop glucoamylases give one-quarter to two-thirds the relative rates of isomaltose formation from glucose compared with glucose formation from maltodextrins at 35, 45 and 55 degrees C, correlating with up to 2% higher peak glucose yields from 30% (w/v) maltodextrin hydrolysis. Conversely, Lys108-->Arg glucoamylase has relative isomaltose formation rates three times higher and glucose yields up to 4% lower than wild-type glucoamylase. Gly137-->Ala and Tyr312-->Trp glucoamylases also give high glucose yields at higher temperatures. Mutated glucoamylases that catalyze high rates of isomaltose formation give higher glucose yields from shorter than from longer maltodextrins, opposite to normal experience with more efficient glucoamylases.

Mutations to alter Aspergillus awamori glucoamylase selectivity. I. Tyr48Phe49-->Trp, Tyr116-->Trp, Tyr175-->Phe, Arg241-->Lys, Ser411-->Ala and Ser411-->Gly.

Glucoamylase mutations to reduce isomaltose formation from glucose condensation and thus increase glucose yield from starch hydrolysis were designed to produce minor changes in the active site at positions not totally conserved. Tyr175-->Phe and Ser411-->Gly glucoamylases had catalytic efficiencies on DP 2-7 maltooligosaccharides like those of wild-type glucoamylase, while the catalytic efficiencies of Tyr116-->Trp, Arg241-->Lys and Ser411-->Ala glucoamylases were reduced by about half and Tyr48Phe49-->Trp glucoamylase had little remaining activity. Tyr175-->Phe, Ser411-->Ala and Ser411-->Gly glucoamylases had decreased ratios of the initial rate of isomaltose formation from glucose condensation to that of glucose formation from maltodextrin hydrolysis at both 35 and 55 degrees C compared with wild-type glucoamylase. Arg241-->Lys glucoamylase had a very similar ratio, while Tyr116-->Trp glucoamylase had a higher ratio. The highest glucose yields from maltodextrin hydrolysis were by the mutant glucoamylases having the lowest ratios of initial rates of isomaltose formation to glucose formation and this predicted high glucose yields better than the ratio of catalytic efficiency for maltose hydrolysis to that for isomaltose hydrolysis.

Mutations to alter Aspergillus awamori glucoamylase selectivity. II. Mutation of residues 119 and 121.

Mutations Ser119-->Glu, Ser119-->Gly, Ser119-->Trp, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly were constructed in glucoamylase to change substrate specificity. Mutation Ser411-->Gly was already known to decrease glucoamylase selectivity toward isomaltose formation and to increase peak glucose yield. All mutated glucoamylases had slightly lower specific activities on maltose than on wild-type glucoamylase. Ser119-->Glu, Ser119-->Gly and Ser119-->Trp glucoamylases were about as active on isomaltose and DP 4-7 maltooligosaccharides as wild-type glucoamylase. Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases were less active. At 55 degrees C Ser119-->Glu, wild-type, Ser119-->Trp, Ser119-->Gly, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases had progressively higher peak glucose yields, generally in the opposite order to their activities. There was also an inverse correlation between peak glucose yield and ratio of initial rate of isomaltose production from glucose condensation to that of glucose production from maltodextrin hydrolysis. The effect of mutations Gly121-->Ala and Ser411-->Gly was not additive in predicting the effect of the double mutation on the ratio or on peak glucose yield.

Glucoamylase structural, functional, and evolutionary relationships.

To correlate structural features with glucoamylase properties, a structure-based multisequence alignment was constructed using information from catalytic and starch-binding domain models. The catalytic domain is composed of three hydrophobic folding units, the most labile and least hydrophobic of them being missing in the most stable glucoamylase. The role of O-glycosylation in stabilizing the most hydrophobic folding unit, the only one where thermostabilizing mutations with unchanged activity have been made, is described. Differences in both length and composition of interhelical loops are correlated with stability and selectivity characteristics. Two new glucoamylase subfamilies are defined by using homology criteria. Protein parsimony analysis suggests an ancient bacterial origin for the glucoamylase gene. Increases in length of the belt surrounding the active site, degree of O-glycosylation, and length of the linker probably correspond to evolutionary steps that increase stability and secretion levels of Aspergillus-related glucoamylases.

Automated docking of glucosyl disaccharides in the glucoamylase active site.

To better understand the molecular basis of glucomylase selectivity, low-energy conformers of glucosyl disaccharides obtained from relaxed-residue conformational mapping were flexibly docked into the glucoamylase active site using AutoDock 2.2. This procedure ensures that significant conformational space is searched and can produce bound structures comparable to those obtained by protein crystallography. alpha-Linked glucosyl disaccharides except alpha,alpha-trehalose dock easily into the active site while exclusively beta-linked disaccharides do not, explaining why only the former are glucoamylase substrates. The optimized docking modes are similar at the nonreducing end of the different substrates. Individual atomic energies of intermolecular interaction allow the definite identification of key hydroxyl groups for each substrate. This approach confirmed the versatility of the second subsite of the glucoamylase active site in binding different substrates.

Automated docking of monosaccharide substrates and analogues and methyl alpha-acarviosinide in the glucoamylase active site.

Glucoamylase is an important industrial glucohydrolase with a large specificity range. To investigate its interaction with the monosaccharides D-glucose, D-mannose, and D-galactose and with the substrate analogues 1-deoxynojirimycin, D-glucono-1,5-lactone, and methyl alpha-acarviosinide, MM3(92)-optimized structures were docked into its active site using AutoDock 2.1. The results were compared to structures of glucoamylase complexes obtained by protein crystallography. Charged forms of some substrate analogues were also docked to assess the degree of protonation possessed by glucoamylase inhibitors. Many forms of methyl alpha-acarviosinide were conformationally mapped by using MM3(92), characterizing the conformational pH dependence found for the acarbose family of glucosidase inhibitors. Their significant conformers, representing the most common states of the inhibitor, were used as initial structures for docking. This constitutes a new approach for the exploration of binding modes of carbohydrate chains. Docking results differ slightly from x-ray crystallographic data, the difference being of the order of the crystallographic error. The estimated energetic interactions, even though agreeing in some cases with experimental binding kinetics, are only qualitative due to the large approximations made by AutoDock force field.

The pathomechanics of chronic, recurrent cervical nerve root neurapraxia. The chronic burner syndrome.

This study defined chronic recurrent cervical nerve root neurapraxia, the chronic burner syndrome, characterized the clinical findings, and described the responsible pathomechanics. We studied a subset of 55 athletes (mean age, 22 years) for evaluation of recurrent burners. Eleven subjects were professional athletes. The mechanism of injury was extension combined with ipsilateral-lateral deviation in 46 patients (83%). Spurling's sign was positive in 39 patients (70%). Twenty-nine patients (53%) had developmentally narrowed cervical canals, and 48 patients (87%) had evidence of disk disease by magnetic resonance imaging. The disk disease was in the form of a disk bulge, disk protrusion, or a frank disk herniation deforming the cord. Fifty-one patients (93%) had disk disease or narrowing of the intervertebral foramina secondary to degenerative disk disease. Although burners may be the result of a brachial plexus stretch injury in high school and collegiate football players seen with acute symptoms, nerve root compression in the intervertebral foramina secondary to disk disease is a more common cause in collegiate and professional players who have recurrent or chronic burner syndromes. There is a high incidence of cervical canal stenosis in football players with recurrent burner syndrome. The combination of disk disease and cervical spinal canal stenosis may lead to an alteration in normal cervical spine mechanics that may make these athletes more prone to chronic burner syndromes.