Effect Of Mechanical Stress Research Articles

Competing effects of temperature and mechanical stress on polar vortex transition in oxide superlattices

Competing effects of temperature and mechanical stress on polar vortex transition in oxide superlattices

Vitreous tissue cryopreservation using a blood vessel model and cryomacroscopy for scale-up studies: Observations and mathematical modeling

Successful long-term cryobanking of multicellular tissues and organs at deep subzero temperatures calls for the avoidance of ice cryoinjury by reliance upon ice-free cryopreservation techniques. However, the quality of the cryopreserved material is the direct result of its ability to survive a host of harmful mechanisms, chief among which is overcoming the trifecta effects of ice crystallization, toxicity, and mechanical stress. This study aims at exploring improved conditions to scale-up ice-free cryopreservation by combining DP6 as a base cryoprotective agent (CPA) solution with an array of synthetic ice modulators (SIMs). This study is conducted by integrating cryomacroscopy techniques, thermal modeling, solid mechanics analysis, and viability and contractility investigation to correlate physical effects, thermal outcomes, and cryobiology results. As an extension of previous work, this study aims at scale-up of established baseline blood vessel models, while comparing the relative toxicity and vitreous stability of 4 ml and 10 ml samples of DP6 containing either sucrose as a SIM, or the commercial synthetic ice blockers (X1000 and Z1000). Using that established protocol, the addition and removal of DP6+0.6M sucrose and DP6 + 1% X1000 + 1% Z1000 were both well tolerated in rabbit carotid and pig femoral artery models, when assessed for metabolic recovery and contractility. Using cryomacroscopy, it was demonstrated that DP6 + 0.6M sucrose provided a stable vitrification medium under marginal cooling and warming conditions that resulted in >50% survival rate. By contrast, DP6 + 1% X1000 + 1% Z1000 was subject to visible ice formation during cooling under the same thermal conditions, resulting in a significantly lower recovery of ∼20%. Thermal modeling is used in this study to verify the actual cooling and rewarming rates in the specimens, while thermomechanics analysis is used to explain why fractures were observed using cryomacroscopy when the specimens were contained in glass vials but not in plastic vials.

Investigating N6-methyladenosin RNA modification as a mediator of microRNA mechanical regulation in the heart

Abstract Background The heart is exposed to mechanical forces and continuously adapts to its environment in a process known as cardiac remodeling. Excessive mechanical load is a primary driver of pathological cardiac remodeling, leading to heart failure (HF), a prominent cause of morbidity and mortality worldwide. A set of microRNAs (miRs), termed mechano-miRs, has been identified to respond to mechanical forces and contribute to heart pathophysiology and HF. N6-methyladenosine (m6A) RNA modification is emerging as a novel regulatory layer impacting gene expression and occurring both in coding and non-coding RNAs. In this context, the N6-adenosine-methyltransferase METTL-3 regulates the processing of miR primary transcripts (pri-miRs) to functional miRs, promoting angiogenesis and cardiac repair after myocardial infarction. On the other side, dysregulation of m6A due to a defect in FTO, a m6A demethylase, has been implicated in HF. However, the role of m6A RNA modifications in heart mechanical responses and mechano-miR regulation remains unexplored. Purpose This project aims to: 1) investigate the impact of mechanical overload on the expression of m6A regulatory machinery components; 2) unveil the role of m6A-mechano-miRs axis in mechanically induced responses driving pathological cardiac remodeling leading to heart failure. Methods and Results Using living myocardial slices (LMS) technology, we modulate mechanical load in a multicellular environment while maintaining physiological behavior. Human LMS, obtained from the left ventricle of human donor non-failing hearts (NHS Blood and Transplant INOAR program, IRAS project ID: 189069) and rat LMS are subjected to two degrees of mechanical preload: physiological sarcomere length (SL) (SL = 2.2 μm) and overload (SL = 2.4 μm). This enables us to mimic the effects of mechanical stress on the heart during HF (volume overload model). LMS are maintained for 48 hours under electrical stimulation in circulating oxygenated media at 37°C. Mechanically overloaded LMS exhibit decreased contractility, accompanied by an increase in global m6A levels compared to physiological controls. Consistent with this finding, the analysis of the expression of m6A machinery reveals a downregulation of m6A demethylases (FTO and ALKBH5) in overloaded LMS. We identified putative cardiac mechano-miRs by small-RNA sequencing in both human and rat LMS and next predicted potential m6A sites on their primary transcripts via SRAMP (sequence-based RNA adenosine methylation site predictor). Conclusions Mechanical overload affects global m6A levels and the expression of m6A demethylases in the heart. Ongoing analysis of the m6A profile on the identified mechano-miR primary transcripts and mRNA targets holds promise for unveiling novel mechanically regulated m6A events, providing insights for transformative therapeutic strategies.

Effects of Stress on Biological Characteristics and Metabolism of Periodontal Ligament Stem Cells of Deciduous Teeth

Introduction and AimsPeriodontal ligament stem cells (PDLSCs) from deciduous teeth (DePDLSCs) can perceive and respond to mechanical signals upon exposure to various environments. The effects of mechanical stress on the biological characteristics and metabolism of DePDLSCs were investigated using in vitro stress loading. MethodsDePDLSCs were subjected to mechanical stresses of different strengths. Cell proliferation, expression of osteogenic/osteoclastic factors, apoptosis, and oxidative stress levels were evaluated using CCK-8 assays, alkaline phosphatase staining, real-time PCR, flow cytometry, and malondialdehyde and superoxide dismutase assays. Liquid chromatography-mass spectrometry was used to perform nontargeted metabolomic detection and analysis. ResultsUnder stresses of 75 and 150 kPa, the expression of osteogenesis-related factors OPG, ALP, and RUNX2 decreased, and the ratio of RANKL/OPG significantly increased. A pressure of 150 kPa induced oxidative stress and caused a significant increase in cell apoptosis. Among the differential metabolites screened from the 150 kPa group, spermine, spermidine, ceramide, phosphatidylethanolamine, lysophosphatidylethanolamine, linoleic acid, and docosatrienoic acid were the most significantly upregulated. The metabolites screened from the 75 kPa group were mainly related to glycerophospholipid and sphingolipid metabolism, oxidative phosphorylation, and mineral absorption, which were common pathways affected in both experimental groups. ConclusionA certain degree of mechanical stress can inhibit the proliferative activity and osteogenic differentiation of DePDLSCs, enhance their osteoclast-inducing ability, and cause elevated levels of cell apoptosis and oxidative stress. The metabolic expression profile of DePDLSCs changed significantly under stress. Understanding changes in cellular activity and metabolic reactions may provide an experimental basis for elucidating the role of mechanical stress in root resorption and periodontal tissue remodelling of deciduous teeth. Clinical RelevanceMechanical stress may affect periodontal tissue remodeling and root resorption of DePDLSc.

Diversity and Composition of Posidonia oceanica-Associated Bacterial and Fungal Communities: Effect of Boat-Induced Mechanical Stress in the Villefranche-sur-Mer Bay (France)

The anchoring and mooring of boats mechanically damage Posidonia oceanica plants; however, no information is available on the effect of this kind of damage on the plant holobiont, i.e., on the associated bacterial and fungal communities. Indeed, bacterial communities are known to change under different plant stress conditions but the dynamics of seagrass-associated fungi remain largely unexplored. We used DNA metabarcoding to profile the bacterial and fungal colonizers of two nearby P. oceanica patches in the Villefranche-sur-Mer bay (France) differing by the amount of exposure to mechanical stress due to boat transit and anchoring. Bacterial communities showed a significant reduction in diversity with an increase in Vibrio sp. in the rhizome and root samples from the impacted site, where the accumulation of dead organic material favors opportunistic heterotrophs. Conversely, fungal communities showed increased diversity in the leaf samples from the impacted site, where a reduction in the dominant P. oceanica host-specific mutualistic endosymbiont, Posidoniomyces atricolor, was found. This change was probably due to the opening up of new colonizable niches for several fungal species. Although this study represents a preliminary assessment of the effect of mechanical stresses on P. oceanica-associated microbial communities, it further supports their putative use as a seagrass descriptor.

The effect of residual mechanical stresses and vacancy defects on the diffusion expansion of the damaged layer during irradiation of BeO ceramics

The paper presents the results of a study on the application of Raman and UV spectroscopy methods to determine the structural damage kinetics in the near-surface layer of BeO ceramics caused by high-dose irradiation with He2+ ions. Interest in this type of ceramics is due to the combination of its structural and thermophysical parameters, making these ceramics one of the promising classes of materials for microelectronics and structural materials for nuclear reactors, with the possibility of operation in conditions of heightened radiation background. According to the conducted studies, it was established that with the irradiation fluence growth, changes in the nature of deformation structural distortions associated with the accumulation of residual mechanical stresses of tensile and compressive types are observed. At irradiation fluences of 1016-5×1016 Не2+/cm2, tensile stresses play a dominant role in structural distortions, while the value of compressive stresses at fluence growth makes up a small share in the overall nature of the deformations. Moreover, an elevation in the irradiation fluence above 5×1016 He2+/cm2 leads to a rise in the concentration of defects caused by the formation of oxygen vacancies, as well as He-VO type complexes, the presence of which is indicated by the halo intensity growth in the Raman spectra, as well as a change in the intensity of the absorption bands. Analysis of changes in thermophysical parameters revealed that a rise in structural distortions associated with the accumulation of complex defects results in thermal conductivity reduction and a deterioration in heat transfer processes associated with partial amorphization of the damaged layer. Moreover, the established direct relationship between the value of residual mechanical stresses and the degradation of thermal conductivity indicates the cumulative effect of destructive changes caused by irradiation, as well as the influence of diffusion mechanisms on the damaged layer thickness growth.

Effects of carbon nanotubes on the isotropic to nematic phase transition in nematic liquid crystal elastomer

The isotropic to nematic phase transition on nematic liquid crystal elastomer - carbon nanotubes composites has been studied theoretically. The model free energy density has been constructed using Flory Huggins theory of isotropic mixing and Landau-de Gennes theory. The effects of carbon nanotubes and external mechanical stress on the isotropic to nematic phase transition of the nematic liquid crystal elastomer-carbon nanotubes composites have been discussed. The volume fraction of carbon nanotubes dependence of the isotropic to nematic transition temperature, mechanical strain and strain birefringence has been calculated. The weak first-order character of the isotropic to nematic phase transition on nematic liquid crystal elastomer-carbon nanotubes composites has been predicted.

The effect of uniaxial mechanical stress on electronic and dynamic properties of silicon

The effect of uniaxial mechanical stress on electronic and dynamic properties of silicon

Synergistic effects of bacteria, enzymes, and cyclic mechanical stresses on the bond strength of composite restorations

Predicting how tooth and dental material bonds perform in the mouth requires a deep understanding of degrading factors. Yet, this understanding is incomplete, leading to significant uncertainties in designing and evaluating new dental adhesives. The durability of dental bonding interfaces in the oral microenvironment is compromised by bacterial acids, salivary enzymes, and masticatory fatigue. These factors degrade the bond between dental resins and tooth surfaces, making the strength of these bonds difficult to predict. Traditionally studied separately, a combined kinetic analysis of these interactions could enhance our understanding and improvement of dental adhesive durability. To address this issue, we developed and validated an original model to evaluate the bond strength of dental restorations using realistic environments that consider the different mechanical, chemical, and biological degradative challenges working simultaneously: bacteria, salivary esterases, and cyclic loading. We herein describe a comprehensive investigation on dissociating the factors that degrade the bond strength of dental restorations. Our results showed that cariogenic bacteria are the number one factor contributing to the degradation of the bonded interface, followed by cyclic loading and salivary esterases. When tested in combinatorial mode, negative and positive synergies towards the degradation of the interface were observed. Masticatory loads (i.e., cycling loading) enhanced the lactic acid bacterial production and the area occupied by the biofilm at the bonding interface, resulting in more damage at the interface and a reduction of 73 % in bond strength compared to no-degraded samples. Salivary enzymes also produced bond degradation caused by changes in the chemical composition of the resin/adhesive. However, the degradation rates are slowed compared to the bacteria and cyclic loading. These results demonstrate that our synergetic model could guide the design of new dental adhesives for biological applications without laborious trial-and-error experimentation.

Physical aspects of epithelial cell-cell interactions: hidden system complexities.

The maintenance of homeostasis and the retention of ordered epithelial cell self-organization are essential for morphogenesis, wound healing, and the spread of cancer across the epithelium. However, cell-cell interactions in an overcrowded environment introduce a diversity of complications. Such interactions arise from an interplay between the cell compressive and shear stress components that accompany increased cell packing density. They can lead to various kinds of cell rearrangement such as: the epithelial-to-mesenchymal cell state transition; live cell extrusion; and cell jamming. All of these scenarios of cell rearrangement under mechanical stress relate to changes in the strengths of the cell-cell and cell-matrix adhesion contacts. The objective of this review study is twofold: first, to provide a comprehensive summary of the biological and physical factors influencing the effects of cell mechanical stress on cell-cell interactions, and the consequences of these interactions for the status of cell-cell and cell-matrix adhesion contacts; and secondly, to offer a bio-physical/mathematical analysis of the aforementioned biological aspects. By presenting these two approaches in conjunction, we seek to highlight the intricate nature of biological systems, which manifests in the form of complex bio-physical/mathematical equations. Furthermore, the juxtaposition of these apparently disparate approaches underscores the importance of conducting experiments to determine the multitude of parameters that contribute to the development of these intricate bio-physical/mathematical models.

Comparison of fatigue characteristics and failure analysis of Ni-based superalloy subjected to thermomechanical fatigue under different phase conditions

In this research, the thermomechanical fatigue (TMF) characteristics of a second-generation Ni-based single-crystal superalloy were investigated. The combined effects of temperature cycling and mechanical stress were examined using a strain-controlled approach under different TMF testing conditions. The results indicated that cyclic softening occurred during the early stages during in-phase (IP) loading. In contrast, out-of-phase (OP) tests exhibited cyclic hardening, as evidenced by an increase in the stress range, which intensified at higher mechanical strain amplitudes. This resulted in a shorter fatigue life compared to the IP TMF case. The crack distribution patterns and damage mechanisms on the cross-sectional and fracture surfaces of the failed specimens were analyzed. In the OP TMF tests, oxidation-assisted cracking was identified as the primary damage mechanism. The dominance of oxide layers was a key distinguishing feature. Additionally, twinning-related deformation and topologically close-packed (TCP) phase particles were observed in specimens subjected to a higher mechanical strain of 0.6%, where they ran parallel to the main crack, contributing to a reduction in lifetime. Conversely, the IP TMF cases were mainly dominated by creep damage mechanisms, with some signs of oxidation penetration observed during testing. The application of the Ostergren model to the fatigue life prediction of a Ni-based single-crystal superalloy under both conditions was evaluated. As this model was insufficient, a modified Ostergren model was proposed by incorporating the maximum and amplitude stresses in the power law forms.

Understanding the Structure-Function Relationship through 3D Imaging and Biomechanical Analysis: A Novel Methodological Approach Applied to Anterior Cruciate Ligaments.

Understanding the microstructure of fibrous tissues, like ligaments, is crucial due to their nonlinear stress-strain behavior from unique fiber arrangements. This study introduces a new method to analyze the relationship between the microstructure and function of anterior cruciate ligaments (ACL). We tested the procedure on two ACL samples, one from a healthy individual and one from an osteoarthritis patient, using a custom tensioning device within a micro-CT scanner. The samples were stretched and scanned at various strain levels (namely 0%, 1%, 2%, 3%, 4%, 6%, 8%) to observe the effects of mechanical stress on the microstructure. The micro-CT images were processed to identify and map fibers, assessing their orientations and volume fractions. A probabilistic mathematical model was then proposed to relate the geometric and structural characteristics of the ACL to its mechanical properties, considering fiber orientation and thickness. Our feasibility test indicated differences in mechanical behavior, fiber orientation, and volume distribution between ligaments of different origins. These indicative results align with existing literature, validating the proposed methodology. However, further research is needed to confirm these preliminary observations. Overall, our comprehensive methodology shows promise for improving ACL diagnosis and treatment and for guiding the creation of tissue-engineered grafts that mimic the natural properties and microstructure of healthy tissue, thereby enhancing integration and performance in biomedical applications.

HIF-1α–dependent regulation of angiogenic factor expression in Müller cells by mechanical stimulation

Mechanical stress regulates various biological processes in cells, tissues, and organs as well as contributes to the pathogenesis of various diseases. The retina is subjected to mechanical stress imposed by intraocular pressure as well as by retinal hemorrhage and edema. Responses to mechanical stress have been studied in retinal pigment epithelial cells and Müller cells of the retina, with the former cells having been found to undergo a stress-induced increase in the expression of vascular endothelial growth factor (VEGF), which plays a key role in physiological and pathological angiogenesis in the retina. We here examined the effects of stretch stimulation on the expression of angiogenic factors in cultured human Müller cells. Reverse transcription and quantitative PCR analysis revealed that expression of the VEGF-A gene was increased by such stimulation in Müller cells, whereas that of the angiopoietin 1 gene was decreased. An enzyme-linked immunosorbent assay showed that stretch stimulation also increased VEGF secretion from these cells. Expression of the transcription factor HIF-1α (hypoxia-inducible factor–1α) was increased at both mRNA and protein levels by stretch stimulation, and the HIF-1α inhibitor CAY10585 prevented the effects of mechanical stress on VEGF-A gene expression and VEGF secretion. Furthermore, RNA-sequencing analysis showed that the expression of angiogenesis-related pathway genes was upregulated by stretch stimulation. Our results thus suggest that mechanical stress induces VEGF production in Müller cells in a manner dependent on HIF-1α, and that HIF-1α is therefore a potential therapeutic target for conditions such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.

Analytical characterization of damage in reverse osmosis membranes caused by components of a chlorine dioxide matrix

Reverse osmosis (RO), an important membrane technology for producing process and tap water, faces challenges due to biofilms formed by viable microorganisms, which significantly impair membrane performance. Disinfection is commonly employed to suppress biofilm formation but can also damage the crucial selective upper layer of the membrane. The present study aims to improve and compare analytical methods for detecting membrane damage to aid the design of targeted disinfection approaches that do not compromise RO efficiency. To achieve this, the accompanying byproducts of in-situ formed chlorine dioxide are analyzed individually with regards to their harmfulness for RO membranes. Combined insights from ATR-FTIR investigations, the methylene blue (MB) dye test, and XPS measurements enable a systematic assessment of the chemical constituents in the chlorine dioxide, ranking their harmfulness as: chlorite < persulfate < purified chlorine dioxide < free chlorine. Further investigations reveal that mechanical stress in the form during disinfection significantly accelerates membrane damage during disinfection. The results of this study pave the way for improved RO processes that account for the effects of chemical byproducts and mechanical stresses to balance disinfection efficacy and membrane integrity for environmentally sustainable water purification solutions.

An enhanced Energy-Based hysteresis model for electrical steel sheets considering mechanical stress

Mechanical stress significantly influences the hysteresis characteristics of silicon steel, which are crucial for the design of electrical equipment cores. This study develops an improved Energy-based hysteresis model that integrates the effects of mechanical stress by modifying the anhysteretic magnetization model to include total average anisotropy energy density and introducing a linear relationship between coercive force and mechanical stress. The model’s performance is demonstrated using 30QG120 oriented silicon steel sheets, where the hysteresis loops under varying stress conditions are successfully simulated and validated against experimental data. The results confirm the model’s accuracy and practical utility in predicting material behavior under mechanical stress, offering a valuable tool for optimizing electrical steel sheet design.

Nonsurgical therapy for lumbar spinal stenosis caused by ligamentum flavum hypertrophy: A review.

Lumbar spinal stenosis (LSS) can cause a range of cauda equina symptoms, including lower back and leg pain, numbness, and intermittent claudication. This disease affects approximately 103 million people worldwide, particularly the elderly, and can seriously compromise their health and well-being. Ligamentum flavum hypertrophy (LFH) is one of the main contributing factors to this disease. Surgical treatment is currently recommended for LSS caused by LFH. For patients who do not meet the criteria for surgery, symptom relief can be achieved by using oral nonsteroidal anti-inflammatory drugs (NSAIDs) and epidural steroid injections. Exercise therapy and needle knife can also help to reduce the effects of mechanical stress. However, the effectiveness of these methods varies, and targeting the delay in LF hypertrophy is challenging. Therefore, further research and development of new drugs is necessary to address this issue. Several new drugs, including cyclopamine and N-acetyl-l-cysteine, are currently undergoing testing and may serve as new treatments for LSS caused by LFH.

Effects of mechanical stress, chemical potential, and coverage on hydrogen solubility during hydrogen-enhanced decohesion of ferritic steel grain boundaries: A first-principles study

Hydrogen-enhanced decohesion (HEDE) is one of the many mechanisms of hydrogen embrittlement, a phenomenon that severely impacts structural materials such as iron and iron alloys. Grain boundaries (GBs) play a critical role in this mechanism, where they can provide trapping sites or act as hydrogen diffusion pathways. The interaction of H with GBs and other crystallographic defects, and thus the solubility and distribution of H in the microstructure, depends on the concentration, chemical potential, and local stress. Therefore, for a quantitative assessment of HEDE, a generalized solution energy in conjunction with the cohesive strength as a function of hydrogen coverage is needed. In this paper, we carry out density functional theory calculations to investigate the influence of H on the decohesion of the Σ5(310)[001] and Σ3(112)[11¯0] symmetrical tilt GBs in bcc Fe, as examples for open and close-packed GB structures. A method to identify the segregation sites at the GB plane is proposed. The results indicate that at higher local concentrations, H leads to a significant reduction of the cohesive strength of the GB planes, significantly more pronounced at the Σ5 than at the Σ3 GB. Interestingly, at finite stress, the Σ3 GB becomes more favorable for H solution, as opposed to the case of zero stress, where the Σ5 GB is more attractive. This suggests that, under certain conditions, stresses in the microstructure can lead to a redistribution of H to the stronger grain boundary, which opens a path to designing H-resistant microstructures. To round up our study, we investigate the effects of typical alloying elements in ferritic steel, C, V, Cr, and Mn, on the solubility of H and the strength of the GBs. Published by the American Physical Society 2024

Mechanical and surface characteristics in laser-assisted machining of NiFeCr alloy: An atomic-scale understanding

This investigation explores mechanical characteristics and subsurface damage of NiFeCr alloy in laser-assisted machining through molecular dynamics simulation. The results of machining force, friction coefficient, local strain/stress, temperature distribution, subsurface damage, surface morphology, and worn volume are comprehensively explored, thereby revealing the mechanism of laser-assisted machining under different working conditions. Compared to conventional processing, average machining force, shear strain, subsurface damage, and dislocated length are all reduced in laser-assisted machining, which improves surface finish quality. Amorphization of removed chips in laser-assisted machining enhances the material removal efficiency. Analysis of various laser powers shows a decreased machining force with increasing laser power. However, residual stress and temperature rise, potentially impacting surface integrity. The subsurface damage depth exhibits nonlinearity with increasing laser energy, highlighting the competition between mechanical stress and thermal effect. The surface morphology and atomic flow demonstrate effective material removal with increasing laser power. The average machining force, friction coefficient, and subsurface damage depth show nonlinear relationships with cutting speed. The number of worn atoms is proportional to machining length and cutting speed, indicating enhanced material removal during high-speed machining.

Can repetitive mechanical motion cause structural damage to axons?

Biological structures have evolved to very efficiently generate, transmit, and withstand mechanical forces. These biological examples have inspired mechanical engineers for centuries and led to the development of critical insights and concepts. However, progress in mechanical engineering also raises new questions about biological structures. The past decades have seen the increasing study of failure of engineered structures due to repetitive loading, and its origin in processes such as materials fatigue. Repetitive loading is also experienced by some neurons, for example in the peripheral nervous system. This perspective, after briefly introducing the engineering concept of mechanical fatigue, aims to discuss the potential effects based on our knowledge of cellular responses to mechanical stresses. A particular focus of our discussion are the effects of mechanical stress on axons and their cytoskeletal structures. Furthermore, we highlight the difficulty of imaging these structures and the promise of new microscopy techniques. The identification of repair mechanisms and paradigms underlying long-term stability is an exciting and emerging topic in biology as well as a potential source of inspiration for engineers.

Germination of &lt;I&gt;Cassia fistula&lt;/I&gt; L. Seeds by Mechanical Stress

The effect of mechanical stress in presence of different kinds of chemical media on seed germination, percentage of germination and rate of imbibition in seeds of Cassia fistula L. have been studied. A unique germination percentage of seedlings free-from fungus attack were observed in cracked and punctured seeds. Modifications raised by this stress in seeds enhances quick germination in presence of suitable moisture media. To make comparative study between the rate of imbibition and percentage of germination and to know the susceptibility of seeds towards fungal attack, heating and roasting in addition to cracking and puncturing, were applied and discussed.