Identification of Schizophrenia Susceptibility Loci in the Urban Taiwanese Population.

Background and Objectives: Genomic studies have identified several SNP loci associated with schizophrenia in East Asian populations. Environmental factors, particularly urbanization, play a significant role in schizophrenia development. This study aimed to identify schizophrenia susceptibility loci and characterize their biological functions and molecular pathways in Taiwanese urban Han individuals. Materials and Methods: Participants with schizophrenia were recruited from the Taiwan Precision Medicine Initiative at Tri-Service General Hospital. Genotype-phenotype association analysis was performed, with significant variants annotated and analyzed for functional relevance. Results: A total of 137 schizophrenia patients and 26,129 controls were enrolled. Ten significant variants (p < 1 × 10-5) and 15 expressed genes were identified, including rs1010840 (SOWAHC and RGPD6), rs11083963 (TRPM4), rs11619878 (LINC00355 and LINC01052), rs117010638 (AGBL1 and MIR548AP), rs1170702 (LINC01680 and LINC01720), rs12028521 (KAZN and PRDM2), rs12859097 (DMD), rs1556812 (ATP11A), rs78144262 (LINC00977), and rs9997349 (ENPEP). These variants and associated genes are involved in immune response, blood pressure regulation, muscle function, and the cytoskeleton. Conclusions: Identified variants and associated genes suggest a potential genetic predisposition to schizophrenia in the Taiwanese urban Han population, highlighting the importance of potential comorbidities, considering population-specific genetic and environmental interactions.

Ultrasonic technologies in imaging and drug delivery.

Ultrasonic technologies show great promise for diagnostic imaging and drug delivery in theranostic applications. The development of functional and molecular ultrasound imaging is based on the technical breakthrough of high frame-rate ultrasound. The evolution of shear wave elastography, high-frequency ultrasound imaging, ultrasound contrast imaging, and super-resolution blood flow imaging are described in this review. Recently, the therapeutic potential of the interaction of ultrasound with microbubble cavitation or droplet vaporization has become recognized. Microbubbles and phase-change droplets not only provide effective contrast media, but also show great therapeutic potential. Interaction with ultrasound induces unique and distinguishable biophysical features in microbubbles and droplets that promote drug loading and delivery. In particular, this approach demonstrates potential for central nervous system applications. Here, we systemically review the technological developments of theranostic ultrasound including novel ultrasound imaging techniques, the synergetic use of ultrasound with microbubbles and droplets, and microbubble/droplet drug-loading strategies for anticancer applications and disease modulation. These advancements have transformed ultrasound from a purely diagnostic utility into a promising theranostic tool.

Prevalence and patterns of tongue deformation in obstructive sleep apnea: A whole-night simultaneous ultrasonographic and polysomnographic study.

Tongue deformation during whole-night natural sleep in adult patients with obstructive sleep apnea has not been well evaluated. Through simultaneous ultrasonography and polysomnography during whole-night sleep, we examined the prevalence and patterns of tongue depth changes and their relationship with the severity of obstructive sleep apnea. Sixty consecutive eligible adults presenting with symptoms suggesting obstructive sleep apnea were enrolled. We observed that 88.4% (38/43) of patients with obstructive sleep apnea exhibited a significant increase in the maximum ultrasonographic tongue depth when hypopnea or apnea occurred during sleep. A mixed-model analysis of variance demonstrated that compared with patients with primary snoring or mild obstructive sleep apnea, those with moderate to severe obstructive sleep apnea have significantly greater maximum ultrasonographic tongue depth during respiratory events (p = .0047). We identified three different ultrasonographic patterns of tongue deformation, namely en bloc, tongue body and tongue base. Approximately 82% (27/33) of patients with moderate to severe obstructive sleep apnea demonstrated an en bloc tongue deformation. By contrast, 70% (19/27) of primary snorers or patients with mild obstructive sleep apnea showed a tongue body obstruction. Recognizing the prevalence and patterns of tongue deformation during sleep may provide insights into pathogenesis and treatment decisions in patients with obstructive sleep apnea. Future studies are warranted to verify the treatment results of various tongue procedures by using this approach.

Dynamic tongue area measurements in ultrasound images for adults with obstructive sleep apnea.

Obstructive sleep apnea is a chronic breathing disorder where the recursive collapse of the upper airway causes cessation of airflow during sleep. Quantitative assessments of dynamic tongue motion may provide a better understanding of the mechanism of obstructive sleep apnea. Tongue area changes, in submental ultrasound images recorded during wakefulness from normal breathing by the Müller manoeuvre, were tracked using the modified optical flow-based method. The results demonstrated that patients with obstructive sleep apnea had a larger mid-sagittal tongue area compared to the control group (during normal breathing, p = .004, during the Müller manoeuvre p = .005, and differences between normal breathing and the Müller manoeuvre, p = .01). Tongue area measurements were significantly different in patients with varying severity of OSA during normal breathing and during the Müller manoeuvre, and had differences between normal breathing and the Müller manoeuvre (p = .005, p = .008 and p = .03, respectively). Patients with moderate or severe obstructive sleep apnea exhibited minimal movement of the tongue. In contrast, normal controls and patients with mild obstructive sleep apnea demonstrated bidirectional tongue motions during a transition from normal breathing to the Müller manoeuvre in wakefulness. Identifying different patterns of deformation and displacement of the tongue may have potential in evaluating the presence and the pathogenesis of OSA.

High-Resolution Shear Wave Imaging of the Human Cornea Using a Dual-Element Transducer.

Estimating the corneal elasticity can provide valuable information for corneal pathologies and treatments. Ophthalmologic pathologies will invariably cause changes to the elasticity of the cornea. For example, keratoconus and the phototoxic effects of ultraviolet radiation usually increase the corneal elasticity. This makes a quantitative estimation of the elasticity of the human cornea important for ophthalmic diagnoses. The present study investigated the use of a proposed high-resolution shear wave imaging (HR-SWI) method based on a dual-element transducer (comprising an 8-MHz element for pushing and a 32-MHz element for imaging) for measuring the group shear wave velocity (GSWV) of the human cornea. An empirical Young's modulus formula was used to accurately convert the GSWV to Young's modulus. Four quantitative parameters, bias, resolution, contrast, and contrast-to-noise ratio (CNR), were measured in gelatin phantoms with two different concentrations (3% and 7%) to evaluate the performance of HR-SWI. The biases of gelatin phantoms (3% and 7%) were 5.88% and 0.78%, respectively. The contrast and CNR were 0.76, 1.31 and 3.22, 2.43 for the two-side and two-layer phantoms, respectively. The measured image resolutions of HR-SWI in the lateral and axial directions were 72 and 140 µm, respectively. The calculated phase SWV (PSWV) and their corresponding Young's modulus from six human donors were 2.45 ± 0.48 m/s (1600 Hz) and 11.52 ± 7.81 kPa, respectively. All the experimental results validated the concept of HR-SWI and its ability for measuring the human corneal elasticity.

Simultaneous recording of ultrasound and polysomnography during natural sleep in patients with obstructive sleep apnea: a pilot study.

Tongue base deformation may play a critical role in the phenomenon of obstructive sleep apnea, but polysomnography provides limited information regarding the effect of tongue motion during natural sleep. We reported on preliminary results of combining a novel ultrasound system and polysomnography for simultaneous recordings during natural sleep in volunteers and patients with obstructive sleep apnea. All participants underwent time-synchronized polysomnography and submental transcutaneous ultrasound examinations. The wearable ultrasound device detected the air-mucosal interface of the tongue surface and automatically determined the maximum tongue base thickness in real time. All participants reported no sensation of heat, no sign of skin allergy, and an average of mild disturbance after the ultrasound recordings. In the individual patient with obstructive sleep apnea, we demonstrated a significant difference (P < 0.001) between the ultrasonic tongue base thickness measured during eupnea and that measured during snoring, hypopnea and apnea. The ultrasonic tongue base thickness increased and remained before the occurrence of obstructive apnea. On average, increased tongue base thicknesses of 2.5 (4.1%), 6.0 (9.8%) and 7.7 mm (12.5%) are associated with snoring, hypopnea and apnea, respectively. Our present data demonstrate that simultaneous examination of ultrasonic tongue base thickness and polysomnography is feasible for prolonged recording during natural sleep. The proposed method also enables the detection of significant differences in ultrasonic tongue base thickness between eupnea and obstructive respiratory events evaluated using polysomnography. This novel technique can be used to generate hypotheses for subsequent investigations of the underlying mechanisms and individualized combined therapy for obstructive sleep apnea. CLINICAL TRIAL REGISTRATION: This study has been registered at the Chinese Clinical Trial Registry website with the registration number of ChiCTR-DDT-13003313. The date of registration was 13 July 2013.

A Coaxial Dual-element Focused Ultrasound Probe for Guidance of Epidural Catheterization: An Experimental Study.

Ultrasound guidance for epidural block has improved clinical blind-trial problems but the design of present ultrasonic probes poses operating difficulty of ultrasound-guided catheterization, increasing the failure rate. The purpose of this study was to develop a novel ultrasonic probe to avoid needle contact with vertebral bone during epidural catheterization. The probe has a central circular passage for needle insertion. Two focused annular transducers are deployed around the passage for on-axis guidance. A 17-gauge insulated Tuohy needle containing the self-developed fiber-optic-modified stylet was inserted into the back of the anesthetized pig, in the lumbar region under the guidance of our ultrasonic probe. The inner transducer of the probe detected the shallow echo signals of the peak-peak amplitude of 2.8 V over L3 at the depth of 2.4 cm, and the amplitude was decreased to 0.8 V directly over the L3 to L4 interspace. The outer transducer could detect the echoes from the deeper bone at the depth of 4.5 cm, which did not appear for the inner transducer. The operator tilted the probe slightly in left-right and cranial-caudal directions until the echoes at the depth of 4.5 cm disappeared, and the epidural needle was inserted through the central passage of the probe. The needle was advanced and stopped when the epidural space was identified by optical technique. The needle passed without bone contact. Designs of the hollow probe for needle pass and dual transducers with different focal lengths for detection of shallow and deep vertebrae may benefit operation, bone/nonbone identification, and cost.

An Acoustic Tracking Approach for Medical Ultrasound Image Simulator.

Ultrasound examinations are a standard procedure in the clinical diagnosis of many diseases. However, the efficacy of an ultrasound examination is highly dependent on the skill and experience of the operator, which has prompted proposals for ultrasound simulation systems to facilitate training and education in hospitals and medical schools. The key technology of the medical ultrasound simulation system is the probe tracking method that is used to determine the position and inclination angle of the sham probe, since this information is used to display the ultrasound images in real time. This study investigated a novel acoustic tracking approach for an ultrasound simulation system that exhibits high sensitivity and is cost-effective. Five air-coupled ultrasound elements are arranged as a 1D array in front of a sham probe for transmitting the acoustic signals, and a 5 × 5 2D array of receiving elements is used to receive the acoustic signals from the moving transmitting elements. Since the patterns of the received signals can differ for different positions and angles of the moving probe, the probe can be tracked precisely by the acoustic tracking approach. After the probe position has been determined by the system, the corresponding ultrasound image is immediately displayed on the screen. The system performance was verified by scanning three different subjects as image databases: a simple commercial phantom, a complicated self-made phantom, and a porcine heart. The experimental results indicated that the tracking and angle accuracies of the presented acoustic tracking approach were 0.7 mm and 0.5°, respectively. The performance of the acoustic tracking approach is compared with those of other tracking technologies.

A gated recurrent unit model based on ultrasound images of dynamic tongue movement for determining the severity of obstructive sleep apnea.

Obstructive sleep apnea (OSA) presents as a respiratory disorder characterized by recurrent upper pharyngeal airway collapse during sleep. Dynamic tongue movement (DTM) analysis emerges as a promising avenue for elucidating the pathophysiological underpinnings of OSA, thereby facilitating its diagnosis. Recent endeavors have utilized artificial intelligence techniques to categorize OSA severity leveraging electrocardiography and blood oxygen saturation data. Nonetheless, the integration of ultrasound (US) imaging of the tongue remains largely untapped in the development of machine learning models aimed at determining the severity of OSA. This study endeavors to bridge this gap by capturing US images of DTM dynamics during wakefulness, encompassing transitions from normal breathing (NB) to the performance of the Müller maneuver (MM) in a cohort of 53 patients. Leveraging the modified optical flow method (MOFM), the trajectories of patients' DTM were tracked, facililtating the extraction of 27 parameters vital for model training. These parameters encompassed nine-point lateral movement, nine-point axial movement, and nine-point total displacement of the tongue, resulting in a dataset of 186,030 samples. The gated recurrent unit (GRU) method, renowned for its efficacy in motion tracking, was employed for model development in this study. Validation of the developed model was conducted via stratified k-fold cross-validation (SCV). The systems' overall performance in classifying OSA severity, as quantified by mean accuracy (MA), yielded a value of 43.49%. This pilot investigation marks an exploratory endeavor into the utilization of artificial intelligence for the classification of OSA severity based on US images and dynamic movement patterns. This novel model holds potential to assist clinicians in categorizing OSA severity and guiding the selection of pertinent treatment modalities tailored to the individual needs of patients afflicted with OSA.

High-Resolution Tissue Doppler and Strain Imaging for Adult Zebrafish Myocardial Tissue Through Ultrafast High-Frequency Ultrasound Vector Doppler Estimation.

Zebrafish has been considered as an essential small-animal model for investigating the mechanism of heart regeneration. Due to the small size of zebrafish heart, high-frequency ultrasound (HFUS) imaging is often required for in vivo evaluations of its dynamic functions. Although commercial HFUS systems are available for myocardial velocity and strain measurement, only the outer myocardial region can be quantified due to the complex structure of zebrafish heart. In this study, a high-resolution 2-D myocardial tissue Doppler and strain imaging based on ultrafast HFUS imaging was developed for zebrafish heart imaging during heart regeneration. The cardiac flow region was first extracted to recognize the myocardial region, and the myocardial velocity and strain were then determined through vector Doppler estimation. Adult AB-line zebrafish was used for in vivo experiments, and cryoinjury was induced in the apical region of the heart. Both the myocardial velocity and strain of the whole ventricle after cryoinjury were directly visualized over 28 days. Myocardial velocity (during later diastolic motion) and strain, respectively, were significantly decreased (anterior wall: -2.0 mm/s and -3.3%; apical region: -2.0 mm/s and -4.5%; and posterior wall (PW): -1.7 mm/s and -4.3%) at the first three days after cryoinjury, which indicates weak myocardial beating due to heart injury. However, these all returned to the baseline values at 14 days after cryoinjury. All of the experimental results indicate that the proposed method is a useful tool for heart regeneration studies in adult zebrafish. In particular, it allows for the noninvasive evaluation of regional dynamic heart function.

Estimating the viscoelastic anisotropy of human skin through high-frequency ultrasound elastography.

BACKGROUND: The skin is the largest organ of the human body and serves distinct functions in protecting the body. The viscoelastic properties of the skin play a key role in supporting the skin-healing process, also it may be changed due to some skin diseases. PROPOSE: In this study, high-frequency ultrasound (HFUS) elastography based on a Lamb wave model was used to noninvasively assess the viscoelastic anisotropy of human skin. METHOD: Elastic waves were generated through an external vibrator, and the wave propagation velocity was measured through 40 MHz ultrafast HFUS imaging. Through the use of a thin-layer gelatin phantom, HFUS elastography was verified to produce highly accurate estimates of elasticity and viscosity. In a human study involving five volunteers, viscoelastic anisotropy was assessed by rotating an ultrasound transducer 360°. RESULTS: An oval-shaped pattern in the elasticity of human forearm skin was identified, indicating the high elastic anisotropy of skin; the average elastic moduli were 24.90 ± 6.63 and 13.64 ± 2.67 kPa along and across the collagen fiber orientation, respectively. The average viscosity of all the recruited volunteers was 3.23 ± 0.93 Pa·s. CONCLUSIONS: Although the examined skin exhibited elastic anisotropy, no evident viscosity anisotropy was observed.

Wearable Ultrasound Imaging Device for Dynamic Dual-Direction Shear Wave Elastography of Shoulder Muscle.

The shoulder is the most mobile joint in the human body, thus requiring intricate coordination of adjacent muscles. Patients suffered from rotator cuff muscle injuries have several typical symptoms including shoulder pain and difficulty raising the arm, thus reducing work efficiency and compromising the quality of life. Ultrasound has been used widely for shoulder soft tissue imaging as well as ultrasound elastography was introduced in shoulder examination for the dilemma of treating degenerative rotator cuff tears. However, most of the ultrasound examination was performed under a static condition. Providing dynamic information from shoulder muscle is important in clinical applications because the pains sometimes come from various positions of the shoulder during moving. In this study, a customized wearable T-shaped ultrasound transducer (128 + 128 elements) was proposed for shoulder dual-direction shear wave elastography (DDSWE), which provides the SWE for both longitudinal (SW along the muscle fiber) and transverse (SW cross the muscle fiber) directions dynamically. An optical tracking system was synchronized with an ultrasound imaging system to capture shoulder movements in 3-D space with their corresponding ultrasound images. The performance of DDSWE and the accuracy of optical tracking were verified by phantom experiments. Human studies were carried out by volunteers as they are moving their arms. The experimental results show that the bias and precision for the proposed DDSWE in elastic phantom were about 6% and 1.2% for both directions, respectively. A high accuracy of optical tracking was observed using a 3-D motor stage experimental setup. Human experiments show that the shear wave velocities (SWVs) were increased with the angles of shoulder abduction, and the average transverse and longitudinal SWVs were increased from 2.24 to 3.35 m/s and 2.95 to 5.95 m/s with abduction angle from 0° to 60°, respectively, which they are anisotropic-dependent. All the experimental results indicate that the proposed wearable ultrasound DDSWE can quantify the mechanical properties of shoulder muscles dynamically, thereby helping surgeons and physical therapists determine whether the intensity of rehabilitation shoulder be tuned down or escalated in the future.

Increasing the Maximum Detectable Flow Velocity in High-Frequency Ultrasound Vector Doppler Imaging.

High-frequency ultrasound (HFUS; >30 MHz) Doppler imaging has been widely used in the imaging of small animals and humans because of its high resolution. Vector Doppler imaging (VDI) has certain advantages for visualizing complex flow patterns independent of the Doppler angle. However, no commercial HFUS VDI system is currently available; therefore, several studies have connected an ultrasound research platform (Verasonics Vantage 256) with an HFUS array transducer for HFUS VDI. Unfortunately, the maximum frame rate of this system is only 10 kHz at an operational frequency of 40 MHz because of limitations related to data transmission hardware, thereby restricting the maximum detectable velocity of Doppler measurements. To address this drawback, in the present study, an electrocardiography (ECG)-gating-based HFUS VDI system was developed to avoid Doppler flow aliasing in data acquisition by ultrasound research platform at its maximum frame rate of 10 kHz. The developed method aligns all tilted plane waves with the ECG R-wave, which avoids the trade-off between frame rate and tilted angles number in conventional VDI. The performance of the proposed data acquisition method in HFUS VDI was verified using a steady-flow phantom, for which estimation errors were less than 10% under different flow settings. In animal studies, peak flow velocities in the carotid artery, left ventricle, and aortic arch of wild-type mice were measured (approximately 55, 655, and 765 mm/s, respectively). Also, the HFUS VDI from the mitral regurgitation mice model was obtained to present the complex flow patterns through the proposed method. In contrast to the conventional method, no Doppler aliasing occurs in the proposed method because the frame rate is sufficient. The experimental results indicate the developed HFUS VDI has the potential to become a useful tool for vector flow visualization in small animals, even under a high flow velocity.

High-Performance PMN-PT Single-Crystal-Based 1-3 Composite Transducer Integrated with a Biopsy Needle.

To address the need for high-resolution imaging in lung nodule detection and overcome the limitations of the shallow imaging depth associated with high-frequency ultrasound and the complex structure of lung tissue, we successfully integrated 50 MHz ultrasound transducers with 18-gauge biopsy needles. Featuring a miniaturized size of 0.6 × 0.5 × 0.5 mm3, the 50 MHz micromachined 1-3 composite transducer was tested to perform mechanical scanning of a nodule within a lung-tissue-mimicking phantom in vitro. The high-frequency transducer demonstrated the ability to achieve imaging with an axial resolution of 30 µm for measuring nodule edges. Moreover, the integrated biopsy needle prototype exhibited high accuracy (1.74% discrepancy) in estimating nodule area compared to actual dimensions in vitro. These results underscore the promising potential of biopsy-needle-integrated transducers in enhancing the accuracy of endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNA) for clinical applications.

Visualization of Human Hand Tendon Mechanical Anisotropy in 3-D Using High- Frequency Dual-Direction Shear Wave Imaging.

High-resolution ultrasound shear wave elastography has been used to determine the mechanical properties of hand tendons. However, because of fiber orientation, tendons have anisotropic properties; this results in differences in shear wave velocity (SWV) between ultrasound scanning cross sections. Rotating transducers can be used to achieve full-angle scanning. However, this technique is inconvenient to implement in clinical settings. Therefore, in this study, high-frequency ultrasound (HFUS) dual-direction shear wave imaging (DDSWI) based on two external vibrators was used to create both transverse and longitudinal shear waves in the human flexor carpi radialis tendon. SWV maps from two directions were obtained using 40-MHz ultrafast imaging at the same scanning cross section. The anisotropic map was calculated pixel by pixel, and 3-D information was obtained using mechanical scanning. A standard phantom experiment was then conducted to verify the performance of the proposed HFUS DDSWI technique. Human studies were also conducted where volunteers assumed three hand postures: relaxed (Rel), full fist (FF), and tabletop (TT). The experimental results indicated that both the transverse and longitudinal SWVs increased due to tendon flexion. The transverse SWV surpassed the longitudinal SWV in all cases. The average anisotropic ratios for the Rel, FF, and TT hand postures were 1.78, 2.01, and 2.21, respectively. Both the transverse and the longitudinal SWVs were higher at the central region of the tendon than at the surrounding region. In conclusion, the proposed HFUS DDSWI technique is a high-resolution imaging technique capable of characterizing the anisotropic properties of tendons in clinical applications.

Using a Flipped Classroom to Compare 2 Ultrasonography Operating Methods to Improve Practice in Ultrasound Detection Acupuncture.

Objective: Ultrasound (US) detection acupuncture (UDA) is an innovative acupuncture technique that uses ultrasonography (USG) to detect the depth of the lung before performing acupuncture on the points around the chest to avoid puncturing the lungs. For acupuncturists to use UDA appropriately, it is crucial to have a good operating method to identify the pleura with USG. This study compared 2 US operating methods through active learning in a "flipped classroom" setting for acupuncture students. Materials and Methods: Students and interns were recruited to complete the UDA flipped classroom course and evaluate the operations of 2 US methods on either of 2 simulation models: (1) a single B-mode or (2) a combined M-mode + B-mode. Participants were interviewed and satisfaction surveys were administered to obtain feedback. Results: A total of 37 participants completed the course and evaluations. The combined mode had better measurement accuracy, acupuncture safety, and operating time (P < 0.05), and no pneumothoraxes occurred. Among both participant groups, the combined mode allowed the student group to learn quickly and the intern group to become more proficient. Both interviews and satisfaction surveys yielded positive feedback. Conclusions: Using a combined mode for UDA can improve its performance greatly. The combined mode is definitely helpful for learning and promotion of UDA.

Estimation of Mouse Carotid Arterial Wall Shear Stress Using High-Frequency Ultrasound Imaging.

Wall shear stress (WSS) is a crucial hemodynamic factor that promotes atherosclerosis (plaque) development in arteries; although the relationship between WSS and arterial atherosclerosis has been explored in many animal studies, it is not fully understood. No suitable tool, however, exists for rapidly estimating dynamic WSS in small-animal studies. This study proposes a 40-MHz high-frequency ultrasound (HFUS) imaging system for dynamic WSS estimation based on mouse carotid artery blood flow velocity gradient measurements by vector Doppler imaging (VDI). Aliasing reduces the accuracy of Doppler measurements, which can be prevented by increasing the imaging frame rate. Conventionally, imaging is performed at two tilted angles by alternating between the angles; in the proposed method, the frame rate was doubled by imaging at each tilted angle sequentially and by then temporally aligning the sequences based on pulsatile flow characteristics. Velocity estimation using this method had low errors for both a steady-flow straight-tube and pulsatile flow 60%-stenosis phantom. The method was tested for wild-type (WT) C57BL/6 mice at 16 weeks old and apolipoprotein E knockout (ApoE KO) mice at 16 and 24 weeks old; differences in time-averaged and oscillatory WSS were observed, and histology confirmed that the 24-week ApoE KO mice with the highest oscillatory WSS had the greatest plaque formation. The proposed HFUS WSS imaging method can predict the location and extent of plaque development; thus, this method is useful for small-animal studies investigating the WSS effect on atherosclerotic plaque development.