Discovery of Widespread Host Protein Interactions with the Pre-replicated Genome of CHIKV Using VIR-CLASP.

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Trueness of 3D-printed cobalt chromium versus titanium removable partial denture clasps.

PURPOSE: The purpose of this in vitro study was to evaluate the trueness of removable partial denture clasps fabricated with titanium (Ti) through the selective laser melting (SLM) technique compared to cobalt-chromium alloys (CoCr). MATERIALS AND METHODS: A virtual Aker clasp was designed on a scanned tooth, and SLM printers were used to print 20 claps using cobalt chromium (n = 10) and titanium alloy (n = 10). The deviation between the printed clasps and reference design was measured using the surface matching software (Geomagic control x) at rest, retentive tip, reciprocal tip, retentive shoulder, and reciprocal shoulder. An Independent t-test was used to determine the influence of 3D-printed material on the trueness (a = 0.05). RESULTS: The gap distance in mm between the reference design and printed in titanium showed an average of 0.0001 ±0.0544, 0.0256 ±0.1309, 0.0230 ±0.1028, 0.0701 ±0.1234, and 0.0013 ±0.0735 mm in rest, reciprocal arm tip, retentive arm tip, retentive arm shoulder, and reciprocal arm shoulder, respectively. The gap distance in mm between the reference design and printed clasps in CoCr alloy showed an average of 0.0316 ±0.0692, 0.2783 ±0.1678, 0.1446 ±0.1528, 0.0315 ±0.0906, and 0.0419 ±0.1088 mm in rest, reciprocal arm tip, retentive arm tip, retentive arm shoulder, and reciprocal arm shoulder, respectively. The difference between titanium and CoCr alloys at each observation site was significant. CONCLUSION: Clasps fabricated from titanium with SLM printing have the least deviation and better trueness compared to those fabricated from cobalt chromium.

Orbit/CLASP determines centriole length by antagonising Klp10A in Drosophila spermatocytes.

After centrosome duplication, centrioles elongate before M phase. To identify genes required for this process and to understand the regulatory mechanism, we investigated the centrioles in Drosophila premeiotic spermatocytes expressing fluorescently tagged centriolar proteins. We demonstrated that an essential microtubule polymerisation factor, Orbit (the Drosophila CLASP orthologue, encoded by chb), accumulated at the distal end of centrioles and was required for the elongation. Conversely, a microtubule-severing factor, Klp10A, shortened the centrioles. Genetic analyses revealed that these two proteins functioned antagonistically to determine centriole length. Furthermore, Cp110 in the distal tip complex was closely associated with the factors involved in centriolar dynamics at the distal end. We observed loss of centriole integrity, including fragmentation of centrioles and earlier separation of the centriole pairs, in Cp110-null mutant cells either overexpressing Orbit or depleted of Klp10A Excess centriole elongation in the absence of the distal tip complex resulted in the loss of centriole integrity, leading to the formation of multipolar spindle microtubules emanating from centriole fragments, even when they were unpaired. Our findings contribute to understanding the mechanism of centriole integrity, disruption of which leads to chromosome instability in cancer cells.

Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites.

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we here show an interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasitophorous vacuolar membrane of the parasite. Expression of specific dominant-negative Arf1 and Rab GTPases, which interfere with the host cell Golgi-linked vesicular machinery, results in developmental delay and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the ability of the parasites to induce Golgi fragmentation. Altogether, we demonstrate that the structural integrity of the host cell Golgi and Golgi-associated vesicular traffic is important for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the Golgi structure of the hepatocyte to optimize its own intracellular development. This article has an associated First Person interview with the first author of the paper.

Three-year outcomes for transcatheter repair in patients with mitral regurgitation from the CLASP study.

BACKGROUND: Mitral valve transcatheter edge-to-edge repair (M-TEER) is an effective option for treatment of mitral regurgitation (MR). We previously reported favorable 2-year outcomes for the PASCAL transcatheter valve repair system. OBJECTIVES: We report 3-year outcomes from the multinational, prospective, single-arm CLASP study with analysis by functional MR (FMR) and degenerative MR (DMR). METHODS: Patients with core-lab determined MR ≥ 3+ were deemed candidates for M-TEER by the local heart team. Major adverse events were assessed by an independent clinical events committee to 1 year and by sites thereafter. Echocardiographic outcomes were evaluated by the core laboratory to 3 years. RESULTS: The study enrolled 124 patients, 69% FMR; 31% DMR (60% NYHA class III-IVa, 100% MR ≥ 3+). The 3-year Kaplan-Meier estimate for survival was 75% (66% FMR; 92% DMR) and freedom from heart failure hospitalization (HFH) was 73% (64% FMR; 91% DMR), with 85% reduction in annualized HFH rate (81% FMR; 96% DMR) (p < 0.001). MR ≤ 2+ was achieved and maintained in 93% of patients (93% FMR; 94% DMR) and MR ≤ 1+ in 70% of patients (71% FMR; 67% DMR) (p < 0.001). The mean left ventricular end-diastolic volume (181 mL at baseline) decreased progressively by 28 mL [p < 0.001]. NYHA class I/II was achieved in 89% of patients (p < 0.001). CONCLUSIONS: The 3-year results from the CLASP study demonstrated favorable and durable outcomes with the PASCAL transcatheter valve repair system in patients with clinically significant MR. These results add to the growing body of evidence establishing the PASCAL system as a valuable therapy for patients with significant symptomatic MR.

The contribution of lysophosphatidic acid receptors in the response of human lower esophageal sphincter under the electrical field stimulation.

BACKGROUND: This study aims to identify the impact on the reaction while the clasp and sling fibers of the human lower esophageal sphincter are under the electrical field stimulation, by adding lysophosphatidic acid receptor subtypes antagonist. METHODS: Between March 2018 to December 2018, muscle strips were isolated from 28 patients who underwent esophagectomy for mid-third esophageal carcinomas. Muscle tension measurement technique in vitro and electrical field stimulation were used to examine the effects of selective lysophosphatidic acid receptor antagonist on the clasp and sling fibers of human lower esophageal sphincter. RESULTS: The optimal frequency of frequency-dependent relaxation in clasp fibers and contraction in sling fibers induced by electrical field stimulation is 64 Hz and 128 Hz respectively. The selective lysophosphatidic acid 1 and 3 receptor antagonist produced no significant difference in the frequency-dependent relaxation in clasp fibers and contraction in sling fibers induced by the electrical field stimulation (P > 0.05). CONCLUSION: The electrical field stimulation induced a frequency-dependent relaxation in clasp fibers and contraction in sling fibers. The lysophosphatidic acid 1 and 3 receptors are not involved in the response of clasp and sling fibers of the human lower esophageal sphincter induced by the electrical field stimulation.

CLASPs at a glance.

CLIP-associating proteins (CLASPs) form an evolutionarily conserved family of regulatory factors that control microtubule dynamics and the organization of microtubule networks. The importance of CLASP activity has been appreciated for some time, but until recently our understanding of the underlying molecular mechanisms remained basic. Over the past few years, studies of, for example, migrating cells, neuronal development, and microtubule reorganization in plants, along with in vitro reconstitutions, have provided new insights into the cellular roles and molecular basis of CLASP activity. In this Cell Science at a Glance article and the accompanying poster, we will summarize some of these recent advances, emphasizing how they impact our current understanding of CLASP-mediated microtubule regulation.

Structural Determinants of Virion Assembly and Release in the C Terminus of the Mason-Pfizer Monkey Virus Capsid Protein.

The transition from an immature to a fully infectious mature retrovirus particle is associated with molecular switches that trigger dramatic conformational changes in the structure of the Gag proteins. A dominant maturation switch that stabilizes the immature capsid (CA) lattice is located downstream of the CA protein in many retroviral Gags. The HIV-1 Gag protein contains a stretch of 5 amino acid residues termed the "clasp motif," important for the organization of the hexameric subunits that provide stability to the overall immature HIV-1 shell. Sequence alignment of the CA C-terminal domains (CTDs) of HIV-1 and Mason-Pfizer monkey virus (M-PMV) highlighted a spacer-like domain in M-PMV that may provide a comparable function. The importance of the sequences spanning the CA-nucleocapsid (NC) cleavage has been demonstrated by mutagenesis, but the specific requirements for the clasp motif in several steps of M-PMV particle assembly and maturation have not been determined in detail. In the present study, we report an examination of the role of the clasp motif in the M-PMV life cycle. We generated a series of M-PMV Gag mutants and assayed for assembly of the recombinant proteins in vitro and for the assembly, maturation, release, genomic RNA packaging, and infectivity of the mutant viruses in vivo. The mutants revealed major defects in virion assembly and release in HEK 293T and HeLa cells and even larger defects in infectivity. Our data identify the clasp motif as a fundamental contributor to CA-CTD interactions necessary for efficient retroviral infection. IMPORTANCE The C-terminal domain of the capsid protein of many retroviruses has been shown to be critical for virion assembly and maturation, but the functions of this region of M-PMV are uncertain. We show that a short "clasp" motif in the capsid domain of the M-PMV Gag protein plays a key role in M-PMV virion assembly, genome packaging, and infectivity.

SOGA1 and SOGA2/MTCL1 are CLASP-interacting proteins required for faithful chromosome segregation in human cells.

CLASPs are key modulators of microtubule dynamics throughout the cell cycle. During mitosis, CLASPs independently associate with growing microtubule plus-ends and kinetochores and play essential roles in chromosome segregation. In a proteomic survey for human CLASP1-interacting proteins during mitosis, we have previously identified SOGA1 and SOGA2/MTCL1, whose mitotic roles remained uncharacterized. Here we performed an initial functional characterization of human SOGA1 and SOGA2/MTCL1 during mitosis. Using specific polyclonal antibodies raised against SOGA proteins, we confirmed their expression and reciprocal interaction with CLASP1 and CLASP2 during mitosis. In addition, we found that both SOGA1 and SOGA2/MTCL1 are phospho-regulated during mitosis by CDK1. Immunofluorescence analysis revealed that SOGA2/MTCL1 co-localizes with mitotic spindle microtubules and spindle poles throughout mitosis and both SOGA proteins are enriched at the midbody during mitotic exit/cytokinesis. GFP-tagging of SOGA2/MTCL1 further revealed a microtubule-independent localization at kinetochores. Live-cell imaging after siRNA-mediated knockdown of SOGA1 and SOGA2/MTCL1 showed that they are independently required for distinct aspects of chromosome segregation. Thus, SOGA1 and SOGA2/MTCL1 are bona fide CLASP-interacting proteins during mitosis required for faithful chromosome segregation in human cells.

[Finite element analyses of retention of removable partial denture circumferential clasps manufactured by selective laser melting].

OBJECTIVE: To compare the retentions of different designs of cobalt-chromium (Co-Cr), pure titanium (CP Ti), and titanium alloy (Ti-6Al-4V) removable partial denture (RPD) circumferential clasps manufactured by selective laser melting (SLM) and to analyze the stress distribution of these clasps during the removal from abutment teeth. METHODS: Clasps with clasp arm size A (1.9 mm width/1.1 mm thickness at the body and 0.8-taper) or B (1.2 times A) and 0.25 mm or 0.50 mm undercut engagement were modeled on a prepared first premolar die, named as designs A1, A2, A3, and A4, respectively. The density and elastic modulus of SLM-built Co-Cr, CP Ti, and Ti-6Al-4V were measured and given to different groups of clasps. The density, elastic modulus, and Poisson ' s ratio of enamel were given to the die. The control group was the cast Co-Cr clasp with design A1, to which the density and elastic modulus of cast Co-Cr alloy were given. The Poisson's ratio of all metals was 0.33. The initial 5 N dislodging force was applied, and the maximum displacement of the clasp along the insertion path was computed. The load was reapplied with an increment of 5 N than in the last simulation until the clasp was completely dislodged. The retentive force range of different groups of clasps was obtained. The retentive forces of the SLM-built Co-Cr, CP Ti, and Ti-6Al-4V clasps with equivalent computed retentive force range to the control group were validated through the insertion/removal experiment. The von Mises stress distributions of these three groups of SLM-built clasps under 15 N loads were analyzed. RESULTS: SLM-built Co-Cr, CP Ti, and Ti-6Al-4V clasps with designs B1 or B2, and Co-Cr clasps with design A2 had higher retentive forces than those of the control group. SLM-built CP Ti and Ti-6Al-4V clasps with design A1 had lower retentive forces than those of the control group. SLM-built Co-Cr clasp with design A1 and SLM-built CP Ti and Ti-6Al-4V clasps with design A2 had equivalent retentive forces to those of the control group. The insertion/removal experiment showed that the measured retentive forces of these three groups of SLM-built clasps were (21.57±5.41) N, (19.75±4.47) N, and (19.32±2.04) N, respectively. No statistically significant measured retentive force difference was found among these three groups of SLM-built clasps (P>0.05). The maximum von Mises stress of these three groups of SLM-built clasps exceeded their responding yield strength except for the Ti-6Al-4V one. CONCLUSION: SLM-built Co-Cr circumferential clasps had higher retention than CP Ti and Ti-6Al-4V ones with the same clasp arm size and undercut engagement. The retention of SLM-built circumferential clasps could be adjusted by changing the undercut engagement and clasp arm size. If SLM-built circumferential clasps are used in clinical practice, the Ti-6Al-4V clasp with clasp arm size A and 0.50 mm undercut engagement is recommended considering the long-term use of RPD in the patient's mouth.

Retentive Forces of Removable Partial Denture Clasp Assemblies Made from Polyaryletherketone and Cobalt-Chromium: A Comparative Study.

PURPOSE: To compare retentive forces of removable partial denture clasps traditionally fabricated with cobalt-chromium (CoCr) material and two computer-aided design and computer-aided manufactured (CAD/CAM) thermoplastic polymers. MATERIALS AND METHODS: Forty-eight clasp assemblies (16 CoCr, 16 polyetheretherketone (PEEK) and 16 polyetherketoneketone (PEKK) thermoplastic polymer) were fabricated for 48 mandibular tooth analogs. Individual clasps were inserted and removed on the tooth analogs utilizing a chewing simulator for 15,000 cycles to simulate 10 years of use. Retentive forces were measured utilizing a mechanical load tester at baseline and intervals of 1500 cycles. Data were analyzed with one-way Analysis of Variance, Tukey post-hoc, and paired T tests. RESULTS: Mean retentive forces between all groups were significantly different (p < 0.001). Retentive forces of CoCr clasps were significantly higher than both polymers (p < 0.001). The mean retentive forces for PEEK were not significantly different from PEKK (p = 0.23). A significant increase in retentive forces was observed for all three clasps after the first period of cycling, followed by continual decrease for the remaining cycles. At the endpoint of 15,000 cycles, no clasp assemblies showed lower retentive forces than at initial baseline. CONCLUSION: Thermoplastic polymer clasps demonstrated lower retentive forces compared to CoCr clasps. All three groups displayed a similar pattern of initial increase, followed by a gradual decrease, of retentive force. Despite this observation, the clasps maintained similar or higher retentive forces than measured at baseline. This resistance to fatigue and ability to fabricate with CAD/CAM technologies provides support for clinical use of these high-performance polymer (HPP) materials.

Analysis of Denture Base Displacement between Conventional Acrylic Removable Partial Dentures and Click Fit Partials for Kennedy's Class I and II Situations: An In Vitro Study.

AIM/OBJECTIVE: The aim of this study was to evaluate the displacement of the denture base of conventional acrylic dentures and Click Fit partials in Kennedy's class I and II situations in the mandibular arch. MATERIALS AND METHODS: Four removable partial dentures-two conventional clasp dentures and two attachment dentures (Click Fit)-were designed. The two conventional clasp dentures were retained by C (conventional) clasps, and the two attachment dentures were retained by rigid precision attachments. The displacement of denture bases and the movements of denture bases were investigated, and the influences of denture design were studied. RESULT: The values obtained were statistically analyzed by using independent t-tests. For all statistical purposes, a p-value of ≤0.001 was considered significant. The results showed that mean vertical displacements (mm) of the conventional acrylic removable partial denture base for Kennedy's class I mandibular arch under 50, 75, and 100 N forces were 0.0317, 0.04377, and 0.06392, respectively, and those for Kennedy's class II mandibular arch under 50, 75, and 100 N forces were 0.04922, 0.09849, and 0.1522, respectively. Vertical displacements (mm) of the Click Fit removable partial denture base for Kennedy's class I mandibular arch under 50, 75, and 100 N forces were 0.02185, 0.03436, and 0.005365, respectively, and those for Kennedy's class II mandibular arch under 50N, 75N, and 100N forces were 0.0445, 0.07851, and 0.14457, respectively. The difference between the groups was statistically significant (p ≤0.001). CONCLUSION: The vertical displacement of the denture base retained by conventional C clasps was more than that of the denture base retained by rigid precision attachment. The displacement of the denture base tended to be less when the denture was designed with a rigid connection for the retainer and with cross-arch stabilization as in Kennedy's class I case. CLINICAL IMPLICATIONS: This research evaluated the vertical denture base displacement using different designs and retention types. Hence, it helped predict the prognosis of different removable partial denture base designs in various clinical conditions.

HIV-1 Exploits CLASP2 To Induce Microtubule Stabilization and Facilitate Virus Trafficking to the Nucleus.

Human immunodeficiency virus type 1 (HIV-1) exploits a number of specialized microtubule (MT) plus-end tracking proteins (commonly known as +TIPs) to induce the formation of stable microtubules soon after virus entry and promote early stages of infection. However, given their functional diversity, the nature of the +TIPs involved and how they facilitate HIV-1 infection remains poorly understood. Here, we identify cytoplasmic linker-associated protein 2 (CLASP2), a +TIP that captures cortical MT plus ends to enable filament stabilization, as a host factor that enables HIV-1 to induce MT stabilization and promote early infection in natural target cell types. Using fixed- and live-cell imaging in human microglia cells, we further show that CLASP2 is required for the trafficking of incoming HIV-1 particles carrying wild-type (WT) envelope. Moreover, both WT CLASP2 and a CLASP2 mutant lacking its C-terminal domain, which mediates its interaction with several host effector proteins, bind to intact HIV-1 cores or in vitro-assembled capsid-nucleocapsid (CA-NC) complexes. However, unlike WT CLASP2, the CLASP2 C-terminal mutant is unable to induce MT stabilization or promote early HIV-1 infection. Our findings identify CLASP2 as a new host cofactor that utilizes distinct regulatory domains to bind incoming HIV-1 particles and facilitate trafficking of incoming viral cores through MT stabilization.IMPORTANCE While microtubules (MTs) have long been known to be important for delivery of incoming HIV-1 cores to the nucleus, how the virus engages and exploits these filaments remains poorly understood. Our previous work revealed the importance of highly specialized MT regulators that belong to a family called plus-end tracking proteins (+TIPs) in facilitating early stages of infection. These +TIPs perform various functions, such as engaging cargos for transport or engaging peripheral actin to stabilize MTs, suggesting several family members have the potential to contribute to infection in different ways. Here, we reveal that cytoplasmic linker-associated protein 2 (CLASP2), a key regulator of cortical capture and stabilization of MTs, interacts with incoming HIV-1 particles, and we identify a distinct C-terminal domain in CLASP2 that promotes both MT stabilization and early infection. Our findings identify a new +TIP acting as a host cofactor that facilitates early stages of viral infection.

Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes.

Insulin-stimulated glucose uptake is known to involve microtubules, although the function of microtubules and the microtubule-regulating proteins involved in insulin action are poorly understood. CLASP2, a plus-end tracking microtubule-associated protein (+TIP) that controls microtubule dynamics, was recently implicated as the first +TIP associated with insulin-regulated glucose uptake. Here, using protein-specific targeted quantitative phosphoproteomics within 3T3-L1 adipocytes, we discovered that insulin regulates phosphorylation of the CLASP2 network members G2L1, MARK2, CLIP2, AGAP3, and CKAP5 as well as EB1, revealing the existence of a previously unknown microtubule-associated protein system that responds to insulin. To further investigate, G2L1 interactome studies within 3T3-L1 adipocytes revealed that G2L1 coimmunoprecipitates CLASP2 and CLIP2 as well as the master integrators of +TIP assembly, the end binding (EB) proteins. Live-cell total internal reflection fluorescence microscopy in adipocytes revealed G2L1 and CLASP2 colocalize on microtubule plus-ends. We found that although insulin increases the number of CLASP2-containing plus-ends, insulin treatment simultaneously decreases CLASP2-containing plus-end velocity. In addition, we discovered that insulin stimulates redistribution of CLASP2 and G2L1 from exclusive plus-end tracking to "trailing" behind the growing tip of the microtubule. Insulin treatment increases α-tubulin Lysine 40 acetylation, a mechanism that was observed to be regulated by a counterbalance between GSK3 and mTOR, and led to microtubule stabilization. Our studies introduce insulin-stimulated microtubule stabilization and plus-end trailing of +TIPs as new modes of insulin action and reveal the likelihood that a network of microtubule-associated proteins synergize to coordinate insulin-regulated microtubule dynamics.

Is the high-performance thermoplastic polyetheretherketone indicated as a clasp material for removable dental prostheses?

OBJECTIVES: To investigate the retention force of polyetheretherketone (PEEK) removable dental prosthesis clasps in comparison with a cobalt-chrome-molybdenum control group after storage in artificial saliva. MATERIALS AND METHODS: Clasps were milled (Dentokeep (PEEKmilled1), NT digital implant technology; breCAM BioHPP Blank (PEEKmilled2), bredent), pressed (BioHPP Granulat for 2 press (PEEKpressed), bredent), or cast (remanium GM 800+ (cobalt-chrome-molybdenum), Dentaurum); N = 60, n = 15/subgroup. Retention force was examined 50 times/specimen in a pull-off test using the universal testing machine (Zwick 1445), where pull-off force was applied with a crosshead speed of 5 mm/minute until the maximum force dropped by 10%, at different aging levels: (1) initial, after storage in artificial saliva for (2) 90 and (3) 180 days. Statistical analysis was performed using one-way ANOVA followed by post hoc Scheffé-test and mixed models (p < 0.05). RESULTS: Cobalt-chrome-molybdenum presented the highest retention force. No differences were observed between polyetheretherketone materials. Cobalt-chrome-molybdenum showed a significant decrease of its values after artificial aging, while polyetheretherketone materials presented similar results over the course of aging. Regarding a repetitive insertion and removal, even though PEEKmilled2 and cobalt-chrome-molybdenum showed an initial increase, ultimately, a decrease in retention force was observed for all tested groups. CONCLUSIONS: Although the control group showed significantly higher results, the retention force of polyetheretherketone materials indicate a potential clinical application. Neither the manufacturing process nor artificial aging showed an impact on the retention force of polyetheretherketone clasps. CLINICAL RELEVANCE: Mechanical properties of novel removable dental prosthesis clasp materials devised to meet the growing esthetic demands of patients need to be investigated to ensure a successful long-term clinical application.

Retention force of polyetheretherketone and cobalt-chrome-molybdenum removable dental prosthesis clasps after artificial aging.

OBJECTIVES: To examine the retention force of removable dental prosthesis (RDP) clasps made from polyetheretherketone (PEEK) and cobalt-chrome-molybdenum (CoCrMo, control group) after storage in water and artificial aging. MATERIALS AND METHODS: For each material, 15 Bonwill clasps with retentive buccal and reciprocal lingual arms situated between the second pre- and first molar were manufactured by milling (Dentokeep [PEEKmilled1], NT digital implant technology; breCAM BioHPP Blank [PEEKmilled2], bredent), pressing (BioHPP Granulat for 2 press [PEEKpressed], bredent), or casting (remanium GM 800+ [CoCrMo], Dentaurum); N = 60, n = 15/subgroup. A total of 50 retention force measurements were performed for each specimen per aging level (initial; after storage [30 days, 37 °C] and 10,000 thermal cycles; after storage [60 days, 37 °C] and 20,000 thermal cycles) in a pull-off test. Data were statistically analyzed using one-way ANOVA, post hoc Scheffé and mixed models (p < 0.05). RESULTS: Initial, PEEKpressed (80.2 ± 35.2) and PEEKmilled1 (98.9 ± 40.3) presented the lowest results, while PEEKmilled2 (170.2 ± 51.8) showed the highest values. After artificial aging, the highest retention force was observed for the control group (131.4 ± 56.8). The influence of artificial aging was significantly higher for PEEK-based materials. While PEEKmilled2 and PEEKpressed showed an initial decline in retention force, all other groups presented no impact or an increase in retention force over a repetitive insertion and removal of the clasps. CONCLUSIONS: Within the tested PEEK materials, PEEKmilled2 presented superior results than PEEKpressed. Although CoCrMo showed higher values after artificial aging, all materials exhibited sufficient retention to recommend usage under clinical conditions. CLINICAL RELEVANCE: As RDPs are still employed for a wide range of indications, esthetic alternatives to conventional CoCrMo clasps are sought.

Surface Microhardness, Flexural Strength, and Clasp Retention and Deformation of Acetal vs Poly-ether-ether Ketone after Combined Thermal Cycling and pH Aging.

AIM: To evaluate the effect of combined thermocycling and artificial saliva pH on flexural strength, surface microhardness, as well as clasp retention and deformation of two different thermoplastic polymers. MATERIALS AND METHODS: Three groups were created, heat-cured polymethyl methacrylate, acetal, and poly-ether-ether ketone (PEEK) resins. Specimens were wrapped in plastic bags containing artificial saliva with three pH values (acidic 5.8, neutral 7.2, and alkaline 8.3). Two Aker clasps materials (acetal and PEEK), for premolar and molar, were stored in neutral salivary pH. Specimens were subjected to 2,000 thermocycles (5-55°C). Surface microhardness, flexural strength, and clasp retention and deformation were evaluated before and after aging. Data were analyzed by ANOVA, Tukey's test, Student's t-test, and paired t-tests (p < 0.05). RESULTS: Thermal cycling at acidic and alkaline pH significantly decreased flexural strength and surface microhardness of acetal. It had no significant effect on PEEK properties. Poly-ether-ether ketone showed statistically significant higher mechanical properties in all groups. Acetal clasps exhibited a statistically significant deformation and a corresponding decrease in retention after thermocycling at neutral pH. CONCLUSION: Mechanical properties of acetal, as well as its clasp retention and deformation, significantly decreased after combining thermal and pH aging and thermal cycling in neutral pH, respectively. Meanwhile, PEEK clasps were not significantly affected. CLINICAL SIGNIFICANCE: Different intraoral variables may significantly affect mechanical performance, retention, and deformation of TMs used for denture base and clasp construction. Some of these TMs may behave better than the other types with recommended improvement of the design like increasing clasp cross-section area.

Staphylococcus aureus-induced endothelial permeability and inflammation are mediated by microtubule destabilization.

Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury. However, the molecular mechanisms of bacterial pathogen-induced EC barrier disruption are incompletely understood. Here, we investigated the role of microtubules (MT) in the mechanisms of EC barrier compromise caused by heat-killed S. aureus (HKSA). Using a customized monolayer permeability assay in human pulmonary EC and MT fractionation, we observed that HKSA-induced barrier disruption is accompanied by MT destabilization and increased histone deacetylase-6 (HDAC6) activity resulting from elevated reactive oxygen species (ROS) production. Molecular or pharmacological HDAC6 inhibition rescued barrier function in HKSA-challenged vascular endothelium. The HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker-associated protein 2 (CLASP2) to the cell periphery, limiting its interaction with adherens junction proteins. HKSA-induced EC barrier dysfunction was also associated with increased Rho GTPase activity via activation of MT-bound Rho-specific guanine nucleotide exchange factor-H1 (GEF-H1) and was abolished by HDAC6 down-regulation. HKSA activated the NF-κB proinflammatory pathway and increased the expression of intercellular and vascular cell adhesion molecules in EC, an effect that was also HDAC6-dependent and mediated, at least in part, by a GEF-H1/Rho-dependent mechanism. Of note, HDAC6 knockout mice or HDAC6 inhibitor-treated WT mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA). Our results indicate that S. aureus-induced, ROS-dependent up-regulation of HDAC6 activity destabilizes MT and thereby activates the GEF-H1/Rho pathway, increasing both EC permeability and inflammation.

The importance of being edgy: cell geometric edges as an emerging polar domain in plant cells.

Polarity is an essential feature of multicellular organisms and underpins growth and development as well as physiological functions. In polyhedral plant cells, polar domains at different faces have been studied in detail. In recent years, cell edges (where two faces meet) have emerged as discrete spatial domains with distinct biochemical identities. Here, we review and discuss recent advances in our understanding of cell edges as functional polar domains in plant cells and other organisms, highlighting conceptual parallels and open questions regarding edge polarity.