Avidity-based biosensors for ubiquitylated PCNA reveal choreography of DNA damage bypass.

In eukaryotes, the posttranslational modifier ubiquitin is used to regulate the amounts, interactions, or activities of proteins in diverse pathways and signaling networks. Its effects are mediated by monoubiquitin or polyubiquitin chains of varying geometries. We describe the design, validation, and application of a series of avidity-based probes against the ubiquitylated forms of the DNA replication clamp, proliferating cell nuclear antigen (PCNA), in budding yeast. Directed against total ubiquitylated PCNA or specifically K63-polyubiquitylated PCNA, the probes are tunable in their activities and can be used either as biosensors or as inhibitors of the PCNA-dependent DNA damage bypass pathway. Used in live cells, the probes revealed the timing of PCNA ubiquitylation during damage bypass and a particular susceptibility of the ribosomal DNA locus to the activation of the pathway. Our approach is applicable to a wide range of ubiquitin-conjugated proteins, thus representing a generalizable strategy for the design of biosensors for specific (poly)ubiquitylated forms of individual substrates.

Use of Systemic Steroids, Hormone Replacement Therapy, or Oral Contraceptives Is Associated with Decreased Implant Survival in Women.

BACKGROUND: Systemic steroids, such as prednisone, hormonal replacement therapies, or oral contraceptives, are commonly prescribed to women who might also be receiving dental implant therapy. However, the effect of these medications on dental implant survival is unknown. METHODS: The medical and dental records of individuals with dental implants (N = 1480 implants) who visited a postgraduate periodontics clinic between 2000 and 2017 were initially considered. Those younger than 21 years old, pregnant, or male were excluded according to the study's exclusion criteria. The presence of systemic diseases and conditions was assessed. Implant failure rates among female patients using systemic steroids, hormone replacement therapy, or oral contraceptives were compared with failure rates among patients not taking those medications. RESULTS: The implant failure rate for the 65 implants in patients taking steroid medications was 7.69%; the failure rate for the 712 implants in patients not taking steroids was 1.54% (p < 0.001). After adjusting for smoking and the presence of diabetes, that relationship persisted, with an 8.47% implant failure rate for the 59 implants in patients taking steroids (vs. 1.54% failure for the 585 implants in patients not taking steroids; p < 0.001). Regression analyses demonstrated that the odds of implant failure versus success were 5.31 times greater in patients taking systemic steroids, hormone replacement therapy, or oral contraceptives (p < 0.05). No statistically significant differences in patient plaque control were found between the experimental and control groups. CONCLUSIONS: Among women, the use of systemic steroids is associated with a five-fold increase in the rate of dental implant failure, regardless of the presence of smoking or diabetes.

Real-Time Deubiquitination Assays Using a Free Ubiquitin Sensor.

Deubiquitinating enzymes cleave ubiquitin (Ub) from its attachment to another Ub, other proteins, peptides, or non-peptide adducts. In all cases, substrate hydrolysis by DUBs releases free Ub or polyubiquitin (polyUb) chains. Whereas most quantitative DUB assays depend on fluorescently labeled artificial substrates, employing a sensor able to detect Ub release in real time makes it possible to monitor DUB activity using virtually any Ub conjugate as a substrate. The protocols here describe the preparation of Atto532-tUI, a high-affinity sensor for free Ub, and its use in real-time deubiquitination assays.

The influence of proton pump inhibitors on tissue attachment around teeth and dental implants: A scoping review.

OBJECTIVES: Proton pump inhibitors, such as omeprazole and pantoprazole, are frequently prescribed for the treatment of acid reflux. However, those medications have been shown to affect a variety of physiologic processes, including bone homeostasis and the gastrointestinal microbiome. The objective of this study was to assess the relationship between proton pump inhibitors and attachment levels around teeth and dental implants. A scoping review was performed to assess the extent and quality of the relevant literature. MATERIALS AND METHODS: We used the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews (PRISMA-ScR) and searched four relevant biomedical literature databases in addition to the grey literature. Keywords in the title and abstract fields, and subject headings for proton pump inhibitors, teeth, and dental implants were included as search terms. RESULTS: Overall search results identified 791 publications which, after applying the inclusion and exclusion criteria, yielded 27 publications that were further analyzed for relevance and quality of scientific evidence. The majority of eligible publications were retrospective cohort studies. Following critical analysis, 13 publications, including six abstracts, were used to assess the effect of proton pump inhibitors on tissue attachment around teeth and dental implants. CONCLUSIONS: There are few high-quality studies describing the effect of proton pump inhibitors on tissue attachment around teeth and dental implants. Nevertheless, among the included papers with the fewest confounding factors, there was a positive relationship between proton pump inhibitors and soft tissue attachment levels around teeth, and a predominantly negative but variable effect of proton pump inhibitors on the bone level around dental implants. Additional well-controlled prospective studies are required to fully elucidate those relationships.

Familial Presentation of Ankyloglossia and Localized Periodontitis.

An association between ankyloglossia and periodontitis has not previously been reported. This case series describes three sisters who each had ankyloglossia and a molar/incisor pattern of localized periodontitis. The concurrent presentation of both conditions within the family suggests that further investigation of genetic factors that might concurrently affect the pathogenesis of both disorders may be warranted.

Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor.

Double-strand breaks (DSBs) are lesions in DNA that, if not properly repaired, can cause genomic instability, oncogenesis, and cell death. Multiple chromatin posttranslational modifications (PTMs) play a role in the DNA damage response to DSBs. Among these, RNF168-mediated ubiquitination of lysines 13 or 15 at the N-terminal tail of histone H2A (H2AK13/15Ub) is essential for the recruitment of effectors of both the non-homologous end joining (NHEJ) and the homologous recombination (HR) repair pathways. Thus, tools and techniques to track the spatiotemporal dynamics of H2AK13/15 ubiquitination at DNA DSBs are important to facilitate studies of DNA repair. Previous work from other groups used the minimal focus-forming region (FFR) of the NHEJ effector 53BP1 to detect H2AK15Ub generated upon damage induced by gamma or laser irradiation in live cells. However, 53BP1-FFR only binds nucleosomes modified with both H2AK15Ub and dimethylation of lysine 20 on histone H4 (H4K20me2); thus, 53BP1-FFR does not recognize H2AK13Ub-nucleosomes or nucleosomes that contain H2AK15Ub but lack methylation of H4K20 (H4K20me0). To overcome this limitation, we developed an avidity-based sensor that binds H2AK13/15Ub without dependence on the methylation status of histone H4K20. This sensor, called Reader1.0, detects DNA damage-associated H2AK13/15Ub in live cells with high sensitivity and selectivity. Here, we present a protocol to detect the formation of H2AK13/15Ub at laser-induced DSBs using Reader1.0 as a live-cell reporter for this histone PTM. Graphic abstract.

Association between proton pump inhibitors and periodontal disease severity.

OBJECTIVES: Proton pump inhibitors (PPIs) are commonly prescribed for the management of acid-related gastrointestinal disorders. PPIs modulate osteoclast function, reduce gastric acid secretion, and are associated with the establishment of a more diverse gastrointestinal microbiota. Periodontitis is characterized by microbe-associated host-mediated inflammation that results in loss of periodontal attachment. The aim of this study was to assess whether a relationship exists between PPIs and periodontal disease. MATERIALS AND METHODS: A retrospective analysis was performed using patient records from a faculty periodontal practice. The proportion of elevated probing depths was used to measure periodontitis severity. Statistical analysis was performed using independent sample t-tests, and Chi-square tests of independence. RESULTS: Records from 1093 patients were initially assessed. Fourteen percent of teeth were associated with ≥6 mm probing depths among PPI users, in contrast to 24% for patients not using PPIs (P = 0.030). Similarly, 27% of teeth exhibited ≥5 mm probing depths among PPI users versus 40% for non-PPI users (P = 0.039). CONCLUSIONS: The results suggest that PPIs are associated with a reduced proportion of elevated probing depths. Future prospective studies are indicated to elucidate possible mechanisms through which PPIs might affect, and potentially be used in the treatment of, periodontitis.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions.

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.

Design of genetically encoded sensors to detect nucleosome ubiquitination in live cells.

Histone posttranslational modifications (PTMs) are dynamic, context-dependent signals that modulate chromatin structure and function. Ubiquitin (Ub) conjugation to different lysines of histones H2A and H2B is used to regulate diverse processes such as gene silencing, transcriptional elongation, and DNA repair. Despite considerable progress made to elucidate the players and mechanisms involved in histone ubiquitination, there remains a lack of tools to monitor these PTMs, especially in live cells. To address this, we combined an avidity-based strategy with in silico approaches to design sensors for specifically ubiquitinated nucleosomes. By linking Ub-binding domains to nucleosome-binding peptides, we engineered proteins that target H2AK13/15Ub and H2BK120Ub with Kd values from 10-8 to 10-6 M; when fused to fluorescent proteins, they work as PTM sensors in cells. The H2AK13/15Ub-specific sensor, employed to monitor signaling from endogenous DNA damage through the cell cycle, identified and differentiated roles for 53BP1 and BARD1 as mediators of this histone PTM.

The Effect of Hypothyroidism on Bone Loss at Dental Implants.

Hypothyroidism (HT) is an endocrine disorder characterized by abnormally reduced thyroid gland activity and is most commonly of autoimmune etiology. HT is associated with alterations in bone metabolism, and HT patients typically experience decreased bone resorption. The objective of this study was to use dental implants as standardized reference markers to compare the extent of alveolar bone loss in implant patients with and without HT. We examined medical and dental history records and radiographic data from 635 patients receiving 1480 implants during 2000-2017. The rate of bone loss was calculated from differences in radiographic bone levels over time, corrected for radiographic distortion. Peri-implant bone loss from patients with HT was significantly lower than for those without HT (t1252= -3.42; 95% confidence interval= 0.47-1.73; P < .001; M = 0.53 and 1.63 mm/yr, respectively). A similar relationship persisted after excluding smokers and diabetics and after additionally excluding those on systemic steroids, hormone replacement therapy, hormone medications, or autoimmune diseases other than HT. Our data suggest that patients with HT have a decreased rate of bone loss around dental implants and may not be at increased risk for dental implant failure. The decreased bone metabolic rate among patients with HT might contribute to those findings.

Lubricant-Impregnated Surfaces for Mitigating Asphaltene Deposition.

Asphaltenes are heavy aromatic components of crude oil. Their complex chemical makeup-an aromatic core surrounded by aliphatic side chains-enables them to adhere to most surfaces. Their buildup in pipes can result in clogging and lead to interruption of production operations and expensive mechanical cleaning. We demonstrate the use of liquid-impregnated surfaces (LIS) to prevent asphaltene deposition and buildup on substrates. Indeed, these surfaces expose a liquid interface to the working fluid, which combines the benefits of a dynamic defect-free surface and tunable interfacial properties. In contrast to bulk additives that are typically mixed into the oil phase, the impregnating liquid also provides the great benefit of protecting the underlying solid surface with a stable and minimal layer of lubricant, thereby reducing costs and eliminating the need for subsequent downstream removal. We first select and confirm the thermodynamic stability of a suitable lubricant and its lack of interaction with asphaltenes. By using a carefully selected system composed of a textured and functionalized solid substrate in conjunction with a fluorinated lubricant, we show that asphaltene adsorption is prevented over long time scales. We further demonstrate the possibility of building such a system with representative industrial materials such as aluminum and expose the resulting substrate to an external shear flow to simulate pipe flow conditions.

Tracking Sulfonated Polystyrene Diffusion in a Chitosan/Carboxymethyl Cellulose Layer-by-Layer Film: Exploring the Internal Architecture of Nanocoatings.

Since chitosan presents the ability to interact with a wide range of molecules, it has been one of the most popular natural polymers for the construction of layer-by-layer thin films. In this study, depth-profiling X-ray photoelectron spectroscopy (XPS) was employed to track the diffusion of sulfonated polystyrene (SPS) in carboxymethyl cellulose/chitosan (CMC/Chi) multilayers. Our findings suggest that the CMC/Chi film does not constitute an electrostatic barrier sufficient to block diffusion of SPS, and that diffusion can be controlled by adjusting the diffusion time and the molecular weight of the polymers that compose the CMC/Chi system. In addition to monitoring the diffusion, it was also possible to observe a process of preferential interaction between Chi and SPS. Thus, the nitrogen N 1s peak, due to functional groups found exclusively in chitosan chains, was the key factor to identifying the molecular interactions involving chitosan and the different polyanions. Accordingly, the presence of a strong polyanion such as SPS shifts the N 1s peak to a higher level of binding energy. Such results highlight that understanding the fundamentals of polymer interactions is a major step to fine-tuning the internal architecture of LbL structures for specific applications (e.g., drug release).

Asphaltene Adsorption on Functionalized Solids.

Asphaltenes, heavy aromatic components of crude oil, are known to adsorb on surfaces and can lead to pipe clogging or hinder oil recovery. Because of their multicomponent structure, the details of their interactions with surfaces are complex. We investigate the effect of the physicochemical properties of the substrate on the extent and mechanism of this adsorption. Using wetting measurements, we relate the initial kinetics of deposition to the interfacial energy of the surface. We then quantify the long-term adsorption dynamics using a quartz crystal microbalance and ellipsometry. Finally, we investigate the mechanism and morphology of adsorption with force spectroscopy measurements as a function of surface chemistry. We determine different adsorption regimes differing in orientation, packing density, and initial kinetics on different substrate functionalizations. Specifically, we find that alkane substrates delay the initial monolayer formation, fluorinated surfaces exhibit fast adsorption but low bonding strength, and hydroxyl substrates lead to a different adsorption orientation and a high packing density of the asphaltene layer.

Relationship between hypothyroidism and periodontitis: A scoping review.

AIM: The objective of this study was to assess the existing literature to determine if a relationship exists between hypothyroidism and periodontitis. METHODS: We used a modified approach to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses by searching five databases in addition to the gray literature. Keywords in the title and abstract fields, as well as subject headings for both periodontal disease and hypothyroidism, were used to search the existing literature for publications relevant to evaluation of the thyroid-periodontitis relationship. RESULTS: The authors screened 847 unique publications which, after applying inclusion and exclusion criteria, yielded 29 publications, which were further analyzed for relevance and applicability. Most of the included papers were cross-sectional studies and retrospective chart reviews. Following critical analysis, four publications, including one abstract, were used to further assess the hypothyroid-periodontitis relationship. CONCLUSIONS: There are very few high-quality studies describing the potential association between hypothyroidism and periodontitis. In general, and among the included papers with the fewest confounding factors, a positive relationship between hypothyroidism and periodontitis was found. Further well-controlled, prospective clinical and immunologic studies will be required to confirm that relationship.

Effect of Proton Pump Inhibitors on Bone Loss at Dental Implants.

PURPOSE: Proton pump inhibitors (PPIs) are prescribed for the treatment of gastric reflux disease, but such medications might also influence bone metabolism. Therefore, the primary goal of this study was to determine if bone loss severity at dental implants could be associated with PPI use. MATERIALS AND METHODS: Dental, medical, and radiographic history records of patients receiving dental implants at the University at Buffalo, School of Dental Medicine from 2000 to 2017 were reviewed in this retrospective clinical study. Bone loss around each implant was evaluated radiographically by direct measurement of crestal bone loss and by counting the number of radiographically evident exposed threads. PPI use was confirmed by medical record examination. The effects of systemic factors were assessed. Confidence intervals (CI) and P values of mean differences between PPI and non-PPI groups were computed via IBM SPSS Statistics v.25. RESULTS: A total of 1,480 implants from 635 patients were used in this study. Greater crestal implant bone loss was associated with patients with a history of PPI medication use. Mean crestal bone loss of 1.60 mm was noted at implants from PPI patients, in contrast to 1.01 mm of crestal implant bone loss at implants from the non-PPI group (group difference = 0.59 mm, 58.40% increase, P = .024, CI [95%] = 0.08 to 1.09 mm). Following adjustment for systemic factors, those effects persisted, with crestal implant bone loss of 1.87 mm from PPI patients, in contrast to 1.04 mm from non-PPI patients (group difference = 0.83 mm, 79.80% increase, P = .028, CI [95%] = 0.09 to 1.56 mm). Similarly, 0.63 exposed threads per implant were found in the PPI group, in contrast to 0.38 supracrestal implant threads in the non-PPI patient group (mean difference = 0.25 exposed threads, 65.8% increase, P = .039, CI [95%] = 0.01 to 0.50 mm). After excluding systemic factors, a similar pattern was observed with 0.79 vs 0.36 threads exposed from subjects taking PPIs, compared with those not taking PPIs, respectively (mean difference = 0.43 exposed threads, 119.4% increase, P = .014, CI [95%] = 0.09 to 0.77 mm). CONCLUSION: The data suggest that PPI medications are related to more loss of crestal bone at implant sites. Patients receiving implant therapy might require more frequent periodontal maintenance.

Fluorescent Sensors That Enable a General Method To Quantify Affinities of Receptor Proteins for Polyubiquitin Ligands.

In all eukaryotic cells, modifications of proteins by polymers of ubiquitin (polyUb) are signals used in diverse biological processes. To better understand how polyUb signals are read and promote their different functions, quantitative measurements of their interactions with receptor proteins are needed. However, affinities and selectivities of different forms of polyUb with various receptors have been difficult to determine because the availability of well-defined polyUb chains can be limiting and there is a lack of general, sensitive methods to assay their interactions. We have addressed this challenge by developing a series of fluorescent protein sensors for polyUb; by competition of the sensors against receptor proteins in vitro for limiting amounts of polyUb, receptor·polyUb affinities can be quantified. Due to the high affinities of the polyUb sensors (Kd ∼ 10-9 M), binding assays using this competition format require much less polyUb (<0.1%) than would be needed in direct titrations of the polyUb ligands. Furthermore, the high sensitivity and large dynamic range of the sensor fluorescence readout allow for precise measurements even for very tight interactions (i.e., nanomolar Kd). Importantly, as demonstrated here with Ub2 and Ub3 ligands, the assay does not require labeling of either the receptor protein or the polyUb, and it can be used with polyUb ligands composed of virtually any Ub-Ub linkage type.

High-affinity free ubiquitin sensors for quantifying ubiquitin homeostasis and deubiquitination.

Ubiquitin (Ub) conjugation is an essential post-translational modification that affects nearly all proteins in eukaryotes. The functions and mechanisms of ubiquitination are areas of extensive study, and yet the dynamics and regulation of even free (that is, unconjugated) Ub are poorly understood. A major impediment has been the lack of simple and robust techniques to quantify Ub levels in cells and to monitor Ub release from conjugates. Here, we describe avidity-based fluorescent sensors that address this need. The sensors bind specifically to free Ub, have dissociation constant Kd values down to 60 pM and, together with a newly developed workflow, allow us to distinguish and quantify the pools of free, protein-conjugated and thioesterified forms of Ub from cell lysates. Alternatively, free Ub in fixed cells can be visualized microscopically by staining with a sensor. Real-time assays using the sensors afford unprecedented flexibility and precision to measure deubiquitination of virtually any (poly)Ub conjugate.

Reversible Self-Healing for Preserving Optical Transparency and Repairing Mechanical Damage in Composites.

This research concentrates on the healing of optical properties, roughness, contact angle hysteresis, and shallow scratches in polymer/nanoparticle composites. A series of ternary composite blends [epoxy/halloysite nanotubes (HNTs)/cellulose acetate butyrate (CAB)] with various CAB concentrations were fabricated and subjected to a series of mechanical damages. The optimized concentration of a nanoparticle is 1.0 vol %, and the CAB concentration is 3.0 vol % based on the mechanical reinforcement and wear resistance. Nanoscale scratching, microlevel falling-sand test, and macrolevel Taber abrasions were utilized to damage the surfaces. The induced damage (roughness and surface scratch up to hundreds of nanometers in depth) healed upon heating. At any temperatures above the softening transition of the semi-interpenetrating network structure of the polymer composites, CAB migrates into the microcracks, and the essential mechanical parameters (modulus, strength, strain to failure) are recovered; in our particular epoxy/HNTs/CAB system, optical transparency is also recovered efficiently. CAB also moves to the macroscopic air/specimen interface and favorably modifies the surface properties, reducing the roll-off angles of water droplets from ∼90° to ∼20°. Through an appropriate choice of CAB additives with different molecular weights, the healing temperature can be tailored.

Incidental Finding of a Suspected Horizontal Root Fracture During Mucogingival Surgery.

Periapical and bitewing radiographs lack the sensitivity to reliably diagnose horizontal root fractures, and, therefore, asymptomatic teeth with root fractures may remain undetected for years. This article reports a case in which a patient presented with a mucogingival defect on tooth No. 24 with no apparent history of dental trauma. During a free gingival graft procedure, a horizontal root fracture was observed in the apical third of the aforementioned tooth. After the clinician communicated this finding to the patient, the patient recollected two instances of trauma that had occurred to this area more than 30 years earlier. The presence of the horizontal root fracture did not affect the postoperative healing from the mucogingival procedure, and the tooth remained stable at the 1-year follow-up.

Investigation of the Internal Chemical Composition of Chitosan-Based LbL Films by Depth-Profiling X-ray Photoelectron Spectroscopy (XPS) Analysis.

Chitosan-based thin films were assembled using the layer-by-layer technique, and the axial composition was accessed using X-ray photoelectron spectroscopy with depth profiling. Chitosan (CHI) samples possessing different degrees of acetylation ([Formula: see text]) and molecular weight ([Formula: see text]) produced via the ultrasound-assisted deacetylation reaction were used in this study along with two different polyanions, namely, sodium polystyrenesulfonate (PSS) and carboxymethylcellulose (CMC). When chitosan, a positively charged polymer in aqueous acid medium, was combined with a strong polyanion (PSS), the total positive charge of chitosan, directly related to its [Formula: see text], was the key factor affecting the film formation. However, for CMC/CHI films, the pH of the medium and [Formula: see text] of chitosan strongly affected the film structure and composition. Consequently, the structure and the axial composition of chitosan-based films can be finely adjusted by choosing the polyanion and defining the chitosan to be used according to its DA and [Formula: see text] for the desired application, as demonstrated by the antibacterial tests.