Oligomeric HIV-1 Integrase Structures Reveal Functional Plasticity for Intasome Assembly and RNA Binding.

Integrase (IN) performs dual essential roles during HIV-1 replication. During ingress, IN functions within an oligomeric "intasome" assembly to catalyze viral DNA integration into host chromatin. During late stages of infection, tetrameric IN binds viral RNA and orchestrates the condensation of ribonucleoprotein complexes into the capsid core. The molecular architectures of HIV-1 IN assemblies that mediate these distinct events remain unknown. Furthermore, the tetramer is an important antiviral target for allosteric IN inhibitors. Here, we determined cryo-EM structures of wildtype HIV-1 IN tetramers and intasome hexadecamers. Our structures unveil a remarkable plasticity that leverages IN C-terminal domains and abutting linkers to assemble functionally distinct oligomeric forms. Alteration of a newly recognized conserved interface revealed that both IN functions track with tetramerization in vitro and during HIV-1 infection. Collectively, our findings reveal how IN plasticity orchestrates its diverse molecular functions, suggest a working model for IN-viral RNA binding, and provide atomic blueprints for allosteric IN inhibitor development.

Effect of superspeed sintering on translucency, opalescence, microstructure, and phase fraction of multilayered 4 mol% yttria-stabilized tetragonal zirconia polycrystal and 6 mol% yttria-stabilized partially stabilized zirconia ceramics.

STATEMENT OF PROBLEM: The optical properties of recently developed multilayer zirconia have mainly been studied for the effects of conventional sintering and speed sintering but not as much for the effect of superspeed sintering. As superspeed sintering protocols typically require a higher sintering temperature and higher heating and cooling rates than speed- and conventional sintering protocols, the optical properties of superspeed sintered zirconia may be affected differently. PURPOSE: The purpose of this in vitro study was to investigate the effect of superspeed sintering on the optical properties, microstructure, and phase fraction of multilayered 4 mol% yttria-stabilized (4Y-) and 6 mol% yttria-stabilized (6Y-) zirconia. MATERIAL AND METHODS: Multilayered 4Y- and 6Y-zirconia were sectioned. After conventional and superspeed sintering, the translucency parameter (TP), and opalescence parameter (OP) were measured with a spectrophotometer (n=10). To obtain the grain sizes from the field emission scanning electron microscopy (FE-SEM) images for each layer (n=2), more than 500 (6Y-zirconia) and 800 grains (4Y-zirconia) were measured by linear intercept methods. The phase fractions were obtained through X-ray diffraction (XRD) analysis by using the Rietveld method (n=1). The results were analyzed by 3-way ANOVA and post hoc Tukey honest significant difference tests (TP and OP) and by 3-way ANOVA and post hoc Scheffé tests (grain size) (α=.05). RESULTS: No layers exhibited a significant difference in TP after superspeed sintering, except the dentin layer (DL) and transition layer 2 (T2) of 4Y- and 6Y-zirconia, respectively. The TP increased (P<.05) in DL for superspeed sintered 4Y-zirconia and decreased (P<.05) in T2 for the superspeed sintered 6Y-zirconia. However, the difference in TP by superspeed sintering was lower than the perceptibility thresholds of 50:50%. The OP decreased (P<.05) in the DL and T2 of 4Y-zirconia after superspeed sintering. For 6Y-zirconia, the OP decreased (P<.05) in all layers except for the transition layer 1 (T1) after superspeed sintering. However, the difference in OP values was minimal, with only a 1.1 difference observed for Zolid Gen-X (4Y) and a range of 1.22 to 1.62 for Katana UTML (6Y) when using superspeed sintering. No significant change was found in the grain size after superspeed sintering of either zirconia. Regardless of the sintering speed, the average grain size of the 6Y-zirconia (conventional: 2.09 to 2.21 µm; superspeed: 2.11 to 2.20 µm) was larger than that of the 4Y-zirconia (conventional: 0.50 to 0.52 µm; superspeed: 0.52 to 0.54 µm). Owing to superspeed sintering, the metastable tetragonal (T') phase content increased while the tetragonal (T) phase decreased in 4Y-zirconia; in 6Y-zirconia, the cubic (C) phase content increased, while the T'-phase content decreased. CONCLUSIONS: Superspeed sintering did not result in any clinically significant changes in the translucency and opalescence of 4Y- or 6Y-zirconia.

Effect of Cooling Rate on Mechanical Properties, Translucency, Opalescence, and Light Transmission Properties of Monolithic 4Y-TZP during Glazing.

A standard cooling rate has not been established for glazing; therefore, the effects of the cooling rate on the properties of zirconia need to be evaluated to predict outcomes in clinical practice. 4Y-TZP glazed at three different cooling rates was analyzed to estimate the effect of cooling rate during glazing on the mechanical and optical properties. Hardness tests, field-emission scanning electron microscopy analysis, X-ray diffraction analysis, flexural strength measurement, and optical property evaluations were performed. When 4Y-TZP was glazed at a higher cooling rate (Cooling-1) than the normal cooling rate (Cooling-2), there was no significant difference in grain size, flexural strength, average transmittance, and translucency parameters. The hardness was slightly reduced. The opalescence parameter was reduced for the 2.03 mm thick specimens. When 4Y-TZP was glazed at a lower cooling rate (Cooling-3) than the normal cooling rate, there was no significant difference in hardness, grain size, flexural strength, and translucency parameters. In addition, the average transmittance and opalescence parameters were slightly reduced for the 0.52 and 2.03 mm specimens, respectively. The effects of the cooling rate during glazing on the mechanical and optical properties of 4Y-TZP appear to be minimal and clinically insignificant. Therefore, even if the cooling rate cannot be strictly controlled during glazing, the clinical outcomes will not be significantly affected.

Bacterial Reverse Mutation Test of Verbenalin.

Objectives: Verbenalin is a compound found in herbs such as Cornus officinalis and Verbena officinalis. This study investigated whether verbenalin is safe by analyzing its mutagenicity. Methods: To examine the mutagenic potential of verbenalin, a bacterial reverse mutation test (Ames test) was conducted with Salmonella typhimurium and Escherichia coli strains. Experiments with and without metabolic activity were performed. Results: The mean colony number was less than double that of the control. Growth inhibition and precipitation of verbenalin were not apparent in all strains at different concentrations regardless of metabolic activity. Conclusion: Verbenalin did not show any signs of mutagenicity in this study. Additional toxicity studies including repeated oral toxicity, reproductive toxicity, and carcinogenicity tests are needed.

Effect of cooling rate on hardness and microstructure of Pd-Ag-In-Sn-Ga alloy during porcelain firing simulation.

This study examined the effect of the cooling rate on the hardness and its effect on the microstructure during porcelain firing simulation of a Pd-Ag-In-Sn-Ga metal-ceramic alloy. In practice, after each firing step for porcelain bonding, the prosthesis is cooled to room temperature before proceeding to the next firing step. The cooling step is known to allow the hardness of the metal substructure to increase. The aim of the study was to determine whether controlling the cooling rate after each porcelain-firing step increases the hardness of the Pd-Ag-based metal-ceramic alloy. The results showed that the hardness of specimens cooled at a higher cooling rate increased after each firing step compared to specimens cooled at a lower cooling rate (p < 0.05). During cooling after the firing simulation the InPd3-based phase of tetragonal structure precipitated from the Pd-Ag-rich matrix of the face-centered cubic structure. Hardening by cooling at a higher cooling rate after firing was the result of the coherency strains that formed at the interface of the Pd-Ag-rich matrix and the metastable phase based on the InPd3 phase. . The reduced hardness obtained in the specimen cooled at a lower cooling rate after firing resulted from the loss of coherency strains as the fine metastable phases based on the InPd3 phase were transformed into the coarser stable phase with decreased (c/a) of 0.88. This finding revealed that controlling the cooling rate during porcelain firing simulation improves the hardness of the Pd-Ag-In-Sn-Ga metal-ceramic alloy without an additional heat treatment of the alloy.

Effect of ice quenching after oxidation with or without vacuum on the hardness of Pd-Ag-Au-In alloy during porcelain firing simulation.

This study examined the effect of ice-quenching after oxidation treatment with or without vacuum, on the change in the hardness of a Pd-Ag-Au-In metal-ceramic alloy during porcelain-firing simulation. The aim of the present study is to determine whether ice quenching after oxidation renders a Pd-Ag metal-ceramic alloy soft enough for easy processing without the need for an additional solution treatment and whether it affects the final hardness of the Pd-Ag metal-ceramic alloy after porcelain firing simulation. The oxidation treatment resulted in the as-cast specimen becoming homogenized, which was progressed further by vacuuming. The bench-cooling to room temperature after the oxidation resulted in precipitation. From the next firing step, the precipitation occurred during the firing process as well as subsequent bench-cooling; this was owing to the presence of a phase boundary wherein the single phase is separated into two phases between the temperatures of the oxidation treatment (1010 °C) and post-oxidation treatment (~960 °C). During the firing process until main bake, the specimen that was ice-quenched after oxidation maintained a higher hardness than the bench-cooled specimen, particularly for the oxidation treated specimen with vacuum (p < 0.05). The mechanism of ice-quenching after oxidation with vacuum to induce further hardening during the firing process involved more active precipitation; this was attributed to the further progress of homogenization during the oxidation. The precipitation reaction in the present study corresponded to the Pd-In binary phase diagram, illustrating that as the firing proceeded, the Pd3(In,Sn,Ga) phase was precipitated from the Pd-Ag-Au-rich matrix. The findings revealed that ice quenching after oxidation makes the alloy soft enough for easy processing without an additional heat treatment and does not affect the final hardness of the Pd-Ag metal-ceramic alloy after porcelain firing simulation.

Effect of ice-quenching after oxidation treatment on hardening of a Pd-Cu-Ga-Zn alloy for bonding porcelain.

This study examined the effect of ice-quenching after oxidation treatment on hardness change of a Pd-Cu-Ga-Zn metal-ceramic alloy during porcelain firing simulation. Although not statistically significant, the alloy was softened slightly during porcelain firing simulation with conventional slow cooling rate. On the other hand, the hardness increased significantly by ice-quenching instead of the slow cooling after oxidation (p<0.001). The gap in the final hardness depending on ice-quenching occurred in the matrix and plate-like precipitates but not in the particle-like structure without plate-like precipitates (p<0.05). The mechanism of ice-quenching after oxidation to prevent softening and induce hardening during porcelain firing simulation involved the more active precipitation and retardation of microstructural coarsening. In conclusion, for practical work on Pd-Cu-Ga-Zn alloys, the oxidation treatment followed by ice-quenching instead of slow cooling is recommended for the simultaneous oxidation and hardening effects on the alloy.

Effect of ice-quenching on the change in hardness of a Pd-Au-Zn alloy during porcelain firing simulation.

This study examined the effect of ice-quenching after degassing on the change in hardness of a Pd-Au-Zn alloy during porcelain firing simulations. By ice-quenching after degassing, the specimens were softened due to homogenization without the need for an additional softening heat treatment. The lowered hardness by ice-quenching after degassing was recovered greatly from the first stage of porcelain firing process by controlling the cooling rate. The increase in hardness during cooling after porcelain firing was attributed to the precipitation of the f.c.t. PdZn phase containing Au, which caused severe lattice strain in the interphase boundary between the precipitates and matrix of the f.c.c. structure. The final hardness was slightly higher in the ice-quenched specimen than in the specimen cooled at stage 0 (the most effective cooling rate for alloy hardening) after degassing. This was attributed to the more active grain interior precipitation during cooling in the ice-quenched specimen after degassing.

IL-1 receptor antagonist (IL-1Ra)-Fc ameliorate autoimmune arthritis by regulation of the Th17 cells/Treg balance and arthrogenic cytokine activation.

INTRODUCTION: IL-1ß signalling has a critical role in the pathogenesis of various types of inflammatory arthritis including rheumatoid arthritis (RA). We aimed to investigate the therapeutic effects of human IL-1 receptor antagonist with Fc fragment (hIL-1Ra-Fc) on autoimmune arthritis and to identify the possible mechanisms by which hIL-1RA-Fc exerts anti-arthritic effects in a murine model of RA and patients with rheumatoid arthritis. METHODS: Collagen-induced arthritis (CIA) murine model was established in DBA/1J mice. The levels of various cytokines were determined by using enzyme-linked immunosorbent assay. The mouse joints were assessed for clinical arthritis score and histologic features. Th17 cells and Treg cells were stained by using antibodies specific for CD4, IL-17, CD25, and FoxP3. Osteoclastogenesis was determined by TRAP staining and real-time PCR. RESULTS: hIL-1RA-Fc reduced the arthritis incidence, histological inflammation and cartilage score in the CIA model. The expression of proinflammatory cytokines, VEGF and RANK, was reduced in the affected joint of hIL-1Ra-Fc-treated mice. hIL-1Ra-Fc-treated mice showed decreased number of Th17 cells with increased number of Treg cells in spleens. hIL-1Ra-Fc reduced Th17 cell differentiation by inactivation of STAT3 signalling, and reciprocally induced Treg cell differentiation through STAT5 signalling. In addition, the expression of IL-17, TNF-α, RANKL, and VEGF was decreased, while Foxp3 gene expression was increased in PBMCs of RA patients after administration of hIL-1Ra-Fc. CONCLUSION: The anti-arthritis effects of hIL-1RA-Fc are associated with regulation of balance between Th17 cells and Treg cells and suppression of osteoclastogenesis and angiogenesis in the affected joints.

Structural features of cephalosporin acylase reveal the basis of autocatalytic activation.

Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through two steps of intramolecular autoproteolysis, the first mediated by a serine residue, and the second by a glutamate, which releases the pro-segment and produces an active enzyme. In this study, we have determined the crystal structures of mutants which could affect primary or secondary auto-cleavage and of sequential intermediates of a slow-processing mutant at 2.0-2.5A resolutions. The pro-segments of the mutants undergo dynamic conformational changes during activation and adopt surprisingly different loop conformations from one another. However, the autoproteolytic site was found to form a catalytically competent conformation with a solvent water molecule, which was essentially conserved in the CA mutants.

The crystal structure of ferritin from Helicobacter pylori reveals unusual conformational changes for iron uptake.

The crystal structure of recombinant ferritin from Helicobacter pylori has been determined in its apo, low-iron-bound, intermediate, and high-iron-bound states. Similar to other members of the ferritin family, the bacterial ferritin assembles as a spherical protein shell of 24 subunits, each of which folds into a four-alpha-helix bundle. Significant conformational changes were observed at the BC loop and the entrance of the 4-fold symmetry channel in the intermediate and high-iron-bound states, whereas no change was found in the apo and low-iron-bound states. The imidazole rings of His149 at the channel entrance undergo conformational changes that bear resemblance to heme configuration and are directly coupled to axial translocation of Fe ions through the 4-fold channel. Our results provide the first structural evidence of the translocation of Fe ions through the 4-fold channel in prokaryotes and the transition from a protein-dominated process to a mineral-surface-dominated process during biomineralization.

Crystallization and X-ray diffraction data collection of topoisomerase IV ParE subunit from Xanthomonas oryzae pv. oryzae.

Topoisomerase IV is involved in topological changes in the bacterial genome using the free energy from ATP hydrolysis. Its functions are the decatenation of daughter chromosomes following replication by DNA relaxation and double-strand DNA breakage. In this study, the N-terminal fragment of the topoisomerase IV ParE subunit from Xanthomonas oryzae pv. oryzae was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.15 A resolution using a synchrotron-radiation source. The crystal belonged to space group P4(2)2(1)2, with unit-cell parameters a = b = 105.30, c = 133.76 A. The asymmetric unit contains one molecule, with a corresponding V(M) of 4.21 A(3) Da(-1) and a solvent content of 69.6%.

Induction of mucosal and systemic immune responses following oral immunization of mice with Lactococcus lactis expressing human papillomavirus type 16 L1.

Human papillomavirus type 16 L1 (HPV16 L1) has shown considerable promise as a parenteral vaccine for prevention of cervical cancers. Here, we report the possibility of oral vaccination for HPV16 L1 using Lactococcus lactis (L. lactis) as a live vector. L. lactis MG1363 was transformed with two types of HPV16 L1-encoding plasmids for intracellular expression or secretion. L. lactis transformed with HPV16 L1-encoding plasmids retained biochemical lactic acid production capability. The mucosal and systemic immune responses were affected by the cellular location of expressed HPV16 L1 proteins in L. lactis. Serum IgG responses were induced after oral immunizations of L. lactis secreting HPV16 L1. Vaginal IgA immune responses were observed following oral immunization with L. lactis expressing HPV16 L1 in an intracellular form, but not with L. lactis secreting HPV16 L1. Furthermore, induction of HPV16 L1-specific mucosal immune responses was affected by immunization frequency. Six immunizations over 5 weeks were required to induce vaginal immune responses. The levels of HPV16 L1-specific vaginal IgA were maintained until 12 weeks after the first vaccination. These results suggest the feasibility of L. lactis as an oral vaccine vehicle of HPV16 L1 and demonstrate the importance of cellular loci of expressed antigen for induction of vaginal and systemic immune responses.

Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries.

Cephalosporin acylase (CA), a member of the N-terminal nucleophile hydrolase family, is activated through sequential primary and secondary autoproteolytic reactions with the release of a pro segment. We have determined crystal structures of four CA mutants. Two mutants are trapped after the primary cleavage, and the other two undergo secondary cleavage slowly. These structures provide a look at pro-segment conformation during activation in N-terminal nucleophile hydrolases. The highly strained helical pro segment of precursor is transformed into a relaxed loop in the intermediates, suggesting that the relaxation of structural constraints drives the primary cleavage reaction. The secondary autoproteolytic step has been proposed to be intermolecular. However, our analysis provides evidence that CA is processed in two sequential steps of intramolecular autoproteolysis involving two distinct residues in the active site, the first a serine and the second a glutamate.