Differences of Host Country-Destination Image Assessment for International Students According to Risk Perception in COVID-19 Tourism.

The study examined the comparative assessment of destination image according to the perception of COVID-19 and travel risk among international students. The online survey was administered to 786 international students enrolled in universities in Korea. Cluster analysis was performed, and three distinct clusters were identified based on risk perception. Destination image attributes were generated into four underlying dimensions: social environment, tourist environment, destination environment, and imagery, using the exploratory factor analysis. A revised Importance-Performance Analysis (IPA) method was utilized to assess the destination image of Korea and used to compare the expected performance of the attributes on each cluster. In addition, a revised IPA grid of each cluster was presented to unveil the satisfaction attributes of the destination image and suggest communication strategies. This study provides practical insights to destination marketers and organizations to design marketing strategies for international students. Further practical and theoretical implications are discussed.

FMCW Radar Estimation Algorithm with High Resolution and Low Complexity Based on Reduced Search Area.

We propose a frequency-modulated continuous wave (FMCW) radar estimation algorithm with high resolution and low complexity. The fast Fourier transform (FFT)-based algorithms and multiple signal classification (MUSIC) algorithms are used as algorithms for estimating target parameters in the FMCW radar systems. FFT-based and MUSIC algorithms have tradeoff characteristics between resolution performance and complexity. While FFT-based algorithms have the advantage of very low complexity, they have the disadvantage of a low-resolution performance; that is, estimating multiple targets with similar parameters as a single target. On the other hand, subspace-based algorithms have the advantage of a high-resolution performance, but have a problem of very high complexity. In this paper, we propose an algorithm with reduced complexity, while achieving the high-resolution performance of the subspace-based algorithm by utilizing the advantages of the two algorithms; namely, the low-complexity advantage of FFT-based algorithms and the high-resolution performance of the MUSIC algorithms. The proposed algorithm first reduces the amount of data used as input to the subspace-based algorithm by using the estimation results obtained by FFT. Secondly, it significantly reduces the range of search regions considered for pseudo-spectrum calculations in the subspace-based algorithm. The simulation and experiment results show that the proposed algorithm achieves a similar performance compared with the conventional and low complexity MUSIC algorithms, despite its considerably lower complexity.

High-Efficiency Super-Resolution FMCW Radar Algorithm Based on FFT Estimation.

This paper proposes a high-efficiency super-resolution frequency-modulated continuous-wave (FMCW) radar algorithm based on estimation by fast Fourier transform (FFT). In FMCW radar systems, the maximum number of samples is generally determined by the maximum detectable distance. However, targets are often closer than the maximum detectable distance. In this case, even if the number of samples is reduced, the ranges of targets can be estimated without degrading the performance. Based on this property, the proposed algorithm adaptively selects the number of samples used as input to the super-resolution algorithm depends on the coarsely estimated ranges of targets using the FFT. The proposed algorithm employs the reduced samples by the estimated distance by FFT as input to the super resolution algorithm instead of the maximum number of samples set by the maximum detectable distance. By doing so, the proposed algorithm achieves the similar performance of the conventional multiple signal classification algorithm (MUSIC), which is a representative of the super resolution algorithms while the performance does not degrade. Simulation results demonstrate the feasibility and performance improvement provided by the proposed algorithm; that is, the proposed algorithm achieves average complexity reduction of 88% compared to the conventional MUSIC algorithm while achieving its similar performance. Moreover, the improvement provided by the proposed algorithm was verified in practical conditions, as evidenced by our experimental results.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Anti-melanogenic effects of hot-water extracts from Torreya nucifera via MAPKs and cAMP signaling pathway on B16F10 cells.

Torreya nucifera is an evergreen tree in the family Taxaceae, the seeds, leaves, and stems of which have long been used as edible products and herbal medicines in Korea. Previous studies of biological activity have shown that T. nucifera has antioxidant and anti-inflammatory effects. However, the effect of T. nucifera leaves on melanogenesis are yet to be studied. In this investigation, we used B16F10 melanoma cells to test the efficacy of T. nucifera leaf hot water extract (TLWE). α-melanocyte stimulating hormone (α-MSH) stimulated B16F10 melanoma cells were treated with various concentrations of TLWE (50, 100, and 200 µg/mL). The results showed that TLWE reduced the melanin content and cellular tyrosinase activity in a concentration-dependent manner. It also inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) signaling pathway. The compounds catechin and ρ-coumaric acid, which are known to have a whitening effect on skin, were detected by HPLC analysis. These results suggest that TLWE has an anti-melanogenic effect. In addition, the safety of TLWE was tested. The results of the skin irritation test showed that TLWE is harmless to the human skin, even at higher concentrations than those used in the experiment. Therefore, we suggest that the water extract of T. nucifera leaves has potential for use as a skin-whitening agent.

Establishment of Conditions for Long-Term Maintenance of Primary Embryonic Cell Cultures from Olive Flounder Paralichthys olivaceus.

Primary cell culture is a sufficient method frequently used to study the cellular properties and mechanisms of isolated cells in a controlled environment. In this study, an embryonic cell line (FGBC8) derived from the blastula stages of embryos of olive flounder Paralichthys olivaceus was developed. Furthermore, conditions for optimal long-term maintenance of this primary embryonic cell culture were investigated. Morphologically, FGBC8 cells were composed primarily of epithelial-like cells. FGBC8 cells were subcultured for >160 passages over ~830 days. The doubling time of FGBC8 cells was 73.8 h, and the modal diploid chromosome number was 48. FGBC8 cells transfected with green fluorescence protein (GFP)-expression plasmid exhibited a strong signal 48 h after transfection. Consequently, we demonstrated that fish serum is a crucial supplement for the long-term survival and maintenance of comparable morphology in these primary embryonic cells. Our results can be used as a guide for primary embryonic cell cultures for other fish species and may be useful for cell biotechnological applications.

Low-Complexity MUSIC-Based Direction-of-Arrival Detection Algorithm for Frequency-Modulated Continuous-Wave Vital Radar.

This paper proposes a low complexity multiple-signal-classifier (MUSIC)-based direction-of-arrival (DOA) detection algorithm for frequency-modulated continuous-wave (FMCW) vital radars. In order to reduce redundant complexity, the proposed algorithm employs characteristics of distance between adjacent arrays having trade-offs between field of view (FOV) and resolution performance. First, the proposed algorithm performs coarse DOA estimation using fast Fourier transform. On the basis of the coarse DOA estimation, the number of channels as input of the MUSIC algorithm are selected. If the estimated DOA is smaller than 30°, it implies that there is an FOV margin. Therefore, the proposed algorithm employs only half of the channels, that is, it is the same as doubling the spacing between arrays. By doing so, the proposed algorithm achieves more than 40% complexity reduction compared to the conventional MUSIC algorithm while achieving similar performance. By experiments, it is shown that the proposed algorithm despite the low complexity is enable to distinguish the adjacent DOA in a practical environment.

7,8-Dimethoxycoumarin stimulates melanogenesis via MAPKs mediated MITF upregulation.

Background: Melanin in the skin is the defense against the harmful UV radiation, which is considered as one of the major risk factors for skin cancer. The compound 7,8-dimethoxycoumarin (DMC, C11H10O4), a natural coumarin molecule present in several medicinal plants, possesses antioxidant and anti-inflammatory activities. However, the mechanism underlying its effects on melanogenesis in melanocytes is unclear. Therefore, we investigated the effect of DMC on melanogenesis activation in B16F10 melanoma cells. Methods: We examined the cytotoxic range of DMC on B16F10 melanoma cells and increased effects of melanogenesis, and intracellular tyrosinase activity. In addition, regulation mechanisms were assessed by Western blot analysis. Results: The results showed that DMC significantly increased melanin content and tyrosinase activity in the cells without being cytotoxic. Furthermore, DMC stimulated the expression of tyrosinase, TRP-1, TRP-2, and MITF thereby activating melanin production and Akt phosphorylation was increased in the Akt signaling pathway. on the contrary, interfering with the phosphorylation of ERK in the MAPKs pathway. Conclusions: These results suggest that DMC may serve as a candidate for potential melanin-producing activator and anti-gray hair applications.

First report of cathepsin E in a teleost (Korean rose bitterling, Rhodeus uyekii): Molecular characterisation and tissue distribution.

We isolated and characterised a cDNA encoding the aspartic protease cathepsin E (CTSE) in Korean rose bitterling, Rhodeus uyekii. The full-length Rhodeus uyekii CTSE (RuCTSE) cDNA (1396 bp) contains an open reading frame of 1218 bp, encoding 405 amino acids. Alignment of multiple CTSE protein sequences revealed that two of the aspartyl protease active site residues and a disulphide bond were well-conserved among the other CTSE sequences. Phylogenetic analysis revealed that RuCTSE is most closely related to freshwater fish cathepsin E. RuCTSE is widely expressed in the liver, spleen, ovary, testis, brain, eye, intestine, muscle, fin, stomach, and kidney. This first report of teleost CTSE will provide important information related to the identification of other cathepsin E genes in various fish species and will serve as a useful molecular tool to help clarify biological activities in other teleosts.

Low-Complexity Joint Range and Doppler FMCW Radar Algorithm Based on Number of Targets.

A low-complexity joint range and Doppler frequency-modulated continuous wave (FMCW) radar algorithm based on the number of targets is proposed in this paper. This paper introduces two low-complexity FMCW radar algorithms, that is, region of interest (ROI)-based and partial discrete Fourier transform (DFT)-based algorithms. We find the low-complexity condition of each algorithm by analyzing the complexity of these algorithms. From this analysis, it is found that the number of targets is an important factor in determining complexity. Based on this result, the proposed algorithm selects a low-complexity algorithm between two algorithms depending the estimated number of targets and thus achieves lower complexity compared two low-complexity algorithms introduced. The experimental results using real FMCW radar systems show that the proposed algorithm works well in a real environment. Moreover, central process unit time and count of float pointing are shown as a measure of complexity.

Horse Oil Mitigates Oxidative Damage to Human HaCaT Keratinocytes Caused by Ultraviolet B Irradiation.

Horse oil products have been used in skin care for a long time in traditional medicine, but the biological effects of horse oil on the skin remain unclear. This study was conducted to evaluate the protective effect of horse oil on ultraviolet B (UVB)-induced oxidative stress in human HaCaT keratinocytes. Horse oil significantly reduced UVB-induced intracellular reactive oxygen species and intracellular oxidative damage to lipids, proteins, and DNA. Horse oil absorbed light in the UVB range of the electromagnetic spectrum and suppressed the generation of cyclobutane pyrimidine dimers, a photoproduct of UVB irradiation. Western blotting showed that horse oil increased the UVB-induced Bcl-2/Bax ratio, inhibited mitochondria-mediated apoptosis and matrix metalloproteinase expression, and altered mitogen-activated protein kinase signaling-related proteins. These effects were conferred by increased phosphorylation of extracellular signal-regulated kinase 1/2 and decreased phosphorylation of p38 and c-Jun N-terminal kinase 1/2. Additionally, horse oil reduced UVB-induced binding of activator protein 1 to the matrix metalloproteinase-1 promoter site. These results indicate that horse oil protects human HaCaT keratinocytes from UVB-induced oxidative stress by absorbing UVB radiation and removing reactive oxygen species, thereby protecting cells from structural damage and preventing cell death and aging. In conclusion, horse oil is a potential skin protectant against skin damage involving oxidative stress.

A Low-Complexity FMCW Surveillance Radar Algorithm Using Two Random Beat Signals.

This paper proposes a low-complexity frequency-modulated continuous wave (FMCW) surveillance radar algorithm using random dual chirps in order to overcome the blind-speed problem and reduce the computational complexity. In surveillance radar algorithm, the most widely used moving target indicator (MTI) algorithm is proposed to effectively remove clutter. However, the MTI algorithm has a so-called 'blind-speed problem' that cannot detect a target of a specific velocity. In this paper, we try to solve the blind-speed problem of MTI algorithm by randomly selecting two beat signals selected for MTI for each frame. To further reduce the redundant complexity, the proposed algorithm first performs one-dimensional fast Fourier transform (FFT) for range detection and performs multidimensional FFT only when it is determined that a target exists at each frame. The simulation results show that despite low complexity, the proposed algorithm detects moving targets well by avoiding the problem of blind speed. Furthermore, the effectiveness of the proposed algorithm was verified by performing an experiment using the FMCW radar system in a real environment.

Starvation-induced Physiological Responses and RNA/DNA Ratios in Rock Bream, Oplegnathus fasciatus, and Olive Flounder, Paralichthys olivaceus.

In a 12-week experiment, the rock bream, Oplegnathus fasciatus, and olive flounder, Paralichthys olivaceus, were investigated to determine the effects of starvation on their physiological parameters. The protein and DNA contents of the starved fish were significantly higher than the initial values and those of the fed fish. The RNA contents and RNA/DNA ratios of the fed fish were significantly higher than those of the other groups (P<0.05). The hematocrit, hemoglobin, red blood cells (RBC), and mean corpuscular volume (MCV) of the fed rock bream were significantly higher than at baseline (P<0.05), whereas the mean corpuscular hemoglobin concentration (MCHC) of the fed fish was lower than at baseline (P<0.05). The hematocrit, hemoglobin, RBC, and MCHC of the starved group were significantly lower than the baseline values, whereas the MCV of the starved group was significantly higher than the baseline value (P<0.05). No significant difference in alanine aminotransferase was observed between the fed fish and baseline, whereas the starved fish value was significantly higher than the baseline value (P<0.05). There were no significant differences in cortisol levels. However, the glucose level in the fed group was significantly higher than the baseline level and that in the starved group was significantly lower than the baseline level (P<0.05).

Landmark-based Morphometric and Meristic Analysis of Serranidae.

The landmark-based morphometric and meristic analysis of the kelp grouper (Epinephelus bruneus), red spotted grouper (E. akaara) and seven-banded grouper (E. septemfasciatus) were performed to compare the differentiation of overall body shape and structure. The measurements of the morphometric dimensions were observed in 25 parts (truss dimension: 16 parts; head part dimension: 9 parts) of 38 morphometric dimensions and also meristic differences observed in 3 parts (dorsal fin, anal fin and caudal fin) of 6 meristic counts (P < 0.05). Observed morphometric characteristics primarily involved in truss and head part dimension, kelp grouper have larger values in caudal part of truss dimension, kelp grouper, red spotted grouper and seven-banded grouper have similar values in pectoral part of truss dimension, in addition to, results of head part dimension showed that red spotted grouper have smaller values in overall dimensions (P < 0.05). As meristic characteristics, kelp grouper have more number of anal fin rays than other fish, red spotted grouper have more number of dorsal soft rays than other fish, and seven spotted grouper have more number of anal soft rays, and caudal fin rays than other fish (P < 0.05). Photographed under the x-ray, kelp grouper have the most curved vertebral column and largest swim bladder than other fishes (P < 0.05). Our results of this study confirmed that 3 subfamily fishes adequately can distinguish with external body shape, and we hope that the results of our study could be used to identify in Serranidae family as taxonomical parameters.

Morphological Development of Embryo, Larvae and Juvenile in Yellowtail Kingfish, Seriola lalandi.

This study monitored the morphological development of embryo, larvae and juvenile yellowtail kingfish, Seriola lalandi, for their aquaculture. The fertilized eggs obtained by natural spawning were spherical shape and buoyant. Fertilized eggs were transparent and had one oil globule in the yolk, with an egg diameter of 1.35 ± 0.04 mm and an oil globule diameter of 0.32 ± 0.02 mm. The fertilized eggs hatched 67-75 h after fertilization in water at 20 ± 0.5°C. The total length (TL) of the hatched larvae was 3.62 ± 0.16 mm. During hatching, the larvae, with their mouth and anus not yet opened. The yolk was completely absorbed 3 days after hatching (DAH), while the TL of post-larvae was 4.72 ± 0.07 mm. At 40 DAH, the juveniles had grown to 30.44 ± 4.07 mm in TL, body depth increased, the body color changed to a black, yellow, and light gray-blue color, and 3-4 vertical stripes appeared. At 45 DAH, the juveniles were 38.67 ± 5.65 mm in TL and 10.10 ± 0.94 mm in body depth. The fish were green with a light orange color, with 7 faint green-brown stripes on the sides of their body. At 87 DAH, the juveniles had grown to 236.11 mm in TL, 217.68 mm in fork length, and 136.5 g in weight. The fish resembled their adult form, with a light yellow-green body color, loss of the pattern on the sides of their body, and a yellow coloration at the tip of the caudal fin.

Management of Sexual Maturation and Natural Spawning of Captive- Reared Yellowtail Kingfish, Seriola lalandi, in an Indoor Rearing Tank.

This study describes results on sexual maturation and characteristics of natural spawned eggs to develop a method for the production of stable, healthy fertilized eggs from captive-reared yellowtail kingfish, Seriola lalandi. A total of 59 yellowtail kingfish were captured off the coast of Jeju Island, after which the broodstock was cultured in indoor culture tank (100 m(3)) until they were 6.1-14.9 kg in body weight. As part of the rearing management for induced sex maturation, the intensity of illumination was maintained at 130 lux. The photoperiod (light/dark; L/D) was set to a 12 L/12 D from October 2013 to January 2014, and 15 L/9 D from February 2014 to June 2014. Feeds comprised mainly EP (Extruded Pellets), with squid cuttlefish added for improvement of egg quality, and was given from April to June 2014. The first spawning of yellowtail kingfish occurred in May 3, 2014, at a water temperature of 17.0°C. Spawning continued until June 12, 2014, with the water temperature set at 20.5°C. Time of spawning was 26 times at this period. The total number of eggs that spawned during the spawning period was 4,449×10(3). The buoyant rate of spawning eggs and fertilization rate of buoyant eggs during the spawned period were 76.1% and 100%, respectively. The diameters of the egg and oil globule were 1.388 ± 0.041 mm and 0.378 ± 0.029 mm, respectively, which was higher in early eggs than in those from late during the spawned period.

Effects of light-emitting diode spectra on the vertebrate ancient long opsin and gonadotropin hormone in the goldfish Carassius auratus.

We determined the molecular mechanism underlying the environmental (photoperiodic) regulation of sexual maturation in fish, we examined the expression of sexual maturation-related hormones and vertebrate ancient long opsin (VAL-opsin) in goldfish (Carassius auratus) exposed to different light spectra (red and green light-emitting diodes). We further evaluated the effect of exogenous gonadotropin hormone (GTH) on the expression of VAL-opsin under different light conditions. Our results demonstrated that the expression of GTHs was higher in the fish exposed to green light, and VAL-opsin levels were increased in the fish receiving GTH injection. Therefore, we have uncovered a molecular mechanism underlying the environmental (light)-induced trigger for sexual maturation: VAL-opsin is activated by green light and GTH, which promotes the expression of sexual maturation genes.

Effects of different light wavelengths from LEDs on oxidative stress and apoptosis in olive flounder (Paralichthys olivaceus) at high water temperatures.

We investigated how different light spectra affect thermal stress in olive flounder (Paralichthys olivaceus), using light emitting diodes (LEDs; blue, 450 nm; green, 530 nm; red, 630 nm) at two intensities (0.3 and 0.5 W/m(2)) at relatively high water temperatures (25 and 30 °C, compared to a control condition of 20 °C). We measured the expression and activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and the levels of plasma hydrogen peroxide (H2O2) and lipid peroxidation (LPO). Furthermore, the levels and mRNA expression of caspase-3 were measured, and terminal transferase dUTP nick end labeling (TUNEL) assays of liver and comet assays were performed. The expression and activity of antioxidant enzymes, as well as plasma H2O2 and LPO levels were significantly higher after exposure to high temperatures, and significantly lower after exposure to green and blue light. Caspase-3 levels and mRNA expression showed a similar pattern. The TUNEL assay showed that apoptosis markedly increased at higher water temperatures, compared with the 20 °C control. In contrast, green light irradiation decreased apoptosis rate. Furthermore, the comet assays showed that nuclear DNA damage was caused by thermal stress, and that green light irradiation played a role in partially preventing this damage. Overall, these results suggest that light with green and blue wavelengths can reduce both high temperature-induced oxidative stress and apoptosis, and that particularly green light is efficient for this. Therefore, green light can play a role in protecting in olive flounder from thermal stress damage.

Cytogenetic study of diploid and induced tetraploid in Korean rose bitterling, Rhodeus uyekii.

In this study, we induced tetraploidy in Korean rose bitterling, Rhodeus uyekii, by applying various hydrostatic pressure shock conditions. Tetraploidy was not induced under 4500 psi pressure treatment in any experimental group. Instead, the induction rate of tetraploidy was highest under 7500 psi hydrostatic pressure treatment. As a result, when the processing method was similar and as the process time increased, the induction rate of each experimental group increased; however, there was no significant difference (P > 0.05). The production rate was 3.1 %, which was highest in all experimental groups exposed to 6000 psi for 10 min after being fertilized for 100 min. The production rate was highest in the experimental groups treated with hydrostatic pressure alone, whereas the production rate was lowest in groups treated under hydrostatic pressure with chemical treatment. The abnormal rate of all experimental groups treated with 7500 psi for 20 min was very high, at about 5 %. Based on these studies, only hydrostatic pressure shock was considered effective at inducing tetraploidy based on the calculated hatching, abnormal, and induction rates. The most effective condition for inducing tetraploidy was 6000 psi of hydrostatic pressure shock for 10 min after being fertilized for 100 min. The chromosome number of the induced tetraploid Korean rose bitterling was 4n = 96, while that of the diploid was 2n = 48. In the diploid, there were 1 or 2 nucleoli in the cells, whereas the induced tetraploids contained 1, 2, 3, or 4. The DNA content of tetraploids and diploids were 3.68 ± 0.009 pg/nucleus and 1.84 ± 0.019 pg/nucleus, respectively, according to flow cytometric analysis. The DNA content and chromosome number of the tetraploids were twice that of the diploids.

Effects of melatonin injection or green-wavelength LED light on the antioxidant system in goldfish (Carassius auratus) during thermal stress.

We tested the mitigating effects of melatonin injections or irradiation from green-wavelength light-emitting diodes (LEDs) on goldfish (Carassius auratus) exposed to thermal stress (high water temperature, 30 °C). The effects of the two treatments were assessed by measuring the expression and activity levels of the antioxidant enzymes, superoxide dismutase and catalase, plasma hydrogen peroxide, lipid hydroperoxide, and lysozyme. In addition, a comet assay was conducted to confirm that high water temperature damaged nuclear DNA. The expression and activity of the antioxidant enzymes, plasma hydrogen peroxide, and lipid hydroperoxide were significantly higher after exposure to high temperature and were significantly lower in fish that received melatonin or LED light than in those that received no mitigating treatment. Plasma lysozyme was significantly lower after exposure to high temperature and was significantly higher after exposure to melatonin or LED light. The comet assay revealed that thermal stress caused a great deal of damage to nuclear DNA; however, treatment with melatonin or green-wavelength LED light prevented a significant portion of this damage from occurring. These results indicate that, although high temperatures induce oxidative stress and reduce immune system strength in goldfish, both melatonin and green-wavelength LED light inhibit oxidative stress and boost the immune system. LED treatment increased the antioxidant and immune system activity more significantly than did melatonin treatment.