Finite element modeling of α-helices and tropocollagen molecules referring to spike of SARS-CoV-2

The newly developed finite element (FE) modeling at the atomic scale was used to predict the static and dynamic response of the α-helix (AH) and tropocollagen (TC) protein fragments, the main building blocks of the spike of the SARS-CoV-2. The geometry and morphology of the spike’s stalk and its connection to the viral envelope were determined from the combination of most recent molecular dynamics (MD) simulation and images of cryoelectron microscopy. The stiffness parameters of the covalent bonds in the main chain of the helix were taken from the literature. The AH and TC were modeled using both beam elements (wire model) and shell elements (ribbon model) in FE analysis to predict their mechanical properties under tension. The asymptotic stiffening features of AH and TC under tensile loading were revealed and compared with a new analytical solution. The mechanical stiffnesses under other loading conditions, including compression, torsion, and bending, were also predicted numerically and correlated with the results of the existing MD simulations and tests. The mode shapes and natural frequencies of the spike were predicted using the built FE model. The frequencies were shown to be within the safe range of 1–20 MHz routinely used for medical imaging and diagnosis by means of ultrasound. These results provide a solid theoretical basis for using ultrasound to study damaging coronavirus through transient and resonant vibration at large deformations.     

Genetic dissection of days to flowering via genome-wide association studies in Turkish common bean germplasm

Common bean is a nutrient‐dense legume crop serving as a source of food for millions of people. Characterization of unexplored common bean germplasm to unlock the phenotypic and genetic variations is still needed to explore the breeding potential of this crop. The current study aimed to dissect the genetic basis having association for days to flowering (DF). A total of 188 common bean accessions collected from 19 provinces of Turkey were used as plant material under five environments and two locations. Analysis of variance (ANOVA) revealed that genotypes and genotype by environment interaction have significant effects on DF. A total of 10 most stable accessions were evaluated from stability analysis. Overall maximum (75) and minimum (54) DF were observed for Hakkari-51 and Mus-46 accessions, respectively. The implemented constellation plot divided studied germplasm according to their DF and growth habit. A total of 7900 DArTseq markers were used for association analysis. Mixed linear model using the Q + K Model resulted a total of 18 DArTseq markers from five environments. DArT-8668385 marker identified in Bolu during 2016 was also associated with DF in Sivas during 2017. Combined data of five years resulted a total of four markers (DArT-22346534, DArT-3369768, DArT-3374613, and DArT-3370801) having significant association ( p  <  0.01 ) for DF. DArT-22346534 present on Pv 08 accounted a maximum of 9.89% variation to the studied trait. A total of four putative candidate genes were predicted from sequences reflecting homology to identified four DArTseq markers. We envisage that exploitation of identified DArTseq markers will hopefully beneficial for the development of new common bean varieties having better adaptation ability to changing climatic conditions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01029-8.     

Experimental validation of a finite element model of a human cadaveric tibia

Finite element (FE) models of long bones are widely used to analyze implant designs. Experimental validation has been used to examine the accuracy of FE models of cadaveric femurs; however, although convergence tests have been carried out, no FE models of an intact and implanted human cadaveric tibia have been validated using a range of experimental loading conditions. The aim of the current study was to create FE models of a human cadaveric tibia, both intact and implanted with a unicompartmental knee replacement, and to validate the models against results obtained from a comprehensive set of experiments. Seventeen strain rosettes were attached to a human cadaveric tibia. Surface strains and displacements were measured under 17 loading conditions, which consisted of axial, torsional, and bending loads. The tibia was tested both before and after implantation of the knee replacement. FE models were created based on computed tomography (CT) scans of the cadaveric tibia. The models consisted of ten-node tetrahedral elements and used 600 material properties derived from the CT scans. The experiments were simulated on the models and the results compared to experimental results. Experimental strain measurements were highly repeatable and the measured stiffnesses compared well to published results. For the intact tibia under axial loading, the regression line through a plot of strains predicted by the FE model versus experimentally measured strains had a slope of 1.15, an intercept of 5.5 microstrain, and an R(2) value of 0.98. For the implanted tibia, the comparable regression line had a slope of 1.25, an intercept of 12.3 microstrain, and an R(2) value of 0.97. The root mean square errors were 6.0% and 8.8% for the intact and implanted models under axial loads, respectively. The model produced by the current study provides a tool for simulating mechanical test conditions on a human tibia. This has considerable value in reducing the costs of physical testing by pre-selecting the most appropriate test conditions or most favorable prosthetic designs for final mechanical testing. It can also be used to gain insight into the results of physical testing, by allowing the prediction of those variables difficult or impossible to measure directly.     

Laboratory evaluation of the rapid diagnostic tests for the detection of Vibrio cholerae O1 using diarrheal samples

BackgroundCholera, an acute diarrheal disease is a major public health problem in many developing countries. Several rapid diagnostic tests (RDT) are available for the detection of cholera, but their efficacies are not compared in an endemic setting. In this study, we have compared the specificity and sensitivity of three RDT kits for the detection of Vibrio cholerae O1 and compared their efficiency with culture and polymerase chain reaction (PCR) methods.MethodsFive hundred six diarrheal stool samples collected from patients from two different hospitals in Kolkata, India were tested using SD Bioline Cholera, SMART-II Cholera O1 and Crystal-VC RDT kits. All the stool samples were screened for the presence of V. cholerae by direct and enrichment culture methods. Stool DNA-based PCR assay was made to target the cholera toxin (ctxAB) and O1 somatic antigen (rfb) encoding genes. Statistical evaluation of the RDTs has been made using STATA software with stool culture and PCR results as the gold standards. The Bayesian latent class model (LCM) was used to evaluate the diagnostic tests in the absence of the gold standard.ResultsInvolving culture technique as gold standard, the sensitivity and specificity of the cholera RDT kits in the direct testing of stools was highest with SAMRT-II (86.1%) and SD-Cholera (94.4%), respectively. The DNA based PCR assays gave very high sensitivity (98.4%) but the specificity was comparatively low (75.3%). After enrichment, the high sensitivity and specificity was detected with SAMRT-II (78.8%) and SD-Cholera (99.1%), respectively. Considering PCR as the gold standard, the sensitivity and specificity of the RDTs remained between 52.3–58.2% and 92.3–96.8%, respectively. In the LCM, the sensitivity of direct and enrichment testing was high in SAMRT-II (88% and 92%, respectively), but the specificity was high in SD cholera for both the methods (97% and 100%, respectively). The sensitivity/specificity of RDTs and direct culture have also been analyzed considering the age, gender and diarrheal disease severity of the patients.ConclusionOverall, the performance of the RDT kits remained almost similar in terms of specificity and sensitivity. Performance of PCR was superior to the antibody-based RDTs. The RTDs are very useful in identifying cholera cases during outbreak/epidemic situations and for making them as a point-of-care (POC) testing tool needs more improvement.     

Mechanical responses of the periodontal ligament based on an exponential hyperelastic model: a combined experimental and finite element method

The V–W exponential hyperelastic model is adopted to describe the instantaneous elastic response of the periodontal ligament (PDL). The general theoretical framework of constitutive modeling is described based on nonlinear continuum mechanics, and the elasticity tensor used to develop UMAT subroutine is formulated. Nanoindentation experiment is performed to characterize mechanical properties of an adult pig PDL specimen. Then the experiment is simulated by using the finite element (FE) analysis. Meanwhile, the optimized material parameters are identified by the inverse FE method. The good agreement between the simulated results and experimental data demonstrates that the V–W model is capable of describing the mechanical behavior of the PDL. Therefore, the model and its implementation into FE code are validated. By using the model, we simulate the tooth movement under orthodontic loading to predict the mechanical responses of the PDL. The results show that local concentrations of stress and strain in the PDL are found.     

Using a multi‐model ensemble approach to determine biodiversity hotspots with limited occurrence data in understudied areas: An example using freshwater mussels in México

Species distribution models (SDMs) are an increasingly important tool for conservation particularly for difficult‐to‐study locations and with understudied fauna. Our aims were to (1) use SDMs and ensemble SDMs to predict the distribution of freshwater mussels in the Pánuco River Basin in Central México; (2) determine habitat factors shaping freshwater mussel occurrence; and (3) use predicted occupancy across a range of taxa to identify freshwater mussel biodiversity hotspots to guide conservation and management. In the Pánuco River Basin, we modeled the distributions of 11 freshwater mussel species using an ensemble approach, wherein multiple SDM methodologies were combined to create a single ensemble map of predicted occupancy. A total of 621 species‐specific observations at 87 sites were used to create species‐specific ensembles. These predictive species ensembles were then combined to create local diversity hotspot maps. Precipitation during the warmest quarter, elevation, and mean temperature were consistently the most important discriminatory environmental variables among species, whereas land use had limited influence across all taxa. To the best of our knowledge, our study is the first freshwater mussel‐focused research to use an ensemble approach to determine species distribution and predict biodiversity hotspots. Our study can be used to guide not only current conservation efforts but also prioritize areas for future conservation and study.     

Spatially resolved free-energy contributions of native fold and molten-globule-like Crambin

The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol⁻¹ due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol⁻¹. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.     

Development and validation of a subject-specific finite element model of a human clavicle

This study aimed to develop and validate a finite element (FE) model of a human clavicle which can predict the structural response and bone fractures under both axial compression and anterior–posterior three-point bending loads. Quasi-static non-injurious axial compression and three-point bending tests were first conducted on a male clavicle followed by a dynamic three-point bending test to fracture. Then, two types of FE models of the clavicle were developed using bone material properties which were set to vary with the computed tomography image density of the bone. A volumetric solid FE model comprised solely of hexahedral elements was first developed. A solid-shell FE model was then created which modelled the trabecular bone as hexahedral elements and the cortical bone as quadrilateral shell elements. Finally, simulations were carried out using these models to evaluate the influence of variations in cortical thickness, mesh density, bone material properties and modelling approach on the biomechanical responses of the clavicle, compared with experimental data. The FE results indicate that the inclusion of density-based bone material properties can provide a more accurate reproduction of the force–displacement response and bone fracture timing than a model with uniform bone material properties. Inclusion of a variable cortical thickness distribution also slightly improves the ability of the model to predict the experimental response. The methods developed in this study will be useful for creating subject-specific FE models to better understand the biomechanics and injury mechanism of the clavicle.     

A nonlinear finite element model validation study based on a novel experimental technique for inducing anterior wedge-shape fractures in human vertebral bodies in vitro

Vertebral compression fracture is a common medical problem in osteoporotic individuals. The quantitative computed tomography (QCT)-based finite element (FE) method may be used to predict vertebral strength in vivo, but needs to be validated with experimental tests. The aim of this study was to validate a nonlinear anatomy specific QCT-based FE model by using a novel testing setup. Thirty-seven human thoracolumbar vertebral bone slices were prepared by removing cortical endplates and posterior elements. The slices were scanned with QCT and the volumetric bone mineral density (vBMD) was computed with the standard clinical approach. A novel experimental setup was designed to induce a realistic failure in the vertebral slices in vitro. Rotation of the loading plate was allowed by means of a ball joint. To minimize device compliance, the specimen deformation was measured directly on the loading plate with three sensors. A nonlinear FE model was generated from the calibrated QCT images and computed vertebral stiffness and strength were compared to those measured during the experiments. In agreement with clinical observations, most of the vertebrae underwent an anterior wedge-shape fracture. As expected, the FE method predicted both stiffness and strength better than vBMD (R² improved from 0.27 to 0.49 and from 0.34 to 0.79, respectively). Despite the lack of fitting parameters, the linear regression of the FE prediction for strength was close to the 1:1 relation (slope and intercept close to one (0.86 kN) and to zero (0.72 kN), respectively). In conclusion, a nonlinear FE model was successfully validated through a novel experimental technique for generating wedge-shape fractures in human thoracolumbar vertebrae.     

Cryo‐EM structures of human ABCA7 provide insights into its phospholipid translocation mechanisms

Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo‐EM structures of lipid‐embedded human ABCA7 in an open state and in a nucleotide‐bound, closed state at resolutions between 3.6 and 4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid‐free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid‐body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.     

Discovery of novel paeonol-based derivatives against skin inflammation in vitro and in vivo

T-LAK-cell-originated protein kinase (TOPK), a novel member of the mitogen-activated protein kinase family, is considered an effective therapeutic target for skin inflammation. In this study, a series (A − D) of paeonol derivatives was designed and synthesised using a fragment growing approach, and their anti-inflammatory activities against lipopolysaccharide (LPS)-induced nitric oxide production in RAW264.7 cells were tested. Among them, compound B12 yielded the best results (IC₅₀ = 2.14 μM) with low toxicity (IC₅₀ > 50 µM). Preliminary mechanistic studies indicated that this compound could inhibit the TOPK-p38/JNK signalling pathway and phosphorylate downstream related proteins. A murine psoriasis-like skin inflammation model was used to determine its therapeutic effect.     

Targeting Wnt/tenascin C-mediated cross talk between pancreatic cancer cells and stellate cells via activation of the metastasis suppressor NDRG1

A major barrier to successful pancreatic cancer (PC) treatment is the surrounding stroma, which secretes growth factors/cytokines that promote PC progression. Wnt and tenascin C (TnC) are key ligands secreted by stromal pancreatic stellate cells (PSCs) that then act on PC cells in a paracrine manner to activate the oncogenic β-catenin and YAP/TAZ signaling pathways. Therefore, therapies targeting oncogenic Wnt/TnC cross talk between PC cells and PSCs constitute a promising new therapeutic approach for PC treatment. The metastasis suppressor N-myc downstream-regulated gene-1 (NDRG1) inhibits tumor progression and metastasis in numerous cancers, including PC. We demonstrate herein that targeting NDRG1 using the clinically trialed anticancer agent di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibited Wnt/TnC-mediated interactions between PC cells and the surrounding PSCs. Mechanistically, NDRG1 and DpC markedly inhibit secretion of Wnt3a and TnC by PSCs, while also attenuating Wnt/β-catenin and YAP/TAZ activation and downstream signaling in PC cells. This antioncogenic activity was mediated by direct inhibition of β-catenin and YAP/TAZ nuclear localization and by increasing the Wnt inhibitor, DKK1. Expression of NDRG1 also inhibited transforming growth factor (TGF)-β secretion by PC cells, a key mechanism by which PC cells activate PSCs. Using an in vivo orthotopic PC mouse model, we show DpC downregulated β-catenin, TnC, and YAP/TAZ, while potently increasing NDRG1 expression in PC tumors. We conclude that NDRG1 and DpC inhibit Wnt/TnC-mediated interactions between PC cells and PSCs. These results further illuminate the antioncogenic mechanism of NDRG1 and the potential of targeting this metastasis suppressor to overcome the oncogenic effects of the PC-PSC interaction.     

Role of vasorin,an anti‐apoptotic,anti‐TGF‐β and hypoxia‐induced glycoprotein in the trabecular meshwork cells and glaucoma

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open‐angle glaucoma patients compared to non‐glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti‐apoptotic and anti‐TGF‐β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF‐β2‐induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride‐induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF‐α‐induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF‐β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.     

Finite element analysis of acetabular fractures—development and validation with a synthetic pelvis

Acetabular fracture presents a challenging situation to trauma surgeons today due to its complexity. Finite element (FE) models can be of great help as they can improve the surgical planning and post surgery patient management for those with acetabular fractures. We have developed a non-linear finite element model of the pelvis and validated its fracture prediction capability with synthetic polyurethane pelves. A mechanical experiment was performed with the synthetic bones and fracture loads and patterns were observed for two different loading cases. Fracture loads predicted by our FE model were within one standard deviation of the experimental fracture loads for both loading cases. The incipient fracture pattern predicted by the model also resembled the actual pattern from the experiment. Although it is not a complete validation with human cadaver bones, the good agreement between model predictions and experimental results indicate the validity of our approach in using non-linear FE formulation along with contact conditions in predicting bone fractures.     

Estimation of conditions evoking fracture in finger bones under pinch loading based on finite element analysis

A finger finite element (FE) model was created from CT images of a Japanese male in order to obtain a shape-biofidelic model. Material properties and articulation characteristics of the model were taken from the literature. To predict bone fracture and realistically represent the fracture pattern under various loading conditions, the ESI-Wilkins-Kamoulakos rupture model in PAM-CRASH (ESI Group S.A., Paris, France) was utilized in this study with parameter values of the rupture model determined by compression testing and simulation of porcine fibula. A finger pinch simulation was then conducted to validate the finger FE model. The force-displacement curve and fracture load from the pinch simulation was compared to the result of finger pinch test using cadavers. Simulation results are coincident with the test result, indicating that the finger FE model can be used in an analysis of finger bone fracture during pinch accident. With this model, several pinch simulations were conducted with different pinching object’s stiffness and pinching energy. Conditions for evoking finger bone fracture under pinch loading were then estimated based on these results. This study offers a novel method to predict possible hazards of manufactured goods during the design process, thus finger injury due to pinch loading can be avoided.     

Quantification of a rat tail vertebra model for trabecular bone adaptation studies

A feedback controlled loading apparatus for the rat tail vertebra was developed to deliver precise mechanical loads to the eighth caudal vertebra (C8) via pins inserted into adjacent vertebrae. Cortical bone strains were recorded using strain gages while subjecting the C8 in four cadaveric rats to mechanical loads ranging from 25 to 100 N at 1 Hz with a sinusoidal waveform. Finite element (FE) models, based on micro computed tomography, were constructed for all four C8 for calculations of cortical and trabecular bone tissue strains. The cortical bone strains predicted by FE models agreed with strain gage measurements, thus validating the FE models. The average measured cortical bone strain during 25-100 N loading was between 298 +/- 105 and 1210 +/- 297 microstrain (muepsilon). The models predicted average trabecular bone tissue strains ranging between 135 +/- 35 and 538 +/- 138 mu epsilon in the proximal region, 77 +/- 23-307 +/- 91 muepsilon in the central region, and 155 +/- 36-621 +/- 143 muepsilon in the distal region for 25-100 N loading range. Although these average strains were compressive, it is also interesting that the trabecular bone tissue strain can range from compressive to tensile strains (-1994 to 380 mu epsilon for a 100 N load). With this novel approach that combines an animal model with computational techniques, it could be possible to establish a quantitative relationship between the microscopic stress/strain environment in trabecular bone tissue, and the biosynthetic response and gene expression of bone cells, thereby study bone adaptation.     

Constrained tibial vibration in mice: a method for studying the effects of vibrational loading of bone

Vibrational loading can stimulate the formation of new trabecular bone or maintain bone mass. Studies investigating vibrational loading have often used whole-body vibration (WBV) as their loading method. However, WBV has limitations in small animal studies because transmissibility of vibration is dependent on posture. In this study, we propose constrained tibial vibration (CTV) as an experimental method for vibrational loading of mice under controlled conditions. In CTV, the lower leg of an anesthetized mouse is subjected to vertical vibrational loading while supporting a mass. The setup approximates a one degree-of-freedom vibrational system. Accelerometers were used to measure transmissibility of vibration through the lower leg in CTV at frequencies from 20 Hz to 150 Hz. First, the frequency response of transmissibility was quantified in vivo, and dissections were performed to remove one component of the mouse leg (the knee joint, foot, or soft tissue) to investigate the contribution of each component to the frequency response of the intact leg. Next, a finite element (FE) model of a mouse tibia-fibula was used to estimate the deformation of the bone during CTV. Finally, strain gages were used to determine the dependence of bone strain on loading frequency. The in vivo mouse leg in the CTV system had a resonant frequency of 60 Hz for +/-0.5 G vibration (1.0 G peak to peak). Removing the foot caused the natural frequency of the system to shift from 60 Hz to 70 Hz, removing the soft tissue caused no change in natural frequency, and removing the knee changed the natural frequency from 60 Hz to 90 Hz. By using the FE model, maximum tensile and compressive strains during CTV were estimated to be on the cranial-medial and caudolateral surfaces of the tibia, respectively, and the peak transmissibility and peak cortical strain occurred at the same frequency. Strain gage data confirmed the relationship between peak transmissibility and peak bone strain indicated by the FE model, and showed that the maximum cyclic tibial strain during CTV of the intact leg was 330+/-82microepsilon and occurred at 60-70 Hz. This study presents a comprehensive mechanical analysis of CTV, a loading method for studying vibrational loading under controlled conditions. This model will be used in future in vivo studies and will potentially become an important tool for understanding the response of bone to vibrational loading.     

Spatial dynamics of Chinese Muntjac related to past and future climate fluctuations

Climate fluctuations in the past and in the future are likely to result in population expansions, shifts, or the contraction of the ecological niche of many species, and potentially leading to the changes in their geographical distributions. Prediction of suitable habitats has been developed as a useful tool for the assessment of habitat suitability and resource conservation to protect wildlife. Here, we model the ancestral demographic history of the extant modern Chinese Muntjac Muntiacus reevesi populations using approximate Bayesian computation (ABC) and used the maximum entropy model to simulate the past and predict the future spatial dynamics of the species under climate oscillations. Our results indicated that the suitable habitats for the M. reevesi shifted to the Southeast and contracted during the Last Glacial Maximum, whereas they covered a broader and more northern position in the Middle Holocene. The ABC analyses revealed that the modern M. reevesi populations diverged in the Middle Holocene coinciding with the significant contraction of the highly suitable habitat areas. Furthermore, our predictions suggest that the potentially suitable environment distribution for the species will expand under all future climate scenarios. These results indicated that the M. reevesi diverged in the recent time after the glacial period and simultaneously as its habitat’s expanded in the Middle Holocene. Furthermore, the past and future climate fluctuation triggered the change of Chinese muntjac spatial distribution, which has great influence on the Chinese muntjac’s population demographic history.