Ligand-bound glutamine binding protein assumes multiple metastable binding sites with different binding affinities.

Protein dynamics plays key roles in ligand binding. However, the microscopic description of conformational dynamics-coupled ligand binding remains a challenge. In this study, we integrate molecular dynamics simulations, Markov state model (MSM) analysis and experimental methods to characterize the conformational dynamics of ligand-bound glutamine binding protein (GlnBP). We show that ligand-bound GlnBP has high conformational flexibility and additional metastable binding sites, presenting a more complex energy landscape than the scenario in the absence of ligand. The diverse conformations of GlnBP demonstrate different binding affinities and entail complex transition kinetics, implicating a concerted ligand binding mechanism. Single molecule fluorescence resonance energy transfer measurements and mutagenesis experiments are performed to validate our MSM-derived structure ensemble as well as the binding mechanism. Collectively, our study provides deeper insights into the protein dynamics-coupled ligand binding, revealing an intricate regulatory network underlying the apparent binding affinity.

Early molecular assessment of osseointegration in humans.

OBJECTIVE: To determine the early temporal-wide genome transcription regulation by the surface topography at the bone-implant interface of implants bearing microroughened or superimposed nanosurface topology. MATERIALS AND METHODS: Four commercially pure titanium implants (2.2 × 5.0 mm) with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed (n = 2/surface) in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole-genome microarray using the Affymetrix Human gene 1.1 ST Array was used to describe the gene expression profiles that were differentially regulated by the implant surfaces. RESULTS: There were no significant differences when comparing the two implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix (ECM), collagen fibril organization, and angiogenesis were upregulated at both surfaces (day7 vs. day3). Abundant upregulation of several differential markers of alternative activated macrophages was observed (e.g., MRC1, MSR1, MS4A4A, SLC38A6, and CCL18). The biological processes involved with the inflammatory/immune response gene expression were concomitantly downregulated. CONCLUSIONS: Gene regulation implicating collagen fibrillogenesis and ECM organization as well as the inflammatory/immune responses involving the alternative activated pathway are observed in implant adherent cells at early (3-7 days) after implantation. These gene expression events may indicate a pivotal role of collagen fibrillogenesis as well as immunomodulation in altering bone accrual and biomechanical physical properties of the implant-bone interface.

Assessment of the validity of a multigene analysis in the diagnostics of inflammatory bowel disease.

OBJECTIVES: The findings of a previous multigene study indicated that the expression of a panel of seven specific genes had strong differential power regarding inflammatory bowel disease (IBD) versus non-IBD, as well as ulcerative colitis (UC) versus Crohn's disease (CD). This prospective confirmatory study based on an independent patient cohort from a national Danish IBD centre was conducted in an attempt to verify these earlier observations. DESIGN, SETTING AND PARTICIPANTS: A total of 119 patients were included in the study (CD, UC and controls). Three mucosal biopsies were retrieved from the left side of the colon of each patient. RNA was extracted, and RT-PCR was performed to retain expression profiles from the seven selected genes. Expression data from the training set (18 CD, 20 UC and 20 controls) were used to build a classification model, using quadratic discriminant analysis, and data from the test set (20 CD, 21 UC and 20 controls) were used to test the validity of the model. RESULTS: The present investigation did not confirm the previous observation that a panel of seven specific genes is able to distinguish between patients with CD and UC, whereas the discriminative power for IBD versus control subjects was substantiated. CONCLUSION: Our results fail to demonstrate that the previously identified seven-gene classification model is able to discriminate between CD and UC but suggest that the gene panel merely discriminates between inflamed and noninflamed colonic tissue. Thus, a reliable and simple diagnostic tool is still warranted for optimal diagnosis and treatment of patients with IBD, especially the subgroup with unclassified disease.