Predicting glycosaminoglycan surface protein interactions and implications for studying axonal growth.

Cell-surface carbohydrates play important roles in numerous biological processes through their interactions with various protein-binding partners. These interactions are made possible by the vast structural diversity of carbohydrates and the diverse array of carbohydrate presentations on the cell surface. Among the most complex and important carbohydrates are glycosaminoglycans (GAGs), which display varied stereochemistry, chain lengths, and patterns of sulfation. GAG-protein interactions participate in neuronal development, angiogenesis, spinal cord injury, viral invasion, and immune response. Unfortunately, little structural information is available for these complexes; indeed, for the highly sulfated chondroitin sulfate motifs, CS-E and CS-D, there are no structural data. We describe here the development and validation of the GAG-Dock computational method to predict accurately the binding poses of protein-bound GAGs. We validate that GAG-Dock reproduces accurately (<1-Å rmsd) the crystal structure poses for four known heparin-protein structures. Further, we predict the pose of heparin and chondroitin sulfate derivatives bound to the axon guidance proteins, protein tyrosine phosphatase σ (RPTPσ), and Nogo receptors 1-3 (NgR1-3). Such predictions should be useful in understanding and interpreting the role of GAGs in neural development and axonal regeneration after CNS injury.

Optimized In Vitro Antibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus.

Staphylococcus aureus small-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST of S. aureus SCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86 S. aureus SCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin; mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.

A Detailed Analysis of Workplace Foot and Ankle Injuries.

INTRODUCTION: This study analyzes the incidence rate and median workdays missed due to foot and ankle injuries across age groups, sexes, and industries. METHODS: Workplace injury data from 2003 to 2019 were obtained using the Nonfatal Cases Involving Day Away from Work: Selected Characteristics database provided by the Bureau of Labor Statistics (BLS). The data were grouped by injury location (ie, foot, ankle), injury type (ie, fracture, sprain), and industry, and reported with injury incidence rates and median workdays missed. RESULTS: The incidence rate of foot and ankle injuries significantly decreased from 2003 to 2019 (P < .001). With increasing age, the incidence rate of foot and ankle injuries decreased (P < .001) and median workdays missed increased (P < .001). Men had significantly higher rates of foot and ankle injuries (P < .001). Agriculture, forestry, fishing, and hunting (foot=10.23%, ankle=10.41%); construction (foot=8.14%, ankle=8.68%); and transportation and warehousing (foot=11.06%, ankle=13.80%) industries had the highest injury incidence rates. Transportation and warehousing (foot=16.8 days, ankle=16.3 days), mining (foot=44.9 days, ankle=17.1 days), and utilities (foot=26.7 days, ankle=24.4 days) industries had the highest median workdays missed. CONCLUSION: Increased incidence and severity of workplace foot and ankle injuries are associated with male sex and heavy labor industries. Age was positively associated with severity and negatively associated with incidence of workplace ankle injuries. LEVELS OF EVIDENCE: Level III, Retrospective cohort study.

Characterizing and predicting the functional and conformational diversity of seven-transmembrane proteins.

The activation of seven-transmembrane receptors (7TMRs) allows cells to sense their environment and convert extracellular signals (like hormone binding) into intracellular signals (through G protein-coupled and/or ß arrestin-coupled pathways). A single 7TMR is capable of transducing a wide spectrum of physiological responses inside a cell by coupling to these pathways. This intracellular pleiotropic action is enabled by multiple conformations exhibited by these receptors. Developments in membrane protein structure determination technologies have led to a rapid increase in crystal structures for many 7TMRs. Majority of these receptors have been crystallized in their inactive conformation and, for some, one of the many active conformations has also been crystallized. Given the topological constraints of a lipid bilayer that results in a single fold of seven almost parallel TM helices connected by mostly unstructured loops, these structures exhibit a diversity of conformations not only across the receptors but also across the different functional forms for receptors with structures for one of the functionally active conformations. Here we present a method to characterize this conformational diversity in terms of transmembrane helix topology (TMHTOP) parameters and how to use these helix orientation parameters to predict functionally-distinct multiple conformations for these receptors. The TMHTOP parameters enable a quantification of the structural changes that underlie 7TMR activation and also sheds a unique mechanistic light on the pleiotropic nature of these receptors. It provides a common language to describe the 7TMR activation mechanisms as well as differences across many receptors in terms of visually intuitive structural parameters. Protein structure prediction methods can use these parameters to describe 7TMR conformational ensembles, which coupled to experimental data can be used to develop testable hypotheses for the structural basis of 7TMR functions.

Outcomes and complications after different surgical techniques for the treatment of chronic distal biceps tendon ruptures: a systematic review and quantitative synthesis.

Hypothesis: The purpose of this study was to perform a systematic review of the available literature evaluating surgical outcomes after chronic distal biceps tendon rupture. Surgical techniques, including primary repair, autograft reconstruction, and allograft reconstruction, were compared, as well as subjective and objective clinical outcomes and complication rates. Methods: A systematic literature search of Level I-IV studies reporting outcomes of surgically treated chronic distal biceps tendon ruptures were performed via PubMed, Cochrane Collaboration, Science Direct, and Google Scholar databases. Twenty-two papers were identified in the review, with 236 patients. A Modified Coleman Methodological Score (CMS) was determined for every article to assess study quality. Patient-reported outcomes, postoperative range of motion, flexion and supination strength, and postoperative complications were recorded. MAYO elbow scores (MEPS) were reported more consistently than the other outcome tools. Results: No Level I or level II studies were identified in our search, and the heterogeneity of outcome measures precluded meta-analysis. Studies demonstrated mean MEPS scores ranging between 86 and 100, regardless of the surgical technique utilized. All studies reported a mean flexion-extension arc equal to or greater than 5-130°. The reported mean postoperative flexion strength was within 10% of the unaffected contralateral side. The most common complication for both direct repair and reconstruction groups was paresthesia of the lateral antebrachial cutaneous nerve [direct repair: 18-16.8%; reconstruction: 8-6.2% (allograft: 4-6%; autograft: 4-7%)]. Rerupture was uncommon and occurred in three patients who had undergone direct repair and in one patient after autograft reconstruction. Conclusions: Surgical treatment of chronic distal biceps injuries yields favorable objective and subjective outcomes. Currently, available evidence suggests that direct repair, autograft reconstruction, or allograft reconstruction are all viable treatment options with similar outcomes.

Predicted 3D structures for adenosine receptors bound to ligands: comparison to the crystal structure.

G protein-coupled receptors (GPCRs) are therapeutic targets for many diseases, but progress in developing active and selective therapeutics has been severely hampered by the difficulty in obtaining accurate structures. We have been developing methods for predicting the structures for GPCR ligand complexes, but validation has been hampered by a lack of experimental structures with which to compare our predictions. We report here the predicted structures of the human adenosine GPCR subtypes (A(1), A(2A), A(2B), and A(3)) and the binding sites for adenosine agonist and eight antagonists to this predicted structure, making no use of structural data, and compare with recent experimental crystal structure for ZM241385 bound human A(2A) receptor. The predicted structure correctly identifies 9 of the 12 crystal binding site residues. Moreover, the predicted binding energies of eight antagonists to the predicted structure of A(2A) correlate quite well with experiment. These excellent predictions resulted when we used Monte Carlo techniques to optimize the loop structures, particularly the cysteine linkages. Ignoring these linkages led to a much worse predicted binding site (identifying only 3 of the 12 important residues). These results indicate that computational methods can predict the three-dimensional structure of GPCR membrane proteins sufficiently accurately for use in designing subtype selective ligands for important GPCR therapeutics targets.

Pilus-mediated epithelial cell death in response to infection with Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic pathogen that can cause serious infections in cystic fibrosis (CF) patients. The ET12 lineage appears particularly virulent in CF; however, its pathogenesis is poorly understood and may be associated with host response. To help characterize this response, the ability of B. cenocepacia to induce cytotoxicity and apoptosis in an epithelial cell model was examined. Upon infection with B. cenocepacia strain K56-2, A549 human lung epithelial cells underwent significant cell death; propidium iodine staining and DNA fragmentation assays suggested apoptosis. Initiation of cell death was independent of the type III secretion system, biofilm formation, and secreted bacterial cytotoxins. However, the frequency of cell death was lower in cells infected with a non-piliated mutant, K56-2 cblA::Tp. Furthermore, purified cbl pili were found to directly induce cytotoxicity in A549 cells and activate caspase-9, -8, -7, and -3, the major cysteine proteinases involved in apoptosis. It appears that B. cenocepacia cbl pili, which are a distinctive feature of the ET12 lineage, act as an initiator of cytotoxicity and apoptosis. Understanding the role of cbl pili in the pathogenesis of B. cenocepacia infections offers the potential for decreasing the virulence of these potentially life-threatening organisms in CF patients.

Salicylate induces an antibiotic efflux pump in Burkholderia cepacia complex genomovar III (B. cenocepacia).

An antibiotic efflux gene cluster that confers resistance to chloramphenicol, trimethoprim, and ciprofloxacin has been identified in Burkholderia cenocepacia (genomovar III), an important cystic fibrosis pathogen. Five open reading frames have been identified in the cluster. There is apparently a single transcriptional unit, with llpE encoding a lipase-like protein, ceoA encoding a putative periplasmic linker protein, ceoB encoding a putative cytoplasmic membrane protein, and opcM encoding a previously described outer membrane protein. A putative LysR-type transcriptional regulatory gene, ceoR, is divergently transcribed upstream of the structural gene cluster. Experiments using radiolabeled chloramphenicol and salicylate demonstrated active efflux of both compounds in the presence of the gene cluster. Salicylate is an important siderophore produced by B. cepacia complex isolates, and both extrinsic salicylate and iron starvation appear to upregulate ceoR promoter activity, as does chloramphenicol. These results suggest that salicylate is a natural substrate for the efflux pump in B. cenocepacia and imply that the environment of low iron concentration in the cystic fibrosis lung can induce efflux-mediated resistance, even in the absence of antibiotic selective pressure.

Functional selectivity of dopamine D1 receptor agonists in regulating the fate of internalized receptors.

Recently, we demonstrated that D(1) agonists can cause functionally selective effects when the endpoints of receptor internalization and adenylate cyclase activation are compared. The present study was designed to probe the phenomenon of functional selectivity at the D(1) receptor further by testing the hypothesis that structurally dissimilar agonists with efficacies at these endpoints that equal or exceed those of dopamine would differ in ability to influence receptor fate after internalization, a functional endpoint largely unexplored for the D(1) receptor. We selected two novel agonists of therapeutic interest that meet these criteria (the isochroman A-77636, and the isoquinoline dinapsoline), and compared the fates of the D(1) receptor after internalization in response to these two compounds with that of dopamine. We found that dopamine caused the receptor to be rapidly recycled to the cell surface within 1h of removal. Conversely, A-77636 caused the receptor to be retained intracellularly up to 48 h after agonist removal. Most surprisingly, the D(1) receptor recovered to the cell surface 48 h after removal of dinapsoline. Taken together, these data indicate that these agonists target the D(1) receptor to different intracellular trafficking pathways, demonstrating that the phenomenon of functional selectivity at the D(1) receptor is operative for cellular events that are temporally downstream of immediate receptor activation. We hypothesize that these differential effects result from interactions of the synthetic ligands with aspects of the D(1) receptor that are distal from the ligand binding domain.

Dermoid cyst of the infratemporal fossa: case report and review of the literature.

Background Intracranial dermoid cysts are rare tumors of congenital origin. We report a case of a large dermoid tumor arising in the infratemporal fossa (ITF) with erosion into the middle cranial fossa. After reviewing the literature, we believe this represents the first reported dermoid tumor of the ITF with extension into the middle cranial fossa. Results A 21-year-old women presented with a large cystic mass involving the left infratemporal fossa and middle cranial fossa that was discovered following a motor vehicle collision. Neurologic examination was normal. The mass was resected through a frontotemporal extradural approach with endoscopic assistance. Imaging studies, gross findings, and histopathology were consistent with a dermoid tumor. Conclusion This is the first report of a dermoid cyst arising in the ITF with extension into the middle cranial fossa. We suggest including dermoid tumor in the differential diagnosis of cystic abnormalities in this region. Complete resection of the cyst remains the preferred treatment with surgical approach guided by preoperative imaging.

Structure prediction of G protein-coupled receptors and their ensemble of functionally important conformations.

G protein-coupled receptors (GPCRs) are integral membrane proteins whose "pleiotropic" nature enables transmembrane (TM) signal transduction, amplification, and diversification via G protein-coupled and ß arrestin-coupled pathways. GPCRs appear to enable this by being structurally flexible and by existing in different conformational states with potentially different signaling and functional consequences. We describe a method for the prediction of the three-dimensional structures of these different conformations of GPCRs starting from their amino acid sequence. It combines a unique protocol of computational methods that first predict the TM regions of these receptors and TM helix shapes based on those regions, which is followed by a locally complete sampling of TM helix packings and their scoring that results in a few (~10-20) lowest energy conformations likely to play a role in binding to different ligands and signaling events. Prediction of the structures for multiple conformations of a GPCR is starting to enable the testing of multiple hypotheses related to GPCR activation and binding to ligands with different signaling profiles.

Attenuated virulence of a Burkholderia cepacia type III secretion mutant in a murine model of infection.

Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells. Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium Burkholderia cepacia, an important pulmonary pathogen in immunocompromised patients and patients with cystic fibrosis. One of the genes, designated bscN, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion. Genetic dissection of the regions flanking the bscN gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria. A defined null mutation was generated in the bscN gene, and the null strain and wild-type parent strain were examined by use of a murine model of B. cepacia infection. Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the bscN null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection. Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic bscN mutant. These results implicate type III secretion as an important determinant in the pathogenesis of B. cepacia.

Correlation between an in vitro invasion assay and a murine model of Burkholderia cepacia lung infection.

Our understanding of the virulence of Burkholderia cepacia complex lung infections in cystic fibrosis patients is incomplete. There is a great deal of variability in the clinical course, from simple colonization to severe and often fatal necrotizing pneumonia, termed cepacia syndrome. Multiple subspecies (called genomovars) have been identified, and these genomovars may hold the key to understanding the variable pathogenicity. Thirty-one B. cepacia complex isolates belonging to five of the seven genomovars were examined by using a gentamicin protection assay of invasion with A549 cells. The level of epithelial cell invasion by B. cepacia in the A549 model was relatively low compared with the data obtained for other pathogens and was often variable from assay to assay. Thus, a statistical approach was used to determine invasiveness. When this model was used, one of four genomovar I strains (25%), three of eight genomovar II strains (37.5%), seven of nine genomovar III strains (77.8%), one of four genomovar IV strains (25%), and none of the four genomovar V strains examined were defined as invasive. All other strains were categorized as either noninvasive or indeterminate. Invasive, noninvasive, and indeterminate isolates belonging to genomovars II and III were subsequently tested for splenic invasion with the mouse agar bead model. Correlation between the models for six strains was demonstrated. Our results indicate that a statistical model used to determine invasiveness in an in vitro invasion assay can be used to predict in vivo invasiveness.

Contribution of Burkholderia cenocepacia flagella to infectivity and inflammation.

Burkholderia cenocepacia is an opportunistic pathogen that can cause severe lung infections in cystic fibrosis patients. To understand the contribution of B. cenocepacia flagella to infection, a strain mutated in the major flagellin subunit, fliCII, was constructed in B. cenocepacia K56-2 and tested in a murine agar bead model of lung infection. C57/BL6 mice infected with approximately 10(8) wild-type K56-2 bacteria exhibited 40% mortality after 3 days, whereas no mortality was noted in mice infected with the fliCII mutant. Among the mice surviving the infection with either strain, there was no significant difference in the bacterial loads in the lungs and spleen, bacteremia, weight loss, or infiltration of immune effector cells at 3 days postinfection. Similar results were observed at 24 h, prior to expression of the lethality phenotype. KC, a murine interleukin-8 (IL-8) homolog, was elevated in both the bronchoalveolar lavage fluid and serum of mice infected with the wild type compared to the fliCII mutant at 24 h, suggesting that flagella stimulated host cells. To demonstrate that flagella contributed to these responses, the interaction between B. cenocepacia and Toll-like receptor 5 (TLR5) was investigated. Infection of HEK293 cells with heat-killed wild-type K56-2, but not infection with the fliCII mutant, resulted in both NF-kappaB activation and IL-8 secretion that was dependent upon expression of TLR5. Together, these results demonstrate that B. cenocepacia flagella contribute to virulence in an in vivo infection model, and that induction of host immune responses through interaction with TLR5 may contribute to its overall pathogenic potential.