The effect of osteocyte-derived RANKL on bone graft remodeling: An in vivo experimental study.

OBJECTIVES: Autologous bone is considered the gold standard for grafting, yet it suffers from a tendency to undergo resorption over time. While the exact mechanisms of this resorption remain elusive, osteocytes have been shown to play an important role in stimulating osteoclastic activity through their expression of receptor activator of NF-κB (RANK) ligand (RANKL). The aim of this study was to assess the function of osteocyte-derived RANKL in bone graft remodeling. MATERIALS AND METHODS: In Tnfsf11fl/fl ;Dmp1-Cre mice without osteocyte-specific RANKL as well as in Dmp1-Cre control mice, 2.6 mm calvarial bone disks were harvested and transplanted into mice with matching genetic backgrounds either subcutaneously or subperiosteally, creating 4 groups in total. Histology and micro-computed tomography of the grafts and the donor regions were performed 28 days after grafting. RESULTS: Histology revealed marked resorption of subcutaneous control Dmp1-Cre grafts and new bone formation around subperiosteal Dmp1-Cre grafts. In contrast, Tnfsf11fl/fl ;Dmp1-Cre grafts showed effectively neither signs of bone resorption nor formation. Quantitative micro-computed tomography revealed a significant difference in residual graft area between subcutaneous and subperiosteal Dmp1-Cre grafts (p < .01). This difference was not observed between subcutaneous and subperiosteal Tnfsf11fl/fl ;Dmp1-Cre grafts (p = .17). Residual graft volume (p = .08) and thickness (p = .13) did not differ significantly among the groups. Donor area regeneration was comparable between Tnfsf11fl/fl ;Dmp1-Cre and Dmp1-Cre mice and restricted to the defect margins. CONCLUSIONS: The results suggest an active function of osteocyte-derived RANKL in bone graft remodeling.

Emulating Clinical Diagnostic Reasoning for Jaw Cysts with Machine Learning.

The detection and classification of cystic lesions of the jaw is of high clinical relevance and represents a topic of interest in medical artificial intelligence research. The human clinical diagnostic reasoning process uses contextual information, including the spatial relation of the detected lesion to other anatomical structures, to establish a preliminary classification. Here, we aimed to emulate clinical diagnostic reasoning step by step by using a combined object detection and image segmentation approach on panoramic radiographs (OPGs). We used a multicenter training dataset of 855 OPGs (all positives) and an evaluation set of 384 OPGs (240 negatives). We further compared our models to an international human control group of ten dental professionals from seven countries. The object detection model achieved an average precision of 0.42 (intersection over union (IoU): 0.50, maximal detections: 100) and an average recall of 0.394 (IoU: 0.50-0.95, maximal detections: 100). The classification model achieved a sensitivity of 0.84 for odontogenic cysts and 0.56 for non-odontogenic cysts as well as a specificity of 0.59 for odontogenic cysts and 0.84 for non-odontogenic cysts (IoU: 0.30). The human control group achieved a sensitivity of 0.70 for odontogenic cysts, 0.44 for non-odontogenic cysts, and 0.56 for OPGs without cysts as well as a specificity of 0.62 for odontogenic cysts, 0.95 for non-odontogenic cysts, and 0.76 for OPGs without cysts. Taken together, our results show that a combined object detection and image segmentation approach is feasible in emulating the human clinical diagnostic reasoning process in classifying cystic lesions of the jaw.

Preoperative buccal bone volume predicts long-term graft retention following augmentation in the esthetic zone: A retrospective case series.

OBJECTIVES: Buccal bone augmentation in the esthetic zone is routinely used to achieve optimal clinical outcomes. Nonetheless, long-term data are sparse, and it is unknown how baseline buccal bone volume affects the retention of the augmented volume over time. MATERIAL AND METHODS: This is a long-term follow-up retrospective case series. After a preoperative computed tomography scan, implants were placed in the anterior maxilla following guided bone regeneration, autogenous block grafting, or both. At the follow-up, patients received a computed tomography scan and a clinical examination. Buccal bone volume was the primary outcome. Buccal bone thickness, peri-implant, and esthetic parameters were secondary outcomes. RESULTS: After a median follow-up of 6.7 years (interquartile range: 4.9-9.4), 28 implants in 19 patients (median age at augmentation: 43.3 years, interquartile range: 34.4-56.7, 53% female) were followed up. Preoperative buccal bone volume at baseline (V0 ) showed a moderate correlation to final buccal bone volume (Vt , rs  = .43) but a strong correlation to the absolute volumetric change (ΔV = Vt -V0 , rs  = -.80). A linear mixed model for Vt had a large intercept of 91.39 (p < .001) and a rather small slope of .11 for V0 (p = .11). Observed differences between treatments were not statistically significant in the mixed model. V0 above 105 mm3 predicted a negative volume change (ΔV < 0) with a specificity of 100% and a sensitivity of 96%. CONCLUSIONS: The results suggest higher gains in sites with lower V0 and point to a cutoff V0 above which the augmented volume is not retained long-term.

The Effect of the COVID-19 Pandemic on Patient Selection, Surgical Procedures, and Postoperative Complications in a Specialized Dental Implant Clinic.

During the coronavirus disease 2019 (COVID-19) pandemic, aerosol-generating procedures, including dental implant treatments, are considered high-risk. With dental implant treatment mostly an elective procedure, we aimed to assess whether the pandemic influenced patient selection, surgical procedures, and postoperative complications. We compared dental implant treatments during (March to December 2020) and before (December 2018 to February 2020) the COVID-19 pandemic based on patient and implant parameters, as well as postoperative complications. For analysis, we used the Chi-squared test with the Holm-Sidak correction for multiple comparisons. The number of implants placed during the COVID-19 pandemic (696 implants in 406 patients, 70 implants per month) was comparable to pre-pandemic levels (1204 implants in 616 patients, 80 implants per month). Regarding patient parameters, there were no significant differences in respiratory (p = 0.69) and cardiovascular conditions (p = 0.06), diabetes (p = 0.69), and smoking (p = 0.68). Regarding implant parameters, there was a significant difference in the distribution of augmentative procedures (no augmentation, guided bone regeneration, and sinus floor elevation, p = 0.01), but no significant differences in the types of edentulous spaces (p = 0.19) and the timing of implant placement (p = 0.52). Regarding complications, there were significantly fewer minor complications (p < 0.001) and early (i.e., before loading) implant failures (p = 0.02) compared with pre-pandemic levels. Our results suggest that the COVID-19 pandemic had no effect on patient selection and only a slight effect on the surgical procedures. However, postoperative complications, including early failures, were significantly less prevalent during the pandemic.

Prediction of post-traumatic neuropathy following impacted mandibular third molar removal.

OBJECTIVES: The extraction of impacted mandibular third molars is a common surgical procedure often associated with complications including post-traumatic neuropathy. Previous work has focused on identifying confounding factors, but a robust preoperative risk prediction model remains elusive. METHODS: Using a dataset of 648 patients and 812 impacted mandibular third molars, we used least absolute shrinkage and selection operator (LASSO) to fit prediction models based on risk factors assessed at both the tooth and patient levels. In addition, we fitted multivariable logistic regression models with the Firth correction for generalized estimating equations (GEE). RESULTS: The LASSO model for post-traumatic neuropathy identified distoangular impaction of ≥ 45° (odds ratio [OR] = 2.9), proximity to the inferior alveolar nerve of ≤ 3 mm (OR = 1.9), disadvantageous curving (OR = 1.4), and psychiatric conditions (OR = 2.1) as predictors [area under the receiving operator characteristic curve (AUC) = 0.75]. Among other complications analyzed, the LASSO model for bleeding identified deep embedding or full impaction (OR = 1.8), psychiatric conditions (OR = 1.3), and age (OR = 0.9) as predictors (AUC = 0.64). These associations between predictors and postoperative complications were fundamentally reinforced by the corresponding GEE models. CONCLUSIONS: Our findings point to the predictability of post-traumatic neuropathy and bleeding based on tooth anatomy and patient characteristics, overall suggesting that preoperatively identifiable factors can predict the risk of adverse outcomes in the extraction of impacted mandibular third molars. CLINICAL SIGNIFICANCE: Mandibular third molar extraction is both a routine procedure and a leading cause of trigeminal neuropathy. Prevention of post-traumatic neuropathy, aided by individualized preoperative risk prediction, is of high clinical relevance.

Resonance frequency analysis of implants placed in condensed bone.

OBJECTIVES: Resonance frequency analysis (RFA) is used to monitor implant stability. Its output, the Implant Stability Quotient (ISQ), supposedly correlates with insertion torque, a common measurement of primary stability. However, the reliability of RFA in condensed bone remains unclear. MATERIAL AND METHODS: In this human cadaver study in edentulous jaws and fresh extraction sockets, implants were inserted using a split-mouth approach into condensed or untreated bone. Mean ISQ, peak insertion torque, and pre- and postoperative bone volume fractions (BV/TV) were assessed. RESULTS: In edentulous jaws, insertion torque and ISQ correlated both in untreated (r = 0.63, p = 0.02) and in condensed (r = 0.82, p  < 0.01) bone. In extraction sockets, insertion torque and ISQ only correlated in untreated (r = 0.78, p < 0.01), but not in condensed bone (r = 0.15, p = 0.58). In all edentulous jaws, preoperative BV/TV correlated with insertion torque (r = 0.90, p < 0.0001), ISQ (r = 0.64, p < 0.001), and changes in BV/TV (r = -0.71, p < 0.01). In all extraction sockets, preoperative BV/TV did not correlate with either insertion torque (r = 0.33, p = 0.15), ISQ (r = 0.38, p = 0.09), or changes in BV/TV (r = -0.41, p = 0.09). Joint analysis identified preoperative BV/TV as a predictor of postoperative BV/TV (p < 0.001), insertion torque (p < 0.001), and ISQ (p < 0.001). CONCLUSIONS: RFA is feasible for monitoring stability after late implant placement into condensed bone, but not after immediate placement into condensed fresh extraction sites.

Osteoconductive properties of upside-down bilayer collagen membranes in rat calvarial defects.

BACKGROUND: Bilayer collagen membranes are routinely used in guided bone/tissue regeneration to serve as osteoconductive scaffolds and prevent the invasion of soft tissues. It is recommended to place the membranes with their dense layer towards the soft tissue and their porous layer towards the bony defect area. However, evidence supporting this recommendation is lacking. This study aimed to determine whether the alignment of bilayer collagen membranes has an effect on bone regeneration. METHODS: In two groups of ten male Sprague-Dawley rats each, a 5-mm calvarial defect was created. Thereafter, the defect was randomly covered with a bilayer, resorbable, pure type I and III collagen membrane placed either regularly or upside-down (i.e., dense layer towards bone defect). After 4 weeks of healing, micro-computed tomography (µCT), histology, and histomorphometry of the inner cylindrical region of interest (4.5 mm in diameter) were performed to assess new bone formation and the consolidation of the collagen membrane in the defect area. RESULTS: Quantitative µCT showed similar bone volume (median 8.0 mm3, interquartile range 7.0-10.0 vs. 6.2 mm3, 4.3-9.4, p = 0.06) and trabecular thickness (0.21 mm, 0.19-0.23 vs. 0.18 mm, 0.17-0.20, p = 0.03) between upside-down and regular placement, both leading to an almost complete bony coverage. Histomorphometry showed comparable new bone areas between the upside-down and regularly placed membranes, 3.9 mm2 (2.7-5.4) vs. 3.8 mm2 (2.2-4.0, p = 0.31), respectively. Both treatment groups revealed the same regeneration patterns and spatial distribution of bone with and without collagen fibers, as well as residual collagen fibers. CONCLUSIONS: Our data support the osteoconductive properties of collagen membranes and suggest that bone regeneration is facilitated regardless of membrane layer alignment.

A volumetric prediction model for postoperative cyst shrinkage.

OBJECTIVES: With only limited information available on dimensional changes after jaw cyst surgery, postoperative cyst shrinkage remains largely unpredictable. We aimed to propose a model for volumetric shrinkage based on time elapsed since cyst surgery. MATERIAL AND METHODS: We used data from patients that underwent cyst enucleation or decompression between 2007 and 2017 and had at least three computed tomography (CT) scans per patient. We fitted one simple exponential decay model [V(t) = V0 · e-ɑt] and one model with a patient-specific decay rate [Vk(t) = V0 · e-ßt + γkt]. RESULTS: Based on 108 CT scans from 36 patients (median age at surgery: 45.5 years, IQR: 32.3-55.3, 44% female), our simple exponential decay model is V(t) = V0 · e-0.0035t where V(t) is the residual cyst volume after time t elapsed since surgery, V0 is the initial cyst volume, and e is the base of the natural logarithm. Considering a patient-specific decay rate, the model is Vk(t) = V0 · e-0.0049t + γkt where γk is normally distributed, with expectation 0 and standard deviation 0.0041. CONCLUSIONS: Using an exponential regression model, we were able to reliably estimate volumetric shrinkage after jaw cyst surgery. The patient-specific decay rate substantially improved the fit of the model, whereas adding specific covariates as interaction effects to model the decay rate did not provide any significant improvement. CLINICAL RELEVANCE: Estimating postoperative cyst shrinkage is relevant for both treatment planning of jaw cyst surgery as well as evaluating the clinical success of the surgical approach.

Angular changes in implants placed in the anterior maxillae of adults: a cephalometric pilot study.

OBJECTIVES: Completion of adolescent growth represents the earliest time point for implant placement, yet craniofacial growth persists into adulthood and may affect implant position. We aimed to assess whether implants placed in the anterior maxillae of adults show angular changes over time. MATERIAL AND METHODS: We conducted a cephalometric pilot study in postpubertal patients with no growth disorders, skeletal malformations, or parafunctions. The patients received a single implant in the anterior maxilla and no orthodontic or orthognathic treatment afterwards. We measured angular changes of implants and central incisors on cephalograms taken immediately and after at least 5 years postoperatively with the Sella-Nasion line (SNL) and the nasal line (NL) as references. Changes in implant-SNL angles were the primary outcome. RESULTS: In 21 patients (30.2 ± 11.5 years at surgery) after a mean follow-up of 8.6 ± 1.3 years, implant-SNL angles and implant-NL angles changed in 81% and 57% of implants, respectively. Implant-SNL changes ranged from 3° counterclockwise to 4° clockwise and were more prevalent in males (100% vs. 58%) and patients under 30 at surgery (85% vs. 63%); mean absolute differences were larger in males (1.8 ± 1.0° vs. 1.3 ± 1.4°) and patients under 30 at surgery (1.5 ± 1.4° vs. 1.1 ± 1.4°). Incisor-SNL angles and incisor-NL angles changed in 89% and 32% of incisors, respectively. CONCLUSIONS: Implants placed in the anterior maxillae of adults show modest angular changes over time. CLINICAL RELEVANCE: Changes in implant angles have potential functional and esthetic consequences.

An advanced prediction model for postoperative complications and early implant failure.

OBJECTIVES: Risk prediction in implant dentistry presents specific challenges including the dependence of observations from patients with multiple implants and rare outcome events. The aim of this study was to use advanced statistical methods based on penalized regression to assess risk factors in implant dentistry. MATERIAL AND METHODS: We conducted a retrospective study from January 2016 to November 2018 recording postoperative complications including bleeding, hematoma, local infection, and nerve damage, as well as early implant failure. We further assessed patient- and implant-related risk factors including smoking and diabetes, as well as treatment parameters including types of gaps and surgical procedures. Univariable and multivariable generalized estimating equation (GEE) models were estimated to assess predictor effects, and a prediction model was fitted using L1 penalized estimation (lasso). RESULTS: In a total of 1,132 patients (mean age: 50.6 ± 16.5 years, 55.4% female) and 2,413 implants, postoperative complications occurred in 71 patients. Sixteen implants were lost prior to loading. Multivariable GEE models showed a higher risk of any complication for diabetes mellitus (p = .006) and bone augmentation (p = .039). The models further revealed a higher risk of local infection for bone augmentation (p = .003), and a higher risk of hematoma formation for diabetes mellitus (p = .007) and edentulous jaws (p = .024). The lasso model did not select any risk factors into the prediction model. CONCLUSIONS: Using novel methodology well-suited to tackle the specific challenges of risk prediction in implant dentistry, we were able to reliably estimate associations of risk factors with outcomes.

Draft Genome Sequence of Mycolicibacterium sp. Strain CH28, a Potential Degrader of Diisopropyl Ether, Isolated from Pharmaceutical Wastewater.

Mycolicibacterium sp. strain CH28 is a novel bacterial isolate belonging to a group of rapidly growing mycobacteria. Here, we report the draft genome sequence of strain CH28 and provide insights into the genetic background of its potential diisopropyl ether-degrading capability.

Age Differences in the Transfer and Maintenance of Practice-Induced Improvements in Task Switching: The Impact of Working-Memory and Inhibition Demands.

Recent aging studies on training in task switching found that older adults showed larger improvements to an untrained switching task as younger adults do. However, less clear is what type of cognitive control processes can explain these training gains as participants were trained with a particular type of switching task including bivalent stimuli, requiring high inhibition demands, and no task cues helping them keeping track of the task sequence, and by this, requiring high working-memory (WM) demands. The aims of this study were first to specify whether inhibition, WM, or switching demands are critical for the occurrence of transfer and whether this transfer depends on the degree of overlap between training and transfer situation; and second to assess whether practiced-induced gains in task switching can be maintained over a longer period of time. To this end, we created five training conditions that varied in switching (switching vs. single task training), inhibition (switching training with bivalent or univalent stimuli), and WM demands (switching training with or without task cues). We investigated 81 younger adults and 82 older adults with a pretest-training-posttest design and a follow-up measurement after 6 months. Results indicated that all training and age groups showed improvements in task switching and a differential effect of training condition on improvements to an untrained switching task in younger and older adults. For younger adults, we found larger improvements in task switching for the switching groups than the single-task training group independently of inhibition and WM demands, suggesting that practice in switching is most critical. However, these benefits disappeared after 6 months. In contrast, for older adults training groups practicing task switching under high inhibition demands showed larger improvements to untrained switching tasks than the other groups. Moreover, these benefits were maintained over time. We also found that the transfer of benefits in task switching was larger with greater overlap between training and transfer situation. However, results revealed no evidence for transfer to other untrained cognitive task. Overall, the findings suggest that training in resolving interference while switching between two tasks is most critical for the occurrence of transfer in the elderly.

The deeper sources of political conflict: evidence from the psychological, cognitive, and neuro-sciences.

Political disputes ruin family reunions, scuttle policy initiatives, and spur violence and even terrorism. We summarize recent research indicating that the source of political differences can be found in biologically instantiated and often subthreshold predispositions as reflected in physiological, cognitive, and neural patterns that incline some people toward innovation and others toward conservatism. These findings suggest the need to revise traditional views that maintain that political opinions are the product of rational, conscious, socialized thought.

Auditory event-related potentials reflect dedicated change detection activity for higher-order acoustic transitions.

The processing of auditory changes at cortical level relies partly on dedicated change-detectors whose activity is reflected in the elicitation of the N1 and P2 event-related potentials (ERPs). In previous studies, N1 and P2 have been found only for first-order frequency transitions (i.e. constant-to-glide) but not for higher-order transitions (i.e. glide-to-constant). We tested whether this asymmetry is due to the complete lack, or the smaller number of dedicated higher-order change detectors compared to first-order change detectors by recording ERPs to constant-to-glide and glide-to-constant frequency transitions within pure and complex tones. For constant-to-glide transitions ERP amplitudes increased with the rate of frequency change and spectral complexity. Importantly, for glide-to-constant transitions, N1 was elicited, even though only for spectrally rich tones when the frequency-change rate was fastest. Thus, the asymmetry in auditory change-related N1 elicitation is attributable not to the lack of higher-order change detectors, but to their relatively low number.

Stimulus-focused attention speeds up auditory processing.

Stimulus-focused attention enhances the processing of auditory stimuli, which is indicated by enhanced neural activity. In situations where fast responses are required, attention may not only serve as a means to gain more information about the relevant stimulus, but it may provide a processing speed gain as well. In two experiments we investigated whether attentional focusing decreased the latency of the auditory N1 event related potential. In Experiment 1 slowly emerging, soft (20dB sensation level) sounds were presented in two conditions, in which participants performed a sound-detection task or watched a silent movie and ignored the sounds. N1 latency was shorter in the sound-detection task in comparison to the ignore condition. In Experiment 2 we investigated whether the attentional N1 latency-decrease was caused by a frequency-specific attentional preparation or not. To this end, tone sequences were presented with a single tone frequency or with four different frequencies. N1 latency was shorter in the sound-detection task in comparison to the ignore condition regardless the number of frequencies. These results suggest that stimulus-focused attention increases stimulus processing speed by generally increasing sensory gain.

Functional interaction of the circadian clock and UV RESISTANCE LOCUS 8-controlled UV-B signaling pathways in Arabidopsis thaliana.

Circadian clocks regulate many molecular and physiological processes in Arabidopsis (Arabidopsis thaliana), allowing the timing of these processes to occur at the most appropriate time of the day in a 24-h period. The accuracy of timing relies on the synchrony of the clock and the environmental day/night cycle. Visible light is the most potent signal for such synchronization, but light-induced responses are also rhythmically attenuated (gated) by the clock. Here, we report a similar mutual interaction of the circadian clock and non-damaging photomorphogenic UV-B light. We show that low-intensity UV-B radiation acts as entraining signal for the clock. UV RESISTANCE LOCUS 8 (UVR8) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are required, but ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) are dispensable for this process. UV-B responsiveness of clock gene expression suggests that photomorphogenic UV-B entrains the plant clock through transcriptional activation. We also demonstrate that UV-B induction of gene expression under these conditions is gated by the clock in a HY5/HYH-independent manner. The arrhythmic early flowering 3-4 mutant showed non-gated, high-level gene induction by UV-B, yet displayed no increased tolerance to UV-B stress. Thus, the temporal restriction of UV-B responsiveness by the circadian clock can be considered as saving resources during acclimation without losing fitness.

Identification of a novel cis-regulatory element for UV-B-induced transcription in Arabidopsis.

Ultraviolet-B light (UV-B) regulates the expression of genes in a wavelength- and fluence rate-dependent fashion. A signaling pathway consisting of CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) and UV RESISTANCE LOCUS 8 (UVR 8) mediates responsiveness to longer wavelength, low intensity UV-B light-activating, for example, HY5 gene expression. By contrast, transcription of another group of genes, including ANAC13, modulated by shorter wavelength, higher intensity UV-B is controlled by a yet unknown and largely COP1-independent signaling cascade. Here we provide evidence by promoter deletion analysis, and characterization of genetic mutants displaying aberrant expression patterns, that two cis-regulatory elements, designated MRE(ANAC13) and UVBox(ANAC13), are required for maximal UV-B induction of the ANAC13 gene in transgenic plants. These elements are located in the proximal 150-bp region of the ANAC13 promoter. They show no significant similarity to each other; the putative MRE(ANAC13) (-AACCTT-) is closely related to MRE(CHS) (-AACCTA-) found in the CHALCONE SYNTHASE (CHS) gene, whereas UVBox(ANAC13) (with core sequence CAAG) represents a novel cis-regulatory element. The novel UVBox(ANAC13) sequence is significantly enriched in the promoter region of a subset of UV-B-induced genes with similar activation properties as ANAC13. In addition, we demonstrate that expression of a chimeric gene containing only the dimerized 12-mer containing UVBox(ANAC13) fused to a minimal CaMV35S promoter/luciferase reporter is (i) efficiently induced by shorter wavelength, higher intensity UV-B, but (ii) does not respond either to longer wavelength UV-B and red light or (iii) to abscisic acid treatment and osmotic, salt, heat and cold stresses.

Predictive modeling of siderphore production by Pseudomonas fluorescens under iron limitation.

Iron is required by many microorganisms for growth. Although it is the most abundant transition metal on earth, its solubility is very low and therefore its bioavailability is poor. To overcome this limitation, many microorganisms have developed iron chelating mechanisms that enable them to bind the metal to organic molecules from which they are later released. In particular, pseudomonads are prominent producers of the chelator pyoverdine that has a high iron binding capability. We present a mathematical model for pyoverdine production by Pseudomonas fluorescens. It is a nonlinear and non-autonomous system of four ordinary differential equations for the dependent variables size of bacterial population, pyoverdine, dissolved iron and chelated iron. The transient adaptation of the average physiological state of the population to the environmental condition is explicitly included in the model formulation. A complete qualitative description of the model solution is given, based on analytical techniques. The model is quantitatively validated against experimental data of pyoverdine and population size. To this end we conduct and discuss a parameter identification study. It is found that the model, if calibrated using pyoverdine data alone is able to predict the population size and vice versa, with some restrictions. Thus the model can be used as an indirect experimental tool.

Arabidopsis thaliana circadian clock is regulated by the small GTPase LIP1.

BACKGROUND: At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative-feedback loops and generate a basic approximately 24 hr oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. RESULTS: The lip1-1 (light insensitive period 1) mutant isolated from the model plant Arabidopsis thaliana displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. In wild-type plants, period length shortens with increasing light fluence rates and the phase of rhythms can be shifted by light pulses administered to dark-adapted plants. In contrast, in lip1-1, period length is nearly insensitive to light intensity and significantly larger phase shifts (delays) can be induced during the subjective night. The mutant also displays elevated photomorphogenic responses to red and blue light, which cannot be explained by the circadian defect, suggesting distinct functions for LIP1 in the circadian light input and photomorphogenesis. The LIP1 gene encodes a functional, plant-specific atypical small GTPase, and therefore we postulate that it acts similarly to ZEITLUPE at postranscriptional level. CONCLUSIONS: LIP1 represents the first small GTPase implicated in the circadian system of plants. LIP1 plays a unique negative role in controlling circadian light input and is required for precise entrainment of the plant clock.

Experimental validation of a predicted feedback loop in the multi-oscillator clock of Arabidopsis thaliana.

Our computational model of the circadian clock comprised the feedback loop between LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1), and a predicted, interlocking feedback loop involving TOC1 and a hypothetical component Y. Experiments based on model predictions suggested GIGANTEA (GI) as a candidate for Y. We now extend the model to include a recently demonstrated feedback loop between the TOC1 homologues PSEUDO-RESPONSE REGULATOR 7 (PRR7), PRR9 and LHY and CCA1. This three-loop network explains the rhythmic phenotype of toc1 mutant alleles. Model predictions fit closely to new data on the gi;lhy;cca1 mutant, which confirm that GI is a major contributor to Y function. Analysis of the three-loop network suggests that the plant clock consists of morning and evening oscillators, coupled intracellularly, which may be analogous to coupled, morning and evening clock cells in Drosophila and the mouse.