Do all ceramic and composite CAD-CAM materials exhibit equal bonding properties to implant Ti-base materials? An Interfacial Fracture Toughness Study.

OBJECTIVES: To compare the interfacial fracture toughness (IFT) with or without aging, of four different classes of CAD-CAM ceramic and composite materials bonded with self-adhesive resin cement to titanium alloy characteristic of implant abutments. METHODS: High translucent zirconia (Katana; KAT), lithium disilicate-based glass-ceramic (IPS. emax.CAD; EMX), polymer-infiltrated ceramic network material (PICN) (Vita Enamic; ENA), and dispersed filler composite (Cerasmart 270; CER) were cut into equilateral triangular prisms and bonded to titanium prisms with identical dimensions using Panavia SA Cement Universal. The surfaces were pretreated following the manufacturers' recommendations and developed interfacial area ratio (Sdr) of the pretreated surfaces was measured. IFT was determined using the Notchless Triangular Prism test in a water bath at 36 °C before and after thermocycling (10,000 cycles) (n = 40 samples/material). RESULTS: IFT of the materials ranged from 0.80 ± 0.25 to 1.10 ± 0.21 MPa.m1/2 before thermocycling and from 0.71 ± 0.24 to 1.02 ± 0.25 MPa.m1/2 after thermocycling. There was a statistical difference between IFT of CER and the two top performers in each scenario: KAT and EMX before aging, and KAT and ENA after aging. Thermocycling significantly decreased IFT of EMX. The Weibull modulus of IFT was similar for all materials and remained so after thermocycling. Sdr measurements revealed that ENA (7.60)>Ti (4.97)>CER (2.85)>KAT (1.09)=EMX (0.96). SIGNIFICANCE: Dispersed filler CAD-CAM composite showed lower performance than the other materials. Aging only affected IFT of Li-Si glass-ceramic, whereas zirconia and PICN performed equally well, probably due to their chemical bonding potential and surface roughness respectively.

Intraoral wear of PICN CAD-CAM composite restorations used in severe tooth wear treatment: 5-year results of a prospective clinical study using 3D profilometry.

OBJECTIVES: To evaluate, in a prospective clinical study over 5 years with ex vivo 3D profilometry analyses, the intraoral wear of Polymer-Infiltrated Ceramic Network (PICN) CAD-CAM composite restorations used in severe tooth wear treatment with the One-Step No-Prep technique. METHODS: 192 PICN (Vita Enamic) restorations on molars and premolars were included in a prospective clinical study involving patients treated according to the One-step No-prep protocol (n = 7). All patients showed clinical signs of bruxism. Replicas of restorations on molars and premolars were realized at each evaluation time (baseline and then every year up to 5 years) and scanned to perform 3D profilometry. Baseline and recall scans were superimposed with Geomagic Control software. The mean material wear was calculated for the full occlusal area (FOA) and for the occlusal contact area (OCA), respectively. Clinical evaluation of restorations was performed at recall. RESULTS: At 5 years, the estimated mean material wear for FOA was inferior to the accuracy threshold of the profilometry measurement chain. For OCA, the estimated mean wear of the material was - 27.97 µm. This material wear was shown to be significantly influenced by time (p < 0.0001) and patient (p = 0.026), while the type of tooth (molar or premolar) had no influence. At 5 years, the survival and the success rates of restorations were 99.48% and 90.62%, respectively. SIGNIFICANCE: The PICN material exhibits a low wear process in the treatment of severe tooth wear despite the presence of clinical signs of bruxism, and it constitutes a suitable material for the One-step No-prep technique.

Structure and Function of BcpE2, the Most Promiscuous GH3-Family Glucose Scavenging Beta-Glucosidase.

Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here, we revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was able to release the glucose moiety of all tested types of plant-derived heterosides (aryl ß-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covered the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material.

Characterisation of the structural, dynamic and aggregation properties of the W64R amyloidogenic variant of human lysozyme.

The accumulation in vital organs of amyloid fibrils made of mutational variants of lysozyme (HuL) is associated with a human systemic amyloid disease. The detailed comparison of the in vitro properties of the I56T and D67H amyloidogenic variants to those of the T70N non-amyloidogenic variant and the wild-type (WT) protein suggested that the deposition of large amounts of aggregated disease-related lysozyme variants is initiated by the formation of transient intermediate species. The ability to populate such intermediates is essentially due to the destabilisation of the protein and the loss of the global structural cooperativity under physiologically relevant conditions. Here, we report the characterisation of a third naturally occurring amyloidogenic lysozyme variant, W64R, in comparison with the I56T and WT proteins. The X-ray crystal structure of the W64R variant at 1.15 Å resolution is very similar to that of the WT protein; a few interactions within the ß-domain and at the interface between the α- and ß-domains differ, however, from those in the WT protein. Consequently, the W64R mutation destabilizes the protein to an extent that is similar to that observed for the I56T and D67H mutations. The ΔG°NU(H2O) is reduced by 24 kJ·mol-1 and the Tm is about 12 °C lower than that of the WT protein. Under native conditions, the W64R and I56T proteins are readily digested by proteinase K, while the WT protein remains intact. These results suggest that the two variant proteins transiently populate similar partially unfolded states in which proteinase K cleavage sites are accessible to the protease. Moreover, the in vitro aggregation properties of the W64R protein are similar to those of the I56T variant. Altogether, these results indicate that the properties of the W64R protein are astonishingly similar to those of the I56T variant. They further corroborate the idea that HuL variants associated with the disease are those whose stability and global structural cooperativity are sufficiently reduced to allow the formation of aggregation prone partially folded intermediates under physiological conditions.

Structures of the free and inhibitors-bound forms of bromelain and ananain from Ananas comosus stem and in vitro study of their cytotoxicity.

The Ananas comosus stem extract is a complex mixture containing various cysteine ​​proteases of the C1A subfamily, such as bromelain and ananain. This mixture used for centuries in Chinese medicine, has several potential therapeutic applications as anti-cancer, anti-inflammatory and ecchymosis degradation agent. In the present work we determined the structures of bromelain and ananain, both in their free forms and in complex with the inhibitors E64 and TLCK. These structures combined with protease-substrate complexes modeling clearly identified the Glu68 as responsible for the high discrimination of bromelain in favor of substrates with positively charged residues at P2, and unveil the reasons for its weak inhibition by cystatins and E64. Our results with purified and fully active bromelain, ananain and papain show a strong reduction of cell proliferation with MDA-MB231 and A2058 cancer cell lines at a concentration of about 1 µM, control experiments clearly emphasizing the need for proteolytic activity. In contrast, while bromelain and ananain had a strong effect on the proliferation of the OCI-LY19 and HL-60 non-adherent cell lines, papain, the archetypal member of the C1A subfamily, had none. This indicates that, in this case, sequence/structure identity beyond the active site of bromelain and ananain is more important than substrate specificity.

The bacterial cell division protein fragment EFtsN binds to and activates the major peptidoglycan synthase PBP1b.

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division, the machinery responsible for PG synthesis localizes mid-cell, at the septum, under the control of a multiprotein complex called the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. Interestingly, a short sequence of FtsN (Leu75-Gln93, known as EFtsN) was shown to be essential and sufficient for its functioning in vivo, but what exactly this sequence is doing remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its biosynthetic glycosyltransferase (GTase) activity. We also report the crystal structure of PBP1b in complex with EFtsN, which demonstrates that EFtsN binds at the junction between the GTase and UB2H domains of PBP1b. Interestingly, mutations to two residues (R141A/R397A) within the EFtsN-binding pocket reduced the activation of PBP1b by FtsN but not by the lipoprotein LpoB. This mutant was unable to rescue the ΔponB-ponAts strain, which lacks PBP1b and has a thermosensitive PBP1a, at nonpermissive temperature and induced a mild cell-chaining phenotype and cell lysis. Altogether, the results show that EFtsN interacts with PBP1b and that this interaction plays a role in the activation of its GTase activity by FtsN, which may contribute to the overall septal PG synthesis and regulation during cell division.

Biochemical and structural characterization of a mannose binding jacalin-related lectin with two-sugar binding sites from pineapple (Ananas comosus) stem.

A mannose binding jacalin-related lectin from Ananas comosus stem (AcmJRL) was purified and biochemically characterized. This lectin is homogeneous according to native, SDS-PAGE and N-terminal sequencing and the theoretical molecular mass was confirmed by ESI-Q-TOF-MS. AcmJRL was found homodimeric in solution by size-exclusion chromatography. Rat erythrocytes are agglutinated by AcmJRL while no agglutination activity is detected against rabbit and sheep erythrocytes. Hemagglutination activity was found more strongly inhibited by mannooligomannosides than by D-mannose. The carbohydrate-binding specificity of AcmJRL was determined in some detail by isothermal titration calorimetry. All sugars tested were found to bind with low affinity to AcmJRL, with Ka values in the mM range. In agreement with hemagglutination assays, the affinity increased from D-mannose to di-, tri- and penta-mannooligosaccharides. Moreover, the X-ray crystal structure of AcmJRL was obtained in an apo form as well as in complex with D-mannose and methyl-α-D-mannopyranoside, revealing two carbohydrate-binding sites per monomer similar to the banana lectin BanLec. The absence of a wall separating the two binding sites, the conformation of ß7ß8 loop and the hemagglutinating activity are reminiscent of the BanLec His84Thr mutant, which presents a strong anti-HIV activity in absence of mitogenic activity.

Crystal structure and kinetic analysis of the class B3 di-zinc metallo-ß-lactamase LRA-12 from an Alaskan soil metagenome.

We analyzed the kinetic properties of the metagenomic class B3 ß-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-ß-lactamases (MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB ß-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B ß-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-ß7 and ß11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 ß-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens.

Structural and Kinetic Insights into the "Ceftazidimase" Behavior of the Extended-Spectrum ß-Lactamase CTX-M-96.

Diversification of the CTX-M ß-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 Å and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the ß3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution.

Crystal structure of the extended-spectrum ß-lactamase PER-2 and insights into the role of specific residues in the interaction with ß-lactams and ß-lactamase inhibitors.

PER-2 belongs to a small (7 members to date) group of extended-spectrum ß-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most ß-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward ß-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singular trans bond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A ß-lactamases. PER ß-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A ("A" indicates an insertion according to Ambler's scheme for residue numbering in PER ß-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different ß-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.