Non-invasive in vivo study of morphology and mechanical properties of the median nerve.

The current literature studied the median nerve (MN) at specific locations during joint motions. As only a few particular parts of the nerve are depicted, the relevant information available is limited. This experiment investigated the morphological and biomechanical properties of the MN. The effects of the shoulder and wrist motions on MN were explored as well. Eight young healthy female individuals were tested with two-dimensional ultrasound and shear wave elastography (SWE). The morphological and biomechanical properties were examined in limb position 1, with the wrist at the neutral position, the elbow extended at 180°, and the shoulder abducted at 60°. In addition, the experiment assessed the differences among the wrist, forearm, elbow, and upper arm with Friedman's test and Bonferroni post hoc analysis. Two groups of limb positions were designed to explore the effects of shoulder movements (shoulder abducted at 90° and 120°) and wrist movements (wrist extended at 45° and flexed at 45°) on the thickness and Young's modulus. Differences among the distributions of five limb positions were tested as well. The ICC3, 1 values for thickness and Young's modulus were 0.976 and 0.996, respectively. There were differences among the MN thicknesses of four arm locations in limb position 1, while Young's modulus was higher at the elbow and wrist than at the forearm and upper arm. Compared to limb position 1, only limb position 4 had an effect on MN thickness at the wrist. Both shoulder and wrist motions affected MN Young's modulus, and the stiffness variations at typical locations all showed a downward trend proximally in all. The distributions of MN thickness and Young's modulus showed fold line patterns but differed at the wrist and the pronator teres. The MN in the wrist is more susceptible to limb positions, and Young's modulus is sensitive to nerve changes and is more promising for the early diagnosis of neuropathy.

The unique biodegradation pathway of benzo[a]pyrene in moderately halophilic Pontibacillus chungwhensis HN14.

Benzo[a]pyrene (BaP), as the typical representative of polycyclic aromatic hydrocarbons (PAHs), is a serious hazard to human health and natural environments. Though the study of microbial degradation of PAHs has persisted for decades, the degradation pathway of BaP is still unclear. Previously, Pontibacillus chungwhensis HN14 was isolated from high salinity environment exhibiting a high BaP degradation ability. Here, based on the intermediates identified, BaP was found to be transformed to 4,5-epoxide-BaP, BaP-trans-4,5-dihydrodiol, 1,2-dihydroxy-phenanthrene, 2-carboxy-1-naphthol, and 4,5-dimethoxybenzo[a]pyrene by the strain HN14. Furthermore, functional genes involved in degradation of BaP were identified using genome and transcriptome data. Heterogeneous co-expression of monooxygenase CYP102(HN14) and epoxide hydrolase EH(HN14) suggested that CYP102(HN14) could transform BaP to 4,5-epoxide-BaP, which was further transformed to BaP-trans-4,5-dihydrodiol by EH(HN14). Moreover, gene cyp102(HN14) knockout was performed using CRISPR/Cas9 gene-editing system which confirmed that CYP102(HN14) play a key role in the initial conversion of BaP. Finally, a novel BaP degradation pathway was constructed in bacteria, which showed BaP could be converted into chrysene, phenanthrene, naphthalene pathways for the first time. These findings enhanced our understanding of microbial degradation process for BaP and suggested the potential of using P. chungwhensis HN14 for bioremediation in PAH-contaminated environments.

Novel insights into the synergetic degradation of pyrene by microbial communities from mangroves in China.

Pyrene is a high molecular weight polycyclic aromatic hydrocarbon (HMW-PAHs). It is a ubiquitous, persistent, and carcinogenic environmental contaminant that has raised concern worldwide. This research explored synergistic bacterial communities for efficient pyrene degradation in seven typical Southern China mangroves. The bacterial communities of seven typical mangroves were enriched by pyrene, and enriched bacterial communities showed an excellent pyrene degradation capacity of > 95% (except for HK mangrove and ZJ mangrove). Devosia, Hyphomicrobium, Flavobacterium, Marinobacter, Algoriphahus, and Youhaiella all have significant positive correlations with pyrene (R>0, p < 0.05) by 16SrRNA gene sequencing and metagenomics analysis, indicated that these genera play a vital role in pyrene metabolism. Meanwhile, the functional genes were involved in pyrene degradation that was enriched in the bacterial communities, including the genes of nagAa, ndoR, pcaG, etc. Furthermore, the analyses of functional genes and binning genomes demonstrated that some bacterial communities as a unique teamwork to cooperatively participate in pyrene degradation. Interestingly, the genes related to biogeochemical cycles were enriched, such as narG , soxA, and cyxJ, suggested that bacterial communities were also helpful in maintaining the stability of the ecological environment. In addition, some novel species with pyrene-degradation potential were identified in the pyrene-degrading bacterial communities, which can enrich the resource pool of pyrene-degrading strains. Overall, this study will help develop further research strategies for pollutant removal.

Bioinspired Structural Composite Flexible Material with High Cushion Performance.

Impacts occur everywhere, and they pose a serious threat to human health and production safety. Flexible materials with efficient cushioning and energy absorption are ideal candidates to provide protection from impacts. Despite the high demand, the cushioning capacity of protective materials is still limited. In this study, an integrated bionic strategy is proposed, and a bioinspired structural composite material with highly cushioning performance is developed on the basis of this strategy. The results demonstrated that the integrated bionic material, an S-spider web-foam, has excellent energy storage and dissipation as well as cushioning performance. Under impact loading, S-spider web-foam can reduce peak impact forces by a factor of 3.5 times better than silicone foam, achieving unprecedented cushioning performance. The results of this study deepen the understanding of flexible cushioning materials and may provide new strategies and inspiration for the preparation of high-performance flexible cushioning materials.

The Fundamental Property of Human Leg During Walking: Linearity and Nonlinearity.

Leg properties have been involved in the broad study of human walking from mechanical energy to motion prediction of robotics. However, the variable leg elasticities and their functions during gait have not been fully explored. This study presented that the fundamental leg properties during human walking comprise axial stiffness, rest leg length, tangential stiffness and force-free leg angles. We measured the axial force-leg length and tangential force-leg angle data in eight participants (mean ± s.d. age 24.6 ± 3.0 years, mass 68.2 ± 6.8 kg, height 177.5 ± 5.2 cm) at three self-selected walking speeds (slow: 1.25 ± 0.22, normal: 1.48 ± 0.28, fast: 1.75 ± 0.32 m/s) on two different contact conditions (fixed and moving). After obtaining these gait measurements, we extracted the linear and nonlinear leg elasticities during human walking by using a minimum root-mean-square fitting. We found that the axial stiffness of nonlinear elasticity (fixed condition: 7.1-8.0, moving condition: 21.3-22.6) is higher than that of the linear elasticity (fixed condition: 5.0-5.7, moving condition: 15.2-16.5). The tangential stiffness behaves different during four stance phases of gait, with the highest (linear: 2.52-3.72, nonlinear: 1.71-2.01, in moving condition) occurred at early stance and second highest at late stance, followed by two stiffnesses in mid-stance. For both linearity and nonlinearity, the axial stiffness and rest length are independent of walking speeds in both contact conditions, while the tangential stiffness and contact angles are independent of walking speeds only in moving condition. Regardless of walking speed, elasticity and contact condition, the force-free contact angle at mid-stance is maintained at average of 82.2 °. This paper first demonstrates the mechanical walking leg property from both axial and tangential aspects. The findings provide insight into the fundamental properties including linearity and nonlinearity of human leg during locomotion for stability analysis and precise motion prediction of robotics and rehabilitation exoskeletons.

Cytochrome P450 for environmental remediation: catalytic mechanism, engineering strategies and future prospects.

Environmental pollution is a global concern. Various organic compounds are released into the environment through wastewater, waste gas, and waste residue, ultimately accumulating in the environment and the food chain. This poses a significant threat to both human health and ecology. Currently, a growing body of research has demonstrated that microorganisms employ their Cytochrome P450 (CYP450) system for biodegradation, offering a crucial approach for eliminating these pollutants in environmental remediation. CYP450, a ubiquitous catalyst in nature, includes a vast array of family members distributed widely across various organisms, including bacteria, fungi, and mammals. These enzymes participate in the metabolism of diverse organic compounds. Furthermore, the rapid advancements in enzyme and protein engineering have led to increased utilization of engineered CYP450s in environmental remediation, enhancing their efficiency in pollutant removal. This article presents an overview of the current understanding of various members of the CYP450 superfamily involved in transforming organic pollutants and the engineering of biodegrading CYP450s. Additionally, it explores the catalytic mechanisms, current practical applications of CYP450-based systems, their potential applications, and the prospects in bioremediation.

Effects of extreme cyclic loading on the cushioning performance of human heel pads under engineering test condition.

Human heel pads commonly undergo cyclic loading during daily activities. Low cyclic loadings such as daily human walking tend to have less effect on the mechanical properties of heel pads. However, the impact of cyclic loading on cushion performance, a vital biomechanical property of heel pads, under engineering test condition remains unexplored. Herein, dynamic mechanical measurements and finite element (FE) simulations were employed to explore this phenomenon. It was found that the wavy collagen fibers in the heel pad will be straightened under cycle compression loading, which resulted in increased stiffness of the heel pad. The stiffness of the heel pads demonstrated an inclination to escalate over a span of 50,000 loading cycles, consequently resulting in a corresponding increase in peak impact force over the same loading cycles. Sustained cyclic loading has the potential to result in the fracturing of the straightened collagen fibers, this collagen breakage may diminish the stiffness of the heel pad, leading to a reduction in peak impact force. This work enhances understanding of the biomechanical functions of human heel pad and may provide potential inspirations for the innovative development of healthcare devices for foot complex.

Assessment of blood flow around the knee joint in patients with knee osteoarthritis by color Doppler ultrasound.

OBJECTIVE: To explore a method for early diagnosis of knee osteoarthritis (OA) by comparing vascular morphology and hemodynamic parameters between healthy controls and patients, and explore the effects of age on the blood flow of healthy subjects. MATERIAL AND METHODS: 80 healthy subjects and 106 patients with knee OA were recruited. Color Doppler ultrasound was used to measure the vascular morphology and hemodynamic parameters of 11 blood vessels around the knee joint. A general linear model was used to analyze the difference of blood flow parameters between healthy controls and patients, and to explore the effects of sex and age on the blood flow of healthy subjects. RESULTS: Age has no significant effect on the vessel diameter of healthy subjects, and it only affect the blood flow velocity (P ＜ 0.05) and blood flow volume (P ＜0.0083) of a few blood vessels. There was no significant difference in vascular morphological parameter between healthy controls, patients with mild OA and moderate/severe OA, but there was significant difference in vascular hemodynamic parameters (P ＜ 0.0167). Specifically, in the early stage of knee OA, hemodynamic parameters of main arteries in patients did not change significantly, but those of branch vessels changed significantly (P ＜ 0.0167). With the development of knee OA, the difference of hemodynamic parameters of branch vessels in patients increased gradually. CONCLUSION: Hemodynamic parameters of branch vessels around the knee joint have changed significantly in the early stage of knee OA, which can be treated as an important index to screening the early patients.

Novosphingobium mangrovi sp. nov., isolated from mangrove sediment.

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain, designated as HK4-1T, was isolated from mangrove sediments in Hong Kong, PR China. Based on 16S rRNA gene sequence data, strain HK4-1T was found to belong to the genus Novosphingobium, family Erythrobacteraceae, and showed high similarity to Novosphingobium chloroacetimidivorans BUT-14T (96.88 %) and Novosphingobium indicum H25T (96.88 %). The G+C content of the whole genome of strain HK4-1T was 64.05 mol%. The major fatty acids were C16 : 0, C18 : 1 ω7c and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid and two unknown lipids. The predominant respiratory quinone was Q-10. Based on genomic, phylogenetic, phenotypic, physiological and chemotaxonomic data, strain HK4-1T should be classified as representing a novel species of the genus Novosphingobium, for which the name Novosphingobium mangrovi sp. nov. is proposed. The type strain of Novosphingobium mangrovi sp. nov. is HK4-1T (=MCCC 1K08252T=JCM 35764T).

The Biomechanical Influence of Defected Cartilage on the Progression of Osteochondral Lesions of the Talus: A Three-dimensional Finite Element Analysis.

OBJECTIVES: Osteochondral lesions of the talus (OLTs) are common injuries in the general population. Abnormal mechanical conditions applied to defected cartilage are believed to be the culprits to deteriorating OLTs. This study aims to investigate the biomechanical effects of defect size of talar cartilage on OLTs during ankle movements. METHODS: A finite element model of the ankle joint was created based on the computed tomography images of a healthy male volunteer. Different defect sizes (S = 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, and 2.0 cm2 ) of talar cartilage were modeled to simulate the progression of OLTs. Mechanical moments were applied to the model to generate different ankle movements, including dorsiflexion, plantarflexion, inversion, and eversion. The effects of varying defect sizes on peak stress and its location were evaluated. RESULTS: The maximum stress on the talar cartilage increased as the area of the defect enlarged. Additionally, as the defect size of OLTs increased, the areas with peak stress on talar cartilage tended to move closer to where the injury was located. High stresses were present in the medial and lateral areas of the talus at the neutral position of the ankle joint. The concentrated stresses were mainly located in the anterior and posterior defect areas. The peak stress in the medial region was higher than on the lateral side. The order of peak stress from highest to lowest was dorsiflexion, internal rotation, inversion, external rotation, plantar flexion, and eversion. CONCLUSIONS: Osteochondral defect size and ankle joint movements significantly modulate the biomechanical features of the articular cartilage in osteochondral lesions of the talus. The progression of osteochondral lesions in a talus deteriorates the biomechanical well-being of the bone tissues of the talus.

Development of a Bionic Tube with High Bending-Stiffness Properties Based on Human Tibiofibular Shapes.

The human tibiofibular complex has undergone a long evolutionary process, giving its structure a high bearing-capacity. The distinct tibiofibular shape can be used in engineering to acquire excellent mechanical properties. In this paper, four types of bionic tubes were designed by extracting the dimensions of different cross-sections of human tibia-fibula. They had the same outer profiles, but different inner shapes. The concept of specific stiffness was introduced to evaluate the mechanical properties of the four tubes. Finite-element simulations and physical bending-tests using a universal testing machine were conducted, to compare their mechanical properties. The simulations showed that the type 2 bionic tube, i.e., the one closest to the human counterpart, obtained the largest specific-stiffness (ε = 6.46 × 104), followed by the type 4 (ε = 6.40 × 104) and the type 1 (ε = 6.39 × 104). The type 3 had the largest mass but the least stiffness (ε = 6.07 × 104). The specific stiffness of the type 2 bionic tube increased by approximately 25.8%, compared with that of the type 3. The physical tests depicted similar findings. This demonstrates that the bionic tube inspired by the human tibiofibular shape has excellent effectiveness and bending properties, and could be used in the fields of healthcare engineering, such as robotics and prosthetics.

Energy Flow and Functional Behavior of Individual Muscles at Different Speeds During Human Walking.

Understanding the distinct functions of human muscles could not only help professionals obtain insights into the underlying mechanisms that we accommodate compromised neuromuscular system, but also assist engineers in developing rehabilitation devices. This study aims to determine the contribution of major muscle and the energy flow in the human musculoskeletal system at four sub-phases (collision, rebound, preload, push-off) during the stance of walking at different speeds. Gait experiments were performed with three self-selected speeds: slow, normal, and fast. Muscle forces and mechanical work were calculated by using a subject-specified musculoskeletal model. The functions of individual muscles were characterized as four functional behaviors (strut, spring, motor, damper), which were determined based on the mechanical energy. The results showed that during collision, hip flexors (iliacus and psoas major) and ankle dorsiflexors (anterior tibialis) were the most dominant muscles in buffering the stride with energy absorption; during rebound, the posterior muscles (gluteus maximus, gastrocnemius, posterior tibialis, soleus) contributed the most to energy generation; during preload, energy for preparing push-off was mainly absorbed by the muscles surrounding knee (vastus, semimembranosus, semitendinosus); during push-off, ankle plantar flexors (gastrocnemius, soleus, posterior tibialis, peroneus muscles, flexor digitorum, flexor hallucis) mainly behaved to generate energy for forward propulsion. With increased walking speed, additional energy (almost 400%) from harder stride was mainly absorbed by the flexor muscles. Hip extensors and adductors transferred more energy (around 150%) to the distal segments during rebound. Soleus and gastrocnemius muscles generated more energy (about 75%) to the proximal segments for propulsion. Along with our previous study of joint-level energy analysis, these findings could assist better understanding of human musculoskeletal behaviors during locomotion and provide principles for the bio-design of related assistive devices from motors performance enhancement to rehabilitation such as exoskeleton and prosthesis.

"Metal-bone" scaffold for accelerated peri-implant endosseous healing.

Restoring bone defects caused by conditions such as tumors, trauma, or inflammation is a significant clinical challenge. Currently, there is a need for the development of bone tissue engineering scaffolds that meet clinical standards to promote bone regeneration in these defects. In this study, we combined the porous Ti6Al4V scaffold in bone tissue engineering with advanced bone grafting techniques to create a novel "metal-bone" scaffold for enhanced bone regeneration. Utilizing 3D printing technology, we fabricated a porous Ti6Al4V scaffold with an average pore size of 789 ± 22.69 µm. The characterization and biocompatibility of the scaffold were validated through in vitro experiments. Subsequently, the scaffold was implanted into the distal femurs of experimental animals, removed after 3 months, and transformed into a "metal-bone" scaffold. When this "metal-bone" scaffold was re-implanted into bone defects in the animals, the results demonstrated that, in comparison to a plain porous Ti6Al4V scaffold, the scaffold containing bone tissue achieved accelerated early-stage bone regeneration. The experimental group exhibited more bone tissue generation in the early stages at the defect site, resulting in superior bone integration. In conclusion, the "metal-bone" scaffold, containing bone tissue, proves to be an effective bone-promoting scaffold with promising clinical applications.

A Piezoresistive Sensor with High Sensitivity and Flexibility Based on Porous Sponge.

Chemical plating has recently been employed for the preparation of flexible piezoresistive sensors; however, plating solutions and processes that affect the sensitivity still need further exploration. In the study, a sponge-based flexible sensor with copper as its conductive material is prepared using electroless plating. The variation in sponge resistance and sensitivity changes with different plating times are studied. It is found that, with the increasing plating time, the conductivity increases and the resistance of sample will decrease. Moreover, the range of resistance difference will decrease under compression, thus the sensitivity decreases. Furthermore, the sensor's applications were assessed, verifying the practicability of the developed preparation method. This study may bring ideas for the new development of flexible pressure sensors.

In Vivo Analysis of the Dynamic Motion Stability Characteristics of Geese's Neck.

The goose's neck is an excellent stabilizing organ with its graceful neck curves and flexible movements. However, the stabilizing mechanism of the goose's neck remains unclear. This study adopts a dynamic in vivo experimental method to obtain continuous and accurate stable motion characteristics of the goose's cervical vertebra. Firstly, the results showed that when the body of a goose was separately moved back and forth along the Y direction (front and back) and Z direction (up and down), the goose's neck can significantly stabilize the head. Then, because of the limitation of the X-ray imaging area, the three-dimensional intervertebral rotational displacements for vertebrae C4-C8 were obtained, and the role that these five segments play in the stabilization of the bird's neck was analyzed. This study reveals that the largest range of the adjacent vertebral rotational movement is around the X-axis, the second is around the Y-axis, and the smallest is around the Z-axis. This kinematic feature is accord with the kinematic feature of the saddle joint, which allows the flexion/around X-axis and lateral bending/around Y-axis, and prevents axial rotation/around Z-axis.

Mechanisms and component design of prosthetic knees: A review from a biomechanical function perspective.

Prosthetic knees are state-of-the-art medical devices that use mechanical mechanisms and components to simulate the normal biological knee function for individuals with transfemoral amputation. A large variety of complicated mechanical mechanisms and components have been employed; however, they lack clear relevance to the walking biomechanics of users in the design process. This article aims to bridge this knowledge gap by providing a review of prosthetic knees from a biomechanical perspective and includes stance stability, early-stance flexion and swing resistance, which directly relate the mechanical mechanisms to the perceived walking performance, i.e., fall avoidance, shock absorption, and gait symmetry. The prescription criteria and selection of prosthetic knees depend on the interaction between the user and prosthesis, which includes five functional levels from K0 to K4. Misunderstood functions and the improper adjustment of knee prostheses may lead to reduced stability, restricted stance flexion, and unnatural gait for users. Our review identifies current commercial and recent studied prosthetic knees to provide a new paradigm for prosthetic knee analysis and facilitates the standardization and optimization of prosthetic knee design. This may also enable the design of functional mechanisms and components tailored to regaining lost functions of a specific person, hence providing individualized product design.

Non-invasive and quantitive analysis of flatfoot based on ultrasound.

Flatfoot is a common foot deformity that seriously affects the quality of life. The aim of this study is to develop an accurate and noninvasive method for the diagnosis of flatfoot based on B-mode ultrasound. In this study, 51 patients (the flatfoot group) and 43 healthy subjects (the control group) were included. The plantar fascia angle, a new measurement for use in the diagnosis of flatfoot is proposed, as determined using B-mode ultrasound. For comparison, the calcaneal pitch angle and medial cuneiform height were also measured using lateral X-radiography, based on traditional diagnostic methods. The intraclass correlation values of the plantar fascia angle, the calcaneal pitch angle, and the medial cuneiform height were all more than 0.95, and there is a moderate correlation (r = 0.51) between the medial cuneiform height and the calcaneal pitch angle, and an excellent correlation (r = 0.85) between the plantar fascia angle and the calcaneal pitch angle. The optimal cutoff value, sensitivity, and specificity for medial cuneiform height in flatfoot diagnosis were 12.8 mm, 93.0%, and 54.9%, respectively. The optimal cutoff value, sensitivity, and specificity for plantar fascia angle in flatfoot diagnosis were 9.8°, 97.7%, and 94.1%, respectively. The proposed plantar fascia angle has good sensitivity and specificity in diagnosing flatfoot, therefore supplying a new approach for the noninvasive diagnosis of flatfoot.

A bioinspired robotic knee with controlled joint surfaces and adjustable ligaments.

The knee joint plays a key role in kinematic and kinetic performances of pedestrain locomotion. The key role of meniscus with matched ligaments in joint stability and movability has not been fully explored in current robotic knee designs. We fabricate a bioinspired robotic knee based on a kinematic model of an anatomical knee in order to reveal the relationship between the meniscus, ligaments and their stability and movability, respectively. The kinematic model was built from magnetic resonance imaging of the human knee with generated contact profiles and customized ligament fibers. Then, the bioinspired knee was designed, and its dynamic stability was maintained by ligaments and specific contact profiles, which were acquired based on the kinematic model. Finally, a monopod robot with the bioinspired knee assembled was developed for dynamic testing. The results show that (1) a smooth rolling-sliding motion can be achieved with the addition of menisci and compatible ligaments; and (2) joint stiffness can be adjusted by changing the springs and activation lengths of ligament fibers. This study gives biomimetic insights into a new design of knee joint for a robotic/prosthetic leg.

Oxidization of benzo[a]pyrene by CYP102 in a novel PAHs-degrader Pontibacillus sp. HN14 with potential application in high salinity environment.

Benzo [a]pyrene (BaP) is a type of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) with potent carcinogenicity; however, there are limited studies on its degradation mechanism. Here, a strain of Pontibacillus sp. HN14 with BaP degradation ability was isolated from mangrove sediments in Dongzhai Port, Hainan Province. Our study showed that biodegradation efficiencies reached 42.15% after Pontibacillus sp. HN14 was cultured with 20 mg L-1 BaP as the sole carbon source for 25 days and still had degradability of BaP at a 25% high salinity level. Moreover, 9,10-dihydrobenzo [a]pyrene-7(8H)-one, an intermediate metabolite, was detected during BaP degradation in the HN14 strain. Genome analysis identified a gene encoding the CYP102(HN14) enzyme. The results showed that the E. coli strain with CYP102(HN14) overexpression could transfer BaP to 9,10-dihydrobenzo [a]pyrene-7(8H)-one with a conversion rate of 43.5%, indicating that CYP102(HN14) played an essential role in BaP degradation in Pontibacillus sp. HN14. Thus, our results provide a novel BaP biodegradation molecule, which could be used in BaP bioremediation in high salinity conditions. This study is the first to show that CYP102(HN14) had the BaP oxidization ability in bacteria. CYP102(HN14) could be essential in removing PAHs in saline-alkali soil and other high salt environments through enzyme immobilization.