Functional connectivity density alterations in children with strabismus and amblyopia based on resting-state functional magnetic resonance imaging (fMRI).

PURPOSE: To explore functional connectivity density (FCD) values of brain areas in children with strabismus and amblyopia (SA) based on blood oxygen level-dependent (BOLD) signals. METHODS: This study recruited 26 children (14 male, 12 females) with SA and 26 healthy children (14 male, 12 female) as healthy controls (HCs). Both groups matched in age, gender, educational level and socioeconomic background. While resting, all participants underwent fMRI scanning and global FCD (gFCD) and local FCD (lFCD) values were calculated. Receiver operating characteristic (ROC) curves were created to investigate whether there was a significant difference between children with SA and healthy controls. RESULTS: When compared with healthy controls, children with SA had significantly lower gFCD values in the right cerebellum, left putamen, and right superior frontal gyrus; however, the same metrics showed opposite changes in the right angular gyrus, left middle cingulate gyrus, left angular gyrus, right superior parietal gyrus, and right middle frontal gyrus. In children with SA, lFCD values were found to be remarkably decreased in regions of the middle right temporal pole, right cerebellum, left putamen, left hippocampus, right hippocampus, left thalamus, left cerebellum; values were increased in the right superior parietal gyrus as compared with healthy controls. CONCLUSION: We noted abnormal neural connectivity in some brain areas of children with SA; detailing such connectivity aberrations is useful in exploring the pathophysiology of SA and providing useful information for future clinical management.

Brain Functional Connectivity Changes in Patients with Acute Eye Pain: A Resting-State Functional Magnetic Resonance Imaging (fMRI) Study.

BACKGROUND By using functional magnetic resonance imaging (fMRI), we aimed to study the changes in potential brain function network activity in patients with acute eye pain. Also, by using the voxel-wise degree centrality (DC) method, we aimed to explore the relationship between spontaneous brain activity and the clinical features of patients with acute eye pain. MATERIAL AND METHODS A total of 15 patients with acute eye pain (5 women and 10 men; EP group) and 15 healthy controls (5 women and 10 men; HC group), were scanned by fMRI. The DC method was used to evaluate changes in spontaneous brain activity. Receiver operating characteristic (ROC) curves were analyzed, and Pearson correlation analysis was used to study the relationship between DC values and clinical manifestations in different regions of brain. RESULTS The area of the left limbic lobe showed a reduction in DC value in patients in the EP group. DC values were elevated in the left cerebellum posterior lobe, left inferior parietal lobule, left inferior temporal gyrus, left precuneus, and right cerebellum posterior lobe in the EP group. The visual analog scale value of the eyes in the EP group was negatively correlated with the left limbic lobe signal value and positively correlated with the left inferior parietal lobule signal value. Further, the scores of the hospital anxiety and depression scale and DC value of the left limbic lobe were negatively correlated. CONCLUSIONS Compared with the HC group, patients with acute eye pain had abnormal patterns of intrinsic brain activity in different brain regions, which may help reveal the potential neural mechanisms involved in eye pain.

Abnormal interhemispheric functional connectivity in patients with strabismic amblyopia: a resting-state fMRI study using voxel-mirrored homotopic connectivity.

BACKGROUND: Previous studies have demonstrated that strabismus amblyopia can result in markedly brain function alterations. However, the differences in spontaneous brain activities of strabismus amblyopia (SA) patients still remain unclear. Therefore, the current study intended to employthe voxel-mirrored homotopic connectivity (VMHC) method to investigate the intrinsic brain activity changes in SA patients. PURPOSE: To investigate the changes in cerebral hemispheric functional connections in patients with SA and their relationship with clinical manifestations using the VMHC method. MATERIAL AND METHODS: In the present study, a total of 17 patients with SA (eight males and nine females) and 17 age- and weight-matched healthy control (HC) groups were enrolled. Based on the VMHC method, all subjects were examined by functional magnetic resonance imaging. The functional interaction between cerebral hemispheres was directly evaluated. The Pearson's correlation test was used to analyze the clinical features of patients with SA. In addition, their mean VMHC signal values and the receiver operating characteristic curve were used to distinguish patients with SA and HC groups. RESULTS: Compared with HC group, patients with SA had higher VMHC values in bilateral cingulum ant, caudate, hippocampus, and cerebellum crus 1. Moreover, the VMHC values of some regions were positively correlated with some clinical manifestations. In addition, receiver operating characteristic curves presented higher diagnostic value in these areas. CONCLUSION: SA subjects showed abnormal brain interhemispheric functional connectivity in visual pathways, which might give some instructive information for understanding the neurological mechanisms of SA patients.

Altered spontaneous brain activity patterns in dysthyroid optic neuropathy: a resting-state fMRI study.

This research investigates the characteristics of spontaneous brain activity in dysthyroid optic neuropathy patients using the regional homogeneity technique. Sixteen patients with dysthyroid optic neuropathy and 16 thyroid-associated ophthalmopathy patients without dysthyroid optic neuropathy were recruited, matched for weight, height, age, sex, and educational level. All participants underwent resting-state functional nuclear resonance imaging, and the characteristics of spontaneous brain activity were evaluated using the regional homogeneity technique. Each participant in the dysthyroid optic neuropathy group also completed the Hospital Anxiety and Depression scale. Receiver operating characteristic curves were used to compare brain activity between the two groups. Pearson correlation analysis evaluated the relationship between regional homogeneity and clinical manifestations in dysthyroid optic neuropathy patients. In addition, we analyzed the correlation between Hospital Anxiety and Depression scale and regional homogeneity. We found that the regional homogeneity values at the corpus callosum/cingulate gyrus and parietal lobe/middle frontal gyrus significantly decreased in dysthyroid optic neuropathy patients. Regional homogeneity values at the corpus callosum/cingulate gyrus and parietal lobe/middle frontal gyrus were negatively correlated with Hospital Anxiety and Depression scale and disease duration. It was found that the regional homogeneity signal values were significantly lower than in thyroid-associated ophthalmopathy without in dysthyroid optic neuropathy, which may indicate a risk of regional brain dysfunction in dysthyroid optic neuropathy. The results show that regional homogeneity has the potential for early diagnosis and prevent dysthyroid optic neuropathy. In addition, the findings suggest possible mechanisms of dysthyroid optic neuropathy optic nerve injury. They may provide a valuable basis for further research on the pathological mechanisms of dysthyroid optic neuropathy.

Altered Brain Network Centrality in Patients with Diabetic Optic Neuropathy: A Resting-State FMRI Study.

OBJECTIVE: Recent studies have suggested that diabetic optic neuropathy (DON) independently increases the incidence of brain diseases like cerebral infarction and hemorrhage. In this study, voxel-level degree centrality (DC) was used to study potential changes in functional network brain activity in DON patients. METHODS: The study included 14 DON patients and 14 healthy controls (HCs) matched by age, sex, and weight. All subjects underwent resting functional magnetic resonance imaging. Receiver operating characteristic curves and Pearson correlation analysis were performed. RESULTS: The DC values of the left frontal mid-orb and right middle frontal gyrus/right frontal sup were significantly lower in DON patients compared to HCs. The DC value of the left temporal lobe was also significantly higher than in HCs. CONCLUSION: Three different brain regions show DC changes in DON patients, suggesting common optic neuropathy in the context of diabetes and providing new ideas for treating optic nerve disease in patients with long-term diabetes. ABBREVIATIONS: AUC = area under the curve; BCVA = best corrected visual acuity; DC = degree centrality; DON = diabetic optic neuropathy; fMRI = functional magnetic resonance imaging; HC = healthy control; LFMO = left frontal mid orb; LTL = left temporal lobe; RFS = right frontal sup; RMFG = right middle frontal gyrus; ROC = receiver operating characteristic.

ALTERED BRAIN ACTIVITY IN PATIENTS WITH DIABETIC RETINOPATHY USING REGIONAL HOMOGENEITY: A RESTING-STATE fMRI STUDY.

Objective: Previous neuroimaging studies have shown that diabetic retinopathy (DR) is accompanied by abnormal spontaneous brain activity. The purpose of the current study was to investigate changes in brain neural homogeneity in patients with DR using regional homogeneity (ReHo). Methods: A total of 56 subjects were recruited, including 28 patients with DR (16 female and 12 male patients) and 28 healthy controls (HCs) (16 female and 12 male patients) approximately matched for age and sex. All subjects underwent resting-state functional magnetic resonance imaging scans. The ReHo method was applied to explore neural homogeneity in the brain. The patients with DR were distinguished from HCs following the construction of receiver operating characteristic curves. The ReHo method was applied to assess changes in synchronous neural activity. Results: Compared to HCs, the ReHo values in the left and right posterior lobes of the cerebellum in patients with DR were significantly increased, whereas ReHo values in the right anterior cingulate gyrus, right cuneus, bilateral precuneus, and left-middle frontal gyrus were significantly decreased. In addition, the ReHo value in the right cuneus showed a positive correlation with the best corrected visual acuity in patients with DR. Conclusion: Dysfunctional brain homology may reveal the pathological mechanisms underlying the visual pathways of patients with DR. Abbreviations: AUC = area under the curve; BA = Brodmann area; DR = diabetic retinopathy; fMRI = functional magnetic resonance imaging; HC = healthy control; MRI = magnetic resonance imaging; rs-fMRI = resting-state fMRI; ReHo = regional homogeneity; ROC = receiver operating characteristic.

Resting cerebral blood flow alterations specific to the comitant exophoria patients revealed by arterial spin labeling perfusion magnetic resonance imaging.

PURPOSE: It has been shown in many previous studies that there were significant changes of the brain anatomy and function in strabismus. However, the significance of the alterations of resting cerebral blood flow (CBF) in comitant exophoria (CE) remains obscure. Arterial spin labeling (ASL) MRI, which is a noninvasive method, could be applied to detect the cerebral blood flow quantitatively. Our study aimed to compare the resting CBF between the comitant exophoria and health controls using pseudo-continuous arterial spin labeling (pCASL) perfusion MRI method. METHODS: 32 patients (25 males and 7 females) with CE (study group), and 32 (25 males and 7 females) healthy individuals with matched age and sex status (control group) underwent a whole-brain pCASL magnetic resonance (MR) examination at the resting state. The resting CBF were voxel-wise compared between the two groups using an analysis of variance designed in a statistical parametric mapping program. The CE patients were distinguishable from the healthy controls (HCs) by receiver operating characteristic (ROC) curves. RESULTS: Compared with the control group, the CE group showed significantly increased resting CBF values in the right parahippocampal regions, bilateral medial frontal gyrus/anterior cingulate cortex, left inferior frontal gyrus, right inferior frontal gyrus, left superior frontal gyrus, bilateral medial cingulate cortex, right middle frontal gyrus, and right paracentral lobule. CONCLUSION: Comitant exophoria showed increased resting CBF in eye movement-related brain areas including supplementary eye field, cingulate eye field and frontal eye field, which could be an explanation of the brain function compensation for the ocular motility disorders in the CE patients.

Melatonin promotes superovulation in sika deer (Cervus nippon).

In this study, the effects of melatonin (MT) on superovulation and reproductive hormones (melatonin, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and PRL) were investigated in female sika deer. Different doses (40 or 80 mg/animal) of melatonin were subcutaneously implanted into deer before the breeding season. Exogenous melatonin administration significantly elevated the serum FSH levels at the time of insemination compared with levels in control animals. During superovulation, the serum LH levels in donor sika deer reached their highest values (7.1±2.04 ng/mL) at the point of insemination, compared with the baseline levels (4.98±0.07 ng/mL) in control animals. This high level of LH was sustained until the day of embryo recovery. In contrast, the serum levels of PRL in the 80 mg of melatonin-treated group were significantly lower than those of control deer. The average number of corpora lutea in melatonin-treated deer was significantly higher than that of the control (p<0.05). The average number of embryos in the deer treated with 40 mg of melatonin was higher than that of the control; however, this increase did not reach significant difference (p>0.05), which may be related to the relatively small sample size. In addition, embryonic development in melatonin-treated groups was delayed.

Targeting scleral remodeling and myopia development in form deprivation myopia through inhibition of EFEMP1 expression.

The role of extracellular matrix (ECM) remodeling in the axial elongation associated with myopia has not been fully elucidated, although it is considered a significant factor. EFEMP1, a regulator of ECM, has been associated with various pathological conditions. This study aimed to examine the involvement of EFEMP1 in scleral remodeling during form deprivation myopia. The results indicate a progressive increase in EFEMP1 expression following prolonged form deprivation treatment, followed by a subsequent decrease upon recovery. To gain a deeper understanding of the mechanism of EFEMP1, we conducted transcriptome sequencing on primary scleral fibroblasts that were subjected to lentivirus-mediated overexpression of EFEMP1. Validation was performed using lentivirus-induced overexpression and shRNA targeting EFEMP1 in combination with LY294002, a PI3K inhibitor. Our findings suggest that EFEMP1 may be involved in the development of FDM by regulating the expression of the PI3K/AKT/MMP2 axis. The AAV-mediated injection of shEFEMP1 under Tenon's capsule in guinea pigs was observed to effectively delay the progression of myopia and posterior scleral remodeling. In contrast, the AAV-mediated overexpression of EFEMP1 exacerbated the development of myopia and resulted in further thinning of collagen fibers in the posterior sclera. In summary, adjusting EFEMP1 concentrations could potentially serve as a viable approach to prevent and treat myopia by influencing the remodeling process of the posterior sclera.

EFEMP1 is a potential biomarker of choroid thickness change in myopia.

Purpose: To explore the possible molecular mechanism by which epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) regulates choroid thickness (CT) in the development of myopia. Methods: In total, 131 subjects were divided into the emmetropia (EM) group, non-high myopia (non-HM) group and high myopia (HM) group. Their age, refraction, intraocular pressure, and other ocular biometric parameters were collected. A 6 × 6 mm area centered on the optic disc was scanned by coherent optical tomography angiography (OCTA) to measure CT, and the tear concentrations of EFEMP1 were quantified using enzyme-linked immunosorbent assay (ELISA) analysis. Twenty-two guinea pigs were divided into the control group and the form-deprivation myopia (FDM) group. The right eye of the guinea pig in the FDM group was covered for 4 weeks, and the diopter and axial length of the right eye of the guinea pig were measured before and after the treatment. After the measurement, the guinea pig was euthanized, and the eyeball was removed. Quantitative reverse transcription polymerase chain reaction, western blotting assays and immunohistochemistry were used to assess the expression of EFEMP1 in the choroid. Results: There were significant differences in CT among the three groups (p < 0.001). CT was positively correlated with age in HM (r = -0.3613, p = 0.0021), but no significant correlation with SE (p > 0.05) was observed. Furthermore, there were increased levels of EFEMP1 in the tears of myopic patients. After 4 weeks of covering the right eye of the FDM guinea pigs, there was a significant increase in axial length and a decrease in diopter (p < 0.05). The mRNA and protein expression of EFEMP1 was significantly increased in the choroid. Conclusion: Choroidal thickness was significantly thinner in myopic patients, and the expression level of EFEMP1 in the choroid increased during the development of FDM. Therefore, EFEMP1 may be involved in the regulation of choroidal thickness in myopia patients.

Spontaneous changes in brain network centrality in patients with pathological myopia: A voxel-wise degree centrality analysis.

BACKGROUND: Myopia has become a worldwide problem that endangers public health and adds a serious socioeconomic burden. Current research has focused on the pathogenesis and manifestations of pathological myopia (PM). However, few studies have been conducted on the spontaneous activity of the patient's brain. PURPOSE: To study the potential brain network activity in patients with PM by the degree centrality (DC) method. MATERIALS AND METHODS: This experiment included 15 PM patients and 15 healthy controls (HCs). Every participant experienced a resting-state functional magnetic resonance imaging (rs-fMRI) scan. Receiver operating characteristic (ROC) curve analysis was used to distinguish between PM patients and HCs. Correlation analysis was used to explore the relationships between mean DC values and clinical performance in different brain regions. RESULTS: It showed that patients with PM had lower DC values in the right fusiform gyrus (FR) and right cingulate (CAR). The ROC curve was used to indicate the accuracy of the correlation. It showed that in PM group, left best corrected visual acuity (BCVA-L) and right best corrected visual acuity (BCVA-R) were negatively correlated with the DC value of FR. CONCLUSION: The occurrence of PM is mainly related to the abnormal activity of the fusiform and cingulum. DC value might be used as a biological marker of abnormal brain activity in PM patients.

Retinal alterations in evaluation of rheumatoid arthritis with chloroquine treatment: A new approach.

OBJECTIVE: To evaluate the effect of hydroxychloroquine on conjunctival and retinal microvascular density in rheumatoid arthritis (RA) patients. METHODS: Ten healthy controls, 10 RA patients who had not been treated with hydroxychloroquine, and 10 RA patients who had been treated with chloroquine for more than 5 years were recruited. Optical coherence tomography (OCTA) was used to examine the conjunctival and superficial and deep retinal microvascular density and compared the differences in microvascular density between the three groups. RESULTS: The vascular density in RA group in superficial microvascular was significantly lower than that in control group (p < 0.001). Compared with RA group, the chloroquine group showed statistically significantly lower microvascular (p < 0.001) and deep microvascular (p = 0.018). Superficial microvascular was positively correlated with conjunctival vessel density in RA patients (r = 0.868, p = 0.0048). CONCLUSIONS: The use of chloroquine could further reduce the vascular density in the absence of statistical difference in the course of the disease.

Effect of potassium fertilization on storage root number, yield, and appearance quality of sweet potato (Ipomoea batatas L.).

Increasing storage root number is a pivotal approach to enhance both storage root (SR) yield and appearance quality of sweet potato. Here, 2-year field experiments were conducted to investigate the effect of 0 (K0), 120 (K1), 240 (K2), and 360 (K3) kg ha-1 potassium fertilizer (K2O) on lignin metabolism, root growth, storage root yield, and uniformity. The results demonstrated that potassium (K) application led to a decrease in the activities of key enzymes involved in lignin biosynthesis, including phenylalanine deaminase (PAL), 4-coumarate coenzyme A ligase (4-CL), cinnamic acid dehydrogenase (CAD), polyphenol oxidase (PPO), and peroxidase (POD). This resulted in a significant reduction in lignin and G-type lignin contents in potential SRs compared to K0 treatment within 10-30 days after planting (DAP). BJ553 exhibited a significant decrease in PAL activity, as well as lignin and G-type contents at 10 DAP, whereas YS25 showed delayed effects until 20 DAP. However, the number and distribution of secondary xylem conduits as well as the mid-column diameter area in roots were increased in K2 treatment. Interestingly, K2 treatment exhibited significantly larger potential SR diameter than other treatments at 15, 20, and 25 DAP. At harvest, K2 treatment increased the SR number, the single SR weight, and overall yield greatly compared with K0 treatment, with an average increase of 19.12%, 16.54%, and 16.92% respectively. The increase of SR number in BJ553 was higher than that of YS25. Furthermore, K2 treatment exhibited the lowest coefficient of variation for both SR length and diameter, indicating a higher yield of middle-sized SRs. In general, appropriate potassium application could effectively suppress lignin biosynthesis, leading to a reduction in the degree of pericycle lignification in potential SRs. This promotes an increase in the number of storage roots and ultimately enhances both yield and appearance quality of sweet potato. The effect of potassium fertilizer on lignin metabolism in BJ553 roots was earlier and resulted in a greater increase in the SR number compared to YS25.

[Factors analysis and genetic parameter estimation of female reproductive traits in pigs].

Reproductive performance of stock sows is one of the important factors of economic impact in pig farms. In this study, 8491 litter records from 2699 sows of Yorkshire, Landrace, and Duroc were analyzed using fixed model to determine the effect of parity, mating season, and breed on total number born (TNB), number healthy birth (NHB), litter birth weight (LWB), number weak birth (NWB), stillbirth, mummy fetus, and deform fetus by the least square analysis. Genetic parameters of the above traits were estimated by restricted maximum likelihood (REML) procedure. In addition, the effectiveness of pure-breeding and cross-breeding on litter performance were compared. The results showed that, parity, mating season, and breed had significant effect on TNB, NHB, and LWB(P < 0.001).The effects of parity and breed were significant on NWB(P < 0.001), while mating season had non-significant effect on NWB. Parity showed significant effect on stillbirth, while the effect of mating season and breed was not significant. Parity, mating season, and breed had no significant effect on mummy fetus and deform fetus. Landrace♂×Large White♀ showed the best litter performance, including TNB, NHB, and LWB. Moreover, LWB of Landrace depicted the highest heritability, while other traits were all bellow 0.2. The genetic correlation between TNB and NHB, NHB and LWB were higher than 0.96 in the three breeds. These results provided reference data for minimizing low-reproductive performance caused by non-infectious factors and improving sow reproductive performance in pig farms.

Altered spontaneous brain activity patterns in patients with diabetic retinopathy using amplitude of low-frequency fluctuation.

BACKGROUND: Diabetes mellitus is a metabolic disorder characterized by prolonged elevation of blood glucose due to various causes. Currently, the relationship between diabetic retinopathy (DR) and altered connectivity of brain function is unclear. AIM: To investigate the relationship between this brain activity and clinical manifestations and behaviors of DR patients by using the amplitude of low-frequency fluctuation (ALFF) technique. METHODS: Twenty-four DR patients and 24 healthy controls (HCs) matched for age and gender were enrolled. We measured and recorded average ALFF values of DR patients and HCs and then classified them using receiver operating characteristic (ROC) curves. RESULTS: ALFF values of both left and right posterior cerebellar lobe and right anterior cingulate gyrus were remarkably higher in the DR patients than in the HCs; however, DR patients had lower values in the bilateral calcarine area. ROC curve analysis of different brain regions demonstrated high accuracy in the area under the curve analysis. There was no significant relationship between mean ALFF values for different regions and clinical presentations in DR patients. Neuronal synchronization abnormalities in some brain regions of DR patients were associated with cognitive and visual disorders. CONCLUSION: Abnormal spontaneous brain activity was observed in many areas of DR patients' brains, which may suggest a possible link between clinical manifestations and behaviors in DR patients.

Gray matter volume alterations in patients with strabismus and amblyopia: voxel-based morphometry study.

This study proposes the use of the voxel-based morphometry (VBM) technique to investigate structural alterations of the cerebral cortex in patients with strabismus and amblyopia (SA). Sixteen patients with SA and sixteen healthy controls (HCs) underwent magnetic resonance imaging. Original whole brain images were analyzed using the VBM method. Pearson correlation analysis was performed to evaluate the relationship between mean gray matter volume (GMV) and clinical manifestations. Receiver operating characteristic (ROC) curve analysis was applied to classify the mean GMV values of the SA group and HCs. Compared with the HCs, GMV values in the SA group showed a significant difference in the right superior temporal gyrus, posterior and anterior lobes of the cerebellum, bilateral parahippocampal gyrus, and left anterior cingulate cortex. The mean GMV value in the right superior temporal gyrus, posterior and anterior lobes of the cerebellum, and bilateral parahippocampal gyrus were negatively correlated with the angle of strabismus. The ROC curve analysis of each cerebral region confirmed the accuracy of the area under the curve. Patients with SA have reduced GMV values in some brain regions. These findings might help to reveal the potential pathogenesis of SA and its relationship with the atrophy of specific regions of the brain.

Abnormal Fractional Amplitude of Low Frequency Fluctuation Changes in Patients With Dry Eye Disease: A Functional Magnetic Resonance Imaging Study.

Purpose: To investigate spontaneous brain activity in patients with dry eye (DE) and healthy control (HC) using the fractional amplitude of low frequency fluctuation (fALFF) technique with the aim of elucidating the relationship between the clinical symptoms of DE and changes in brain function. Material and Methods: A total of 28 patients with DE and 28 matched healthy volunteers (10 males and 18 females in each group) were enrolled. Resting-state functional magnetic resonance imaging scans were performed in both groups. Then all subjects were required to complete a comprehensive Hospital Anxiety and Depression Scale (HADS). Receiver operating characteristic (ROC) curve analysis was used to evaluate the differences in fALFF values between the two groups and their diagnostic value. Linear correlations between HADS and fALFF values in different brain regions of DE patients were analyzed using the Pearson correlation coefficient. Results: Patients with DE had significantly higher fALFF values in the left calcarine sulcus (CS) than the HC group, while fALFF values in the bilateral middle frontal gyrus (MFG) and right MFG/right inferior frontal gyrus (IFG) were significantly lower in DE patients than in HC group. fALFF values had a high diagnostic value for differentiating patients with DE from the HC group (P < 0.001). Right MFG and right MFG/IFG were significantly correlated with HADS values. Conclusion: Our study found that DE mainly involved functional disorders in the brain areas of the left CS, bilateral MFG and right MFG/right IFG, which helped us to find possible clinical features of DE disease and reflected the potential pathological mechanism of DE.