Comparison of calculation methods used for the determination of anthelmintic resistance in sheep in a temperate continental climate.

This study compared results obtained with five different fecal egg count reduction (FECR) calculation methods for defining resistance to ivermectin, fenbendazole, and levamisole in gastrointestinal nematodes of sheep in a temperate continental climate: FECR1 and FECR2 used pre-and posttreatment fecal egg count (FEC) means from both treated and control animals, but FECR1 used arithmetic means, whereas FECR2 used geometric means; FECR3 used arithmetic means for pre- and posttreatment FECs from treated animals only; FECR4 was calculated using only arithmetic means for posttreatment FECs from treated and control animals; and FECR5 was calculated using mean FEC estimates from a general linear mixed model. The classification of farm anthelmintic resistance (AR) status varied, depending on which FECR calculation method was used and whether a bias correction term (BCT, i.e., half the minimum detection limit) was added to the zeroes or not. Overall, agreement between all methods was higher when a BCT was used, particularly when levels of resistance were low. FECR4 showed the highest agreement with all the other FECR methods. We therefore recommend that small ruminant clinicians use the FECR4 formula with a BCT for AR determination, as this would reduce the cost of the FECRT, while still minimizing bias and allowing for comparisons between different farms. For researchers, we recommend the use of FECR1 or FECR2, as the inclusion of both pre- and posttreatment FECs and use of randomly allocated animals in treatment and control groups makes these methods mathematically more likely to estimate the true anthelmintic efficacy.

Comparison of calculation methods used for the determination of anthelmintic resistance in sheep in a temperate continental climate.

This study compared results obtained with five different fecal egg count reduction (FECR) calculation methods for defining resistance to ivermectin, fenbendazole, and levamisole in gastrointestinal nematodes of sheep in a temperate continental climate: FECR1 and FECR2 used pre- and posttreatment fecal egg count (FEC) means from both treated and control animals, but FECR1 used arithmetic means, whereas FECR2 used geometric means; FECR3 used arithmetic means for pre- and posttreatment FECs from treated animals only; FECR4 was calculated using only arithmetic means for posttreatment FECs from treated and control animals; and FECR5 was calculated using mean FEC estimates from a general linear mixed model. The classification of farm anthelmintic resistance (AR) status varied, depending on which FECR calculation method was used and whether a bias correction term (BCT, i.e., half the minimum detection limit) was added to the zeroes or not. Overall, agreement between all methods was higher when a BCT was used, particularly when levels of resistance were low. FECR4 showed the highest agreement with all the other FECR methods. We therefore recommend that small ruminant clinicians use the FECR4 formula with a BCT for AR determination, as this would reduce the cost of the FECRT, while still minimizing bias and allowing for comparisons between different farms. For researchers, we recommend the use of FECR1 or FECR2, as the inclusion of both pre- and posttreatment FECs and use of randomly allocated animals in treatment and control groups makes these methods mathematically more likely to estimate the true anthelmintic efficacy.

Evaluation of a short training course of chest X-ray interpretation for the diagnosis of paediatric TB.

BACKGROUND: Chest X-ray (CXR) interpretation is challenging for the diagnosis of paediatric TB. We assessed the performance of a three half-day CXR training module for healthcare workers (HCWs) at low healthcare levels in six high TB incidence countries. METHODS: Within the TB-Speed Decentralization Study, we developed a three half-day training course to identify normal CXR, CXR of good quality and identify six TB-suggestive features. We performed a pre-post training assessment on a pre-defined set of 20 CXR readings. We compared the proportion of correctly interpreted CXRs and the median reading score before and after the training using the McNemar test and a linear mixed model. RESULTS: Of 191 HCWs, 43 (23%) were physicians, 103 (54%) nurses, 18 (9.4%) radiology technicians and 12 (6.3%) other professionals. Of 2,840 CXRs with both assessment, respectively 1,843 (64.9%) and 2,277 (80.2%) were correctly interpreted during pre-training and post-training (P < 0.001). The median reading score improved significantly from 13/20 to 16/20 after the training, after adjusting by country, facility and profession (adjusted ß = 3.31, 95% CI 2.44-4.47). CONCLUSION: Despite some limitations of the course assessment that did not include abnormal non-TB suggestive CXR, study findings suggest that a short CXR training course could improve HCWs' interpretation skills in diagnosing paediatric TB.

CONTEXTE: L'interprétation de la radiographie thoracique (CXR) est un défi pour le diagnostic de la TB pédiatrique. Nous avons évalué la performance d'un module de formation de trois demi-journées sur la CXR destiné aux agents de santé (HCWs) dans six pays où l'incidence de la TB est élevée et où les ressources en services de santé sont limitées. MÉTHODES: Dans le cadre de l'étude de décentralisation TB-Speed, nous avons mis au point un cours de formation de trois demi-journées pour identifier une CXR normale, une CXR de bonne qualité et six caractéristiques suggestives de la TB. Nous avons effectué une évaluation avant et après la formation sur un ensemble prédéfini de 20 clichés radiologiques. Nous avons comparé la proportion de CXR correctement interprétées et le score médian de lecture avant et après la formation à l'aide du test de McNemar et d'un modèle linéaire mixte. RÉSULTATS: Sur les 191 HCWs, 43 (23%) étaient des médecins, 103 (54%) des infirmières, 18 (9,4%) des techniciens en radiologie et 12 (6,3%) d'autres professionnels. Sur 2 840 CXR avec les deux évaluations, respectivement 1 843 (64,9%) et 2 277 (80,2%) ont été correctement interprétées avant et après la formation (P < 0,001). Le score médian de lecture s'est amélioré de manière significative, passant de 13/20 à 16/20 après la formation, après ajustement par pays, établissement et profession (ß ajusté = 3,31; IC 95% 2,44­4,47). CONCLUSION: Malgré certaines limites de l'évaluation du cours qui n'incluait pas de CXR anormale non évocatrice de TB, les résultats de l'étude suggèrent qu'une formation courte sur la CXR pourrait améliorer les compétences d'interprétation des HCWs dans le diagnostic de la TB pédiatrique.

Systematic review with meta-analysis: enteral nutrition therapy for the induction of remission in paediatric Crohn's disease.

BACKGROUND: Despite potential adverse-events in a paediatric population, corticosteroids are used to induce remission in paediatric Crohn's disease. Exclusive enteral nutrition also induces remission, but is infrequently used in the USA because corticosteroids are considered the superior therapy. New data have become available since the publication of the most recent meta-analysis in 2007. AIM: To see if current literature supports the use of EEN versus CS in paediatric populations. METHODS: All studies with comparator arms of exclusive enteral nutrition and an exclusive corticosteroids, with remission clearly defined were identified by searching eight online databases. RESULTS: Of 2795 identified sources, nine studies met our inclusion criteria. Eight of these (n = 451), had data that could be abstracted into our meta-analysis. Exclusive enteral nutrition was as effective as corticosteroids in inducing remission (OR = 1.26 [95% CI 0.77, 2.05]) in paediatric Crohn's disease. There was no difference between Exclusive enteral nutrition and corticosteroids efficacy when comparing newly diagnosed Crohn's (OR = 1.61 [95% CI .87, 2.98]) or relapsed (OR = 0.76 [95% CI .29-1.98]). Intestinal healing was significantly more likely among patients receiving Exclusive enteral nutrition compared to corticosteroids (OR = 4.5 [95% CI 1.64, 12.32]). There was no difference in the frequency of biomarker normalisation including CRP (OR = 0.85 [95% CI .44, 1.67]) and faecal calprotectin (OR 2.79 [95% CI .79-10.90]). CONCLUSIONS: There is no difference in efficacy between exclusive enteral nutrition and corticosteroids in induction of remission in Crohn's disease in a paediatric population. Exploratory analyses suggest that a greater proportion of patients treated with exclusive enteral nutrition achieved mucosal healing.

A census to determine the prevalence and risk factors for caprine arthritis-encephalitis virus and visna/maedi virus in the Swiss goat population.

In Switzerland, viruses belonging to two different phylogenetic groups of small ruminant lentiviruses (SRLV) are currently circulating: the caprine arthritis-encephalitis virus (CAEV) and visna/maedi virus (VMV). In the past two decades, a mandatory national control program has led to a very low prevalence of seropositivity, while completely eliminating CAE as a clinical manifestation. However, in order to reduce the high costs and effort associated with this program, adjustments based on the most recent epidemiological knowledge are needed. The purpose of this study was to estimate the seroprevalence of CAEV and VMV using the newest diagnostic tools available, and to identify potential risk factors for infection with these viruses in Switzerland. For the prevalence estimation, a census was carried out including 10,696 farms with a total of 85,454 goats. Blood samples were analysed using a 3-step serological testing algorithm consisting of Chekit ELISA, Western Blot and SU5 ELISA. A risk factor analysis was conducted using logistic regression models built with data obtained from a mail questionnaire, and serological results from the census. The apparent herd-level prevalences were 0.38%, 2.77%, and 3.04% for CAEV, VMV and SRLV, respectively. Animal-level prevalences were 0.06% for CAEV, 0.55% for VMV, and 0.61% for SRLV. No statistically significant risk factors associated with CAEV or VMV infection were identified. However, the proportional high number of CAEV seropositive dwarf goats, in relation to their population size, could indicate that these hobby breeds may slip through some of the official controls. For an infection with SRLV, a medium herd size (7-40 goats) was found to be protective, compared with smaller (OR=1.90, p=0.034) and larger herds (OR=1.95, p=0.038). In conclusion, considering that all CAEV positive animals were culled, these results imply that CAEV is no longer actively spreading and has successfully been controlled in Switzerland. However, given the uncertain pathogenic potential of VMV in goats, future surveillance should also be taking into account the not insignificant number of VMV circulating in the Swiss goat population.

Use of a modified Delphi panel to identify and weight criteria for prioritization of zoonotic diseases in Switzerland.

Zoonotic diseases have a significant impact on public health globally. To prevent or reduce future zoonotic outbreaks, there is a constant need to invest in research and surveillance programs while updating risk management strategies. However, given the limited resources available, disease prioritization based on the need for their control and surveillance is important. This study was performed to identify and weight disease criteria for the prioritization of zoonotic diseases in Switzerland using a semi-quantitative research method based on expert opinion. Twenty-eight criteria relevant for disease control and surveillance, classified under five domains, were selected following a thorough literature review, and these were evaluated and weighted by seven experts from the Swiss Federal Veterinary Office using a modified Delphi panel. The median scores assigned to each criterion were then used to rank 16 notifiable and/or emerging zoonoses in Switzerland. The experts weighted the majority of the criteria similarly, and the top three criteria were Severity of disease in humans, incidence and prevalence of the disease in humans and treatment in humans. Based on these weightings, the three highest ranked diseases were Avian Influenza, Bovine Spongiform Encephalitis, and Bovine Tuberculosis. Overall, this study provided a preliminary list of criteria relevant for disease prioritization in Switzerland. These were further evaluated in a companion study which involved a quantitative prioritization method and multiple stakeholders.

PCR generation of large amounts of purified DNA.

The preparation and purification of PCR generated DNA fragments suitable for footprinting and classical drug binding studies is described. One of the fragments, a 214-mer derived from pBR322 DNA exhibits a biphasic melting profile. This behavior appears to be due to a non-random distribution of base pairs within the fragment causing a region rich in AT base pairs to melt prior to a segment having a high concentration of GC base pairs. The usefulness of large amounts of PCR generated DNA for footprinting and optical binding studies involving drugs is also presented and discussed.

A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep.

BACKGROUND: Anthelmintic drugs have been widely used in sheep as a cost-effective means for gastro-intestinal nematode (GIN) control. However, growing anthelmintic resistance (AHR) has created a compelling need to identify evidence-based management recommendations that reduce the risk of further development and impact of AHR. OBJECTIVE: To identify, critically assess, and synthesize available data from primary research on factors associated with AHR in sheep. METHODS: Publications reporting original observational or experimental research on selected factors associated with AHR in sheep GINs and published after 1974, were identified through two processes. Three electronic databases (PubMed, Agricola, CAB) and Web of Science (a collection of databases) were searched for potentially relevant publications. Additional publications were identified through consultation with experts, manual search of references of included publications and conference proceedings, and information solicited from small ruminant practitioner list-serves. Two independent investigators screened abstracts for relevance. Relevant publications were assessed for risk of systematic bias. Where sufficient data were available, random-effects Meta-Analyses (MAs) were performed to estimate the pooled Odds Ratio (OR) and 95% Confidence Intervals (CIs) of AHR for factors reported in ≥2 publications. RESULTS: Of the 1712 abstracts screened for eligibility, 131 were deemed relevant for full publication review. Thirty publications describing 25 individual studies (15 observational studies, 7 challenge trials, and 3 controlled trials) were included in the qualitative synthesis and assessed for systematic bias. Unclear (i.e. not reported, or unable to assess) or high risk of selection bias and confounding bias was found in 93% (14/15) and 60% (9/15) of the observational studies, respectively, while unclear risk of selection bias was identified in all of the trials. Ten independent studies were included in the quantitative synthesis, and MAs were performed for five factors. Only high frequency of treatment was a significant risk factor (OR=4.39; 95% CI=1.59, 12.14), while the remaining 4 variables were marginally significant: mixed-species grazing (OR=1.63; 95% CI=0.66, 4.07); flock size (OR=1.02; 95% CI=0.97, 1.07); use of long-acting drug formulations (OR=2.85; 95% CI=0.79, 10.24); and drench-and-shift pasture management (OR=4.08; 95% CI=0.75, 22.16). CONCLUSIONS: While there is abundant literature on the topic of AHR in sheep GINs, few studies have explicitly investigated the association between putative risk or protective factors and AHR. Consequently, several of the current recommendations on parasite management are not evidence-based. Moreover, many of the studies included in this review had a high or unclear risk of systematic bias, highlighting the need to improve study design and/or reporting of future research carried out in this field.

Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: comparison of detection methods for drug resistance.

In 2011, a field study was conducted to assess drug resistance of gastro-intestinal nematodes in sheep flocks in Ontario, Canada. Benzimidazole resistance in Haemonchus contortus was assessed by genetic analysis of eggs; measurement of resistant allele percentages at codons 167, 198 and 200 in the ß-tubulin gene was determined on pools of H. contortus eggs using pyrosequencing. Susceptibility to benzimidazoles in gastro-intestinal nematodes was also determined using a Faecal Egg Count Reduction Test (FECRT) and a Larval Development Assay (LDA). In total, 16 farms were assessed with the genetic test. Based on resistant allele frequencies, all of the farms (16/16) tested had benzimidazole resistance in H. contortus; the overall percentage of benzimidazole-resistant H. contortus (estimated prior to treatment using the Hardy-Weinberg formula) was 68.5%. The FECRT and LDA were performed on 11 and 13 farms, respectively. Resistance to fenbendazole was detected on 100% (11/11) of the farms where the FECRT was performed. The LDA revealed the presence of thiabendazole resistance in H. contortus in 92% (12/13) of the farms. Estimated percentages of resistant parasites in H. contortus populations obtained with the two biological tests and the genetic test were compared. The results of the genetic test were in agreement with the biological tests and confirmed that benzimidazole resistance in H. contortus is present in Ontario sheep flocks. Differences between the different methods of drug resistance detection are discussed in terms of cost, time and sampling.

A longitudinal study on the effect of lambing season on the periparturient egg rise in Ontario sheep flocks.

The epidemiology of the periparturient egg rise (PPER) of gastrointestinal nematodes (GINs) in sheep remains unclear, and may be influenced by the lambing season. This longitudinal study was performed to determine the effect of out-of-season lambing on the PPER in ewes in Ontario, and whether total plasma protein (TPP) and packed cell volume (PCV) were associated with the PPER. Six farms that practiced out-of-season lambing were enrolled, and sampled for three consecutive lambing seasons (winter, spring and autumn). For each lambing season, all farms were visited five times. On the first visit for each lambing season, 15-20 pregnant ewes and 15-20 non-pregnant/early gestation ewes were randomly selected. At each visit, fecal samples were collected from all selected animals and processed individually to measure GIN fecal egg counts (FECs). Blood samples were collected on three visits in each lambing period and processed to measure TPP and PCV. The ewes were classified into one of five production stages (maintenance [i.e. not pregnant], early or late gestation [<120 d and ≥ 120 d, respectively], and early or late lactation [<40 d and ≥ 40 d, respectively]) based on information collected during farm visits. Linear mixed models were developed for the TPP, PCV and logarithmic-transformed FEC (lnFEC). During the winter and spring lambing season, the FECs increased gradually over the gestation period and peaked during lactation, with these increases being larger in ewes with a low PCV (three-way interaction in the final model). In the autumn lambing season, the FECs started off higher in early gestation, and increased rapidly to peak in late gestation, particularly for animals with low PCV levels. In the TPP model, PCV and lnFEC were positively associated with TPP. During both autumn and winter lambing seasons, the TPP decreased from maintenance throughout gestation and early lactation, followed by an increase in late lactation, except for when there were high FECs. During the spring lambing season, TPP peaked at early gestation, and then decreased in late gestation, to increase more gradually over lactation. In the PCV model, PCV increased with TPP and decreased exponentially with increases in lnFEC. The PPER occurred during all three lambing seasons, and its magnitude and distribution varied with the lambing season, suggesting that the PPER in ewes depends on both environmental and animal physiological factors, an important consideration when implementing preventive parasite control strategies on sheep farms that practice out-of-season lambing.

Anthelmintic resistance in sheep flocks in Ontario, Canada.

Gastrointestinal nematodes (GIN) are a significant constraint to pasture-based sheep production worldwide. Anthelmintic resistance (AR) has been reported in most sheep-raising areas in the world, yet little is known about the AR status in Canada. This study was conducted to determine the frequency of AR in GIN in sheep flocks in Ontario, Canada. Forty-seven sheep flocks were enrolled in the study, and their level of parasitism was monitored monthly throughout a grazing season by analyzing owner-acquired fecal samples from 15 grazing lambs per flock. When the mean GIN fecal egg count (FEC) reached a threshold of 200 eggs per gram (epg), oral ivermectin was supplied to producers to check ivermectin efficacy; the reduction in mean FEC 14 days after ivermectin treatment was calculated. 'Drench failure' was defined as a reduction in mean FEC of <95%. In those flocks with apparent drench failure, researchers performed a Fecal Egg Count Reduction Test (FECRT), dividing sheep into 4 treatment groups (n=10-15): control (i.e. untreated), ivermectin, and, if sufficient numbers of animals - fenbendazole and levamisole. AR was defined as a reduction in mean FEC <95% and a lower 95% confidence interval <90%. Larval cultures were performed on pooled post-treatment FECRT samples. Larval Development Assays (LDAs) to detect the presence of resistance to thiabendazole and levamisole were performed prior to the ivermectin drench check on pooled owner-acquired fecal samples that reached the 200 epg threshold. Approximately 89% (42/47) of the farms reached the FEC threshold of 200 epg; 93% (39/42) of these farms performed an ivermectin drench check, and 88% (34/39) of these farms had drench failure. The FECRT was performed on 29 of the 34 farms. Resistance to ivermectin, fenbendazole and levamisole was demonstrated on 97% (28/29), 95% (19/20) and 6% (1/17) of the farms tested, respectively, with considerable variability in resistance levels among farms. Haemonchus sp. was the most commonly cultured parasite from post-treatment fecal samples. LDA results for 21 farms were available; of these, 14% (3/21) and 62% (13/21) had low and high levels of thiabendazole resistance, respectively, while none of the farms exhibited resistance to levamisole. Amongst these tested farms, resistance to both ivermectin and benzimidazoles was very common. These findings strongly suggest that AR, particularly in Haemonchus sp., is a serious problem in these sheep flocks. Thus, marked changes in GIN management need to be instituted immediately to mitigate a worsening situation.

The Cochrane review of assistive technology for rheumatoid arthritis.

AIM: The aim of this systematic review is to summarise the available evidence on the effectiveness of assistive technology for adults with rheumatoid arthritis in terms of improving functional ability and reducing pain, and to assess potential adverse effects related to device use. METHODS: In this review, randomised controlled trials, clinical controlled trials, controlled before and after studies and interrupted time series available through systematic searches (electronic databases, grey literature, contact with authors, reference lists) up to October 2008 were included. Two reviewers independently selected trials for inclusion, assessed the validity of included trials, and extracted data. Investigators were contacted to obtain missing information. RESULTS: Out of 7177 hits, 13 articles were reviewed in full text and only one trial was finally included (N.=29). The study was a randomised crossover trial, in which the use of an eye drop device was compared to a standard bottle in people with rheumatoid arthritis suffering from persistent dry eyes. The results show that the eye drop device improved application of eye drops and prevented adverse effects in terms of touching the eye with the bottle tip. The study was considered to have low quality of evidence. CONCLUSION: Since only one trial met the inclusion criteria for this review, there is very limited evidence for the effect of assistive technology for adults with rheumatoid arthritis. There is an urgent need for high-quality research in this field, in order to reach sufficient evidence on the effectiveness of this commonly used intervention.

REALISE-ing their potential?: Implementing local library projects to support evidence-based health care.

Librarian involvement in Evidence-based Health Care provides many opportunities at a local level. Unfortunately, the potential for innovative projects to inform future developments is generally lost by a failure to 'pass the baton'--to identify lessons learnt and transferable principles. The 'Library Support for Evidence-based Health Care' Project, funded by the NHS Executive Northern and Yorkshire, resulted in the implementation of locally responsive packages of hardware and software in six of the Region's libraries. The opportunity to evaluate the collective experience of these sites, and to synthesize principles of good practice, was provided by a separately funded post-hoc evaluation, the Research Evaluation to Audit Library and Information Support for EBHC (REALISE). This paper reports on how this evaluation was conducted, documents the strengths and weaknesses of the Project itself, and attempts to provide a checklist for use in similar projects. The paper concludes by outlining the relevance of the findings to the introduction of planned organizational approaches to quality (clinical governance) and the development of local implementation strategies across the UK, required by the NHS Information Strategy, Information for Health.

Kinetic model for the regulation by substrate of intramolecular electron transfer in trimethylamine dehydrogenase.

The reaction of trimethylamine dehydrogenase (TMADH) with trimethylamine has been studied by rapid-scanning stopped-flow spectroscopy and steady-state kinetics. The covalently bound 6-S-cysteinylflavin mononucleotide (FMN) cofactor is initially reduced by substrate and exhibits a limiting first order rate constant of 230 s(-1) at pH 7.5 and 30 degrees C. One electron is then transferred intramolecularly from the reduced FMNH2 to the oxidized [4Fe-4S]2+ center. This reaction is biphasic, and the extent of the reaction which corresponds to the faster and slower rates is dependent upon the concentration of trimethylamine. The limiting first order rate constants are 160 and 4 s(-1). At low substrate concentrations, the faster rate is dominant, and at high substrate concentrations, the slower rate is dominant. These results are used to develop a model for the reductive half-reaction of TMADH in which two molecules of substrate bind to TMADH. One binds at the active site of oxidized TMADH and is converted to products. A second molecule binds but is not converted to products and influences the rate of intramolecular electron transfer. Analysis of the transient kinetic data yielded apparent dissociation constants for trimethylamine of 36 and 148 mu M, respectively, for binding to the catalytic and noncatalytic sites. Steady-state kinetic studies indicated substrate inhibition which was best described by a model in which binding of a second molecule of trimethylamine causes a 10-fold reduction in k(cat) from 11 to 1.1 s(-1). This suggests that, at high substrate concentrations, the rate of the intramolecular electron transfer reaction has become sufficiently slow to be at least partially rate-limiting for the steady-state reaction. These kinetic data are interpreted in the context of the known crystal structure of TMADH. The mechanistic implications regarding long range electron transfer and possible physiologic significance of these findings are discussed.

Intramolecular electron transfer in trimethylamine dehydrogenase: a thermodynamic analysis.

Within the enzyme trimethylamine dehydrogenase [TMADH], intramolecular electron transfer occurs between a fully reduced covalently bound 6-S-cysteinylflavin [FMN] cofactor, and an oxidized iron-sulfur [4Fe-4S]2+ center. When the enzyme is reduced by substrate trimethylamine, the kinetics of this intramolecular electron transfer [ET] reaction are biphasic, suggesting that ET occurs via two alternative processes [Falzon, L., & Davidson, V.L. (1996) Biochemistry 35, 2445-2452]. The formation of the FMN semiquinone was monitored by stopped-flow spectroscopy, and the two rate constants for the biphasic reaction were determined at temperatures ranging from 12 to 37 degrees C. Analysis of these rate constants by ET theory yielded values of 2.2 eV for the reorganizational energy [lambda] associated with each reaction and electronic coupling [H(AB)] of 5.9 and 47 cm-1 for the slower and faster ET reactions, respectively. The analysis also predicted average theoretical distance between the two redox centers of 12.3 A for the slower reaction and 8.1 A for the faster reaction. These predicted distances correlate well with the known crystal structure of TMADH and the most efficient pathways for ET that were predicted from the known structure using the Greenpath program. This analysis suggests that for each reaction the ET event is rate-limiting, but coupled to a highly unfavorable non-ET process, and that binding of a second molecule of substrate to reduced TMADH decreases the efficiency of the intramolecular ET.