3D Visualisation of Additive Occlusion and Tunable Full-Spectrum Fluorescence in Calcite

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallisation processes. There is growing evidence that these additives are often occluded within the crystal lattice, where this promises an elegant means of creating nanocomposites and tuning physical properties. Here, we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO3), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy was then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required

Intraoperative device closure of perimembranous ventricular septal defects in the young children under transthoracic echocardiographic guidance; initial experience

<p>Abstract</p> <p>Objectives</p> <p>This study aimed to assess the safety and feasibility of intraoperative device closure of perimembranous ventricular septal defects (VSD) in young children guided by transthoracic echocardiography (TTE).</p> <p>Methods</p> <p>We enrolled 18 patients from our hospital to participate in the study from June 2011 to September 2011. A minimal inferior median incision was performed after full evaluation of the perimembranous VSD by real-time TTE, and a domestically made device was inserted to occlude the perimembranous VSD. The proper size of the device was determined by means of transthoracic echocardiographic analysis.</p> <p>Results</p> <p>Implantation was ultimately successful in 16 patients using TTE guidance. In these cases, the complete closure rate immediately following the operation and on subsequent follow-up was 100%. Symmetric devices were used in 14 patients, and asymmetric devices were used in two patients. Two patient were transformed to surgical treatment, one for significant residual shunting, and the other for unsuccessful wire penetration of the VSD. The follow-up periods were less than nine months, and only one patient had mild aortic regurgitation. There were no instances of residual shunt, noticeable aortic regurgitation, significant arrhythmia, thrombosis, or device failure.</p> <p>Conclusions</p> <p>Minimally invasive transthoracic device closure of perimembranous VSDs is safe and feasible, using a domestically made device under transthoracic echocardiographic guidance, without the need for cardiopulmonary bypass. This technique should be considered an acceptable alternative to surgery or device closure guided by transesophageal echocardiography in selected young children. However, a long-term evaluation of outcomes is necessary.</p

Atrioventricular block of intraoperative device closure perimembranous ventricular septal defects; a serious complication

<p>Abstract</p> <p>Background</p> <p>Atrioventricular block (AVB) is a well-reported complication after closure of perimembranous ventricular septal defects (VSDs). To report the occurrence of AVB either during or following closure of perimembranous VSDs using a novel "hybrid" method involving a minimal inferior median incision and of intraoperative device closure of the perimembranous VSDs.</p> <p>Methods</p> <p>Between January 2009 and January 2011, patients diagnosed with perimembranous VSDs eligible for intraoperative device closure with a domestic occluder were identified. All patients were assessed by real-time transesophageal echocardiography (TEE) and electrocardiography.</p> <p>Results</p> <p>Of the 97 included patients, 94 were successfully occluded using this approach. Complete AVB occurred in only one case and one case of Mobitz type II AVB was diagnosed intraoperatively. In both patients, the procedure was aborted and the AVBs quickly resolved. Glucocorticosteroids were administered to another two patients who developed Mobitz type II AVB intraoperatively. Those two patients converted to Mobitz type I AVB 3 days and 5 days postsurgically. During the follow-up period (range, 6-24 months), one patient developed complete AVB 1 week following device insertion. Surgical device removal was followed by a rapid and complete recovery of atrioventricular conduction.</p> <p>Conclusions</p> <p>Intraoperative device closure of perimembranous VSDs with a domestic occluder resulted in excellent closure rates; however, AVB is a serious complication that can occur either during or any time after device closure of perimembranous VSDs. The technique described herein may reduce the incidence of perioperative AVB complications. Surgeons are encouraged to closely monitor all patients postsurgically to ensure AVB does not occur in their patients. Additional long-term data to better identify the prevalence and risk factors for AVB in treated patients are needed.</p

MHC Class I Bound to an Immunodominant Theileria parva Epitope Demonstrates Unconventional Presentation to T Cell Receptors

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1214–224 epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design

Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

BACKGROUND:Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS:Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE:These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein

Effects of Hydrographic Variability on the Spatial, Seasonal and Diel Diving Patterns of Southern Elephant Seals in the Eastern Weddell Sea

Weddell Sea hydrography and circulation is driven by influx of Circumpolar Deep Water (CDW) from the Antarctic Circumpolar Current (ACC) at its eastern margin. Entrainment and upwelling of this high-nutrient, oxygen-depleted water mass within the Weddell Gyre also supports the mesopelagic ecosystem within the gyre and the rich benthic community along the Antarctic shelf. We used Conductivity-Temperature-Depth Satellite Relay Data Loggers (CTD-SRDLs) to examine the importance of hydrographic variability, ice cover and season on the movements and diving behavior of southern elephant seals in the eastern Weddell Sea region during their overwinter feeding trips from Bouvetøya. We developed a model describing diving depth as a function of local time of day to account for diel variation in diving behavior. Seals feeding in pelagic ice-free waters during the summer months displayed clear diel variation, with daytime dives reaching 500-1500 m and night-time targeting of the subsurface temperature and salinity maxima characteristic of CDW around 150–300 meters. This pattern was especially clear in the Weddell Cold and Warm Regimes within the gyre, occurred in the ACC, but was absent at the Dronning Maud Land shelf region where seals fed benthically. Diel variation was almost absent in pelagic feeding areas covered by winter sea ice, where seals targeted deep layers around 500–700 meters. Thus, elephant seals appear to switch between feeding strategies when moving between oceanic regimes or in response to seasonal environmental conditions. While they are on the shelf, they exploit the locally-rich benthic ecosystem, while diel patterns in pelagic waters in summer are probably a response to strong vertical migration patterns within the copepod-based pelagic food web. Behavioral flexibility that permits such switching between different feeding strategies may have important consequences regarding the potential for southern elephant seals to adapt to variability or systematic changes in their environment resulting from climate change

Genetic and Structural Basis for Selection of a Ubiquitous T Cell Receptor Deployed in Epstein-Barr Virus Infection

Despite the ∼1018 αβ T cell receptor (TCR) structures that can be randomly manufactured by the human thymus, some surface more frequently than others. The pinnacles of this distortion are public TCRs, which exhibit amino acid-identical structures across different individuals. Public TCRs are thought to result from both recombinatorial bias and antigen-driven selection, but the mechanisms that underlie inter-individual TCR sharing are still largely theoretical. To examine this phenomenon at the atomic level, we solved the co-complex structure of one of the most widespread and numerically frequent public TCRs in the human population. The archetypal AS01 public TCR recognizes an immunodominant BMLF1 peptide, derived from the ubiquitous Epstein-Barr virus, bound to HLA-A*0201. The AS01 TCR was observed to dock in a diagonal fashion, grasping the solvent exposed peptide crest with two sets of complementarity-determining region (CDR) loops, and was fastened to the peptide and HLA-A*0201 platform with residue sets found only within TCR genes biased in the public response. Computer simulations of a random V(D)J recombination process demonstrated that both TCRα and TCRβ amino acid sequences could be manufactured easily, thereby explaining the prevalence of this receptor across different individuals. Interestingly, the AS01 TCR was encoded largely by germline DNA, indicating that the TCR loci already comprise gene segments that specifically recognize this ancient pathogen. Such pattern recognition receptor-like traits within the αβ TCR system further blur the boundaries between the adaptive and innate immune systems

pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst

Homology modeling and molecular dynamics simulations of MUC1-9/H-2Kb complex suggest novel binding interactions

International audienceHuman MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K. The structure of MUC1-9 in complex with H-2K has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide's central region, partially characterized as a -turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K