A novel flexible nerve guidance conduit promotes nerve regeneration while providing excellent mechanical properties

Autografting is the gold standard for surgical repair of nerve defects>5 mm in length;however,autografting is associated with potential complications at the nerve donor site.As an alternative,nerve guidance conduits may be used.The ideal conduit should be flexible,resistant to kinks and lumen collapse,and provide physical cues to guide nerve regeneration.We designed a novel flexible conduit using electrospinning technology to create fibers on the innermost surface of the nerve guidance conduit and employed melt spinning to align them.Subsequently,we prepared disordered electrospun fibers outside the aligned fibers and helical melt-spun fibers on the outer wall of the electrospun fiber lumen.The presence of aligned fibers on the inner surface can promote the extension of nerve cells along the fibers.The helical melt-spun fibers on the outer surface can enhance resistance to kinking and compression and provide stability.Our novel conduit promoted nerve regeneration and functional recovery in a rat sciatic nerve defect model,suggesting that it has potential for clinical use in human nerve injuries.

Contribution of mechanical forces to structural synaptic plasticity:insights from 3D cellular motility mechanisms

Cells,tissues,and organs are constantly subjected to the action of mechanical forces from the extracellular environment-and the nervous system is no exception.Cell-intrinsic properties such as membrane lipid composition,abundance of mechanosensors,and cytoskeletal dynamics make cells more or less likely to sense these forces.Intrinsic and extrinsic cues are integrated by cells and this combined information determines the rate and dynamics of membrane protrusion growth or retraction(Yamada and Sixt,2019).Cell protrusions are extensions of the plasma membrane that play crucial roles in diverse contexts such as cell migration and neuronal synapse formation.In the nervous system,neurons are highly dynamic cells that can change the size and number of their pre-and postsynaptic elements(called synaptic boutons and dendritic spines,respectively),in response to changes in the levels of synaptic activity through a process called plasticity.Synaptic plasticity is a hallmark of the nervous system and is present throughout our lives,being required for functions like memory formation or the learning of new motor skills(Minegishi et al.,2023;Pillai and Franze,2024).

Silk-based nerve guidance conduits with macroscopic holes modulate the vascularization of regenerating rat sciatic nerve

Peripheral nerve injuries induce a severe motor and sensory deficit. Since the availability of autologous nerve transplants for nerve repair is very limited, alternative treatment strategies are sought, including the use of tubular nerve guidance conduits(tNGCs). However, the use of tNGCs results in poor functional recovery and central necrosis of the regenerating tissue, which limits their application to short nerve lesion defects(typically shorter than 3 cm). Given the importance of vascularization in nerve regeneration, we hypothesized that enabling the growth of blood vessels from the surrounding tissue into the regenerating nerve within the tNGC would help eliminate necrotic processes and lead to improved regeneration. In this study, we reported the application of macroscopic holes into the tubular walls of silk-based tNGCs and compared the various features of these improved silk^(+) tNGCs with the tubes without holes(silk^(–) tNGCs) and autologous nerve transplants in an 8-mm sciatic nerve defect in rats. Using a combination of micro-computed tomography and histological analyses, we were able to prove that the use of silk^(+) tNGCs induced the growth of blood vessels from the adjacent tissue to the intraluminal neovascular formation. A significantly higher number of blood vessels in the silk^(+) group was found compared with autologous nerve transplants and silk^(–), accompanied by improved axon regeneration at the distal coaptation point compared with the silk^(–) tNGCs at 7 weeks postoperatively. In the 15-mm(critical size) sciatic nerve defect model, we again observed a distinct ingrowth of blood vessels through the tubular walls of silk^(+) tNGCs, but without improved functional recovery at 12 weeks postoperatively. Our data proves that macroporous tNGCs increase the vascular supply of regenerating nerves and facilitate improved axonal regeneration in a short-defect model but not in a critical-size defect model. This study suggests that further optimization of the macroscopic holes silk^(+) tNGC approach containing macroscopic holes might result in improved grafting technology suitable for future clinical use.

The role of axon guidance molecules in the pathogenesis of epilepsy

Current treatments for epilepsy can only manage the symptoms of the condition but cannot alter the initial onset or halt the progression of the disease. Consequently, it is crucial to identify drugs that can target novel cellular and molecular mechanisms and mechanisms of action. Increasing evidence suggests that axon guidance molecules play a role in the structural and functional modifications of neural networks and that the dysregulation of these molecules is associated with epilepsy susceptibility. In this review, we discuss the essential role of axon guidance molecules in neuronal activity in patients with epilepsy as well as the impact of these molecules on synaptic plasticity and brain tissue remodeling. Furthermore, we examine the relationship between axon guidance molecules and neuroinflammation, as well as the structural changes in specific brain regions that contribute to the development of epilepsy. Ample evidence indicates that axon guidance molecules, including semaphorins and ephrins, play a fundamental role in guiding axon growth and the establishment of synaptic connections. Deviations in their expression or function can disrupt neuronal connections, ultimately leading to epileptic seizures. The remodeling of neural networks is a significant characteristic of epilepsy, with axon guidance molecules playing a role in the dynamic reorganization of neural circuits. This, in turn, affects synapse formation and elimination. Dysregulation of these molecules can upset the delicate balance between excitation and inhibition within a neural network, thereby increasing the risk of overexcitation and the development of epilepsy. Inflammatory signals can regulate the expression and function of axon guidance molecules, thus influencing axonal growth, axon orientation, and synaptic plasticity. The dysregulation of neuroinflammation can intensify neuronal dysfunction and contribute to the occurrence of epilepsy. This review delves into the mechanisms associated with the pathogenicity of axon guidance molecules in epilepsy, offering a valuable reference for the exploration of therapeutic targets and presenting a fresh perspective on treatment strategies for this condition.

Netrin-1 signaling pathway mechanisms in neurodegenerative diseases

Netrin-1 and its receptors play crucial roles in inducing axonal growth and neuronal migration during neuronal development.Their profound impacts then extend into adulthood to encompass the maintenance of neuronal survival and synaptic function.Increasing amounts of evidence highlight several key points:(1)Diminished Netrin-1 levels exacerbate pathological progression in animal models of Alzheimer’s disease and Parkinson’s disease,and potentially,similar alterations occur in humans.(2)Genetic mutations of Netrin-1 receptors increase an individuals’susceptibility to neurodegenerative disorders.(3)Therapeutic approaches targeting Netrin-1 and its receptors offer the benefits of enhancing memory and motor function.(4)Netrin-1 and its receptors show genetic and epigenetic alterations in a variety of cancers.These findings provide compelling evidence that Netrin-1 and its receptors are crucial targets in neurodegenerative diseases.Through a comprehensive review of Netrin-1 signaling pathways,our objective is to uncover potential therapeutic avenues for neurodegenerative disorders.

Decoding molecular mechanisms:brain aging and Alzheimer's disease

The complex morphological,anatomical,physiological,and chemical mechanisms within the aging brain have been the hot topic of research for centuries.The aging process alters the brain structure that affects functions and cognitions,but the worsening of such processes contributes to the pathogenesis of neurodegenerative disorders,such as Alzheimer's disease.Beyond these observable,mild morphological shifts,significant functional modifications in neurotransmission and neuronal activity critically influence the aging brain.Understanding these changes is important for maintaining cognitive health,especially given the increasing prevalence of age-related conditions that affect cognition.This review aims to explore the age-induced changes in brain plasticity and molecular processes,differentiating normal aging from the pathogenesis of Alzheimer's disease,thereby providing insights into predicting the risk of dementia,particularly Alzheimer's disease.

Pyroptosis,ferroptosis,and autophagy in spinal cord injury:regulatory mechanisms and therapeutic targets

Regulated cell death is a form of cell death that is actively controlled by biomolecules.Several studies have shown that regulated cell death plays a key role after spinal cord injury.Pyroptosis and ferroptosis are newly discovered types of regulated cell deaths that have been shown to exacerbate inflammation and lead to cell death in damaged spinal cords.Autophagy,a complex form of cell death that is interconnected with various regulated cell death mechanisms,has garnered significant attention in the study of spinal cord injury.This injury triggers not only cell death but also cellular survival responses.Multiple signaling pathways play pivotal roles in influencing the processes of both deterioration and repair in spinal cord injury by regulating pyroptosis,ferroptosis,and autophagy.Therefore,this review aims to comprehensively examine the mechanisms underlying regulated cell deaths,the signaling pathways that modulate these mechanisms,and the potential therapeutic targets for spinal cord injury.Our analysis suggests that targeting the common regulatory signaling pathways of different regulated cell deaths could be a promising strategy to promote cell survival and enhance the repair of spinal cord injury.Moreover,a holistic approach that incorporates multiple regulated cell deaths and their regulatory pathways presents a promising multi-target therapeutic strategy for the management of spinal cord injury.

Dynamics and intermittent stochastic stabilization of a rumor spreading model with guidance mechanism in heterogeneous network

We propose a novel rumor propagation model with guidance mechanism in hetero geneous complex networks.Firstly,the sharp threshold of rumor propagation,global stability of the information-equilibrium and information-prevailingequilibrium under R_(0)<1 and R_(0)>1 is carried out by Lyapunov method and LaSalle's invariant principle.Next,we design an aperiodically intermittent stochastic stabilization method to suppress the rumor propagation.By using the Ito formula and exponential martingale inequality,the expression of the minimum control intensity is calculated.This method can effectively stabilize the rumor propagation by choosing a suitable perturb intensity and a perturb time ratio,while minimizing the control cost.Finally,numerical examples are given to illustrate the analysis and method of the paper.

Study on Numerical Comparison Method of Four-bar Straight-line Guidance Mechanism

In order to solve four-bar straight-line guidance mechanism synthesis problem for the arbitrarily given straight-line’s"angle requirement"and"point-position requirement",a numerical comparison synthesis method for single and double straight-line guidance mechanism is presented,which is convenient to realize by computer program.The basic idea of this method is:to select a four-bar linkage whose relative bar length of crank is 1 as a basic four-bar linkage.Then the other three relative bars’length is changed,and a lot of basic four-bar linkage can be obtained.There are many single and double ball-points of each basic four-bar linkage.With the motion of a basic four-bar linkage,there is straight-line segment of each Ball-point’s path.The data of these basic four-bar linkages is saved to a database.When designing a four-bar straight-line guidance mechanism,the design data is compared with the data in database and a satisfactory four-bar linkage can be obtained.The method effectively solves the straight-line guidance mechanism synthesis problem.

Microdynamic mechanical properties and fracture evolution mechanism of monzogabbro with a true triaxial multilevel disturbance method

The far-field microdynamic disturbance caused by the excavation of deep mineral resources and underground engineering can induce surrounding rock damage in high-stress conditions and even lead to disasters.However,the mechanical properties and damage/fracture evolution mechanisms of deep rock induced by microdynamic disturbance under three-dimensional stress states are unclear.Therefore,a true triaxial multilevel disturbance test method is proposed,which can completely simulate natural geostress,excavation stress redistribution(such as stress unloading,concentration and rotation),and subsequently the microdynamic disturbance triggering damaged rock failure.Based on a dynamic true triaxial test platform,true triaxial microdynamic disturbance tests under different frequency and amplitudes were carried out on monzogabbro.The results show that increasing amplitude or decreasing frequency diminishes the failure strength of monzogabbro.Deformation modulus gradually decreases during disturbance failure.As frequency and amplitude increase,the degradation rate of deformation modulus decreases slightly,disturbance dissipated energy increases significantly,and disturbance deformation anisotropy strengthens obviously.A damage model has been proposed to quantitatively characterize the disturbance-induced damage evolution at different frequency and amplitude under true triaxial stress.Before disturbance failure,the micro-tensile crack mechanism is dominant,and the micro-shear crack mechanism increases significantly at failure.With the increase of amplitude and frequency,the micro-shear crack mechanism increases.When approaching disturbance failure,the acoustic emission fractal dimension changes from a stable value to local large oscillation,and finally increases sharply to a high value at failure.Finally,the disturbance-induced failure mechanism of surrounding rock in deep engineering is clearly elucidated.

Fabrication Techniques and Sensing Mechanisms of Textile‑Based Strain Sensors:From Spatial 1D and 2D Perspectives

The intelligent textile sensors based on fiber(1D)and fabric(2D)are the ideal candidates for wearable devices.Their flexible weaving and unique structure endow them with flexibility,lightweight,good air permeability,and feasible integration with garments.In view of the spring-up of novel textile-based strain sensors,the novel materials and fabrication approaches were elaborated from spatial perspectives,i.e.,1D fibers/yarn and 2D fabric.The intrinsic sensing mechanism is the primary fac-tor affecting sensor sensitivity,and the variation trend of the sensing signal is closely related to it.Although existing studies have involved various sensing mechanisms,there is still lacking systematic classification and discussion.Hence,the sensing mechanisms of textile-based sensors were elaborated from spatial perspectives.Considering that strain sensors were mostly based on resistance variation,the sensing mechanisms of resistive textile-based strain sensors were mainly focused,mainly including fiber deformation,tunneling effect,crack propagation,fabric deformation,electrical contact and bridge connec-tion.Meanwhile,the corresponding resistance prediction models,usually used as important data fitting methodology,were also comprehensively discussed,which can reproduce the resistance trend and provide guidance for the sensor performance.Finally,the multifunctionality of textile-based strain sensors was summarized,namely multi-mode signal detection,visual interaction,energy collection,thermal management and medical treatment were discussed.It was expected to provide research insights into the multifunctional integration of textile sensors.

Failure mechanism and infrared radiation characteristic of hard siltstone induced by stratification effect

The deformation in sedimentary rock induced by train loads has potential threat to the safe operation of tunnels. This study investigated the influence of stratification structure on the infrared radiation and temporal damage mechanism of hard siltstone. The uniaxial compression tests, coupled with acoustic emission(AE) and infrared radiation temperature(IRT) were conducted on siltstones with different stratification effects. The results revealed that the stratigraphic structure significantly affects the stress-strain response and strength degradation characteristics. The mechanical parameters exhibit anisotropy characteristics, and the stratification effect exhibits a negative correlation with the cracking stress and peak stress. The failure modes caused by the stratification effect show remarkable anisotropic features, including splitting failure(Ⅰ: 0°-22.50°, Ⅱ: 90°), composite failure(45°), and shearing failure(67.50°). The AE temporal sequences demonstrate a stepwise response characteristic to the loading stress level. The AE intensity indicates that the stress sensitivity of shearing failure and composite failure is generally greater than that of splitting failure. The IRT field has spatiotemporal migration and progressive dissimilation with stress loading and its dissimilation degree increases under higher stress levels. The stronger the stratification effect, the greater the dissimilation degree of the IRT field. The abnormal characteristic points of average infrared radiation temperature(AIRT) variance at local stress drop and peak stress can be used as early and late precursors to identify fracture instability. Theoretical analysis shows that the competitive relationship between compaction strengthening and fracturing damage intensifies the dissimilation of the infrared thermal field for an increasing stress level. The present study provides a theoretical reference for disaster warnings in hard sedimentary rock mass.

Regeneration of the heart:f rom molecular mechanisms to clinical therapeutics

Heart injury such as myocardial infarction leads to cardiomyocyte loss,fibrotic tissue deposition,and scar formation.These changes reduce cardiac contractility,resulting in heart failure,which causes a huge public health burden.Military personnel,compared with civilians,is exposed to more stress,a risk factor for heart diseases,making cardiovascular health management and treatment innovation an important topic for military medicine.So far,medical intervention can slow down cardiovascular disease progression,but not yet induce heart regeneration.In the past decades,studies have focused on mechanisms underlying the regenerative capability of the heart and applicable approaches to reverse heart injury.Insights have emerged from studies in animal models and early clinical trials.Clinical interventions show the potential to reduce scar formation and enhance cardiomyocyte proliferation that counteracts the pathogenesis of heart disease.In this review,we discuss the signaling events controlling the regeneration of heart tissue and summarize current therapeutic approaches to promote heart regeneration after injury.

The underlying mechanism of variety–water–nitrogen–stubble damage interactions on yield formation in ratoon rice with low stubble height under mechanized harvesting

Agronomic measures are the key to promote the sustainable development of ratoon rice by reducing the damage from mechanical crushing to the residual stubble of the main crop, thereby mitigating the impact on axillary bud sprouting and yield formation in ratoon rice. This study used widely recommended conventional rice Jiafuzhan and hybrid rice Yongyou 2640 as the test materials to conduct a four-factor block design field experiment in a greenhouse of the experimental farm of Fujian Agricultural and Forestry University, China from 2018 to 2019.The treatments included fertilization and no fertilization, alternate wetting and drying irrigation and continuous water flooding irrigation, and plots with and without artificial crushing damage on the rice stubble. At the same time, a 13C stable isotope in-situ detection technology was used to fertilize the pot experiment. The results showed significant interactions among varieties, water management, nitrogen application and stubble status.Relative to the long-term water flooding treatment, the treatment with sequential application of nitrogen fertilizer coupled with moderate field drought for root-vigor and tiller promotion before and after harvesting of the main crop, significantly improved the effective tillers from low position nodes. This in turn increased the effective panicles per plant and grains per panicle by reducing the influence of artificial crushing damage on rice stubble and achieving a high yield of the regenerated rice. Furthermore, the partitioning of 13C assimilates to the residual stubble and its axillary buds were significantly improved at the mature stage of the main crop, while the translocation rate to roots and rhizosphere soil was reduced at the later growth stage of ratooning season rice. This was triggered by the metabolism of hormones and polyamines at the stem base regulated by the interaction of water and fertilizer at this time. We therefore suggest that to achieve a high yield of ratoon rice with low stubble height under mechanized harvesting, the timely application of nitrogen fertilizer is fundamental,coupled with moderate field drying for root-vigor preservation and tiller promotion before and after the mechanical harvesting of the main crop.

Antagonism effect of residual S triggers the dual-path mechanism for water oxidation

Transition metal chalcogenides(TMCs)are recognized as pre-catalysts,and their(oxy)hydroxides derived from electrochemical reconstruction are the active species in the water oxidation.However,understanding the role of the residual chalcogen in the reconstructed layer is lacking in detail,and the corresponding catalytic mechanism remains controversial.Here,taking Cu_(1-x)Co_(x)S as a platform,we explore the regulating effect and existence form of the residual S doped into the reconstructive layer for oxygen evolution reaction(OER),where a dual-path OER mechanism is proposed.First-principles calculations and operando~(18)O isotopic labeling experiments jointly reveal that the residual S in the reconstructive layer of Cu_(1-x)Co_(x)S can wisely balance the adsorbate evolution mechanism(AEM)and lattice oxygen oxidation mechanism(LOM)by activating lattice oxygen and optimizing the adsorption/desorption behaviors at metal active sites,rather than change the reaction mechanism from AEM to LOM.Following such a dual-path OER mechanism,Cu_(0.4)Co_(0.6)S-derived Cu_(0.4)Co_(0.6)OSH not only overcomes the restriction of linear scaling relationship in AEM,but also avoids the structural collapse caused by lattice oxygen migration in LOM,so as to greatly reduce the OER potential and improved stability.

Drug resistance mechanisms in cancers:Execution of prosurvival strategies

One of the quintessential challenges in cancer treatment is drug resistance.Several mechanisms of drug resistance have been described to date,and new modes of drug resistance continue to be discovered.The phenomenon of cancer drug resistance is now widespread,with approximately 90% of cancer-related deaths associated with drug resistance.Despite significant advances in the drug discovery process,the emergence of innate and acquired mechanisms of drug resistance has impeded the progress in cancer therapy.Therefore,understanding the mechanisms of drug resistance and the various pathways involved is integral to treatment modalities.In the present review,I discuss the different mechanisms of drug resistance in cancer cells,including DNA damage repair,epithelial to mesenchymal transition,inhibition of cell death,alteration of drug targets,inactivation of drugs,deregulation of cellular energetics,immune evasion,tumor-promoting inflammation,genome instability,and other contributing epigenetic factors.Furthermore,I highlight available treatment options and conclude with future directions.

Extracellular vesicles in anti-tumor drug resistance: Mechanisms and therapeutic prospects

Drug resistance presents a significant challenge to achieving positive clinical outcomes in anti-tumor therapy.Prior research has illuminated reasons behind drug resistance,including increased drug efflux,alterations in drug targets,and abnormal activation of oncogenic pathways.However,there's a need for deeper investigation into the impact of drug-resistant cells on parental tumor cells and intricate crosstalk between tumor cells and the malignant tumor microenvironment(TME).Recent studies on extracellular vesicles(EVs)have provided valuable insights.EVs are membrane-bound particles secreted by all cells,mediating cell-to-cell communication.They contain functional cargoes like DNA,RNA,lipids,proteins,and metabolites from mother cells,delivered to other cells.Notably,EVs are increasingly recognized as regulators in the resistance to anti-cancer drugs.This review aims to summarize the mechanisms of EV-mediated anti-tumor drug resistance,covering therapeutic approaches like chemo-therapy,targeted therapy,immunotherapy and even radiotherapy.Detecting Ev-based biomarkers to predict drug resistance assists in bypassing anti-tumor drug resistance.Additionally,targeted inhibition of EV biogenesis and secretion emerges as a promising approach to counter drug resistance.We highlight the importance of conducting in-depth mechanistic research on EVs,their cargoes,and functional ap-proaches specifically focusing on EV subpopulations.These efforts will significantly advance the devel-opment of strategies to overcome drug resistance in anti-tumor therapy.

To explore the mechanism of Yigong San anti-gastric cancer and immune regulation

BACKGROUND Yigong San(YGS)is a representative prescription for the treatment of digestive disorders,which has been used in clinic for more than 1000 years.However,the mechanism of its anti-gastric cancer and regulate immunity are still remains unclear.AIM To explore the mechanism of YGS anti-gastric cancer and immune regulation.METHODS Firstly,collect the active ingredients and targets of YGS,and the differentially expressed genes of gastric cancer.Secondly,constructed a protein-protein interaction network between the targets of drugs and diseases,and screened hub genes.Then the clinical relevance,mutation and repair,tumor microenvironment and drug sensitivity of the hub gene were analyzed.Finally,molecular docking was used to verify the binding ability of YGS active ingredient and hub genes.RESULTS Firstly,obtained 55 common targets of gastric cancer and YGS.The Kyoto Encyclopedia of Genes and Genomes screened the microtubule-associated protein kinase signaling axis as the key pathway and IL6,EGFR,MMP2,MMP9 and TGFB1 as the hub genes.The 5 hub genes were involved in gastric carcinogenesis,staging,typing and prognosis,and their mutations promote gastric cancer progression.Finally,molecular docking results confirmed that the components of YGS can effectively bind to therapeutic targets.CONCLUSION YGS has the effect of anti-gastric cancer and immune regulation.

Trajectory tracking guidance of interceptor via prescribed performance integral sliding mode with neural network disturbance observer

This paper investigates interception missiles’trajectory tracking guidance problem under wind field and external disturbances in the boost phase.Indeed,the velocity control in such trajectory tracking guidance systems of missiles is challenging.As our contribution,the velocity control channel is designed to deal with the intractable velocity problem and improve tracking accuracy.The global prescribed performance function,which guarantees the tracking error within the set range and the global convergence of the tracking guidance system,is first proposed based on the traditional PPF.Then,a tracking guidance strategy is derived using the integral sliding mode control techniques to make the sliding manifold and tracking errors converge to zero and avoid singularities.Meanwhile,an improved switching control law is introduced into the designed tracking guidance algorithm to deal with the chattering problem.A back propagation neural network(BPNN)extended state observer(BPNNESO)is employed in the inner loop to identify disturbances.The obtained results indicate that the proposed tracking guidance approach achieves the trajectory tracking guidance objective without and with disturbances and outperforms the existing tracking guidance schemes with the lowest tracking errors,convergence times,and overshoots.

Deep Insight of Design,Mechanism,and Cancer Theranostic Strategy of Nanozymes

Since the discovery of enzyme-like activity of Fe3O4 nanoparticles in 2007,nanozymes are becoming the promising substitutes for natural enzymes due to their advantages of high catalytic activity,low cost,mild reaction conditions,good stability,and suitable for large-scale production.Recently,with the cross fusion of nanomedicine and nanocatalysis,nanozyme-based theranostic strategies attract great attention,since the enzymatic reactions can be triggered in the tumor microenvironment to achieve good curative effect with substrate specificity and low side effects.Thus,various nanozymes have been developed and used for tumor therapy.In this review,more than 270 research articles are discussed systematically to present progress in the past five years.First,the discovery and development of nanozymes are summarized.Second,classification and catalytic mechanism of nanozymes are discussed.Third,activity prediction and rational design of nanozymes are focused by highlighting the methods of density functional theory,machine learning,biomimetic and chemical design.Then,synergistic theranostic strategy of nanozymes are introduced.Finally,current challenges and future prospects of nanozymes used for tumor theranostic are outlined,including selectivity,biosafety,repeatability and stability,in-depth catalytic mechanism,predicting and evaluating activities.