Seasonal nitrate variations, risks, and sources in groundwater under different land use types in a thousand-year-cultivated region, northwestern China.

The global public health concern of nitrate (NO3-) contamination in groundwater is particularly pronounced in irrigated agricultural regions. This paper aims to analyze the spatial distribution of groundwater NO3-, assess potential health risks for local residents, and quantitatively identify nitrate sources during different seasons and land use types in the Jinghuiqu Irrigation District, a region in northwestern China with a longstanding agricultural history. The investigation utilizes hydrochemical parameters, dual isotopic data, and the Bayesian stable isotope mixing model (MixSIAR). The findings underscore significant seasonal variations in the average concentrations of NO3-, with values of 87.72 mg/L and 101.87 mg/L during the wet and dry seasons, respectively. Furthermore, distinct fluctuations in nitrate concentration were observed across different land use types, whereby vegetable lands manifested the maximum concentration. Prolonged exposure to elevated nitrate concentrations may pose potential health risks to residents, especially in the dry season when the non-carcinogenic groundwater nitrate risk surges past its wet season counterpart. The MixSIAR analysis revealed that chemical fertilizers accounted for the majority of nitrate pollution in vegetable lands, both during the dry season (49.6%) and wet season (41.2%). In contrast, manure and sewage contributed significantly to NO3-concentrations in residential land during the wet (74.9%) and dry seasons (67.6%). For croplands, soil nitrogen emerged as a dominant source during the wet season (42.2%), while chemical fertilizers prevailed in the dry season (38.7%). In addition to source variations, the nitrate concentration of groundwater is further affected by hydrogeological conditions, with more permeable aquifers tending to display higher nitrate concentrations. Thus, targeted measures were proposed to modify or impede the nitrogen migration pathway, taking into consideration hydrogeological conditions and incorporating domestic sewage, organic fertilizer, and agricultural management practices.

Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves.

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.

Qualitative and quantitative analysis of phenolic compounds in blueberries and protective effects on hydrogen peroxide-induced cell injury.

This work was conducted to optimize an accelerated solvent extraction for ultra high performance liquid chromatography-mass spectrometry/mass spectrometry analysis of blueberry phenolic compounds. The conditions for accelerated solvent extraction were verified using response surface methodology to obtain the following optimized conditions: ethanol concentration (pH = 3), 48%; temperature, 50℃, and static cycle times, 3. Further, ultra high performance liquid chromatography with quadrupole Exactive Orbitrap mass spectrometry and ultra high performance liquid chromatography with triple-quadrupole tandem mass methods for determination of the detailed phenolic composition were developed and validated. Total of 81 phenolic compounds were identified by ultra high performance liquid chromatography with quadrupole Exactive Orbitrap mass spectrometry including 23 anthocyanins, 32 flavonols, 11 proanthocyanidins, 2 other flavonoids, and 13 phenolic acids. Fifty-one of these compounds have been simultaneously quantified by ultra high performance liquid chromatography with triple-quadrupole tandem mass including 31 anthocyanins, 8 flavonols, 6 proanthocyanidins, 2 other flavonoids, and 8 phenolic acids. Malvidin-dinhexoside has, for the first time, been detected in wild. Moreover, by verifying the protection on PC12 cells against oxidative damage, it was showed that the phenolic extracts (500 µg/mL) can improve significantly the viability (9.26-24.78%) of hydrogen peroxide-induced PC12 cells, activities of superoxide dismutase (34.59-37.90 U/mg) and glutathione peroxidase (6.87-14.42 mU/mg) and decrease the content of malonic dialdehyde (13.27-24.62 nmol/mg). Correlation analysis suggested that anthocyanins might contribute most to these activities.

Geometric optics applied to drops passing through a focused Gaussian beam.

The present study examines the scattered light intensity from a drop passing through a Gaussian beam of a diameter comparable to or smaller than the drop. This is the situation encountered when using the time-shift technique, an optical technique used to characterize drops and aerosols according to the size and the velocity. In simulating the signals received by such an instrument, the computational effort involved when using, for instance, the Generalized Lorenz-Mie Theory or vector ray-tracing, is immense and hardly practical for use in instrument design and/or optimization. In this study theoretical expressions based on geometric optics are derived as an alternative, and they are shown to adequately capture the main features of the time-shift signals. These solutions require little computational effort and can be effectively used to explore the dependencies of the signals on various input factors, thus allowing further instrument development. On the other hand, these relations are also of general interest in the field of light scattering from drops and aerosols.

Simulation of light scattering from a colloidal droplet using a polarized Monte Carlo method: application to the time-shift technique.

This study is devoted to the development and application of a Monte Carlo ray-tracing model to simulate light scattering when a colloid suspension droplet passes through a highly focused Gaussian laser sheet. Within this study, a colloidal suspension droplet refers to a spherical droplet containing multiple spherical inclusions. Such scattering scenarios arise when using the time-shift measurement technique for particle sizing. The incident laser sheet is treated as a large number of polarized light rays: the Stokes vector of each light ray is tracked, achieved by multiplication of the rotation matrix and the Mueller matrix after each scattering event. For the Monte Carlo simulation of light scattering, a very important issue is to generate the deflection angle and azimuthal angle after each scattering event. The scattering from embedded inclusions is computed using the Lorenz-Mie theory and by employing the rejection sampling technique to update the new propagation direction. Multi-reflection and refraction within the droplet is accounted for, as is total reflection at the drop interface. For this, the Mueller matrix formulation is invoked at the drop surface to update the Stokes vector. To validate this simulation code, the scattering diagram from a nanoparticle is computed with this Monte Carlo method and compared with the scattering diagram computed with the Lorenz-Mie theory, the agreement is excellent. This Monte Carlo code is then applied to simulate signals arising from a time-shift device, when a colloid suspension droplet passes through a focused polarized laser sheet, with the objective of measuring the concentration of colloidal particles in the droplet. Measurements verify the ability of the code to properly simulate this light scattering scenario.

Grape and wine polymeric polyphenols: Their importance in enology.

Phenolic compounds are important constituents of red wine, contributing to its sensory properties and antioxidant activity. Owing to the diversity and structural complexity, study of these compounds was mainly limited, during the last three decades, on their low-molecular-mass compounds or simple phenolic compounds. Only in recent years, much attention has been paid to highly polymerized polyphenols in grape and red wines. The reason for this is largely due to the development of analytical techniques, especially those of HPLC-ESI-MS, permitting the structural characterization of highly polymerized polyphenols. Furthermore, the knowledge on the biological properties of polymeric polyphenols of red wine is very limited. Grape polyphenols mainly consist of proanthocyanidins (oligomers and polymers) and anthocyanins, and low amount of other phenolics. Red wine polyphenols include both grape polyphenols and new phenolic products formed from them during winemaking process. This leads to a great diversity of new polyphenols and makes wine polyphenol composition more complex. The present paper summarizes the advances in the research of polymeric polyphenols in grape and red wine and their important role in Enology. Scientific results indicate that polymeric polyphenols, as the major polyphenols in grape and red wine, play a major role in red wine sensory properties, color stability and antioxidant activities.

Preparation and Antioxidant Activity of Ethyl-Linked Anthocyanin-Flavanol Pigments from Model Wine Solutions.

Anthocyanin-flavanol pigments, formed during red wine fermentation and storage by condensation reactions between anthocyanins and flavanols (monomers, oligomers, and polymers), are one of the major groups of polyphenols in aged red wine. However, knowledge of their biological activities is lacking. This is probably due to the structural diversity and complexity of these molecules, which makes the large-scale separation and isolation of the individual compounds very difficult, thus restricting their further study. In this study, anthocyanins (i.e., malvidin-3-glucoside, cyanidin-3-glucoside, and peonidin-3-glucoside) and (⁻)-epicatechin were first isolated at a preparative scale by high-speed counter-current chromatography. The condensation reaction between each of the isolated anthocyanins and (⁻)-epicatechin, mediated by acetaldehyde, was conducted in model wine solutions to obtain ethyl-linked anthocyanin-flavanol pigments. The effects of pH, molar ratio, and temperature on the reaction rate were investigated, and the reaction conditions of pH 1.7, molar ratio 1:6:10 (anthocyanin/(⁻)-epicatechin/acetaldehyde), and reaction temperature of 35 °C were identified as optimal for conversion of anthocyanins to ethyl-linked anthocyanin-flavanol pigments. Six ethyl-linked anthocyanin-flavanol pigments were isolated in larger quantities and collected under optimal reaction conditions, and their chemical structures were identified by HPLC-QTOF-MS and ECD analyses. Furthermore, DPPH, ABTS, and FRAP assays indicate that ethyl-linked anthocyanin-flavanol pigments show stronger antioxidant activities than their precursor anthocyanins.

Protocol to identify fungal profile from fecal metagenomes in cancer patients prior to immunotherapy.

Identifying fungal profiles from metagenomes is challenging due to an incomplete fungal database and limitations in the understanding of software. Here, we describe a protocol for analyzing the fecal metagenome of cancer patients prior to immunotherapy. We describe steps for raw metagenomic sequencing data retrieval, human genome and contaminants elimination, and assigning taxonomy labels to fungal reads. We then detail measures for assessing fungal alpha diversity and beta diversity, along with differential analysis. For complete details on the use and execution of this protocol, please refer to Xiaowen Huang et al.1.

Unveiling the hidden players: exploring the role of gut mycobiome in cancer development and treatment dynamics.

The role of gut fungal species in tumor-related processes remains largely unexplored, with most studies still focusing on fungal infections. This review examines the accumulating evidence suggesting the involvement of commensal and pathogenic fungi in cancer biological process, including oncogenesis, progression, and treatment response. Mechanisms explored include fungal influence on host immunity, secretion of bioactive toxins/metabolites, interaction with bacterial commensals, and migration to other tissues in certain types of cancers. Attempts to utilize fungal molecular signatures for cancer diagnosis and fungal-derived products for treatment are discussed. A few studies highlight fungi's impact on the responsiveness and sensitivity to chemotherapy, radiotherapy, immunotherapy, and fecal microbiota transplant. Given the limited understanding and techniques in fungal research, the studies on gut fungi are still facing great challenges, despite having great potentials.

Not a passive learner but an active one: a focus on the efficacy of philosophy-based language instruction and its consequences on EFL learners' critical thinking, engagement, and academic achievement.

This study investigates the impact of Philosophy-Based Language Teaching (PBLT) on the critical thinking skills and learner engagement of English as a Foreign Language (EFL) students. A concurrent mixed-methods approach, including semi-structured interviews, document analysis, and academic achievement tests, was employed with an experimental group (EG) exposed to PBLT and a control group (CG) receiving traditional instruction. The results reveal a substantial positive effect of PBLT on the critical thinking abilities of EFL learners, as evidenced by thematic analyses of interviews and document content. Themes include heightened critical thinking awareness, collaborative knowledge construction, and increased learner engagement. Academic achievement tests further demonstrate significant improvement in the experimental group's performance. A comparison with existing literature underscores the novelty of our mixed-methods approach. Implications for language teachers, materials developers, syllabus designers, and policy-makers are discussed, highlighting the potential of PBLT in cultivating critical thinking and learner engagement.

UHPLC-MS/MS analysis and protective effects on neurodegenerative diseases of phenolic compounds in different parts of Diospyros kaki L. cv. Mopan.

Persimmon (Diospyros kaki L. cv. Mopan.), an important commercial crop belonging to the genus of Diospyros in the Ebenaceae family, is rich in bioactive phenolic compounds. In this study, the phenolic compounds from fruits, leaves, and calyces of persimmon were qualitatively and quantitatively determined by UPLC-Q-Exactive-Orbitrap/MS and UPLC-QqQ-MS/MS, respectively. Furthermore, the role of phenolic extract from different parts of persimmon on neuroprotective activity in vitro, through against oxidative stress and anti-neuroinflammation effect was firstly evaluated. The results showed that 75 phenolic compounds, and 3 other kinds of compounds were identified, among which 44 of phenolic compounds were quantified from different parts of persimmon. It is the first time that epicatechin-epigallocatechin, catechin-epigallocatechin, catechin-epigallocatechin (A-type), and glycoside derivatives of laricitrin were identified in persimmon extract. The dominated phenolic compounds in three parts of persimmon were significantly different. All phenolic extracts from each part of persimmon showed strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells and LPS-induced BV2 cells. The fruit extract presented the strongest activity, followed by calyx and leaf extract. The systematic knowledge on the phytochemical composition along with activity evaluation of different parts of persimmon could contribute to their targeted selection and development.

Novel l-Cysteine Incomplete Degradation Method for Preparation of Procyanidin B2-3'-O-Gallate and Exploration of its in Vitro Anti-inflammatory Activity and in Vivo Tissue Distribution.

In this study, an effective method for preparation of bioactive galloylated procyanidin B2-3'-O-gallate (B2-3'-G) was first developed by incomplete depolymerization of grape seed polymeric procyanidins (PPCs) using l-cysteine (Cys) in the presence of citric acid. The structure-activity relationship of B2-3'-G was further evaluated in vitro through establishing lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. The results suggested that the better protective effects of B2-3'-G against inflammation were attributed to its polymerization degree and the introduction of the galloyl group, compared to its four corresponding structural units. In vivo experiments demonstrated that the B2-3'-G prototype was distributed in plasma, small intestine, liver, lung, and brain. Remarkably, B2-3'-G was able to penetrate the blood-brain barrier and appeared to play an important role in improving brain health. Furthermore, a total of 18 metabolites were identified in tissues. Potential metabolic pathways, including reduction, methylation, hydration, desaturation, glucuronide conjugation, and sulfation, were suggested.

Mechanism of improving anaerobic fermentation performance of kitchen waste pretreated by ionizing irradiation-part 1: rice.

Ionizing irradiation, as a new pretreatment method for the anaerobic fermentation of organic pollutants, is featured with fast reaction speed, good treatment effect, no need to add any chemical reagents, and no secondary pollution. This study explores the mechanism of improving anaerobic fermentation performance of rice samples pretreated by cobalt-60 gamma irradiation through the influence on fermentation substrate, acidogenic phase and methanogenic phase. The results reveal that the soluble chemical oxygen demand of the irradiated rice sample at an absorbed dose of 9.6 kGy increases by 12.4 times due to the dissolution of small molecules of fat-soluble organic matter. The yield of biogas in the acidogenic phase increases by 22.2% with a slight increase in hydrogen gas content. The yield of biogas and methane gas content in the methanogenic phase increases by 27.3% and 15%, respectively. Microbial genome analysis, performed with MiSeq high-throughput sequencing and metagenomic methods, suggests the microbial abundance and metabolic functions in the anaerobic fermentation process change significantly as a result of the pretreatment by gamma irradiation.

Effects of Different Brewing Technologies on Polyphenols and Aroma Components of Black Chokeberry Wine.

The black chokeberry is a shrub of the Rosaceae family, which is characterized by strong acidity and astringency and is widely processed into wine and alcoholic beverages. However, due to the characteristics of black chokeberries, the wine brewed by traditional methods often has a strong sour taste, weak aroma, and poor sensory quality. In order to improve the sensory quality and explore the effects of different brewing technologies on polyphenols of black chokeberry wine, five brewing technologies (traditional fermentation, frozen fruit fermentation, co-fermentation, carbonic maceration, and co-carbonic maceration) were used in this study. The results showed that compared with the traditional method, the four alternative brewing technologies could reduce acidity, increase the contents of several major polyphenols, and enrich floral scents and fruity aroma, thus significantly improving the sensory qualities of black chokeberry wine. The proposed brewing technologies would be applied to the production of quality black chokeberry or other fruit wines.

Novel flavan-3-ol-dithiothreitol conjugates derived from the degradation of grape seed proanthocyanidins and their neuroprotective potential.

Dithiothreitol (DTT) was adopted as a nucleophile to develop a new acid-catalyzed degradation method for grape seed proanthocyanidin extraction (GSPE). Backpropagation neural network and Box-Behnken design were employed and compared to establish the optimized degradation conditions. GSPE was reacted with DTT at a ratio of 1:1 under mild conditions with 0.14 M HCl at 40.8 °C for 60 min. Three monomeric proanthocyanidins and six novel flavan-3-ol-DTT conjugates consisting of three pairs of diastereomers were simultaneously obtained with a high yield (929 mg/g). All the degradation products showed protective effects against Aß25-35-induced neurotoxicity in PC-12 cells and prevented Aß25-35 aggregation based on the results from MTT and thioflavin T fluorescence assays, respectively. Detailed intermolecular interactions leading to the prevention of Aß25-35 aggregation were elucidated using molecular docking. This work would provide new compounds from functional foods that can be explored for their neuroprotective potential.

Large Scale BN-perovskite Nanocomposite Aerogel Scintillator for Thermal Neutron Detection.

State-of-the-art thermal neutron scintillation detectors rely on rare isotopes for neutron capture, lack stability and scalability of solid-state scintillation devices, and poorly discriminate between the neutron and gamma rays. The boron nitride (BN)-CsPbBr3 perovskite nanocomposite aerogel scintillator enables discriminative detection of thermal neutrons, features the largest known size (9 cm across), the lowest density (0.17 g cm-3 ) among the existing scintillation materials, high BN (50%) perovskite (1%) contents, high optical transparency (85%), and excellent radiation stability. The new detection mechanism relies on thermal neutron capture by 10 B and effective energy transfer from the charged particles to visible-range scintillation photons between the densely packed BN and CsPbBr3 nanocrystals. Low density minimizes the gamma ray response. The neutrons and gamma rays are discriminated by complete decoupling of the respective single pulses in time and intensity. These outcomes open new avenues for neutron detection in resource exploration, clean energy, environmental, aerospace, and homeland security applications.

High-speed countercurrent chromatography as an efficient technique for large separation of plant polyphenols: A review.

Polyphenols, which are commonly found in fruits, vegetables, cereals, and legumes, are the most abundant dietary antioxidants. To date, numerous studies have demonstrated the ability of polyphenols to prevent the development of several diseases and established the corresponding structure-activity relationships. However, polyphenol standards are either not commercially available or very expensive, mainly because the structural complexity and diversity of polyphenols complicate their fractionation and isolation from plant extracts by conventional separation techniques. High-speed countercurrent chromatography (HSCCC) is based on continuous liquid-liquid partitioning, which enables one to eliminate irreversible adsorption on solid supports. This technique has been extensively used for natural product isolation and is well suited to the effective large-scale separation of polyphenols and their derivatives, achieving high purities and yields of up to several hundred milligrams per run within several hours. The present review briefly introduces briefly HSCCC technology and summarizes its applications in the separation and purification of plant polyphenols, including the recent achievements in the large-scale preparation of proanthocyanidins and anthocyanins by our laboratory.

Impact of Different Oak Chips' Aging on the Volatile Compounds and Sensory Characteristics of Vitis amurensis Wines.

In this work, different oak chips were used to age Vitis amurensis wine, and the effects on sensory properties were observed. Twenty-one different oak chips were added to a one-year-old wine made by a traditional technique. The wine was aged for 6 months before analysis by CIELab for color parameters, GC-MS for volatile compounds, and electronic tongue and a tasting panel for sensory properties. The results showed that the addition of any tested oak chip could significantly strengthen the wine's red color. Among 61 volatile compounds, alcohols presented the highest concentrations (873 to 1401 mg/L), followed by esters (568 to 1039 mg/L) and organic acids (157 to 435 mg/L), while aldehydes and volatile phenols occurred at low concentrations. Different oak species with different toasting levels could affect, to varying degrees, the concentrations of esters, alcohols, and volatile phenols, but to a lesser extent those of aldehydes. Sensory analysis by a tasting panel indicated that non- and moderately roasted oak chips gave the wines higher scores than those with heavy toasting levels. The major mouthfeel descriptors determined by electronic tongue were in good agreement with those from the tasting panel.

Integrated LC-MS/MS method and network pharmacology for exploring the characterization and mechanism of neuroprotective effect of Vitis amurensis Rupr. wine polyphenol.

Polyphenols are the main active components in Vitis amurensis Rupr. wine, which show good protective effects on the nervous system, but their compositions in Vitis amurensis Rupr. wine and the molecular mechanism underlying their neuroprotection remains unclear. The purpose of this study is to investigate the potential mechanism of the neuroprotective effect of Vitis amurensis Rupr. wine polyphenols on the basis of the specific composition of polyphenols in Vitis amurensis Rupr. wine. In this study, 40 phenolic compounds which include 15 anthocyanins, 10 flavonoids, 10 phenolic acids, 3 proanthocyanidins and 2 resveratrols were identified by UPLC Q-Exactive Orbitrap MS. Furthermore, An UPLC-QqQ/MS method was established to simultaneously determine the phenolic compounds in Vitis amurensis Rupr. Wine, and analyze the content differences of phenolic compounds between Vitis amurensis Rupr. and Vitis vinifera wine. Finally, network pharmacology was employed for the first time to predict the possible pharmacological mechanisms of Vitis amurensis wine polyphenols against nervous damage. Multivariate network analysis indicated that quercetin, (-)-epigallocatechin and various anthocyanins were found as prominent compounds for the treatment of nervous system diseases. Vitis amurensis Rupr. wine polyphenols mainly acted on these key targets, including AKT1, EGFR, ESR1, and SRC, and further regulate the PI3K-AKT and Rap1 signaling pathway for treating nervous system diseases. Our findings suggested that polyphenols in Vitis amurensis Rupr. wine possess neuroprotective effect through multicomponent, multitarget, and multichannel. PRACTICAL APPLICATIONS: Studies have revealed that Vitis amurensis Rupr. has higher levels of phenolic compounds than Vitis vinifera wine. However, due to the few and limited study of Vitis amurensis Rupr., their compositions in Vitis amurensis Rupr. wine and the molecular mechanism underlying their neuroprotection remains unclear. This research uses a holistic network pharmacology strategy to investigate the potential targets and pharmacological mechanisms of Vitis amurensis Rupr. wine polyphenols against nervous damage. To some things up, The finding elucidates the relationships between signaling pathways, targets, and compounds in Vitis amurensis Rupr. wine polyphenols, which may provide guidance and foundations for further application of medicinal food.

Early-Life Stress Induces Prodromal Features of Parkinsonism in Ageing Rats.

Early-life stress (ELS) can cause long-term effects on human health, ranging from adolescence to adulthood, and even to gerontic. Although clinical retrospective data suggest that ELS may be related to senile neurodegenerative diseases such as Parkinson's disease (PD), there are few prospective investigations to explore its real contribution to PD. Here, we investigated the behavioral, histochemical, neuromorphological, and transcriptional changes induced by maternal separation (MS), an ELS model. Without neurotoxin, MS rats showed behavioral alterations in olfaction, locomotion, and gait characters after depression compared with control rats. Based on neuroimaging and histochemistry, although we found that the dopaminergic system in the striatum was impaired after MS, the decrease of striatal dopamine level was ~33%. Consistently, tyrosine hydroxylase immunostaining positive neurons of MS rats in the substantia nigra showed deficit by about 20% in cell counting. Furthermore, using transcriptome sequencing, we discovered many differentially expressed genes (DEGs) of MS rats in the striatum significantly enriched in the pathway of dopaminergic synapse, and the biological process of locomotion and neuromuscular process controlling balance. Encouragingly, some representative DEGs relating to PD were singled out. These results suggest that ELS-depression rats potentially mimic some key features of prodromal stage of PD during natural senescence. In conclusion, our findings provide some novel insights into the future pathogenesis and therapeutic studies for PD related to depression.