The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 Å crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar b-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

Crystallization has proven to be the most significant bottleneck to high-throughput protein structure determination using diffraction methods. We have used the large-scale, systematically generated experimental results of the Northeast Structural Genomics Consortium to characterize the biophysical properties that control protein crystallization. Datamining of crystallization results combined with explicit folding studies lead to the conclusion that crystallization propensity is controlled primarily by the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability. These analyses identify specific sequence features correlating with crystallization propensity that can be used to estimate the crystallization probability of a given construct. Analyses of entire predicted proteomes demonstrate substantial differences in the bulk amino acid sequence properties of human versus eubacterial proteins that reflect likely differences in their biophysical properties including crystallization propensity. Finally, our thermodynamic measurements enable critical evaluation of previous claims regarding correlations between protein stability and bulk sequence properties, which generally are not supported by our dataset. NIH Public Access Author ManuscriptNat Biotechnol. Author manuscript; available in PMC 2010 January 1. Published in final edited form as:Nat Biotechnol. 2009 January ; 27(1): 51-57. doi:10.1038/nbt.1514. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe ability to determine the atomic structures of macromolecules represents a great achievement in molecular biology because of the unparalleled value of this information in understanding the fundamental chemistry of life [1][2][3][4][5] . While nuclear magnetic resonance represents an invaluable source of structural information, especially for small proteins, most macromolecular structures are determined using x-ray crystallography. Capitalizing on the recent proliferation of genomic sequence data, "structural genomics" consortia have been organized worldwide to develop methods and infrastructure for high-throughput protein structure determination. These groups have contributed to improvements in expression and structure determination methods 6 , and the four largest U.S. consortia accounted for 45% of all novel structures deposited in the Protein Data Bank (PDB) in 2007 7 . While these efforts contribute to the impressive progress of the structural biology community in characterizing the full repertoire of protein structures, the rate of growth in sequence information nonetheless far out-paces that of structural information. Given the ongoing acceleration of whole-genome sequencing, the gap between the two will continue to expand without a breakthrough in macromolecular structure determination methods.The systematic efforts of structural genomics projects show that crystallization is the major bottleneck to protein structure determinati...

Patient motion during an MRI exam can result in major degradation of image quality, and is of increasing concern due to the aging population and its associated diseases. This work presents a general strategy for real-time, intraimage compensation of rigidbody motion that is compatible with multiple imaging sequences. Image quality improvements are established for structural brain MRI acquired during volunteer motion. A headband integrated with three active markers is secured to the forehead. Prospective correction is achieved by interleaving a rapid track-and-update module into the imaging sequence. For every repetition of this module, a short tracking pulse-sequence remeasures the marker positions; during head motion, the rigid-body transformation that realigns the markers to their initial positions is fed back to adaptively update the image-plane-maintaining it at a fixed orientation relative to the head-before the next imaging segment of k-space is acquired. In cases of extreme motion, corrupted lines of kspace are rejected and reacquired with the updated geometry. Patient movement is a fundamental problem in virtually all in vivo MR applications. Motion induces local field variations, causes erroneous positional encoding of k-space data, and corrupts the spin-excitation history between slices; these phenomena manifest in image-space as misregistrations, blurring, and ghosting. Even a few millimeters of movement during scanning can produce severe artifacts in reconstructed data, thus rendering images unusable. Often, it is subject populations with the highest potential diagnostic benefit in which the utility of MRI is curtailed by motion artifacts. In a study of 17 patients with frontoparietal tumors, data from five had to be rejected due to gross motion artifacts (1). Even among a healthy elderly population, our experience suggests that significant artifacts may appear in 10% to 20% of high-resolution structural brain scans; typically used for diagnostic and morphological analysis, such scans are especially prone to motion artifact due to their longer duration. To address these concerns, a motion-correction strategy for brain MRI is presented.The fact that the head is a rigid-body (to a very close approximation) allows an arbitrary motion to be described by six degrees-of-freedom (6-DOF)-three rotations about a three-dimensional (3D) orthogonal coordinate-system, and three translations. Retrospective motion-compensation methods, such as those used to coregister multiple image volumes in functional MRI (fMRI) studies, are well established. The most popular algorithms (2) determine the 6-DOF via minimization of a least-squares cost function and only correct for interimage motion. Retrospective correction involves interpolation, which can cause image blurring, and is further limited by its inability to fully correct for the influences of through-plane motion on local spin-history.In contrast, prospective strategies compensate for motion in the acquisition stage by keeping the image-plane at a fixed orientation ...

A new MRI method is proposed for separately quantifying the two principal forms of tissue storage (nonheme) iron: ferritin iron, a dispersed, soluble fraction that can be rapidly mobilized, and hemosiderin iron, an aggregated, insoluble fraction that serves as a long-term reserve. The method utilizes multiple spin echo sequences, exploiting the fact that aggregated iron can induce nonmonoexponential signal decay for multiple spin echo sequences. The method is validated in vitro for agarose phantoms, simulating dispersed iron with manganese chloride, and aggregated iron with iron oxide microspheres. To demonstrate feasibility for human studies, preliminary in vivo data from two healthy controls and six patients with transfusional iron overload are presented. For both phantoms and human subjects, conventional R 2 and R 2 * relaxation rates are also measured in order to contrast the proposed method with established MRI iron quantification techniques. Quantification of dispersed (ferritin-like) iron may provide a new means of monitoring the risk of iron-induced toxicity in patients with iron overload and, together with quantification of aggregated (hemosiderin-like) iron, improve the accuracy of estimates for total storage iron.

Aqueous outflow in mice is segmental. SPARC-null mice demonstrated a more uniform outflow pattern and decreased collagen fibril diameter. Areas of high flow had less compact juxtacanalicular connective tissue ECM, and IOP was inversely correlated with PEFL. Our data show a correlation between morphology, aqueous outflow, and IOP, indicating a modulatory role of SPARC in IOP regulation.

A prominent risk factor of primary open-angle glaucoma is ocular hypertension, a pathologic state caused by impaired outflow of aqueous humor through the trabecular meshwork within the iridocorneal angle. The juxtacanalicular region of the trabecular meshwork and the inner wall of Schlemm canal have been identified as the main contributors to aqueous outflow resistance, and both extracellular matrix within the trabecular meshwork and trabecular meshwork cell shape have been shown to affect outflow. Overexpression of multiple ECM proteins in perfused cadaveric human eyes has led to increased outflow resistance and elevated IOP. Pharmacologic agents targeting trabecular meshwork cytoskeletal arrangements have been developed after multiple studies demonstrated the importance of cell shape on outflow. Several groups have shown that aqueous outflow occurs only at certain segments of the trabecular meshwork circumferentially, a concept known as segmental flow. This is based on the theory that aqueous outflow is dependent on the presence of discrete pores within the Schlemm canal. Segmental flow has been described in the eyes of multiple species, including primate, bovine, mouse, and human samples. While the trabecular meshwork appears to be the major source of resistance, trabecular meshwork bypass procedures have been unable to achieve the degree of IOP reduction observed with trabeculectomy, reflecting the potential impact of distal flow, or flow through Schlemm canal and collector channels, on outflow. Multiple studies have demonstrated that outflow occurs preferentially near collector channels, suggesting that these distal structures may be more important to aqueous outflow than previously believed.

onjunctival melanoma (CM) is a rare but potentially deadly ocular malignant condition, with a 10-year disease-specific mortality of 9% to 35%. 1 Primary treatment of CM consists of local surgical excision with wide margins and adjuvant therapy (cryotherapy, brachytherapy, and/or topical application of mitomycin C). However, regional and systemic metastasis occurs in approximately 30% of patients within 3 years, and there are no effective treatments for metastatic disease. 1 Conjunctival melanoma appears to be a distinct entity compared with other mucosal melanomas. In contrast to these malignant conditions, CM incidence is often associated with UV sunlight exposure. Conjunctival melanoma is also associated with a higher 5-year survival rate (86%) compared with melanomas of the gastrointestinal tract (4%-33%), urogenital tract (7%-22%), and respiratory mucosal tissues (0%-31%); this difference is possibly related to earlier detection or differences in the innate aggressiveness of the tumor. 2 The molecular attributes of CM remain poorly characterized, which is a problem that has hindered the development of novel therapies. One study 3 reported mutations in BRAF and NRAS in 29% and 18% of CMs, respectively, but the technology used in this study did not allow for a comprehensive assessment of driver mutations, chromosome copy number aberrations (CNAs), and mutational signatures. In the present study, whole-exome sequencing (WES) permits more comprehensive characterization of the molecular biology of CM. MethodsFive formalin-fixed, paraffin-embedded, archival CM specimens were selected based on the availability of sufficient tissue for testing. Tumor DNA was extracted from all 5 and prepared for WES. Ethical approval was obtained from the University of Miami institutional review board for this study, IMPORTANCE Conjunctival melanoma (CM) is a highly aggressive ocular cancer for which treatment options are limited; the molecular pathogenesis is poorly understood. OBJECTIVE To identify the molecular characteristics of CM using next-generation whole-exome sequencing (WES).DESIGN, SETTING, AND PARTICIPANTS Whole-exome sequencing was performed on tumor DNA extracted from the archived specimens of 5 patients with CM who had been treated with surgical excision between 2006 and 2011. These samples were analyzed at a tertiary academic ocular oncology referral center using a customized bioinformatic pipeline.MAIN OUTCOMES AND MEASURES Sample analyses were designed to detect driver mutations, chromosome copy number aberrations, and mutation signatures. RESULTSThe study's 5 patients ranged in age from 51 to 77 years. Four of the 5 were female, and all were white. Mutations were detected in known oncogenes, including BRAF, NRAS, NF1, EGFR, ALK, TERT, and APC. None of the mutations associated with uveal melanoma were found. All samples demonstrated a C→T mutation signature typical of UV-induced DNA damage. The most common CNA was a gain in chromosome 6p. CONCLUSIONS AND RELEVANCEIn these 5 patients, WES allowed identification of m...

A total of 187 male baseball players between 14 and 42 years of age (mean, 20.7 years) had an ulnar collateral ligament reconstruction by 1 surgeon. Eight (4%) of 187 baseball players were treated for the combined flexor-pronator/ulnar collateral ligament injury. There was a statistically significant difference in age between the ulnar collateral ligament group (20.1 years) and the flexor-pronator/ulnar collateral ligament group (33.4 years) (P < .001). Age > or =30 years was a statistically significant age limit to predict the presence of a combined flexor-pronator/ulnar collateral ligament injury (88%) compared with an isolated ulnar collateral ligament injury (1%) (P < .001). Outcomes were 1 excellent (12.5%), 2 fair (25%), and 5 poor (62.5%). Conclusion Combined fflexor-pronator and ulnar collateral ligament injuries in baseball players may portend a worse prognosis, with a 12.5% return to prior level of play. Older age (> or =30 years) is a risk factor in the development of this combined injury. When combined flexor-pronator/ulnar collateral ligament injury is suspected preoperatively, patients should be counseled on expected outcomes appropriately.