Insertion Orientation Research Articles

Do Dual-Thread Orthodontic Mini-Implants Improve Bone/Tissue Mechanical Retention?

The aim of this study was to understand whether the pitch relationship between micro and macro thread designs with a parametrical relationship in a dual-thread mini-implant can improve primary stability. Three types of mini-implants consisting of single-thread (ST) (0.75 mm pitch in whole length), dual-thread A (DTA) with double-start 0.375 mm pitch, and dual-thread B (DTB) with single-start 0.2 mm pitch in upper 2-mm micro thread region for performing insertion and pull-out testing. Histomorphometric analysis was performed in these specimens in evaluating peri-implant bone defects using a non-contact vision measuring system. The maximum inserted torque (Tmax) in type DTA was found to be the smallest significantly, but corresponding values found no significant difference between ST and DTB. The largest pull-out strength (Fmax) in the DTA mini-implant was found significantly greater than that for the ST mini-implant regardless of implant insertion orientation. Mini-implant engaged the cortical bone well as observed in ST and DTA types. Dual-thread mini-implant with correct micro thread pitch (parametrical relationship with macro thread pitch) in the cortical bone region can improve primary stability and enhanced mechanical retention.

Path planning for robot-assisted active flexible needle using improved Rapidly-Exploring Random trees.

In robot-assisted needle-based medical procedures, path planning for a flexible needle is challenging with regard to time consumption and searching robustness for the solution due to the nonholonomic motion of the needle tip and the presence of anatomic obstacles and sensitive organs in the intended needle path. We propose a novel and fast path planning algorithm for a robot-assisted active flexible needle. The algorithm is based on Rapidly-Exploring Random Trees combined with reachability-guided strategy and greedy heuristic strategy. Linear segments are taken into consideration to the paths, and insertion orientations are relaxed by the introduction of the linear segments. The proposed algorithm yields superior results as compared to the commonly used algorithm in terms of computational speed, form of path and robustness of searching ability, which potentially can make it suitable for the real-time intraoperative planning for clinical procedures.

Fluoroscopy-based laser guidance system for linear surgical tool insertion depth control.

In most orthopedic surgeries, knowing how far to insert surgical tools is crucial. The objective of this study was to provide guidance information on depth without tracking surgical tools. A previously developed laser guidance system for linear surgical tool insertion uses two laser beams that display the insertion point and orientation on the skin surface. However, the system only provides 4 degrees of freedom guidance (an entry point on the planned pathway line and the orientation) but do not inform surgeons on the ideal insertion depth. A 5-DOF guidance method was developed to provide guidance information by direct projection onto the surgical area using laser beams without tracking markers. A position and orientation guidance performed by two laser beams and depth guidance performed by a single laser beam are appeared on the surgical area in turn. However, depth point appears on the surgical tool side face with some error because of tool radius. Using the actual depth position, insertion path vector and location of the laser sources, the correct depth point on the tool's surface is calculated by the proposed method. So, this system can indicate and navigate the 5-DOF which is planning path and the correct depth point. An evaluation of the accuracy of depth guidance revealed a depth guidance error of 0.55±0.29 mm and results from phantom target insertions revealed overall system accuracies of 1.44 ± 1.09 mm, 0.91° ± 0.82°. In addition, overall system accuracies of application feasibility experiment under the X-ray condition were 1.94 ± 0.98mm, 1.39° ± 1.30°. A new surgical tool depth insertion method was developed using a fluorolaser guidance system. This tool informs surgeons of the surgical tool tip depth assuming that the insertion point and orientation are correct. The new method was tested successfully in vitro.

The morphology and clinical significance of the dorsal meningovertebra ligaments in the cervical epidural space

Background contextThe dural sac is anchored within the vertebral canal by connective tissue called meningovertebral ligaments in the epidural space. During flavectomy and laminectomy, inadvertent disruption of the dorsal meningovertebral ligaments may lead to dura laceration and cerebrospinal fluid (CSF) leaks. All the described dorsal meningovertebral ligaments were located in the lumbar region. A rare study is available about dorsal meningovertebral ligaments of the cervical spinal dura to the adjacent vertebrae. PurposeTo identify and describe the dorsal meningovertebral ligaments at each cervical level and discuss their clinical significance. Study designA dissection-based study of 22 embalmed cadavers. MethodsThe anatomy was studied in 22 whole cervical cadavers (11 females, 11males), prepared with formaldehyde, whose ages at the time of death ranged from 55 to 78 years. The vertebral canal was divided to expose the dural sac and the spinal nerve roots. At all levels of the cervical vertebra, the morphology, quantity, origin, insertion, and spatial orientation of the dorsal meningovertebral ligaments were determined and the length, width or diameter, and thickness of the ligaments were measured with vernier calipers. ResultsThe dorsal meningovertebral ligaments in the cervical region anchored the posterior dural sac to the ligamentum flavum or laminae. The number of attachment points on the ligamentum flavum was relatively larger than that on the lamina, and the occurrence rate of dorsal meningovertebral ligaments was 100% at C1–C2 and C4––C5. The thickest ligaments were observed at the C1 and C2 vertebrae. The length of the ligaments varied from 1.50 to 35.22 mm, and the orientation of the ligaments mostly was craniocaudal. The morphology of the dorsal meningovertebral ligaments was divided into four types: strip type, cord type, grid type, and thin slice type. ConclusionsIn the cervical spine, the dorsal meningovertebral ligaments exist between the posterior dural sac and the ligamentum flavum or lamina. The dorsal meningovertebral ligaments may be of clinical importance to surgeons. Dissecting the dorsal meningovertebral ligaments before the cervical flavectomy and laminectomy may be an important step in reducing postoperative dura laceration and CSF leaks, which may result in significant benefits for patients and health-care organizations.

Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices.

The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analyzed via coarse grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations, we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of a family of helix sequences. We illustrate this software via analyses of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analyses of these ensembles of simulations, we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application, we use CGMD simulations to examine the self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase and analyze the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers a proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins.

Heat transfer and thermodynamic performance of a parabolic trough receiver with centrally placed perforated plate inserts

In this paper, a numerical investigation of thermal and thermodynamic performance of a receiver for a parabolic trough solar collector with perforated plate inserts is presented. The analysis was carried out for different perforated plate geometrical parameters including dimensionless plate orientation angle, the dimensionless plate spacing, and the dimensionless plate diameter. The Reynolds number varies in the range 1.02×104⩽Re⩽7.38×105 depending on the heat transfer fluid temperature. The fluid temperatures used are 400K, 500K, 600K and 650K. The porosity of the plate was fixed at 0.65. The study shows that, for a given value of insert orientation, insert spacing and insert size, there is a range of Reynolds numbers for which the thermal performance of the receiver improves with the use of perforated plate inserts. In this range, the modified thermal efficiency increases between 1.2% and 8%. The thermodynamic performance of the receiver due to inclusion of perforated plate inserts is shown to improve for flow rates lower than 0.01205m3/s. Receiver temperature gradients are shown to reduce with the use of inserts. Correlations for Nusselt number and friction factor were also derived and presented.

Abnormal Structure and Function of the Esophagogastric Junction and Proximal Stomach in Gastroesophageal Reflux Disease

This study applies concurrent magnetic resonance imaging (MRI) and high-resolution manometry (HRM) to test the hypothesis that structural factors involved in reflux protection, in particular, the acute insertion angle of the esophagus into the stomach, are impaired in gastroesophageal reflux disease (GERD) patients. A total of 24 healthy volunteers and 24 patients with mild-moderate GERD ingested a test meal. Three-dimensional models of the esophagogastric junction (EGJ) were reconstructed from MRI images. Measurements of the esophagogastric insertion angle, gastric orientation, and volume change were obtained. Esophageal function was assessed by HRM. Number of reflux events and EGJ opening during reflux events were assessed by HRM and cine-MRI. Statistical analysis applied mixed-effects modeling. The esophagogastric insertion angle was wider in GERD patients than in healthy subjects (+7° ± 3°; P=0.03). EGJ opening during reflux events was greater in GERD patients than in healthy subjects (19.3 mm vs. 16.8 mm; P=0.04). The position of insertion and gastric orientation within the abdomen were also altered (both P<0.05). Median number of reflux events was 3 (95% CI: 2.5-4.6) in GERD and 2 (95% CI: 1.8-3.3) in healthy subjects (P=0.09). Lower esophageal sphincter (LES) pressure was lower (-11 ± 2 mm Hg; P<0.0001) and intra-abdominal LES length was shorter (-1.0 ± 0.3 cm, P<0.0006) in GERD patients. GERD patients have a wider esophagogastric insertion angle and have altered gastric morphology; structural changes that could compromise reflux protection by the "flap valve" mechanism. In addition, the EGJ opens wider during reflux in GERD patients than in healthy volunteers: an effect that facilitates volume reflux of gastric contents.

Detection of Structural Variants Involving Repetitive Regions in the Reference Genome

Next-generation sequencing techniques are now commonly used to characterize structural variations (SVs) in population genomics and elucidate their associations with phenotypes. Many of the computational tools developed for detecting structural variations work by mapping paired-end reads to a reference genome and identifying the discordant read-pairs whose mapped loci in the reference genome deviate from the expected insert size and orientation. However, repetitive regions in the reference genome represent a major challenge in SV detection, because the paired-end reads from these regions may be mapped to multiple loci in the reference genome, resulting in spuriously discordant read-pairs. To address this issue, we have developed an algorithmic approach for read mapping and SV detection based on the framework of A-Bruijn graphs. Instead of mapping reads to a linear sequence of the reference genome, we propose to map reads onto the A-Bruijn graph constructed from the reference genome in which all instances of the same repeat are collapsed into a single edge. As a result, any given read, either from repetitive regions or not, will be mapped to a unique location in the A-Bruijn graph, and each discordant read-pair in the A-Bruijn graph indicates a potentially true SV event. We also developed a simple clustering algorithm to derive valid clusters of these discordant read-pairs, each supporting a different SV event. Finally, we demonstrate the performance of this approach, compared to existing approaches, by identifying transposition events of insertion sequence (IS) elements, a class of simple mobile genetic elements (MGEs), in E. coli by using simulated and real paired-end sequence data acquired from E. coli mutation accumulation lines.

Linear decay of retrotransposon antisense bias across genes is contingent upon tissue specificity.

Retrotransposons comprise approximately half of the human genome and contribute to chromatin structure, regulatory motifs, and protein-coding sequences. Since retrotransposon insertions can disrupt functional genetic elements as well as introduce new sequence motifs to a region, they have the potential to affect the function of genes that harbour insertions as well as those nearby. Partly as a result of these effects, the distribution of retrotransposons across the genome is non-uniform and there are observed imbalances in the orientation of insertions with respect to the transcriptional direction of the containing gene. Although some of the factors underlying the observed distributions are understood, much of the variability remains unexplained. Detailed characterization of retrotransposon density in genes could help inform predictions of the functional consequence of de novo as well as polymorphic insertions. In order to characterize the relationship between genes and inserted elements, we have examined the distribution of retrotransposons and their internal motifs within tissue-specific and housekeeping genes. We have identified that the previously established retrotransposon antisense bias decays at a linear rate across genes, resulting in an equal density of sense and antisense retrotransposons near the 3′-UTR. In addition, the decay of antisense bias across genes is less pronounced among tissue-specific genes. Our results provide support for the scenario in which this linear decay in antisense bias is established by natural selection shortly after retrotransposon integration, and that total antisense bias observed is above and beyond any bias introduced by the integration process itself. Finally, we provide an example of a retrotransposon acting as an eQTL on a coincident gene, highlighting one of several possible avenues through which insertions may modulate gene function.

Mammalian Prion protein expression in yeast; a model for transmembrane insertion

The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the ER translocon and substrates in transit. Most inserted TM1 has its N-terminus in the ER lumen (Ntm orientation), as expected for arrest of normal secretion. However, 20% is found in inverted Ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased Ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between CtmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a C-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. However, secretion of PrP by the mammalian translocon requires the TRAP complex, absent in yeast, where essentially all PrP ends up as TM species, 85–90% Ntm and 10–15% Ctm. Although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on Ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. Electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and Ctm orientation. An increase in ΔCh from −5 to −7 caused a marked increase in Ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.

Engineered resistance in potato against potato leafroll virus, potato virus A and potato virus Y

Transgenic potato plants of Solanum tuberosum cultivar Vales Sovereign were generated that expressed fused, tandem, 200 bp segments derived from the capsid protein coding sequences of potato virus Y (PVY strain O) and potato leafroll virus (PLRV), as well as the cylindrical inclusion body coding sequences of potato virus A (PVA), as inverted repeat double-stranded RNAs, separated by an intron. The orientation of the expressed double-stranded RNAs was either sense-intron-antisense or antisense-intron-sense RNAs, and the double-stranded RNAs were processed into small RNAs. Four lines of such transgenic potato plants were assessed for resistance to infection by PVY-O, PLRV, or PVA, all transmitted by a natural vector, the green-peach aphid, Myzus persicae. Resistance was assessed by the absence of detectable virus accumulation in the foliage. All four transgenic potato lines tested showed 100% resistance to infection by either PVY-O or PVA, but variable resistance to infection by PLRV, ranging from 72 to 96% in different lines. This was regardless of the orientation of the viral inserts in the construct used to generate the transgenic plants and the gene copy number of the transgene. This demonstrates the potential for using tandem, fused viral segments and the inverted-repeat expression system to achieve multiple virus resistance to viruses transmitted by aphids in potato.

Molecular dynamics simulation of nonsteroidal antiinflammatory drugs, naproxen and relafen, in a lipid bilayer membrane

Naproxen and relafen, as nonsteroidal antiinflammatory drugs, were simulated in neutral and charged forms and their effects on a lipid bilayer membrane were investigated by molecular dynamics simulation using Groningen machine for chemical simulations software (GROMACS). Simulation of 10 systems was performed, which included different dosages of the drug molecules, naproxen and Relafen, in charged and neutral forms, and a mixture of naproxen and Relafen in neutral forms. The effects of the mixture and the individual drugs' dosages on membrane properties, such as electrostatic potential, order parameter, diffusion coefficients, and hydrogen bond formation, were analyzed. Hydration of the drugs in the membrane system was investigated using radial distribution function analysis. Using fully hydrated dimyristoylphosphatidylcholine (DMPC) as a reference system, 128 lipid molecules and water molecules were simulated exclusively, and the same simulation technique was performed on 10 other systems, including drug mixtures and a DMPC membrane. Angular distributions of lipid chains of the membrane were calculated, and the effects of the drug insertion and chain orientation in the membrane were evaluated. © 2013 Wiley Periodicals, Inc.

Identification and characterization of transposable elements inserted into the coding sequences of horse genes

Transposable elements (TEs) are repetitive sequences dispersed throughout mammalian genomes, and they occupy important genetic positions. TEs have been shown to have both harmful and beneficial effects such as exonization, polyadenylation, and/or altering transcription rates in various vertebrate genomes. However, to the best of our knowledge, no study has yet considered the relationship between TEs and horse genes. In this study, we examined the contribution of TEs to the horse genome by collecting TEs inserted within mRNA genes. By screening the abundance, distribution, and orientation of TEs, we found that the majority of TE insertions belong to retroelements and DNA elements, most of which exist in the coding sequences of horse genes. In addition, the MIR, L1, L2, ERVL, and ERVL-MaLR subfamilies were found to be the most abundant in both non-LTR and LTR elements. Retroelements (LTRs, LINEs, and SINEs) among the TEs inserted in the coding sequences showed a preference for antisense orientation. The most pronounced imbalance in insertional orientation was observed in LINEs, which represent 40 % of all TEs in antisense orientation. Through these analyses, we identified that a total of 1310 TEs have been integrated into horse mRNA genes and small fractions of them have been exonized into coding sequences.

All-Atom Molecular Dynamic Simulations of Piscidin 1 and Piscidin 3 in Lipid Bilayers

Piscidin 1 (p1) and the less active piscidin 3 (p3) are α-helical, amphipathic, and cationic antimicrobial peptides (AMPs). They adsorb onto the anionic outer membrane of pathogenic species and induce leakage beyond a threshold peptide concentration. While different mechanisms of membrane disruption have been proposed, an atomic-level description of the events leading to cell death is lacking and elucidating these mechanisms aid in the antibiotic design. Here, p1 and p3 in three different lipid bilayers (3:1 DMPC:DMPG, 1:1 POPE:POPG, and 4:1 POPC:Cholesterol) are studied by solid-state NMR spectroscopy and all-atom molecular dynamics (MD) simulations to identify factors that differentiate the structure, orientation, and depth of insertion of piscidin. 15N-H dipolar coupling calculated for the membrane-bound peptides are in good agreement with values measured by NMR. The tilts of the peptides in the bilayer (τ) determined by fitting dipolar waves to the coupling data agree with those of the NMR-derived and MD-average structures when deviation from an ideal α-helix is included; when ideal values are assumed for the dipolar wave fitting, deviations range from 4° to 6°. While the instantaneous tilt fluctuates approximately ± 10° in the simulation, averaging over a time series of structures yields the same tilt as one for an average structure (as might be obtained from NMR-based structure determination). This is because τ ≈ 90. For transmembrane helices (τ ≈ 0), the average over a MD time series may be significantly different from a single averaged structure. Correlation between τ and the depth of insertion for p1 shows that as the peptide becomes more buried, the peptide tilts to bury the C-terminus. Moreover, the depth of insertion is 0.5 to 1.5A greater for p3 than p1, which likely reduces the activity of p3.

Temporal and spatial Bean pod mottle virus‐induced gene silencing in soybean

Virus-induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)-based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in-depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3' sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near-complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross-sections of stems and leaf petioles, near-complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post-virus inoculation in leaves to 7 weeks post-virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.

Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σ(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

3′ Self-Inactivating Long Terminal Repeat Inserts for the Modulation of Transgene Expression from Lentiviral Vectors

Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.

Coordination of Bending and Wedging in Membrane Fission

Protein complexes controlling intracellular membrane scission generally utilize multiple membrane insertions to enforce structural rearrangements of lipid monolayers. In general, the deformation fields created by individual insertions might interact destructively or constructively so that the resulting stress distribution depends strongly on the spatial organization and orientation of the individual insertions. We show here that in cylindrical topology the interaction between two ring-like membrane insertions can be both attractive and repulsive, dependently on the ratio between the curvatures of the membrane and the ring as well as on the insertion parameters. The attractive interaction drives clusterization of the rings leading to substantial stored elastic stress. This stored energy can be further used for localized membrane remodeling if the rings can interact changing the system geometry and boundary constraints. The model was applied to calculate energy barriers for dynamin-mediated membrane fission, substantial reduction of the barrier was found for a particular parameters range, indicating that the molecular design of the fission machinery involves optimization of the membrane insertion geometry.

Molecular Basis for Immune Recognition of Exposed Phosphatidylserine via the Tim Family of Proteins

The immune system recognizes a vast array of chemical signatures as antigens although historically most research has focused more exclusively on protein/protein recognition. More recently it has been appreciated that lipids can also be a potent stimulus for an immune response, both in the context of lipid-presenting molecules (CD1), and as a component of the cell's plasma membrane(phosphatidylserine receptors). We are studying the molecular mechanisms by which a family of 3 unique phosphatidylserine receptors (Tim family) can directly recognize “out of context” components of the lipid membrane and how this recognition process then stimulates an appropriate immune response. In these results we have employed a variety of biophysical techniques to elucidate the molecular details of how the Tims recognize immunogenic cellular membranes. Specifically, we are investigating protein orientation and depth of membrane insertion via x-ray reflectometry measurements in combination with advanced analysis techniques to utilize existing protein crystal structure information for improved fitting refinement. Additionally, fluorescence spectroscopy is employed to explore both strength of binding and specificity with respect to membrane composition.

Membrane protein structure and dynamics from NMR spectroscopy.

We review the current state of membrane protein structure determination using solid-state nuclear magnetic resonance (NMR) spectroscopy. Multidimensional magic-angle-spinning correlation NMR combined with oriented-sample experiments has made it possible to measure a full panel of structural constraints of membrane proteins directly in lipid bilayers. These constraints include torsion angles, interatomic distances, oligomeric structure, protein dynamics, ligand structure and dynamics, and protein orientation and depth of insertion in the lipid bilayer. Using solid-state NMR, researchers have studied potassium channels, proton channels, Ca(2+) pumps, G protein-coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action. Many of these membrane proteins have also been investigated in detergent micelles using solution NMR. Comparison of the solid-state and solution NMR structures provides important insights into the effects of the solubilizing environment on membrane protein structure and dynamics.