High-resolution temporal transcriptome sequencing unravels ERF and WRKY as the master players in the regulatory networks underlying sesame responses to waterlogging and recovery.

Major crops are generally sensitive to waterlogging, but our limited understanding of the waterlogging gene regulatory network hinders the efforts to develop waterlogging-tolerant cultivars. We generated high-resolution temporal transcriptome data from root of two contrasting sesame genotypes over a 48 h period waterlogging and drainage treatments. Three distinct chronological transcriptional phases were identified, including the early-waterlogging, late-waterlogging and drainage responses. We identified 47 genes representing the core waterlogging-responsive genes. Waterlogging/drainage-induced transcriptional changes were mainly driven by ERF and WRKY transcription factors (TF). The major difference between the two genotypes resides in the early transcriptional phase. A chronological transcriptional network model predicting putative causal regulations between TFs and downstream waterlogging-responsive genes was constructed and some interactions were validated through yeast one-hybrid assay. Overall, this study unveils the architecture and dynamic regulation of the waterlogging/drainage response in a non-model crop and helps formulate new hypotheses on stress sensing, signaling and sophisticated adaptive responses.

Root diversity in sesame (Sesamum indicum L.): insights into the morphological, anatomical and gene expression profiles.

MAIN CONCLUSION: Sesame harbors a large diversity in root morphological and anatomical traits and a high root biomass improves the plant aboveground biomass as well as the seed yield. Sesame provides one of the most nutritious and healthy vegetable oils, sparking an increasing demand of its seeds. However, with the low yield and productivity of sesame, there is still a huge gap between the seed demand and supply. Improving the root system has a high potential to increase crop productivity, but information on the diversity of the sesame root systems is still lacking. In this study, 40 diverse sesame varieties were grown in soil and hydroponics systems and the diversity of the root system was investigated. The results showed that sesame holds a large root morphological and anatomical diversity, which can be harnessed in breeding programmes. Based on the clustering of the genotypes in hydroponics and soil culture systems, we found that similar genotypes were commonly clustered either in the small-root or in the big-root group, indicating that the hydroponics system can be employed for a large-scale root phenotyping. Our results further revealed that the root biomass positively contributes to increased seed yield in sesame, based on multi-environmental trials. By comparing the root transcriptome of two contrasting genotypes, 2897 differentially expressed genes were detected and they were enriched in phenylpropanoid biosynthesis, starch and sucrose metabolism, stilbenoid, diarylheptanoid and gingerol biosynthesis, flavonoid biosynthesis, suggesting that these pathways are crucial for sesame root growth and development. Overall, this study sheds light on the diversity of sesame root system and offers the basis for improving root traits and increasing sesame seed yield.

Advanced Time-Stepping Interpretation of Fly-Scan Continuous Rotation Synchrotron Tomography of Dental Enamel Demineralization.

High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 µm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials.

The DIAD Approach to Correlative Synchrotron X-ray Imaging and Diffraction Analysis of Human Enamel.

The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source (Didcot, U.K.) implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales. Namely, the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging (radiography and tomography) and reciprocal space scattering (diffraction). The switching between the two principal modes is made smooth and rapid by design, so that the data collected is interlaced to obtain near-simultaneous multimodal characterization. Different specific photon energies are used for each mode, and the interlacing of acquisition steps allows conducting static and dynamic experiments. Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice, namely, the methods for gauge volume collocation under different modes of beam-sample interaction. To address this challenge, experiments were conducted at DIAD devoted to the study of human dental enamel, a hierarchical structure composed of hydroxyapatite mineral nanocrystals, as a static sample previously affected by dental caries (tooth decay) as well as under dynamic conditions simulating the process of acid demineralization. Collocation and correlation were achieved between WAXS (wide-angle X-ray scattering), 2D (radiographic), and 3D (tomographic) imaging. While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure, X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation (texture). Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities. Diffraction gauge volumes were identified and visualized within the tomographic data sets, revealing the underlying local information to support the interpretation of the diffraction patterns. The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.

Synchrotron X-ray Studies of the Structural and Functional Hierarchies in Mineralised Human Dental Enamel: A State-of-the-Art Review.

Hard dental tissues possess a complex hierarchical structure that is particularly evident in enamel, the most mineralised substance in the human body. Its complex and interlinked organisation at the Ångstrom (crystal lattice), nano-, micro-, and macro-scales is the result of evolutionary optimisation for mechanical and functional performance: hardness and stiffness, fracture toughness, thermal, and chemical resistance. Understanding the physical-chemical-structural relationships at each scale requires the application of appropriately sensitive and resolving probes. Synchrotron X-ray techniques offer the possibility to progress significantly beyond the capabilities of conventional laboratory instruments, i.e., X-ray diffractometers, and electron and atomic force microscopes. The last few decades have witnessed the accumulation of results obtained from X-ray scattering (diffraction), spectroscopy (including polarisation analysis), and imaging (including ptychography and tomography). The current article presents a multi-disciplinary review of nearly 40 years of discoveries and advancements, primarily pertaining to the study of enamel and its demineralisation (caries), but also linked to the investigations of other mineralised tissues such as dentine, bone, etc. The modelling approaches informed by these observations are also overviewed. The strategic aim of the present review was to identify and evaluate prospective avenues for analysing dental tissues and developing treatments and prophylaxis for improved dental health.

Time-Lapse In Situ 3D Imaging Analysis of Human Enamel Demineralisation Using X-ray Synchrotron Tomography.

Caries is a chronic disease that causes the alteration of the structure of dental tissues by acid dissolution (in enamel, dentine and cementum) and proteolytic degradation (dentine and cementum) and generates an important cost of care. There is a need to visualise and characterise the acid dissolution process on enamel due to its hierarchical structure leading to complex structural modifications. The process starts at the enamel surface and progresses into depth, which necessitates the study of the internal enamel structure. Artificial demineralisation is usually employed to simulate the process experimentally. In the present study, the demineralisation of human enamel was studied using surface analysis carried out with atomic force microscopy as well as 3D internal analysis using synchrotron X-ray tomography during acid exposure with repeated scans to generate a time-lapse visualisation sequence. Two-dimensional analysis from projections and virtual slices and 3D analysis of the enamel mass provided details of tissue changes at the level of the rods and inter-rod substance. In addition to the visualisation of structural modifications, the rate of dissolution was determined, which demonstrated the feasibility and usefulness of these techniques. The temporal analysis of enamel demineralisation is not limited to dissolution and can be applied to other experimental conditions for the analysis of treated enamel or remineralisation.

Multi-resolution Correlative Ultrastructural and Chemical Analysis of Carious Enamel by Scanning Microscopy and Tomographic Imaging.

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.

Development and characterisation of dental composites containing anisotropic fluorapatite bundles and rods.

OBJECTIVES: To develop dental composites incorporating fluorapatite (FA) crystals as a secondary filler and to characterise degree of conversion, key mechanical properties and fluoride release. METHODS: FA rod-like crystals and bundles were hydrothermally synthesised and characterised by scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS), X-ray diffraction (XRD) and 19F MAS-NMR. Composites were formulated containing BisGMA/TEGDMA/BisEMA and barium-aluminium-silicate glass (0FA). FA crystals were incorporated at 10 (10FA), 20 (20FA), 30 (30FA) and 40wt% (40FA) maintaining a filler content of 80wt% (63-67vol%). Degree of conversion (DC), flexural strength (FS), flexural modulus (FM), fracture toughness (K1C), Vickers hardness (HV) and 2-body wear were measured. Fluoride release was measured in neutral and acidic buffers. RESULTS: XRD and 19F MAS-NMR confirmed that only FA was formed, whilst SEM revealed the presence of single rods and bundles of nano-rods. DC ranged between 56-60% (p>0.05). FA composites showed lower FM and lower FS (p<0.05), but comparable wear resistance and HV (p>0.05) to 0FA. 30FA and 40FA showed similar K1C to 0FA (p>0.05), with SEM showing evidence of toughening mechanisms, whereas 10FA and 20FA showed lower K1C (p<0.05). FA containing composites released fluoride that was proportional to the amount of FA incorporated (p<0.05) but only under acidic conditions. SIGNIFICANCE: The addition of FA to the experimental composites reduced strength and stiffness but not the DC, hardness or wear rate. 30FA and 40FA had a higher K1C compared to other FA groups. Fluoride release occurred under an accelerated acidic regime, suggesting potential as a bioactive 'smart' composite.

Deformation and retentive force following in vitro cyclic fatigue of cobalt-chrome and aryl ketone polymer (AKP) clasps.

OBJECTIVE: To compare the retention force of individual clasps made from cobalt chromium (CoCr) or new aryl ketone polymer (AKP) material, Ultaire™ AKP, following prolonged fatigue testing along ideal and non-ideal paths of removal and to assess 3D deformation of the active and passive clasp tips. METHODS: CoCr and AKP clasps were manufactured in their standard, respective processes, digitally scanned prior to testing, then cycled 15,000 times over an e.max analogue crown in artificial saliva. Retentive load was measured in situ, as a function of cycles. Clasps were rescanned to assess deformation and along with their antagonists subjected to SEM to assess localised wear. RESULTS: Distortion of the CoCr clasps was consistently larger than Ultaire™ AKP clasps, irrespective of removal path. CoCr clasps had significantly higher retentive forces than AKP clasps, for both removal paths. Ultaire™ AKP clasps showed a lower but relatively constant retentive force. The non-ideal path of removal affected retentive forces for both clasp materials. SEM showed localised removal of glaze for e.max crowns used with CoCr clasps. SIGNIFICANCE: Ultaire™ AKP clasps showed significantly less permanent deformation and lower retentive force than CoCr clasps. Unlike CoCr, the Ultaire™ AKP clasps did not work harden, nor had as large a reduction in retentive force and accompanying permanent deformation; the retentive force for the Ultaire™ AKP clasps was consistent over 15,000 cycles of fatigue mimicking prolonged clinical use. The AKP material was more robust; showing minimal deformation even in non-ideal paths of removal, as many patients would routinely use.

The contrasting response to drought and waterlogging is underpinned by divergent DNA methylation programs associated with transcript accumulation in sesame.

DNA methylation is a heritable epigenetic mechanism that participates in gene regulation under abiotic stresses in plants. Sesame (Sesamum indicum) is typically considered a drought-tolerant crop but highly susceptible to waterlogging, probably because of its origin in Africa or India. Understanding DNA methylation patterns under drought and waterlogging conditions can provide insights into the regulatory mechanisms underlying sesame contrasting responses to these abiotic stresses. We combined Methylation-Sensitive Amplified Polymorphism and transcriptome analyses to profile cytosine methylation patterns, transcript accumulation, and their interplay in drought-tolerant and waterlogging-tolerant sesame genotypes. Drought stress strongly induced de novo methylation (DNM) whereas most of the loci were demethylated (DM) during the recovery phase. In contrast, waterlogging stress decreased the level of methylation but during the recovery phase, both DM and DNM were concomitantly deployed. In both stresses, the levels of the differentially accumulated transcripts (DATs) highly correlated with the methylation patterns. We observed that DM was associated with an increase of DAT levels while DNM was correlated with a decrease of DAT levels. Altogether, sesame has divergent epigenetic programs that respond to drought and waterlogging stresses and an interplay among DNA methylation and transcript accumulation may partly modulate the contrasting responses to these stresses.

Identification and characterization of the bZIP transcription factor family and its expression in response to abiotic stresses in sesame.

Basic leucine zipper (bZIP) gene family is one of the largest transcription factor families in plants, and members of this family play important roles in multiple biological processes such as light signaling, seed maturation, flower development as well as abiotic and biotic stress responses. Nonetheless, genome-wide comprehensive analysis of the bZIP family is lacking in the important oil crop sesame. In the present study, 63 bZIP genes distributed on 14 linkage groups were identified in sesame, and denominated as SibZIP01-SibZIP63. Besides, all members of SibZIP family were divided into nine groups based on the phylogenetic relationship of Arabidopsis bZIPs, which was further supported by the analysis of their conserved motifs and gene structures. Promoter analysis showed that all SibZIP genes harbor cis-elements related to stress responsiveness in their promoter regions. Expression analyses of SibZIP genes based on transcriptome data showed that these genes have different expression patterns in different tissues. Additionally, we showed that a majority of SibZIPs (85.71%) exhibited significant transcriptional changes in responses to abiotic stresses, including drought, waterlogging, osmotic, salt, and cold, suggesting that SibZIPs may play a cardinal role in the regulation of stress responses in sesame. Together, these results provide new insights into stress-responsive SibZIP genes and pave the way for future studies of SibZIPs-mediated abiotic stress response in sesame.

Functional Characterization of the Versatile MYB Gene Family Uncovered Their Important Roles in Plant Development and Responses to Drought and Waterlogging in Sesame.

The MYB gene family constitutes one of the largest transcription factors (TFs) modulating various biological processes in plants. Although genome-wide analysis of this gene family has been carried out in some species, only three MYB members have been functionally characterized heretofore in sesame (Sesamum indicum L.). Here, we identified a relatively high number (287) of sesame MYB genes (SIMYBs) with an uncommon overrepresentation of the 1R-subfamily. A total of 95% of SIMYBs was mapped unevenly onto the 16 linkage groups of the sesame genome with 55 SIMYBs tandemly duplicated. In addition, molecular characterization, gene structure, and evolutionary relationships of SIMYBs were established. Based on the close relationship between sesame and Arabidopsis thaliana, we uncovered that the functions of SIMYBs are highly diverse. A total of 65% of SIMYBs were commonly detected in five tissues, suggesting that they represent key TFs modulating sesame growth and development. Moreover, we found that SIMYBs regulate sesame responses to drought and waterlogging, which highlights the potential of SIMYBs towards improving stress tolerance in sesame. This work presents a comprehensive picture of the MYB gene family in sesame and paves the way for further functional validation of the members of this versatile gene family.

Use of ventilator associated pneumonia bundle and statistical process control chart to decrease VAP rate in Syria.

OBJECTIVE: Implementation of ventilator associated pneumonia (VAP) bundle as a performance improvement project in the critical care units for all mechanically ventilated patients aiming to decrease the VAP rates. MATERIALS AND METHODS: VAP bundle was implemented in 4 teaching hospitals after educational sessions and compliance rates along with VAP rates were monitored using statistical process control charts. RESULTS: VAP bundle compliance rates were steadily increasing from 33 to 80% in hospital 1, from 33 to 86% in hospital 2 and from 83 to 100% in hospital 3 during the study period. The VAP bundle was not applied in hospital 4 therefore no data was available. A target level of 95% was reached only in hospital 3. This correlated with a decrease in VAP rates from 30 to 6.4 per 1000 ventilator days in hospital 1, from 12 to 4.9 per 1000 ventilator days in hospital 3, whereas VAP rate failed to decrease in hospital 2 (despite better compliance) and it remained high around 33 per 1000 ventilator days in hospital 4 where VAP bundle was not implemented. CONCLUSION: VAP bundle has performed differently in different hospitals in our study. Prevention of VAP requires a multidimensional strategy that includes strict infection control interventions, VAP bundle implementation, process and outcome surveillance and education.