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Objective: To ascertain if monitoring over-the-counter (OTC) drug sales could provide a timely syndromic surveillance method of detecting outbreaks of gastrointestinal illness.Method: This study evaluated the potential of a syndromic surveillance system by comparing retrospective pharmacy OTC sales of anti-nauseants and anti-diarrheals to emergency room visits and case numbers from two Canadian outbreaks related to water contamination by Cryptosporidium, and E.coli O157:H7 and Campylobacter.Results: Local sales trends of weekly aggregate OTC products were comparable to the outbreak epidemic curves. Statistical control tests on the sales data indicated the start of the outbreak periods.Conclusions: An automated monitoring tool based on spatial and temporal trend analyses of daily OTC sales would provide supplemental community health information for public health officials that is timelier than currently available laboratory-based surveillance systems. [ABSTRACT FROM AUTHOR]

Abstract Background An extensive West Nile virus surveillance program of dead birds, mosquitoes, horses, and human infection has been launched as a result of West Nile virus first being reported in Canada in 2001. Some desktop and web GIS have been applied to West Nile virus dead bird surveillance. There have been urgent needs for a comprehensive GIS services and real-time surveillance. Results A pilot system was developed to integrate real-time surveillance, real-time GIS, and Open GIS technology in order to enhance West Nile virus dead bird surveillance in Canada. Driven and linked by the newly developed real-time web GIS technology, this integrated real-time surveillance system includes conventional real-time web-based surveillance components, integrated real-time GIS components, and integrated Open GIS components. The pilot system identified the major GIS functions and capacities that may be important to public health surveillance. The six web GIS clients provide a wide range of GIS tools for public health surveillance. The pilot system has been serving Canadian national West Nile virus dead bird surveillance since 2005 and is adaptable to serve other disease surveillance. Conclusion This pilot system has streamlined, enriched and enhanced national West Nile virus dead bird surveillance in Canada, improved productivity, and reduced operation cost. Its real-time GIS technology, static map technology, WMS integration, and its integration with non-GIS real-time surveillance system made this pilot system unique in surveillance and public health GIS.

Cases of high pathogenicity avian influenza (HPAI) in Canada are upon us again and with reports of infection in US dairy cattle and a dairy farmer in the United States, concern has been raised. Although panic isn't helpful, this heightened level of concern is appropriate, given that reports of human infections with the H5N1 virus often indicate high mortality rates. These can range from 14 to 50%. The current devastating impact of the virus on the poultry industry, as well as its propensity to mutate are also reasons for concern. At the same time, HPAI provides an opportunity for the poultry and livestock industries to align and organize coherently for the management of all zoonotic diseases and other industry issues. To manage HPAI more effectively, it is essential to align all stakeholders under Outbreak Response Best Practices using a formal Quality Management System (QMS). The objective of this article is to describe this approach with examples drawn from management of the Walkerton waterborne disease crisis. We urge the veterinary profession to rise to the challenge of HPAI and use it as a context in which to align more coherently with national stakeholders for the prevention and management of all priority issues within the areas of Agri-food and Public Health., Competing Interests: Disclosure statement: Novometrix Research Inc. specializes in consulting, research, data analytics, and training to tackle complex One Health problems, encompassing human, animal, and environmental health. Based in Guelph, Canada, Novometrix has applied its Community Network Integration (CNI) process to various issues, such as pollinator sustainability, global poultry industry optimization, and outbreak management, including the Walkerton water crisis., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Multiple sclerosis is an autoimmune demyelinating disorder of the CNS, characterized by inflammatory lesions and an underlying neurodegenerative process, which is more prominent in patients with progressive disease course. It has been proposed that mitochondrial dysfunction underlies neuronal damage, the precise mechanism by which this occurs remains uncertain. To investigate potential mechanisms of neurodegeneration, we conducted a functional screening of mitochondria in neurons exposed to the CSF of multiple sclerosis patients with a relapsing remitting (n = 15) or a progressive (secondary, n = 15 or primary, n = 14) disease course. Live-imaging of CSF-treated neurons, using a fluorescent mitochondrial tracer, identified mitochondrial elongation as a unique effect induced by the CSF from progressive patients. These morphological changes were associated with decreased activity of mitochondrial complexes I, III and IV and correlated with axonal damage. The effect of CSF treatment on the morphology of mitochondria was characterized by phosphorylation of serine 637 on the dynamin-related protein DRP1, a post-translational modification responsible for unopposed mitochondrial fusion in response to low glucose conditions. The effect of neuronal treatment with CSF from progressive patients was heat stable, thereby prompting us to conduct an unbiased exploratory lipidomic study that identified specific ceramide species as differentially abundant in the CSF of progressive patients compared to relapsing remitting multiple sclerosis. Treatment of neurons with medium supplemented with ceramides, induced a time-dependent increase of the transcripts levels of specific glucose and lactate transporters, which functionally resulted in progressively increased glucose uptake from the medium. Thus ceramide levels in the CSF of patients with progressive multiple sclerosis not only impaired mitochondrial respiration but also decreased the bioavailability of glucose by increasing its uptake. Importantly the neurotoxic effect of CSF treatment could be rescued by exogenous supplementation with glucose or lactate, presumably to compensate the inefficient fuel utilization. Together these data suggest a condition of 'virtual hypoglycosis' induced by the CSF of progressive patients in cultured neurons and suggest a critical temporal window of intervention for the rescue of the metabolic impairment of neuronal bioenergetics underlying neurodegeneration in multiple sclerosis patients., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A 2A R) that are deuterated apart from 1 H/ 13 C NMR probes at isoleucine δ1 methyl groups, which facilitated 1 H/ 13 C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na + ] is required to allow large agonist-induced structural changes in A 2A R, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

Introduction: This paper briefly describes the use of Canada Post Unaddressed Admail and a geographic information system (GIS) for survey distribution to a specific target population in a large, sparsely populated geographic area, and the effectiveness of this approach., Methods: Surveys were sent as Unaddressed Admail via Canada Post to a target population of people living within 5 km of a wind turbine in southwestern Ontario., Results: The overall response rate from 8 wind farms (in 8 counties) was 8.1%., Conclusion: This approach has the potential to save time and money, but low response rates are common, distribution is not precise and there is potential for selection bias. Despite these flaws, Unaddressed Admail is worth consideration for delivery of information, study-recruitment materials and surveys to rural, remote and specific target populations.

While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding., (© 2013.)

In this chapter, we concentrate on the production of high-quality protein samples for nuclear magnetic resonance (NMR) studies. In particular, we provide an in-depth description of recent advances in the production of NMR samples and their synergistic use with recent advancements in NMR hardware. We describe the protein production platform of the Northeast Structural Genomics Consortium and outline our high-throughput strategies for producing high-quality protein samples for NMR studies. Our strategy is based on the cloning, expression, and purification of 6×-His-tagged proteins using T7-based Escherichia coli systems and isotope enrichment in minimal media. We describe 96-well ligation-independent cloning and analytical expression systems, parallel preparative scale fermentation, and high-throughput purification protocols. The 6×-His affinity tag allows for a similar two-step purification procedure implemented in a parallel high-throughput fashion that routinely results in purity levels sufficient for NMR studies (>97% homogeneity). Using this platform, the protein open reading frames of over 17,500 different targeted proteins (or domains) have been cloned as over 28,000 constructs. Nearly 5000 of these proteins have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html), resulting in more than 950 new protein structures, including more than 400 NMR structures, deposited in the Protein Data Bank. The Northeast Structural Genomics Consortium pipeline has been effective in producing protein samples of both prokaryotic and eukaryotic origin. Although this chapter describes our entire pipeline for producing isotope-enriched protein samples, it focuses on the major updates introduced during the last 5 years (Phase 2 of the National Institute of General Medical Sciences Protein Structure Initiative). Our advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are suitable for implementation in a large individual laboratory or by a small group of collaborating investigators for structural biology, functional proteomics, ligand screening, and structural genomics research., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples. The platform is centered on the cloning, expression and purification of 6X-His-tagged proteins using T7-based Escherichia coli systems. The 6X-His tag allows for similar purification procedures for most targets and implementation of high-throughput (HTP) parallel methods. In most cases, the 6X-His-tagged proteins are sufficiently purified (>97% homogeneity) using a HTP two-step purification protocol for most structural studies. Using this platform, the open reading frames of over 16,000 different targeted proteins (or domains) have been cloned as>26,000 constructs. Over the past 10 years, more than 16,000 of these expressed protein, and more than 4400 proteins (or domains) have been purified to homogeneity in tens of milligram quantities (see Summary Statistics, http://nesg.org/statistics.html). Using these samples, the NESG has deposited more than 900 new protein structures to the Protein Data Bank (PDB). The methods described here are effective in producing eukaryotic and prokaryotic protein samples in E. coli. This paper summarizes some of the updates made to the protein production pipeline in the last 5 years, corresponding to phase 2 of the NIGMS Protein Structure Initiative (PSI-2) project. The NESG Protein Production Platform is suitable for implementation in a large individual laboratory or by a small group of collaborating investigators. These advanced automated and/or parallel cloning, expression, purification, and biophysical screening technologies are of broad value to the structural biology, functional proteomics, and structural genomics communities.

Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins.

To validate the utility of a chief complaint-based emergency department surveillance system, we compared it with respiratory diagnostic data and calls to Telehealth Ontario about respiratory disease. This local syndromic surveillance system accurately monitored status of respiratory diseases in the community and contributed to early detection of respiratory disease outbreaks.

Background: Gastrointestinal illness (GI) remains a leading cause of morbidity and mortality worldwide. In Canada, research has already demonstrated a rate in excess of one episode per person-year. National passive surveillance programs may be enhanced by information from hospitalizations for acute gastrointestinal disease. The objective of this report is to explore the incidence of acute GI in hospital administrative data collected by the Canadian Institute for Health Information (CIHI)--specifically the hospital morbidity database (HMDB)., Methods: Data from acute care facilities and select chronic care and rehabilitation facilities across Canada were analyzed using standardized rates, and age- and sex-adjusted rates for the years 1995-2004., Results: The results indicate that GI causes at least 92,765 hospital admissions per year in Canada. In the majority (78.3%) of gastrointestinal hospitalizations, no specific etiology was recorded. Of the remaining diagnoses, 11.6% were due to viruses, 9.7% to bacteria and 0.3% to parasites. Age-standardized rates of hospitalizations for acute GI appear to have declined over the 10-year period., Conclusion: Gastrointestinal illness is still present in the Canadian population and presents a significant burden to the health care system. Whereas the HMDB likely underestimates the true rate of GI, it does capture cases that are serious enough to require hospitalization. This is a unique source of data and highlights other pathogen-specific disease data not currently collected through national surveillance tools (e.g., viruses).

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-beta mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3' end processing of all cellular pre-mRNAs. Here we report the 1.95-A resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-beta pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.

In the spring of 2003, the Public Health Agency of Canada (then, Health Canada) partnered with several provincial/territorial and regional public health stakeholders to improve pan-Canadian public health surveillance, communications and response through the application of new technologies. This resulted in the creation of the Canadian Network for Public Health Intelligence (CNPHI), a comprehensive framework of applications and resources designed to fill critical gaps in Canada's national public health infostructure. Over the past four years, the CNPHI has evolved into Canada's only pan-Canadian public health information management system. With over 2000 registered users, the current CNPHI environment consists of more than 30 integrated applications and systems that can be loosely categorized into four functional groups: data exchange; data analysis and integration; communication, collaboration and coordination; and knowledge management. Despite poor data repositories, legacy information management systems, and the lack of standards and agreements, the CNPHI has demonstrated that much can be accomplished in these areas. Over the next decade, significant barriers impeding additional advances will be bridged through the implementation of the Electronic Health Record, and through ongoing efforts to address gaps in standards, and data- and information-sharing agreements. Together with new technologies coming on-line, opportunities to further enhance public health surveillance and response will be limited only by one's imagination.

Twenty-eight research dogs were enrolled to determine the prevalence of salmonellae shedding after consumption of 1 Salmonella-contaminated commercial raw food diet meal. Sixteen dogs were exposed to Salmonella-contaminated commercial raw food diets and 12 to Salmonella-free commercial raw food diets. Seven of the exposed dogs shed salmonellae 1-7 days after consumption of Salmonella-contaminated raw food diets. None of the dogs fed Salmonella-free diets shed salmonellae. No clinical signs were observed in either group. Five of the 7 dogs shed the same serotypes as those recovered from food samples used for feeding. Results showed the same serotypes and antimicrobial resistance pattern in 2 of the 7 shedders. Dogs fed Salmonella-contaminated raw food diets can shed salmonellae and may, therefore, be a source of environmental contamination potentially leading to human or animal illness.

Porcine proliferative enteropathy caused by Lawsonia intracellularis is an important enteric disease in swine throughout the world. Information regarding the distribution of this pathogen in Canadian swine herds would be beneficial for the creation of control protocols. Pigs from Ontario, Quebec, and Alberta were tested by using an indirect immunofluorescence assay for antibodies to L. intracellularis. Pig seroprevalence was calculated as the proportion of pigs positive from total pigs tested in the targeted population. Seroprevalence (+/- standard error [s(x)]) in market hogs in Ontario from farrow-finish (FF) farms and finishing (FIN) farms were significantly different at 77% (s(x) = 7%) and 29% (s(x) = 15%), respectively. Seroprevalence for sows and gilts in FF and farrowing and nursery (FAR + NUR) farms in Ontario were 90% (s(x) = 3%) and 93% (s(x) = 6%), respectively. Seroprevalence in breeding females in Quebec from FF and FAR farms was 82% (s(x) = 5%) and 87% (s(x) = 3%), respectively. Seroprevalence (57%, s(x) = 8%) in finishing pigs in Alberta from FF farms was significantly different from that of multisite (MS) farms and FIN farms, 6% (s(x) = 6%) and 9% (s(x) = 5%), respectively. Lawsonia intracellularis appears to be widespread in Canada and the seroprevalence on FF farms is higher than that on FIN and MS farms, possibly due to the presence of breeding females or management differences.

This study evaluated the anti-inflammatory properties of Surolan with the use of a pinna model of inflammation in 80 mice and a randomized complete block design. Within each of 8 blocks, 10 treatments, consisting of different combinations of the constituents of Surolan with and without the prednisolone acetate component, were randomly assigned to the 9-wk-old CD-1 mice. The treatments were administered as a single dose 30 min after pinna inflammation was induced with tetradecanoylphorbol acetate. Ear-skin edema and erythema were assessed and measured 4 and 6 h thereafter. Treatment effects were evaluated with repeated-measures analysis of variance. Inclusion of prednisolone acetate with the Surolan vehicle, either alone or in combination with the nonsteroidal constituents of the suspension, resulted in a significant reduction in mean ear-skin thickness and erythema. The inflammation-reducing properties of prednisolone were not significantly affected by the other components of Surolan.

Background: An extensive West Nile virus surveillance program of dead birds, mosquitoes, horses, and human infection has been launched as a result of West Nile virus first being reported in Canada in 2001. Some desktop and web GIS have been applied to West Nile virus dead bird surveillance. There have been urgent needs for a comprehensive GIS services and real-time surveillance., Results: A pilot system was developed to integrate real-time surveillance, real-time GIS, and Open GIS technology in order to enhance West Nile virus dead bird surveillance in Canada. Driven and linked by the newly developed real-time web GIS technology, this integrated real-time surveillance system includes conventional real-time web-based surveillance components, integrated real-time GIS components, and integrated Open GIS components. The pilot system identified the major GIS functions and capacities that may be important to public health surveillance. The six web GIS clients provide a wide range of GIS tools for public health surveillance. The pilot system has been serving Canadian national West Nile virus dead bird surveillance since 2005 and is adaptable to serve other disease surveillance., Conclusion: This pilot system has streamlined, enriched and enhanced national West Nile virus dead bird surveillance in Canada, improved productivity, and reduced operation cost. Its real-time GIS technology, static map technology, WMS integration, and its integration with non-GIS real-time surveillance system made this pilot system unique in surveillance and public health GIS.

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

This project addresses two important issues relevant to the health of Canadians: the risk of waterborne illness and the health impacts of global climate change. The Canadian health burden from waterborne illness is unknown, although it presumably accounts for a significant proportion of enteric illness. Recently, large outbreaks with severe consequences produced by E. coli O157:H7 and Cryptosporidium have alarmed Canadians and brought demands for political action. A concurrent need to understand the health impacts of global climate changes and to develop strategies to prevent or prepare for these has also been recognized. There is mounting evidence that weather is often a factor in triggering waterborne disease outbreaks. A recent study of precipitation and waterborne illness in the United States found that more than half the waterborne disease outbreaks in the United States during the last half century followed a period of extreme rainfall. Projections of international global climate change scenarios suggest that, under conditions of global warming most of Canada may expect longer summers, milder winters, increased summer drought, and more extreme precipitation. Excess precipitation, floods, high temperatures, and drought could affect the risk of waterborne illness in Canada. The existing scientific information regarding most weather-related adverse health impacts and on the impacts of global climate change on health in Canada is insufficient for informed decision making. The results of this project address this need through the investigation of the complex systemic interrelationships between disease incidence, weather parameters, and water quality and quantity, and by projecting the potential impact of global climate change on those relationships.

Salmonella enterica var. Heidelberg was isolated from an unusual food source during routine case follow-up, prompting a case control investigation of frozen chicken nuggets and strips. Most frozen nuggets and strips are raw; however, par-frying lends a cooked appearance. As such, suitable food preparation precautions might not be undertaken by consumers. Cases were confirmed in the laboratory between 1 January and 1 April 2003. Controls were generated through forward-digit dialing and individually matched by age category. Telephone interviews were conducted, and limited sampling of unopened product was performed. Eighteen matched pairs were interviewed. The odds of infection were 11 times higher in individuals who had consumed frozen processed chicken nuggets and strips (95% confidence interval, 1.42 < odds ratio < 85.20). One-third of cases and controls considered frozen nuggets and strips to be precooked, and one quarter used the microwave, an ill-advised cooking method. Consumer misconceptions contributed to the risk of infection. Clear labels identifying nuggets and strips as raw poultry are needed.

Objective: This study was conducted to investigate the characteristics of public water works (PWW) in southern Ontario with respect to their water sources and treatment regimes., Methods: Data from 481 PWW covering the period 1992-1999 were collected and cartographic manipulations as well as descriptive analyses of the PWW attributes were performed. Tests of associations between different PWW attributes were done using Fisher's Exact test and Cochran-Mantel-Haenszel statistics., Results: Water sources for the PWW included surface water (SW) (21% lakes; 13% rivers), ground water (GW) (64%) and mixed sources (2%). Most (81%) of the population was supplied with SW. Filtration was performed by 84% and 8% of the PWW using SW and GW, respectively. Similarly, disinfection was performed by 99% and 91% of the PWW using SW and GW respectively. There was no significant difference in treatment regimes between PWW in urban and those in rural areas but treatment regime was a function of water source. Overall, most PWW (87.8%) met the minimum treatment requirements of the then Ontario Drinking Water Objectives (ODWO)., Discussion: The study shows that most PWW complied with the minimum treatment requirements of the then ODWO. The minimum treatment required by the ODWO was disinfection for GW and both disinfection and filtration for SW. The non-compliant PWW will need to comply for continued provision of safe drinking water. Suffice it to say that both watershed protection and improved water treatment will be imperative for the continued provision of safe drinking water and control of waterborne diseases.

Determination of precise and accurate protein structures by NMR generally requires weeks or even months to acquire and interpret all the necessary NMR data. However, even medium-accuracy fold information can often provide key clues about protein evolution and biochemical function(s). In this article we describe a largely automatic strategy for rapid determination of medium-accuracy protein backbone structures. Our strategy derives from ideas originally introduced by other groups for determining medium-accuracy NMR structures of large proteins using deuterated, (13)C-, (15)N-enriched protein samples with selective protonation of side-chain methyl groups ((13)CH(3)). Data collection includes acquiring NMR spectra for automatically determining assignments of backbone and side-chain (15)N, H(N) resonances, and side-chain (13)CH(3) methyl resonances. These assignments are determined automatically by the program AutoAssign using backbone triple resonance NMR data, together with Spin System Type Assignment Constraints (STACs) derived from side-chain triple-resonance experiments. The program AutoStructure then derives conformational constraints using these chemical shifts, amide (1)H/(2)H exchange, nuclear Overhauser effect spectroscopy (NOESY), and residual dipolar coupling data. The total time required for collecting such NMR data can potentially be as short as a few days. Here we demonstrate an integrated set of NMR software which can process these NMR spectra, carry out resonance assignments, interpret NOESY data, and generate medium-accuracy structures within a few days. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data was illustrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A.