Insertion Of Strand Research Articles

P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA–LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.

Safety and efficacy of self-expandable metallic stent combined with 125I brachytherapy for the treatment of malignant obstructive jaundice

BackgroundSeveral previous studies demonstrated that the combination of self-expandable metallic stents (SEMS) and 125I seed implantation might prolong stent patency and obtain survival benefits for malignant obstructive jaundice (MOJ) patients. However, these studies rarely mentioned a comparison between CT-guided intratumoral 125I seed implantation and intraluminal 125I seed strand insertion combined with stenting for the management of MOJ. This study aimed to further evaluate the safety and efficacy of SEMS combined with 125I brachytherapy in the management of unresectable MOJ.MethodsFifty-nine patients with unresectable MOJ were retrospectively included from March 2018 to June 2021. The main therapeutic outcomes were evaluated in terms of stent patency, and overall survival. Cumulative stent patency and overall survival rates were calculated by Kaplan–Meier survival analysis. Both clinical and treatment factors associated with survival were analyzed.ResultsTechnical success was achieved in all patients. The clinical success rate was 94% (32/34) in the seeds group and 92% (23/25) in the control group, no significant difference was found (p =1.000). The median duration of stent patency was significantly longer in the 125I brachytherapy group compared with the control group (289 days vs. 88 days, respectively, p =0.001). The 125I brachytherapy group demonstrated a significantly better median overall survival rate than the control group (221 days vs. 78 days, respectively, p =0.001). In multivariate analysis, stents with 125I brachytherapy (p =0.004) was a significant favorable prognostic factor that affected patient survival. No significant difference was observed between CT-guided 125I seed implantation and 125I seed strand insertion in stent patency (p =0.268), and overall survival (p =0.483).ConclusionSEMS combined with 125I brachytherapy is safe and effective for treating MOJ. 125I brachytherapy may help to maintain stent patency and prolong overall survival. There was no significant difference between CT-guided 125I seed implantation with SEMS and 125I seed strand insertion with SEMS in stent patency and overall survival.

Safety and efficacy of iodine-125 seed strand for intraluminal brachytherapy on ureteral carcinoma.

Our aim is to evaluate the safety and efficacy of iodine-125 seed strand for intraluminal brachytherapy on ureteral carcinoma. From November 2014 to November 2021, 22 patients with ureteral cancer not suitable for surgical resection were enrolled. Iodine-125 seed strand was inserted under c-arm CT and fluoroscopic guidance. The technical success rate, complications, disease control rate, and survival time were evaluated. Hydronephrosis Girignon grade and ureteral cancer sizes before and after treatment were compared. A total of 46 seed strands were successfully inserted and replaced, with a technical success rate of 100% and median procedure time of 62min. No procedure-related death, ureteral perforation, infection, or severe bleeding occurred. Minor complications were observed in eight (36.4%) patients, and migration of seed strand was the most common complication. Six months after seed strand brachytherapy, one complete response, three partial responses, and five stable diseases were evaluated, and the disease control rate was 64.3%. The Girignon grade of hydronephrosis was significantly improved 1 to 3 months after seed strand insertion. Disease control rates were 94.4, 62.5, and 64.3% at 1-, 3-, and 6-month follow-up. Twenty patients were successfully followed up, with a mean follow-up of 18.0 ± 14.5 months. The median overall survival and progress-free survival were 24.7 and 13.0 months, respectively. Iodine-125 seed strand is safe and effective for intraluminal brachytherapy and can be used as an alternative to patients with ureteral carcinoma who are not suitable for surgical resection or systemic combined therapy.

Unzipping Mechanism of Free and Polyarginine-Conjugated DNA-PNA Duplexes, Preconfined Inside the α-Hemolysin Nanopore.

In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore's vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore's β-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping. This study identifies key particularities of DNA-PNA duplex unzipping as it takes place inside the nanopore and being preceded by entrapment in the vestibule domain of the α-HL. Our results are a crucial step toward understanding the nucleic acids duplexes unzipping kinetics variability, in confined, variable geometries.

Stent with radioactive seeds strand insertion for malignant hilar biliary obstruction

Purpose This study aimed to assess clinical efficacy and long-term patient outcomes in individuals with malignant hilar biliary obstruction (MHBO) that had been treated via insertion of a stent with a radioactive seed strand (RSS). Material and methods A total of 84 MHBO patients were treated via either normal stent insertion (n = 48) or stent with RSS insertion (n = 36) from January 2015 to December 2018. Results The technical success rates of normal stent insertion and stent with RSS insertion were 93.8% (45/48) and 97.2% (35/36), respectively (p = .632), with clinical success rates of 93.3% (42/45) and 100% (35/35), respectively (p = .252). In these two patient groups, 11 and seven patients, respectively, suffered from stent dysfunction (p = .637). In the normal and RSS groups, median stent patency was 165 and 225 days, respectively (p < .001). All patients in the present study died due to tumor progression, with median survival times of 188 and 250 days in the normal and RSS stent groups, respectively (p < .001). Conclusion Relative to normal stent insertion, combined stent with RSS insertion can effectively prolong both stent patency and patient survival in patients with MHBO.

Analysis of Orthodontic Prescriptions in Molars with Ideal Torque - a Laboratory Study

INTRODUCTION: There is a diverse range of orthodontic appliances available in clinical practice. However, there is also a lack of evidence regarding the effectiveness of different prescriptions on the final positioning of these teeth following rectangular strand insertion into orthodontic molar tubes. This study aimed to determine whether the torque present in the tubes of pre-adjusted devices of different prescriptions would alter the ideal inclination of the first and second lower and upper molars. METHODS: This study utilized plaster models from 30 patients who presented with ideal torque. Four prescriptions with tubes of 0.22x0.028" slot were used: MBT, Roth, Damon, and Edgewise. Tubes were glued on to plaster models on the vestibular side of teeth in the center of the clinical crown. In addition to this gluing position, the position of 1 millimeter below the center of crowns on lower models was evaluated. Gaps between tubes and steel rectangular wires of 0.019x0.025"or 0.021x0.025" thickness were measured, and the effective torque was calculated from these values. CONCLUSIONS: For 0.019x0.025" wires, a higher number of optimal torque maintenances was observed, regardless of the prescription tested, compared to 0.021x0.025" wires. The Roth prescription obtained the values closest to zero in the evaluation of the effective torque, indicating a small average torque change.

Estimation of Friction Coefficient Using Smart Strand

Friction in a post-tensioning system has a significant effect on the distribution of the prestressing force of tendons in prestressed concrete structures. However, attempts to derive friction coefficients using conventional electrical resistance strain gauges do not usually lead to reliable results, mainly due to the damage of sensors and lead wires during the insertion of strands into the sheath and during tensioning. In order to overcome these drawbacks of the existing measurement system, the Smart Strand was developed in this study to accurately measure the strain and prestressing force along the strand. In the Smart Strand, the core wire of a 7-wire strand is replaced with carbon fiber reinforced polymer in which the fiber Bragg grating sensors are embedded. As one of the applications of the Smart Strand, friction coefficients were evaluated using a full-scale test of a 20 m long beam. The test variables were the curvature, diameter, and filling ratio of the sheath. The analysis results showed the average wobble and curvature friction coefficients of 0.0038/m and 0.21/radian, respectively, which correspond to the middle of the range specified in ACI 318-08 in the U.S. and Structural Concrete Design Code in Korea. Also, the accuracy of the coefficients was improved by reducing the effective range specified in these codes by 27–34 %. This study shows the wide range of applicability of the developed Smart Strand system.

Intramolecular Donor Strand Complementation in the E. coli Type 1 Pilus Subunit FimA Explains the Existence of FimA Monomers As Off-Pathway Products of Pilus Assembly That Inhibit Host Cell Apoptosis

Type 1 pili are filamentous organelles mediating the attachment of uropathogenic Escherichia coli to epithelial cells of host organisms. The helical pilus rod consists of up to 3000 copies of the main structural subunit FimA that interact via donor strand complementation, where the incomplete Ig-like fold of FimA is completed by insertion of the N-terminal extension (donor strand) of the following FimA subunit. Recently, it was shown that FimA also exists in a monomeric, assembly-incompetent form and that FimA monomers act as inhibitors of apoptosis in infected host cells. Here we present the NMR structure of monomeric wild-type FimA with its natural N-terminal donor strand complementing the Ig fold. Compared to FimA subunits in the assembled pilus, intramolecular self-complementation in the monomer stabilizes the FimA fold with significantly less interactions, and the natural FimA donor strand is inserted in the opposite orientation. In addition, we show that a motif of two glycine residues in the FimA donor strand, separated by five residues, is the prerequisite of the alternative, parallel donor strand insertion mechanism in the FimA monomer and that this motif is preserved in FimA homologs of many enteroinvasive pathogens. We conclude that FimA is a unique case of a protein with alternative, functionally relevant folding possibilities, with the FimA polymer forming the highly stable pilus rod and the FimA monomer promoting pathogen propagation by apoptosis suppression of infected epithelial target cells.

Dynamic local unfolding in the serpin α-1 antitrypsin provides a mechanism for loop insertion and polymerization

The conformational plasticity of serpins underlies both their activities as protease inhibitors and their susceptibility to pathogenic misfolding. Here, we structurally characterize a sheet-opened state of the serpin alpha-1 antitrypsin (α1AT) and show how local unfolding allows functionally essential strand insertion. Mutations in α1AT that cause polymerization-induced serpinopathies map to the labile region, suggesting that the evolution of serpin function required them to sample conformations on a dynamic energy landscape that increased risk of aggregation.

Infinite Kinetic Stability against Dissociation of Supramolecular Protein Complexes through Donor Strand Complementation

Adhesive type 1 pili from uropathogenic Escherichia coli strains are heat and denaturant resistant, filamentous protein complexes. Individual pilus subunits associate through "donor strand complementation," whereby the incomplete immunoglobulin-like fold of each subunit is completed by the N-terminal extension of a neighboring subunit. We show that antiparallel donor strand insertion generally causes nonequilibrium behavior in protein folding and extreme activation energy barriers for dissociation of subunit-subunit complexes. We identify the most kinetically stable, noncovalent protein complex known to date. The complex between the pilus subunit FimG and the donor strand peptide of the subunit FimF shows an extrapolated dissociation half-life of 3 x 10(9) years. The 15 residue peptide forms ideal intermolecular beta sheet H-bonds with FimG over 10 residues, and its hydrophobic side chains strongly interact with the hydrophobic core of FimG. The results show that kinetic stability and nonequilibrium behavior in protein folding confers infinite stability against dissociation in extracellular protein complexes.

Serpin Polymerization Is Prevented by a Hydrogen Bond Network That Is Centered on His-334 and Stabilized by Glycerol

Polymerization of serpins commonly results from mutations in the shutter region underlying the bifurcation of strands 3 and 5 of the A-sheet, with entry beyond this point being barred by a H-bond network centered on His-334. Exposure of this histidine in antithrombin, which has a partially opened sheet, allows polymerization and peptide insertion to occur at pH 6 or less when His-334 will be predictably protonated with disruption of the H-bond network. Similarly, thermal stability of antithrombin is pH-dependent with a single unfolding transition at pH 6, but there is no such transition when His-334 is buried by a fully closed A-sheet in heparin-complexed antithrombin or in alpha(1)-antitrypsin. Replacement of His-334 in alpha(1)-antitrypsin by a serine or alanine at pH 7.4 results in the same polymerization and loop-peptide acceptance observed with antithrombin at low pH. The critical role of His-334 and the re-formation of its H-bond network by the conserved P8 threonine, on the full insertion of strand 4, are relevant for the design of therapeutic blocking agents. This is highlighted here by the crystallographic demonstration that glycerol, which at high concentrations blocks polymerization, can replace the P8 threonine and re-form the disrupted H-bond network with His-334.

The Nuclease A Inhibitor Represents a New Variation of the Rare PR-1 Fold

Nuclease A (NucA) from Anabaena sp. is a non-specific endonuclease able to degrade single and double-stranded DNA and RNA. The endonucleolytic activity is inhibited by the nuclease A inhibitor (NuiA), which binds to NucA with 1:1 stoichiometry and picomolar affinity. In order to better understand the mechanism of inhibition, the solution structure of NuiA was determined by NMR methods. The fold of NuiA is an α–β–α sandwich but standard database searches by DALI and TOP revealed no structural homologies. A visual inspection of α–β–α folds in the CATH database revealed similarities to the PR-1-like fold (SCOP nomenclature). The similarities include the ordering of secondary structural elements, a single helix on one face of the α–β–α sandwich, and three helices on the other face. However, a major difference is in the IV helix, which in the PR-1 fold is short and perpendicular to the I and III helices, but in NuiA is long and parallel to the I and III helices. Additionally, a strand insertion in the β-sheet makes the NuiA β-sheet completely antiparallel in organization. The fast time-scale motions of NuiA, characterized by enhanced flexibility of the extended loop between helices III and IV, also show similarities to P14a, which is a PR-1 fold. We propose that the purpose of the PR-1 fold is to form a stable scaffold to present this extended structure for biological interactions with other proteins. This hypothesis is supported by data that show that when NuiA is bound to NucA significant changes in chemical shift occur in the extended loop between helices III and IV.

Cell division and peptidoglycan assembly in Escherichia coli.

Research on bacterial cell division has recently gained renewed impetus because of new information about peptidoglycan assembly and about specific cell-division genes and their products. This paper concerns aspects of cell division that specifically concern the peptidoglycan. It is shown that upon division, peptidoglycan assembly switches from lateral wall location to the cell centre, that assembly takes place at the leading edge of the invaginating constriction, that the mode of glycan strand insertion changes from a single-stranded mode to a multi-stranded mode, and that the initiation of division (in contrast to its continuation) requires penicillin-insensitive peptidoglycan synthesis (PIPS). A membrane component X (possibly FtsQ) is proposed to coordinate PIPS with the cell division-initiating protein FtsZ. It is suggested that a largely proteinaceous macromolecular complex (divisome) at the leading edge of constriction encompasses three compartments (cytoplasm, membrane and periplasm). The composition of this complex is proposed to vary depending on whether division is being initiated or completed.

Relationship between the acceptor/donor radioactivity ratio and cross-linking in bacterial peptidoglycan: application to surface synthesis during the division cycle

The relationship between the experimental measurement of the cross-linking of bacterial peptidoglycan and the mode of its insertion is analyzed. The cross-linking value, in practice and in theory, is independent of the pattern of strand insertion. Since the measure of the mode or pattern of insertion is the acceptor/donor radioactivity ratio (ADRR), no correction need be made for the ADRR. The measurement of cross-linking using radioactivity is independent of the labeling time, the specific activity of the label, and the mode of strand insertion. It is not concluded, however, that cross-linking does increase during the division cycle.