Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here, we adapted a biosensor for glycolysis, HYlight, for use in Caenorhabditis elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons cell-autonomously perform glycolysis and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function and uncovers unique relationships between neuronal identities and metabolic landscapes in vivo.

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate.

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.

Pharmacological bypass of NAD+ salvage pathway protects neurons from chemotherapy-induced degeneration.

Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD+) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD+, nicotinamide mononucleotide (NMN), rather than loss of NAD+, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration. We hypothesized that protection could similarly be achieved by reducing NMN production pharmacologically. To achieve this, we took advantage of an alternative pathway for NAD+ generation that goes through the intermediate nicotinic acid mononucleotide (NAMN), rather than NMN. We discovered that nicotinic acid riboside (NAR), a precursor of NAMN, administered in combination with FK866, an inhibitor of the enzyme nicotinamide phosphoribosyltransferase that produces NMN, protected dorsal root ganglion (DRG) axons against vincristine-induced degeneration as well as NMN deamidase. Introducing a different bacterial enzyme that converts NAMN to NMN reversed this protection. Collectively, our data indicate that maintaining NAD+ is not sufficient to protect DRG neurons from vincristine-induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection.

Aseptic Loosening at the Tibia in Total Knee Arthroplasty: A Function of Cement Mantle Quality?

BACKGROUND: Aseptic loosening remains one of the leading causes for failure of total knee arthroplasty (TKA). We sought to identify early radiographic measures that may associate with aseptic tibial component loosening, emphasizing systematic evaluation of the cement mantle. METHODS: All TKA revisions from 2007 to 2015 with the primary indication of tibial aseptic loosening were identified using in an institutional implant retrieval database. After exclusion criteria, 61 TKAs comprised the study group. A matched control group of 59 TKAs that had not failed at a minimum of 3 years was identified for comparison. Radiographic analysis on all 6-week postoperative radiographs included angulation of components, cement penetration depth, and presence of radiolucency at the implant-cement and bone-cement interfaces. Groups were compared with Student's t-test, chi-squared test, and Mann-Whitney U-test. A final multivariable logistic regression model was formed for the outcome of aseptic loosening. RESULTS: On multivariable analysis, failure was associated with a greater number of zones with cement penetration <2 mm (5.6 vs 3.4 zones, odds ratio [OR] 1.89, P < .001), increasing percent involvement of radiolucency at the implant-cement interface (8.7% vs 3.1%, OR = 1.15, P = .001), and increased varus alignment of the tibial component (1.5° vs 0°, OR = 1.35, P = .014). A greater number of zones with a radiolucent line at the bone-cement interface did not significantly associate (1.1 vs 0.3, P = .091). CONCLUSION: Our results suggest that radiographic indicators of poor cement mantle quality associate with later aseptic loosening. This emphasizes the need for surgeons to perform careful cement technique in order to reduce the risk of TKA failure. LEVEL OF EVIDENCE: III (Case-control).

The prevalence of enamel and dentine caries lesions and their determinant factors among children living in fluoridated and non-fluoridated areas.

OBJECTIVE: To determine the prevalence and severity of dental caries (at dentine and enamel levels of diagnosis) amongst Malaysian children and to investigate determinant factors associated with caries detection at these different thresholds. METHODS: This study involved life-long residents aged 12 years-old in fluoridated and non-fluoridated areas in Malaysia (n=595). The survey was carried out in 16 public schools by a calibrated examiner, using ICDAS-II criteria. A questionnaire on socio-demographic and oral hygiene practices was self-administered by parents/guardians. Data were analysed using Mann-Whitney U tests and logistic regression. RESULTS: The overall response rate was 74.4%. Caries prevalence at the dentine level or at the dentine and enamel level was significantly (p⟨0.001) higher among children in the non-fluoridated area (D1₋6MFT⟩0 = 82.4%, D4₋6MFT⟩0 = 53.5%) than in the fluoridated area (D1₋6MFT⟩0 = 68.7%, D4₋6MFT⟩0 = 25.5%). Considering only the decayed component of the index, no significant differences were observed between the two areas when the detection threshold was set at enamel caries (D1₋3) (p=0.506). However, when the detection criteria were elevated to the level of caries into dentine (D4₋6) there were clear differences between the fluoridated and non-fluoridated areas (p=0.006). Exposure to fluoridated water proved a significant predictor for lower caries prevalence in the statistical model. Children whose father and mother had a low monthly income had a significantly higher dentine caries prevalence. CONCLUSION: Results confirmed existing evidence of the benefit of water fluoridation in caries prevention. Detection criteria set at caries into dentine shows clear differences between fluoridated and non-fluoridated areas. Exposure to fluoridated water and socio-economic status were associated with caries prevalence.

The acceptability of fluoride varnish and fissure sealant treatments in children aged 6-9 delivered in a school setting.

OBJECTIVE: To assess the acceptability of fluoride varnish and fissure sealant treatments for children. To investigate the acceptability of delivering this treatment in a school setting for children, parents, clinicians and school staff. BASIC RESEARCH DESIGN: Semi-structured interviews (with children, parents, clinicians and school staff) and a questionnaire (for school staff) as part of a two-arm, randomised clinical trial. PARTICIPANTS: Children aged 6-9, their parents, clinical staff and school staff. INTERVENTIONS: Fluoride varnish or fissure sealant was delivered to children from the ages of 6 to 9 years for 36 months, by a community dental service in a school setting. Fluoride varnish was re-applied every 6 months; fissure sealant was applied once to first permanent molars and re-applied as required. RESULTS: Interviews with children a few days after treatment indicated little difference in preference; acceptability at this point was driven by factors such as finding it fun to visit 'the van' (i.e. mobile dental unit) and receiving a "sticker" rather than specific treatment received. Interviews with parents, clinicians and school staff indicated high acceptability of delivering this type of intervention in a school setting; this may have been partly due to the service being delivered by a well-established, child-oriented community dental service which delivered the clinical trial. CONCLUSIONS: Preventive fluoride varnish and fissure sealant treatments in a school setting has high overall acceptability.

Single-trial decoding of intended eye movement goals from lateral prefrontal cortex neural ensembles.

Neurons in the lateral prefrontal cortex (LPFC) encode sensory and cognitive signals, as well as commands for goal-directed actions. Therefore, the LPFC might be a good signal source for a goal-selection brain-computer interface (BCI) that decodes the intended goal of a motor action previous to its execution. As a first step in the development of a goal-selection BCI, we set out to determine if we could decode simple behavioral intentions to direct gaze to eight different locations in space from single-trial LPFC neural activity. We recorded neuronal spiking activity from microelectrode arrays implanted in area 8A of the LPFC of two adult macaques while they made visually guided saccades to one of eight targets in a center-out task. Neuronal activity encoded target location immediately after target presentation, during a delay epoch, during the execution of the saccade, and every combination thereof. Many (40%) of the neurons that encoded target location during multiple epochs preferred different locations during different epochs. Despite heterogeneous and dynamic responses, the neuronal feature set that best predicted target location was the averaged firing rates from the entire trial and it was best classified using linear discriminant analysis (63.6-96.9% in 12 sessions, mean 80.3%; information transfer rate: 21-59, mean 32.8 bits/min). Our results demonstrate that it is possible to decode intended saccade target location from single-trial LPFC activity and suggest that the LPFC is a suitable signal source for a goal-selection cognitive BCI.

Examiner reliability in fluorosis scoring: a comparison of photographic and clinical methods.

OBJECTIVE: To assess examiner reliability when scoring dental fluorosis in Malaysian children using clinical (Dean's Index) and photographic methods. METHOD: The upper central incisors of 111 children were examined both clinically and photographically for fluorosis status using Dean's index. Twenty children were re-examined after a two-week interval for intra-examiner reliability by a single examiner. In addition, two independent examiners and the clinical examiner scored 111 photographic images of the same children in a standardized manner. Fluorosis scores were compared individually between examiners for both clinical and photographic scoring. Examiner reliability was assessed using both simple and weighted kappa statistics at tooth level. Sensitivity, specificity, positive-negative predictive values and a Receiver Operating Characteristic (ROC) curve were also calculated to determine the accuracy of the test. RESULTS: Across the three examiners, the prevalence of fluorosis (Dean's score ≥ 2) using photographs was lower (ranged from 23% to 26%) than the prevalence recorded by clinical examination (30%). The kappa score for intra-examiner reliability for the duplicate clinical examination was excellent (0.89). Inter-examiner reliability between the photographic method and the clinical examination (gold standard) for each examiner was substantial with weighted kappa values ranging from 0.74 to 0.77. The photographic method indicated higher specificity (99%) than sensitivity (79%) and the area under the ROC curve was also high (0.89) which suggests good accuracy of the diagnostic test. CONCLUSION: These results suggest that photographic examination of fluorosis on central incisors can be recorded with good examiner reliability. The recorded fluorosis prevalence was lower using the photographic scores.

Electrocorticographic activity over sensorimotor cortex and motor function in awake behaving rats.

Sensorimotor cortex exerts both short-term and long-term control over the spinal reflex pathways that serve motor behaviors. Better understanding of this control could offer new possibilities for restoring function after central nervous system trauma or disease. We examined the impact of ongoing sensorimotor cortex (SMC) activity on the largely monosynaptic pathway of the H-reflex, the electrical analog of the spinal stretch reflex. In 41 awake adult rats, we measured soleus electromyographic (EMG) activity, the soleus H-reflex, and electrocorticographic activity over the contralateral SMC while rats were producing steady-state soleus EMG activity. Principal component analysis of electrocorticographic frequency spectra before H-reflex elicitation consistently revealed three frequency bands: µß (5-30 Hz), low γ (γ1; 40-85 Hz), and high γ (γ2; 100-200 Hz). Ongoing (i.e., background) soleus EMG amplitude correlated negatively with µß power and positively with γ1 power. In contrast, H-reflex size correlated positively with µß power and negatively with γ1 power, but only when background soleus EMG amplitude was included in the linear model. These results support the hypothesis that increased SMC activation (indicated by decrease in µß power and/or increase in γ1 power) simultaneously potentiates the H-reflex by exciting spinal motoneurons and suppresses it by decreasing the efficacy of the afferent input. They may help guide the development of new rehabilitation methods and of brain-computer interfaces that use SMC activity as a substitute for lost or impaired motor outputs.

Volitional control of beta activities in Parkinson's disease patients.

Patients diagnosed with Parkinson's disease (PD) have difficulty initiating and executing movements due to an acquired imbalance of the basal ganglia thalamocortical circuit secondary to loss of dopaminergic input into the striatum. The unbalanced circuit is hyper-synchronized, presenting as larger and longer bursts of beta-band (13-30 Hz) oscillations in the subthalamic nucleus (STN). As a first step toward a novel PD therapy that aims to improve symptoms through beta desynchronization, we sought to determine if individuals with PD could acquire volitional control of STN beta power in a neurofeedback task. We found a significant difference in STN beta power between task conditions, and relevant brain signal features could be detected and decoded in real time. This demonstration of volitional control of STN beta motivates development of a neurofeedback therapy to modulate PD symptom severity.

Intracortical brain-computer interfaces in primates: a review and outlook.

Brain-computer interfaces (BCI) translate brain signals into artificial output to restore or replace natural central nervous system (CNS) functions. Multiple processes, including sensorimotor integration, decision-making, motor planning, execution, and updating, are involved in any movement. For example, a BCI may be better able to restore naturalistic motor behaviors if it uses signals from multiple brain areas and decodes natural behaviors' cognitive and motor aspects. This review provides an overview of the preliminary information necessary to plan a BCI project focusing on intracortical implants in primates. Since the brain structure and areas of non-human primates (NHP) are similar to humans, exploring the result of NHP studies will eventually benefit human BCI studies. The different types of BCI systems based on the target cortical area, types of signals, and decoding methods will be discussed. In addition, various successful state-of-the-art cases will be reviewed in more detail, focusing on the general algorithm followed in the real-time system. Finally, an outlook for improving the current BCI research studies will be debated.

Ensembles code for associative learning in the primate lateral prefrontal cortex.

The lateral prefrontal cortex (LPFC) of primates is thought to play a role in associative learning. However, it remains unclear how LPFC neuronal ensembles dynamically encode and store memories for arbitrary stimulus-response associations. We recorded the activity of neurons in LPFC of two macaques during an associative learning task using multielectrode arrays. During task trials, the color of a symbolic cue indicated the location of one of two possible targets for a saccade. During a trial block, multiple randomly chosen associations were learned by the subjects. A state-space analysis indicated that LPFC neuronal ensembles rapidly learn new stimulus-response associations mirroring the animals' learning. Multiple associations acquired during training are stored in a neuronal subspace and can be retrieved hours after learning. Finally, knowledge of old associations facilitates learning new, similar associations. These results indicate that neuronal ensembles in the primate LPFC provide a flexible and dynamic substrate for associative learning.

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use in C. elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapes in vivo.

A Sort-Seq Approach to the Development of Single Fluorescent Protein Biosensors.

Motivated by the growing importance of single fluorescent protein biosensors (SFPBs) in biological research and the difficulty in rationally engineering these tools, we sought to increase the rate at which SFPB designs can be optimized. SFPBs generally consist of three components: a circularly permuted fluorescent protein, a ligand-binding domain, and linkers connecting the two domains. In the absence of predictive methods for biosensor engineering, most designs combining these three components will fail to produce allosteric coupling between ligand binding and fluorescence emission. While methods to construct diverse libraries with variation in the site of GFP insertion and linker sequences have been developed, the remaining bottleneck is the ability to test these libraries for functional biosensors. We address this challenge by applying a massively parallel assay termed "sort-seq," which combines binned fluorescence-activated cell sorting, next-generation sequencing, and maximum likelihood estimation to quantify the brightness and dynamic range for many biosensor variants in parallel. We applied this method to two common biosensor optimization tasks: the choice of insertion site and optimization of linker sequences. The sort-seq assay applied to a maltose-binding protein domain-insertion library not only identified previously described high-dynamic-range variants but also discovered new functional insertion sites with diverse properties. A sort-seq assay performed on a pyruvate biosensor linker library expressed in mammalian cell culture identified linker variants with substantially improved dynamic range. Machine learning models trained on the resulting data can predict dynamic range from linker sequences. This high-throughput approach will accelerate the design and optimization of SFPBs, expanding the biosensor toolbox.

Tracking and Assessing Oil Spill Toxicity to Aquatic Organisms: A Novel Approach.

An in situ exposure and effects bioassay system was developed for assessing the toxicity of oil spills to aquatic organisms. The assessment tool combines components of 2 previously developed systems, the sediment ecotoxicity assessment ring (SEA Ring) and the drifting particle simulator. The integrated drifting exposure and effects assessment ring (DEEAR) is comprised of a Global Positioning System (GPS) float, a drifter drogue, the SEA Ring, and the Cyclops-7 fluorescent sensor. Polyethylene passive sampling devices (PED) were mounted for an additional means to characterize water quality conditions and exposures. The DEEAR is optimized for evaluating oil exposure and toxicity in the shallow surface mixing layer of marine waters. A short-term preliminary test was conducted in San Diego, California, USA, to verify the operation of the GPS tracking, the iridium communications, and the integrated SEA Ring exposure system. Further, a proof-of-concept demonstration was conducted offshore in the Santa Barbara Channel, where natural oil seeps produce surface slicks and sheens. Two DEEAR units were deployed for 24 h-one within the oil slick and one in an area outside observable slicks. An aerial drone provided tracking of the surface oil and optimal sites for deployment. The DEEAR proof-of-concept demonstrated integrated real-time tracking and characterization of oil exposures by grab samples, PED, and fluorescent sensors. Oil exposures were directly linked to toxic responses in fish and mysids. This novel integrated system shows promise for use in a variety of aquatic sites to more accurately determine in situ oil exposure and toxicity. Environ Toxicol Chem 2021;40:1452-1462. © 2021 SETAC.

A competing risk survival analysis model to assess the efficacy of filling carious primary teeth.

In recent years a strategy of selective, symptom-based intervention of carious primary teeth has been developed amongst some British general dental practitioners. Practice-based studies appear to provide evidence that policies of restoration of symptomless carious primary teeth do not confer any significant benefits above those associated with non-restorative care. However, results from these studies contrast with those of many clinical trials and prospective studies of primary molar restorations. In the current investigation, cohort study data from 5,168 carious primary molar teeth from 2,654 British children aged 4-5 years at baseline, augmented with Dental Practice Board treatment data, was utilised to assess the effect of restorative treatment on the likelihood of carious teeth subsequently progressing to either exfoliation or extraction. The effect of demographic and tooth level covariates on the fate of these teeth was also assessed. Multivariate multilevel parametric survival models were applied to the analysis of the carious-exfoliation and carious-extraction transitions to which the teeth were subject, assuming an underlying data hierarchy with teeth nested within individuals. Time of occurrence of caries affected survival experience, with teeth in which caries occurred later in life being associated with higher survival rates to extraction. Amongst filled teeth, later fillings were also associated with higher survival rates to extraction. Demographic and tooth level variables had a limited effect on survival experience. Treatment was found to be significantly associated with survival with respect to extraction, with survival rates of over 80% at 14 years, double those of untreated teeth.

Modelling childhood caries using parametric competing risks survival analysis methods for clustered data.

Caries in primary teeth is an ongoing issue in children's dental health. Its quantification is affected by clustering of data within children and the concurrent risk of exfoliation of primary teeth. This analysis of caries data of 103,776 primary molar tooth surfaces from a cohort study of 2,654 British children aged 4-5 years at baseline applied multilevel competing risks survival analysis methodology to identify factors significantly associated with caries occurrence in primary tooth surfaces in the presence of the concurrent risk of exfoliation, and assessed the effect of exfoliation on caries development. Multivariate multilevel parametric survival models were applied at surface level to the analysis of the sound-carious and sound-exfoliation transitions to which primary tooth surfaces are subject. Socio-economic class, fluoridation status and surface type were found to be the strongest predictors of primary caries, with the highest rates of occurrence and lowest median survival times associated with occlusal surfaces of children from poor socio-economic class living in non-fluoridated areas. The concurrent risk of exfoliation was shown to reduce the distinction in survival experience between different types of surfaces, and between surfaces of teeth from children of different socio-economic class or fluoridation status. Clustering of data had little effect on inferences of parameter significance.

Student perspectives and opinions on their experience at an undergraduate outreach dental teaching centre at Cardiff: a 5-year study.

AIM: Outreach teaching is now regarded as a desirable component of undergraduate dental teaching programmes in the UK. A purpose-built undergraduate dental outreach-training centre was opened in Cardiff in 2002. The aim of this paper is to report student perspectives and opinions on their experience at this unit over a 5-year period. METHODS: Final year dental students at Cardiff University were invited to report their comments on the St David's Primary Care Unit at various times during their placement there. Information was recorded for undergraduate students who commenced final year in 2003, 2004, 2005, 2006 and 2007 (n = 257). RESULTS: After 1 year, the most common favourable aspects reported by students included the availability of a suitably trained nurse for all procedures (n = 191), ready access to helpful/approachable teaching staff (n = 145), and closeness of learning experience to subsequent practice (n = 122). Many students commented on their growing confidence in their own abilities whilst in the unit. CONCLUSION: Overwhelmingly, students reported their enthusiasm for training in an outreach teaching unit, preferring it to traditional dental school environments. Inherent in the comments recorded for each student was a sense of growing confidence in their abilities and development of reflective practice. Further work is needed to identify the impact of this form of dental student training on subsequent practices in Vocational Training and independent clinical careers.

Child Caries Management: A Randomized Controlled Trial in Dental Practice.

This multicenter 3-arm, parallel-group, patient-randomized controlled trial compared clinical effectiveness of 3 treatment strategies over 3 y for managing dental caries in primary teeth in UK primary dental care. Participants aged 3 to 7 y with at least 1 primary molar with dentinal carious lesion were randomized across 3 arms (1:1:1 via centrally administered system with variable-length random permuted blocks): C+P, conventional carious lesion management (complete carious tooth tissue removal and restoration placement) with prevention; B+P, biological management (sealing in carious tooth tissue restoratively) with prevention; and PA, prevention alone (diet, plaque removal, fluorides, and fissure sealants). Parents, children, and dentists were not blind to allocated arm. Co-primary outcomes were 1) the proportion of participants with at least 1 episode of dental pain and/or infection and 2) the number of episodes of dental pain and/or infection during follow-up (minimum, 23 mo). In sum, 1,144 participants were randomized (C+P, n = 386; B+P, n = 381; PA, n = 377) by 72 general dental practitioners, of whom 1,058 (C+P, n = 352; B+P, n = 352; PA, n = 354) attended at least 1 study visit and were included in the primary analysis. The median follow-up was 33.8 mo (interquartile range, 23.8 to 36.7). Proportions of participants with at least 1 episode of dental pain and/or infection were as follows: C+P, 42%; B+P, 40%; PA, 45%. There was no evidence of a difference in incidence of dental pain and/or infection when B+P (adjusted risk difference [97.5% CI]: -2% [-10% to 6%]) or PA (4% [-4% to 12%]) was compared with C+P. The mean (SD) number of episodes of dental pain and/or infection were as follows: C+P, 0.62 (0.95); B+P, 0.58 (0.87); and PA, 0.72 (0.98). Superiority could not be concluded for number of episodes between B+P (adjusted incident rate ratio (97.5% CI): 0.95 [0.75 to 1.21]) or PA (1.18 [0.94 to 1.48]) and C+P. In conclusion, there was no evidence of a difference among the 3 treatment approaches for incidence or number of episodes of dental pain and/or infection experienced by these participants with high caries risk and established disease (trial registration: ISRCTN77044005).

A novel chromatin protein, distantly related to histone H2A, is largely excluded from the inactive X chromosome.

Chromatin on the mammalian inactive X chromosome differs in a number of ways from that on the active X. One protein, macroH2A, whose amino terminus is closely related to histone H2A, is enriched on the heterochromatic inactive X chromosome in female cells. Here, we report the identification and localization of a novel and more distant histone variant, designated H2A-Bbd, that is only 48% identical to histone H2A. In both interphase and metaphase female cells, using either a myc epitope-tagged or green fluorescent protein-tagged H2A-Bbd construct, the inactive X chromosome is markedly deficient in H2A-Bbd staining, while the active X and the autosomes stain throughout. In double-labeling experiments, antibodies to acetylated histone H4 show a pattern of staining indistinguishable from H2A-Bbd in interphase nuclei and on metaphase chromosomes. Chromatin fractionation demonstrates association of H2A-Bbd with the histone proteins. Separation of micrococcal nuclease-digested chromatin by sucrose gradient ultracentrifugation shows cofractionation of H2A-Bbd with nucleosomes, supporting the idea that H2A-Bbd is incorporated into nucleosomes as a substitute for the core histone H2A. This finding, in combination with the overlap with acetylated forms of H4, raises the possibility that H2A-Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome. The distribution of H2A-Bbd thus distinguishes chromatin on the active and inactive X chromosomes.