Imaging and 3D morphological analysis of collagen fibrils.

The recent booming of multiphoton imaging of collagen fibrils by means of second harmonic generation microscopy generates the need for the development and automation of quantitative methods for image analysis. Standard approaches sequentially analyse two-dimensional (2D) slices to gain knowledge on the spatial arrangement and dimension of the fibrils, whereas the reconstructed three-dimensional (3D) image yields better information about these characteristics. In this work, a 3D analysis method is proposed for second harmonic generation images of collagen fibrils, based on a recently developed 3D fibre quantification method. This analysis uses operators from mathematical morphology. The fibril structure is scanned with a directional distance transform. Inertia moments of the directional distances yield the main fibre orientation, corresponding to the main inertia axis. The collaboration of directional distances and fibre orientation delivers a geometrical estimate of the fibre radius. The results include local maps as well as global distribution of orientation and radius of the fibrils over the 3D image. They also bring a segmentation of the image into foreground and background, as well as a classification of the foreground pixels into the preferred orientations. This accurate determination of the spatial arrangement of the fibrils within a 3D data set will be most relevant in biomedical applications. It brings the possibility to monitor remodelling of collagen tissues upon a variety of injuries and to guide tissues engineering because biomimetic 3D organizations and density are requested for better integration of implants.

Fibrillogenesis in dense collagen solutions: a physicochemical study.

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength=150 mM, 20 degrees C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH approximately 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.

Cooperative ordering of collagen triple helices in the dense state.

Extracellular matrixes such as bone, skin, cornea, and tendon have ordered structures comprised for the most part of collagen, an elongated protein of well-defined dimensions and composition. Here we show how the cooperative ordering of collagen triple helices in the dense fluid state is exploited to produce dense ordered collagen matrixes. The spontaneous formation of a birefringent phase occurs at critical concentrations that increase from 50-60 to 80-85 mg/mL as the acetic acid concentration of the solvent increases from 5 to 500 mM. We studied by small-angle X-ray scattering (SAXS) the local liquidlike positional order across the isotropic/anisotropic phase transition by unwinding the cholesteric phase with moderate shearing stress. Interparticle scattering gives rise to a broad interference peak. The average distance between triple helices, dav, is thus estimated and decreases linearly as a function of phi-1/2 from 12.7 +/- 0.9 nm (22.5 mg/mL) to 5.0 +/- 0.6 nm (166.4 mg/mL). Equilibrium concentrations and the order parameter of the nematic phase agree reasonably well with theoretical predictions for semiflexible macromolecules. Striated fibrils with a high degree of alignment were obtained by fine-tuning the delicately balanced electrostatic interactions, which yielded strong elastic gels with a hierarchical organization very similar to that of major biological tissues. Typical Bragg reflections corresponding to the 67 nm period characteristic of collagen fibrils in biological tissues were recorded by SAXS with ordered collagen matrixes reconstituted in vitro.

Two-dimensional crystallogenesis of transmembrane proteins.

Two-dimensional crystallogenesis is a crucial step in the long road that leads to the determination of macromolecules structure via electron crystallography. The necessity of having large and highly ordered samples can hold back the resolution of structural works for a long time, and this, despite improvements made in electron microscopes or image processing. Today, finding good conditions for growing two-dimensional crystals still rely on either "biocrystallo-cooks" or on lucky ones. The present review presents the field by first describing the different crystals that one can encounter and the different crystallisation methods used. Then, the effects of different components (such as protein, lipids, detergent, buffer, and temperature) and the different methods (dialysis, hydrophobic adsorption) are discussed. This discussion is punctuated by correspondences made to the world of three-dimensional crystallogenesis. Finally, a guide for setting up 2D crystallogenesis experiments, built on the discussion mentioned before, is proposed to the reader. More than giving recipes, this review is meant to open up the discussions in this field.

Lessons from experienced guideline implementers: attend to many factors and use multiple strategies.

BACKGROUND: Studies of clinical guideline implementation have focused almost entirely on changing individual clinician behavior with single intervention strategies and without much attention to the situational context. The goal of this project was to learn from clinic leaders, seasoned in the guideline implementation process, what contextual variables they viewed as important and whether implementation success could be expected if only a single implementation strategy was used. METHODS: In 1998, 12 people with extensive experience in leading clinical guideline implementation were identified who were thought to have particularly keen insight into the process. They were interviewed to generate variables they considered important, as well as strategies they considered effective when used appropriately. A modified nominal group/Delphi process was then used for rating these variables and strategies, and the reactions of international experts were obtained to add perspective to this information. RESULTS: Eighty-seven variables and 25 strategies were identified, clustering in 6 categories (ranked in order of importance by the panel): organizational capabilities for change, infrastructure for implementation, implementation strategies, medical group characteristics, guideline characteristics, and external environment. All six categories were considered to be important, key, or essential by the experienced implementers, although variables within a medical group that directly affect its ability to undertake planned change were rated as much more important than either guideline characteristics or the external environment. DISCUSSION: Although the opinions of those experienced in the process of guideline implementation are primarily of value for generating hypotheses, panel members believe that implementation efforts focusing on the individual physician with a single strategy are unlikely to be successful. Rather, implementation efforts must use multiple strategies that take account of multiple characteristics of the guideline, practice organization, and external environment.

A practical approach to evidence grading.

BACKGROUND: The Institute for Clinical Systems Improvement (ICSI) is a collaboration of 17 Minnesota medical groups. Among other activities, ICSI develops health care guidelines and technology assessment reports. To maintain focus on the underlying evidence, ICSI has developed an evidence and conclusion grading system for use by the practicing clinicians who write the documents and use them in making decisions about patient care. THE EVIDENCE GRADING SYSTEM IN DETAIL: The centerpiece of the evidence grading system is the conclusion grading worksheet, which calls for statement of a conclusion, summarization of research reports that support or dispute the conclusion, assignment of classes and quality markers to the research reports, and assignment of a grade to the conclusion. EXPERIENCE AND RESULTS: The system has been used in the writing of more than 40 guidelines and numerous technology assessment reports. An example of a worksheet from the congestive heart failure guideline is presented. The system has helped the drafting groups to attend to the evidence. The methods have proven to be well accepted by practicing physicians and to be practical, although staff expertise in epidemiology is needed to support the system. Grading of conclusions appears to be reliable, although this characteristic of the system has not been rigorously tested. The outputs are valued by users of the documents. DISCUSSION: Although some residual problems remain to be solved, the system appears to be successful in overcoming the complexity of some published systems for grading evidence while still yielding a defensible classification of conclusions based on the strength of the underlying evidence.

Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins.

A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni(2+)-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F(0)F(1)-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein.

The 9 A projection structure of cytochrome b6f complex determined by electron crystallography.

Thin three-dimensional crystals of the cytochrome b6 f complex from the unicellular algae Chlamydomonas reinhardtii have been grown by BioBeads-mediated detergent removal from a mixture of protein and lipid solubilized in Hecameg. Frozen-hydrated crystals, exhibiting p22121 plane group symmetry, were studied by electron crystallography and a projection map at 9 A resolution was calculated. The crystals (unit cell dimensions of a=173.5 A, b=70.0 A and gamma=90.0 degrees) showed the presence of dimers, and within each monomer 14 domains of electron density were observed. The combination of the projection map obtained from ice-embedded crystals of cytochrome b6 f with a previous map obtained from negatively stained samples brings new insight in the organization of the complex. For example, it distinguishes some peaks and/or domains that are only extramembrane or transmembrane, and reveals the possible localization of single-stranded transmembrane alpha-helices (Pet subunits). Furthermore, the cross-correlation of our projection map from frozen hydrated samples with the atomic model of the transmembrane part of the cytochrome bc1 complex has allowed us to localize the cytochrome b6 at the dimer interface and to reveal structural differences between the two complexes.

Collaborating outside the box: three years later.

BACKGROUND: In 1992, 15 employers in Minneapolis-St Paul, operating as the Business Health Care Action Group (BHCAG), combined their self-insured plans. To successfully bid for the BHCAG contract, three competing group practices and a health plan cooperated, operating like a fully integrated care system to measure outcomes, develop practice guidelines, and meet other BHCAG requirements. To accomplish this, a new organization, the Institute for Clinical Systems Integration (ICSI), was conceived. ICSI IN THE EVOLVING MINNEAPOLIS MARKETPLACE: From a business standpoint, ICSI members stood to gain market share by being members of ICSI and the "chosen" consortium. From a professional standpoint, they could realize the fulfillment and satisfaction of knowing that they were innovating, improving care, reducing waste, and sharing their knowledge with others. A NEW MARKET MODEL: To drive the same kind of change for the entire care delivery system in the region, not just for the subset that happened to win the original bid, BHCAG changed the purchase model in February 1995--enrollees could now choose among 16 to 20 discrete care delivery systems instead of preferentially channeling them to the ICSI-HealthPartners network of group practices. All the care systems had become competitors on every level, including quality of care. The "special" customer-supplier relationship between BHCAG and the ICSI medical groups was no longer present. LESSONS LEARNED: Despite major changes in the market dynamics, with the marked decline in the business reason for collaboration which had prompted ICSI to form in the first place, physicians, nurses, and administrative staff from participating medical groups continue to devote massive effort to the development and implementation of best practices.

Projection map of cytochrome b6 f complex at 8 A resolution.

The structure of the cytochrome b6 f complex has been investigated by electron microscopy and image analysis of thin three-dimensional crystals. Electron micrographs of negatively stained specimens were recorded and showed optical diffraction peaks to 10 A resolution. A projection map was calculated at 8 A resolution and showed the presence of cytochrome b6 f dimers. The extramembrane part of each monomer featured a C shape, with mean external diameter approximately of 53 A and an internal groove approximately 14 A long and approximately 9 A wide. Within each monomer, strong features were clearly resolved and tentatively attributed to some of the subunits of the cytochrome b6 f complex. The data are consistent with the Rieske iron-sulfur protein lying close to the monomer-monomer interface and the heme-bearing domain of cytochrome f far from it.

Bio-Beads: an efficient strategy for two-dimensional crystallization of membrane proteins.

This work establishes the potential of Bio-Beads as a simple alternative to conventional dialysis for removing detergent and for obtaining 2D crystals of integral membrane proteins useful for structure analysis by electron crystallography. Kinetic and equilibrium aspects of removal of different detergents by adsorption onto hydrophobic Bio-Beads SM2 have been systematically investigated and extended to 2D crystallization of different prototypic membrane proteins, including: (a) Ca2+ ATPase from sarcoplasmic reticulum; (b) melibiose permease from Escherichia coli; (c) cytochrome b6f from Chlamydomonas reinhardtii. Different crystals could be produced from all protein preparations, with optical diffraction down to 20-25 A in negative stain.

The three faces of performance measurement: improvement, accountability, and research.

In the current climate of public accountability, many clinicians have become uncomfortable with any efforts to create measurement systems. That is unfortunate because measurements are absolutely essential to efforts for improving the processes of medical care. In their guideline implementation and measurement efforts, ISCI and the IMPROVE Project in Minnesota have gradually learned how to distinguish between measurement for improvement and that for accountability. Both approaches are different from the approach that physicians are used to in their encounters with medical research. Understanding these differences and respecting the confidentiality of individual medical groups has been crucial to moving past confusion and suspicion to genuine improvement actions involving multiple medical groups and their contracting managed care plans.

Mechanism of action and impact of a cystitis clinical practice guideline on outcomes and costs of care in an HMO.

BACKGROUND: A study was conducted in 1995 at five primary care clinics of a staff-model health maintenance organization in the Midwest to assess the impact of a cystitis clinical guideline and to help elucidate the guideline implementation process. METHODOLOGY: Two hundred one eligible women with uncomplicated cystitis were treated in a three-month period before the guideline, and 241 similar cases were treated in a three-month period after the guideline. Nursing supervisors and clinic managers at each clinic were interviewed about how the cystitis guideline was implemented at each clinic. RESULTS: Use of a recommended three-day antibiotic treatment increased from 28% to 52% of cases (chi-square = 25.01, p < 0.001). Use of urine cultures decreased from 70% to 37% of cases (chi-square = 48.19, p < 0.001). The proportion of eligible cystitis cases coordinated primarily by the nurse increased from 21% to 78% (chi-square = 142.93, p < 0.001). However, desired changes in use of antibiotics and urine cultures were limited to nurse-coordinated cases. There was no increase in hospital admissions, emergency room visits, repeat office visits (p > 0.05), or repeat antibiotic courses (p > 0.05) after cystitis guideline implementation. Cost of cystitis care delivered after guideline implementation was 35% lower than before guideline implementation. CONCLUSIONS: Use of the guideline was associated with desirable changes in antibiotic use, nurse coordination of care, costs of care, and comparable clinical outcomes. Clinics that used clinical systems and tools to support nurse-coordinated cystitis care had greater guideline adherence than clinics that did not support nurse-coordinated care.

Establishing a central structure for supporting guideline implementation.

Devising practice guidelines is one thing, implementing them is another, perhaps more difficult task. The key to quickest acceptance and greatest utility is to provide the support structure that will create buy-in on the part of clinicians.

Clinical process improvement: engage first, measure later.

The Institute for Clinical Systems Integration (ICSI) is a non-profit quality improvement organization that bridges 20 medical groups and integrated health care systems in the Minneapolis-St. Paul area. Created in 1992 by HealthPartners, HealthSystem Minnesota, and Mayo Clinic as a part of a contract awarded by the Buyers Health Care Action Group, a purchaser coalition, ICSI's aim is collaborative, continual improvement of the clinical care provided by its member institutions. ICSI's main focus is health care guideline developement and implementation. This article describes this process, as well as evidence of program effectiveness and "lessons learned."

Technology assessment at the Institute for Clinical Systems Integration and HealthPartners.

The Technology Assessment Committee of the Institute for Clinical Systems Integration (ICSI) draws upon the resources of many different groups and in turn, keeps the groups informed about new medical treatments, devices, and procedures. This new Minneapolis-based technology assessment committee uses a collaborative effort to serve the information needs of many employer groups, patients, providers, and health care organizations in the area. This committee serves as the principal mechanism by which new medical and surgical procedures, devices, and treatments, as well as new applications of old but unproved or doubtful applications of existing technologies are reviewed for medical appropriateness at ICSI (a nonprofit quality improvement organization) and at HealthPartners (a combined staff and group model HMO in Minnesota).