Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors.

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis.

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase ß-DNA complexes.

Eukaryotic DNA polymerase ß (Pol ß) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol ß has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol ß. First, using a doubly labeled DNA construct, we show that Pol ß bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol ß and donor-labeled DNA, we visualized dynamic fingers closing in single Pol ß-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol ß, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol ß reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices.

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

Influence of sub-nanosecond time of flight resolution for online range verification in proton therapy using the line-cone reconstruction in Compton imaging.

Online ion range monitoring in hadron therapy can be performed via detection of secondary radiation, such as promptγ-rays, emitted during treatment. The promptγemission profile is correlated with the ion depth-dose profile and can be reconstructed via Compton imaging. The line-cone reconstruction, using the intersection between the primary beam trajectory and the cone reconstructed via a Compton camera, requires negligible computation time compared to iterative algorithms. A recent report hypothesised that time of flight (TOF) based discrimination could improve the precision of theγfall-off position (FOP) measured via line-cone reconstruction, where TOF comprises both the proton transit time from the phantom entrance untilγemission, and the flight time of theγ-ray to the detector. The aim of this study was to implement such a method and investigate the influence of temporal resolution on the precision of the FOP. Monte Carlo simulations of a 160 MeV proton beam incident on a homogeneous PMMA phantom were performed using GATE. The Compton camera consisted of a silicon-based scatterer and CeBr3scintillator absorber. The temporal resolution of the detection system (absorber + beam trigger) was varied between 0.1 and 1.3 ns rms and a TOF-based discrimination method applied to eliminate unlikely solution(s) from the line-cone reconstruction. The FOP was obtained for varying temporal resolutions and its precision obtained from its shift across 100 independentγemission profiles compared to a high statistics reference profile. The optimal temporal resolution for the given camera geometry and 108primary protons was 0.2 ns where a precision of 2.30 ± 0.15 mm (1σ) on the FOP was found. This precision is comparable to current state-of-the-art Compton imaging using iterative reconstruction methods or 1D imaging with mechanically collimated devices, and satisfies the requirement of being smaller than the clinical safety margins.

Design principles of a minimal auxin response system.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices.

Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.

Comparison between direct vs indirect anchorage in two miniscrew-supported distalizing devices.

OBJECTIVE: To compare two distalizing devices supported by palatal miniscrews, the MGBM System (MGBM) and the Distal Screw appliance (DS), in dental Class II patients. MATERIALS AND METHODS: Pretreatment (T1) and postdistalization (T2) lateral cephalograms of 53 Class II malocclusion subjects were examined. MGBM consisted of 29 patients (16 males, 13 females) with a mean pretreatment age of 12.3 ± 1.5 years; DS consisted of 24 patients (11 males, 13 females) with a mean pretreatment age of 11.3 ± 1.2 years. The mean distalization time was 6 ± 2 months for MGBM and 9 ± 2 months for DS. Initial and final measurements and treatment changes were compared by means of a Student's t-test. RESULTS: Maxillary superimpositions showed that the maxillary first molar distalized an average of 5.5 mm in the MGBM and 3.2 mm in the DS between T1 and T2; distal molar tipping was greater in the MGBM (10.3°) than in the DS (3.0°). First premolar showed a mean mesial movement of 1.4 mm, with a mesial tipping of 4.4° in the MGBM; on the contrary, first premolar showed a distal movement of 2.2 mm, with a distal tipping of 6.2°, in the DS. CONCLUSIONS: The MGBM system resulted in greater distal molar movement and less treatment time, resulting in more efficient movement than was associated with the DS; DS showed less molar tipping during distalization.

Comparative evaluation of molar distalization therapy using pendulum and distal screw appliances.

OBJECTIVE: To compare dentoalveolar and skeletal changes produced by the pendulum appliance (PA) and the distal screw appliance (DS) in Class II patients. METHODS: Forty-three patients (19 men, 24 women) with Class II malocclusion were retrospectively selected for the study. Twenty-four patients (mean age, 12.2 ± 1.5 years) were treated with the PA, and 19 patients (mean age, 11.3 ± 1.9 years) were treated with the DS. The mean distalization time was 7 months for the PA group and 9 months for the DS group. Lateral cephalograms were obtained at T1, before treatment, and at T2, the end of distalization. A Mann-Whitney U test was used for statistical comparisons of the two groups between T1 and T2. RESULTS: PA and DS were equally effective in distalizing maxillary molars (4.7 mm and 4.2 mm, respectively) between T1 and T2; however, the maxillary first molars showed less distal tipping in the DS group than in the PA group (3.2° vs. 9.0°, respectively). Moreover, significant premolar anchorage loss (2.7 mm) and incisor proclination (5.0°) were noted in the PA group, whereas premolar distal movement (1.9 mm) and no significant changes at the incisor (0.1°) were observed in the DS group. No significant sagittal or vertical skeletal changes were detected between the two groups during the distalization phase. CONCLUSIONS: PA and DS seem to be equally effective in distalizing maxillary molars; however, greater distal molar tipping and premolar anchorage loss can be expected using PA.

Comparative evaluation of molar distalization therapy with erupted second molar: Segmented versus Quad Pendulum appliance.

BACKGROUND: There are controversial opinions about the effect of erupted second molars on distalization of the first molars. Most of the distalizing devices are anchored on the first molars, without including second molars; so, differences between sequentially distalize maxillary molars (second molar followed by the first molar) or distalize second and first molars together are not clear. The aim of the study was to compare sequential versus simultaneous molar distalization therapy with erupted second molar using two different modified Pendulum appliances followed by fixed appliances. METHODS: The treatment sample consisted of 35 class II malocclusion subjects, divided in two groups: group 1 consisted of 24 patients (13 males and 11 females) with a mean pre-treatment age of 12.9 years, treated with the Segmented Pendulum (SP) and fixed appliances; group 2 consisted of 11 patients (6 males and 5 females) with a mean pre-treatment age of 13.2 years, treated with the Quad Pendulum (QP) and fixed appliances. Lateral cephalograms were obtained before treatment (T1), at the end of distalization (T2), and at the end of orthodontic fixed appliance therapy (T3). A Student t test was used to identify significant between-group differences between T1 to T2, T2 to T3, and T1 to T3. RESULTS: QP and SP were equally effective in distalizing maxillary molars (3.5 and 4 mm, respectively) between T1 and T2; however, the maxillary first molar showed less distal tipping (4.6° vs. 9.6°) and more extrusion (1.1 vs. 0.2 mm) in the QP group than in the SP group, as well as the vertical facial dimension, which increased more in the QP group (1.2°) than in the SP group (0.7°). At T3, the QP group maintained greater increase in lower anterior facial height and molar extrusion and decrease in overbite than the SP group. CONCLUSION: Quad Pendulum seems to have greater increase in vertical dimension and molar extrusion than the Segmented Pendulum.

Long-term evaluation of the molar movements following Pendulum and fixed appliances.

OBJECTIVE: To describe the molar movements and skeletal changes associated with Pendulum-fixed appliance treatment and the long-term postretention period. SUBJECTS AND METHODS: The treatment sample consisted of 76 Class II patients, 35 males and 41 females. Lateral cephalograms were obtained at the start of treatment (T1); the end of distalization (T2); the end of orthodontic fixed appliance therapy (T3); and long-term observation (7 years 2 months later; T4). Mean age was 12 years 11 months at T1, 13 years 8 months at T2, 15 years 4 months at T3, and 22 years 5 months at T4. The average amount of Class II molar relationship was 3.1 mm, with a mean overjet of 5.9 mm at the beginning of treatment. A paired t-test was used to identify significant between-group differences between T2-T4 and T3-T4. RESULTS: Distal molar movement was obtained during the distalization phase (T2), and more than half of the distalizing effect was maintained at the end of maxillary growth (T4). Most of the relapse occurred during fixed appliance therapy (T3), whereas no significant change was detected in the postretention period (T4). The molar relationship did not show any significant difference between T2 and T4. The vertical facial dimension increased during the distalization phase (T2) and fixed appliance therapy (T3) but returned to the initial values during the postretention period (T4). CONCLUSIONS: The Pendulum appliance induces significant dentoalveolar effects, which can be partially maintained during the long-term period. The Class I molar relationship does not change during completion of individual growth. Increase in vertical facial dimension represents a temporary effect.

Macrodontic maxillary incisor in alagille syndrome.

This case report describes the surgical-orthodontic guided-eruption of a deeply impacted macrodontic maxillary central incisor in a 10-year-old patient with Alagille syndrome (ALGS). In the first stage, orthodontic treatment with fixed appliance on deciduous teeth allowed to create enough space for the eruption of the maxillary right central incisor. The second stage included closed surgical exposure and vertical traction. After impacted tooth erupted in the proper position, accessory periodontal treatment and dental reshaping procedures may be indicated to camouflage macrodontic incisor with the adjacent teeth. This is the first report that presents a patient with ALGS undergoing orthodontic and surgical treatment.

Non-compliance maxillary molar distalizing appliances: an overview of the last decade.

OBJECTIVES: To perform a literature review focusing on the use of non-compliance intraoral appliances for molar distalization therapy. MATERIALS AND METHODS: A literature search ranged from January 1999 to December 2009 in order to describe dento-alveolar changes and skeletal vertical modifications following the use of conventional anchorage intraoral distalizing appliances. The quality of the retrieved studies was ranked on a 12-points scale, from low to high quality. RESULTS: A total of 214 articles were found and only 24 were considered eligible for the critical examination. Maxillary first molar distalization ranged from 6.4mm to 0.5mm with a concomitant distal tipping from 18.5° to bodily distalization. A smaller amount of distal movement and a greater amount of crown tipping can be noted at second molars. Premolar anchorage loss and incisor proclination represent an unavoidable side-effect and ranged from 4.33 mm to 0.73 mm and from 13.7° to 0.6° respectively. Skeletal vertical modifications were noted. Increase in vertical facial dimension ranged from 1.5° to -1.8° and lower anterior facial height ranged from an increase of 3.2 to a decrease of 0.6 mm. The assessment of study quality showed that 19 studies were of low quality, 3 of medium quality and 2 of medium-high quality. CONCLUSIONS: Maxillary molar distalization can be effectively performed with the use of noncompliance intraoral appliances. As a consequence, premolar anchorage loss, incisor proclination and a slight increase in vertical facial dimension can occur. Because of the lack of high-quality studies the findings of this review should be carefully interpreted.

Soft tissue, skeletal and dentoalveolar changes following conventional anchorage molar distalization therapy in class II non-growing subjects: a multicentric retrospective study.

OBJECTIVES: The purpose of this retrospective prolective study is to evaluate soft tissue, dentoalveolar and skeletal vertical changes following conventional anchorage molar distalization therapy in adult patients. MATERIALS AND METHODS: Forty-six patients (34 females, mean age 25 years 6 months; and 12 males, mean age 28 years 4 months) were recruited from 4 specialists Board Certified. All subjects underwent molar distalization therapy according different distalization mechanics. Cephalometric headfilms were available for all subjects before (T0) and at the end of comprehensive treatment (T1). The initial and final measurements and treatment changes were compared by means of a paired t-test or a paired Wilcoxon test. RESULTS: Mean total treatment time was 3 years 3 months ± 8 months. Maxillary first and second molars distalized 2.16±0.84 mm and 2.01±0.69 mm respectively, but also maintained a slight distal tipping of 1.45° (min 2.22°, max -6.45°) and 3.35° (min 0.47°, max -15.48°) at the end of treatment. Distal movement of maxillary first molar contributed 57.6% to molar correction, and 42.4% was due to a mesial movement of mandibular first molar (1.59±0.46 mm). Dentoalveolar changes contributed to overjet correction; maxillary incisors retroclined 5.78°±3.17°, lower incisors proclined 7.49°±4.52° and occlusal plane rotated down and backward 2.32°±2.10°. A significant clockwise rotation of the mandible (1.97°±1.32°) and a significant increase in lower facial height (3.35±1.48) mm were observed. Upper lip slightly retruded (-1.76±1.70 mm) and lower lip protruded (0.96±0.99 mm) but these changes had a negligible impact on clinical appearance. CONCLUSIONS: Although maxillary molar distalization therapy can be performed in adult patients, significant proclination of the lower incisors, clockwise rotation of the occlusal plane and increase in vertical facial dimension should be expected. Nevertheless, in absence of maxillary third molars and in presence of mandibular third molars this procedure could be recommended.

Failure evaluation after a 6-year retention period: a comparison between glass fiber-reinforced (GFR) and multistranded bonded retainers.

OBJECTIVES: The aim of this study was to compare bond failure and breakage rates of two types of bonded lingual orthodontic retainers (GFR and .0175" multistranded stainless steel wire [MST]) after a 6-year retention period. SUBJECTS AND METHODS: Eighty-five young adults were randomly divided into two groups: 40 subjects received 48 GFR retainers (14 maxillary and 34 mandibular retainers), and 45 subjects received 50 MST retainers (18 maxillary and 32 mandibular retainers). A Fisher's exact test was performed in order to identify significant differences in the percentage of detachment and breakage of the retainers between the groups. RESULTS: The maxillary detachment rates were 21.42% for the GFR group and 22.22% for the MST group; the mandibular detachment rates were 11.76% for the GFR group and 15.62% for the MST group. The maxillary breakage rates were 7.14% for the GFR group and 16.66% for the MST group; the mandibular breakage rates were 8.82% for the GFR group and 15.62% for the MST group. The differences were not statistically significant. CONCLUSION: GFR and multistranded stainless steel retainers showed similar results in terms of bond failure and breakage after 6 years of retention. The use of GFR retainers as a retention strategy should not be discouraged and could be considered a viable esthetic alternative to stainless steel wire retainers.

Cast Haas-type RME appliance: A case report.

Rapid maxillary expansion (RME) in the initial stages of occlusal development has become a routine procedure in orthodontic practice. The increase of the transverse dimensions of the maxilla in the mixed dentition can be carried out by a rapid palatal expander that exploits primary teeth as anchorage to minimize any negative effects on permanent teeth. This case report demonstrates the use of a modified Haas-type RME appliance in a Caucasian girl 7 years 6 months of age with a maxillary transverse deficiency, unilateral crossbite, dental midline deviation, and maxillary anterior dental crowding. The patient was treated with a modified Haas-type RME appliance composed of a six-band metal-cast structure with a partial occlusal covering that was bonded to the primary teeth using glass-ionomer cement. Clinicians see advantages in terms of speed of application and patient compliance by taking a single impression. Stability and retention of the appliance improve thanks to custom-made metal casting and the risks of decementation minimize via the use of glass-ionomer cement. Moreover, the fabrication in inert titanium, hypoallergenic resin, and laser soldering means patients with allergies can use it. The results demonstrate that the expansion carried out on primary teeth is followed by permanent molars and remains stable.