Development and Validation of a Decision Tree Analysis Model for Predicting Home Discharge in a Convalescent Ward: A Single Institution Study.

OBJECTIVES: Accurately predicting the likelihood of inpatients' home discharge in a convalescent ward is crucial for assisting patients and families in decision-making. While logistic regression analysis has been commonly used, its complexity limits practicality in clinical settings. We focused on decision tree analysis, which is visually straightforward. This study aimed to develop and validate the accuracy of a prediction model for home discharge for inpatients in a convalescent ward using a decision tree analysis. METHODS: The cohort consisted of 651 patients admitted to our convalescent ward from 2018 to 2020. We collected data from medical records, including disease classification, sex, age, duration of acute hospitalization, discharge destination (home or nonhome), and Functional Independence Measure (FIM) subitems at admission. We divided the cohort data into training and validation sets and developed a prediction model using decision tree analysis with discharge destination as the target and other variables as predictors. The model's accuracy was validated using the validation data set. RESULTS: The decision tree model identified FIM grooming as the first single discriminator of home discharge, diverging at four points and identifying subsequent branching for the duration of acute hospitalization. The model's accuracy was 86.7%, with a sensitivity of 0.96, specificity of 0.52, positive predictive accuracy of 0.88, and negative predictive accuracy of 0.80. The area under the receiver operating characteristic curve was 0.75. CONCLUSION: The predictive model demonstrated more than moderate predictive accuracy, suggesting its utility in clinical practice. Grooming emerged as a variable with the highest explanatory power for determining home discharge.

Involvement of inflammatory responses in the brain to the onset of major depressive disorder due to stress exposure.

Major depressive disorder (MDD) is a multifactorial disease affected by several environmental factors. Although several potential onset hypotheses have been identified, the molecular mechanisms underlying the pathogenesis of this disorder remain unclear. Several recent studies have suggested that among many environmental factors, inflammation and immune abnormalities in the brain or the peripheral tissues are associated with the onset of MDDs. Furthermore, several stress-related hypotheses have been proposed to explain the onset of MDDs. Thus, inflammation or immune abnormalities can be considered stress responses that occur within the brain or other tissues and are regarded as one of the mechanisms underlying the stress hypothesis of MDDs. Therefore, we introduce several current advances in inflammation studies in the brain that might be related to the pathophysiology of MDD due to stress exposure in this review.

Sulforaphane suppresses the activity of sterol regulatory element-binding proteins (SREBPs) by promoting SREBP precursor degradation.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate various genes involved in cholesterol and fatty acid synthesis. In this study, we describe that naturally occurring isothiocyanate sulforaphane (SFaN) impairs fatty acid synthase promoter activity and reduces SREBP target gene (e.g., fatty acid synthase and acetyl-CoA carboxylase 1) expression in human hepatoma Huh-7 cells. SFaN reduced SREBP proteins by promoting the degradation of the SREBP precursor. Amino acids 595-784 of SREBP-1a were essential for SFaN-mediated SREBP-1a degradation. We also found that such SREBP-1 degradation occurs independently of the SREBP cleavage-activating protein and the Keap1-Nrf2 pathway. This study identifies SFaN as an SREBP inhibitor and provides evidence that SFaN could have major potential as a pharmaceutical preparation against hepatic steatosis and obesity.

Coactivator-associated arginine methyltransferase 1 controls oligodendrocyte differentiation in the corpus callosum during early brain development.

Protein arginine methylation has been recognized as one of key posttranslational modifications for refined protein functions, mediated by protein arginine methyltransferases (Prmts). Coactivator-associated arginine methyltransferase (Carm1, also known as Prmt4) participates in various cellular events, such as cell survival, proliferation, and differentiation through its protein arginine methylation activities. Carm1 regulates cell proliferation of a neuronal cell line and is reportedly expressed in the mammalian brain. However, its detailed function in the central nervous system, particularly in glial cells, remains largely unexplored. In this study, Carm1 exhibited relatively high expression in oligodendrocyte (OL) lineage cells present in the corpus callosum of the developing brain, followed by a remarkable downregulation after active myelination. The suppression of Carm1 activity by inhibitors in isolated oligodendrocyte precursor cells (OPCs) reduced the number of Ki67-expressing and BrdU-incorporated proliferating cells. Furthermore, Carm1 inactivation attenuated OL differentiation, as determined by the expression of Plp, a reliable myelin-related marker. It also impaired the extension of OL processes, accompanied by a significant reduction in gene expression related to OL differentiation and myelination, such as Sox10, Cnp, Myrf, and Mbp. In addition, OLs co-cultured with embryonic dorsal root ganglia neurons demonstrated that Carm1 activity is required for the appropriate formation of myelin processes and myelin sheaths around neuronal axons, and the induction of the clustering of Caspr, a node of Ranvier structural molecule. Thus, we propose that Carm1 is an essential molecule for the development of OPCs and OLs during brain development.

Effects of 3 Weeks of Water Immersion and Restraint Stress on Sleep in Mice.

Repeated stress is a risk factor for mental disorders and can also lead to sleep disturbances. Although the effects of stress on sleep architecture have been investigated in rodents, the length of the stress exposure period in most studies has been limited to about 10 days, and few studies have analyzed the effects of chronic stress over a longer period. Here we investigated how sleep is affected in a mouse model of depression induced by 3 weeks of daily water immersion and restraint stress (WIRS). Sleep was recorded after 1, 2, and 3 weeks of stress exposure. Some stress-induced changes in several sleep measures were maintained across the 3 weeks, whereas other changes were most prominent during the 1st week. The total amount of non-rapid eye movement sleep (NREMS) was increased and the total amount of time spent awake was decreased across all 3 weeks. On the other hand, the amount of REMS during the dark phase was significantly increased in the 1st week compared with that at baseline or the 2nd and 3rd weeks. Electroencephalogram (EEG) power in the delta range was decreased during NREMS, although the total amount of NREMS was increased. These findings indicate that repeated WIRS, which eventually leads to a depression-like phenotype, differentially affects sleep between the early and subsequent periods. The increase in the amount of REMS during the dark phase in the 1st week significantly correlated with changes in body weight. Our results show how sleep changes throughout a long period of chronic stress in a mouse model of depression.

Antidepressive Effects of Kamishoyosan through 5-HT1AReceptor and PKA-CREB-BDNF Signaling in the Hippocampus in Postmenopausal Depression-Model Mice.

Females are well known to suffer disproportionately more than males from stress-related neuropsychiatric disorders, especially during perimenopausal and postmenopausal periods. In addition to a decline in serum estradiol levels, environmental stress and social stress likely contribute to the development of neuropsychiatric symptoms in perimenopausal and postmenopausal women. Kamishoyosan (KSS) is a traditional Japanese Kampo medicine, composed of a specified mixture of 10 crude compounds derived from plant sources, widely used for various neuropsychiatric symptoms in perimenopausal and postmenopausal women. However, the molecular mechanisms underlying KSS-mediated attenuation of neuropsychological symptoms and stress-response behaviors in perimenopausal and postmenopausal women remain unknown. In the present study, we first established a mouse model for postmenopausal depression-like signs using chronic water-immersion and restraint-stressed ovariectomized (OVX) mice to investigate the underlying molecular mechanism of KSS. We found that continuous administration of KSS to these mice normalized the activation of the hypothalamic-pituitary-adrenal (HPA) axis, ameliorated stress-induced depressive behavior, and prevented a decrease of neurogenesis in the hippocampus. As previous studies have implicated dysfunction of the hippocampal 5-HT1A receptor (5-HT1AR) in depressive disorders, we also evaluated the effect of KSS on 5-HT1AR expression and the protein kinase A- (PKA-) cAMP response element-binding- (CREB-) brain-derived neurotrophic factor (BDNF) signaling pathway in the hippocampus in this model. The level of 5-HT1AR in the hippocampus decreased in chronic stress-exposed OVX mice, while KSS treatment normalized the stress-induced decrease in 5-HT1AR expression in the hippocampus of chronic stress-exposed OVX mice. Furthermore, we found that KSS treatment upregulated the expression levels of phosphorylated PKA (p-PKA), phosphorylated CREB (p-CREB), and BDNF in the hippocampus in chronic stress-exposed OVX mice. These results suggest that KSS improves neuropsychiatric symptoms through 5-HT1AR and PKA-CREB-BDNF signaling in the hippocampus in postmenopausal women.

Cytoskeletal Signal-Regulated Oligodendrocyte Myelination and Remyelination.

Myelination and remyelination in the central nervous system (CNS) are essential for rapid conduction of action potentials and for appropriate neuronal communications supporting higher brain functions. Myelination is dependent on developmental stage and is controlled by neuronal axons-oligodendrocyte (OL) signaling. Numerous studies of the initial myelination and remyelination stages in the CNS have demonstrated several key cytoskeletal signals in axons and OLs. In this review, we focus on cytoskeletal signal-regulated OL myelination and remyelination, with particular attention to neuronal Notch proteins, bidirectional Eph/ephrin signaling, OL integrin and cadherin superfamily proteins, OL actin rearrangement, and OL tyrosine kinase Fyn substrate proteins during the initial myelination and remyelination stages in the CNS.

Isoxanthohumol stimulates ubiquitin-proteasome-dependent degradation of precursor forms of sterol regulatory element-binding proteins.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate a wide variety of genes involved in fatty acid and cholesterol synthesis. In the present study, we identified that isoxanthohumol (IXN) suppressed SREBP activity. Low concentrations of IXN (10 and 30 µM) reduced the amount of mature forms of SREBPs, while high concentration of IXN (100 µM) reduced both precursor and mature forms of SREBPs in Huh-7 cells. The IXN-mediated decrease in the precursor forms of SREBPs in Huh-7 cells was completely abolished by culturing cells under sterol-supplemented conditions and was partly abolished by treatment with a proteasome inhibitor, MG132, but not a lysosome inhibitor, NH4Cl. Moreover, IXN accelerated the ubiquitination of the precursor forms of SREBP-1a. These results suggest that IXN suppresses SREBP activity, at least in part, via ubiquitin-proteasome-dependent degradation of the precursor forms of SREBPs. ABBREVIATIONS: ACC1: acetyl-CoA carboxylase 1; DMEM: Dulbecco's modified Eagle's medium; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 25-HC: 25-hydroxycholesterol; HMGCR: HMG-CoA reductase; HMGCS: HMG-CoA synthase; Insig: insulin-induced gene; IXN: isoxanthohumol; LPDS: lipoprotein-deficient serum; SCAP: SREBP cleavage-activating protein; SCD1: stearoyl-CoA desaturase; SREBPs: sterol regulatory element-binding proteins; XN: xanthohumol.

NDE1 positively regulates oligodendrocyte morphological differentiation.

Oligodendrocytes, the myelin-forming cells in the central nervous system (CNS), undergo morphological differentiation characterized by elaborated branched processes to enwrap neuronal axons. However, the basic molecular mechanisms underlying oligodendrocyte morphogenesis remain unknown. Herein, we describe the essential roles of Nuclear Distribution E Homolog 1 (NDE1), a dynein cofactor, in oligodendrocyte morphological differentiation. In the mouse corpus callosum, Nde1 mRNA expression was detected in oligodendrocyte lineage cells at the postnatal stage. In vitro analysis revealed that downregulation of NDE1 by siRNA impaired the outgrowth and extensive branching of oligodendrocyte processes and led to a decrease in the expression of myelin-related markers, namely, CNPase and MBP. In myelinating co-cultures with dorsal root ganglion (DRG) neurons, NDE1-knockdown oligodendrocyte precursor cells (OPCs) failed to develop into MBP-positive oligodendrocytes with multiple processes contacting DRG axons. Immunoprecipitation studies showed that NDE1 interacts with the dynein intermediate chain (DIC) in oligodendrocytes, and an overexpressed DIC-binding region of NDE1 exerted effects on oligodendrocyte morphogenesis that were similar to those following NDE1 knockdown. Furthermore, NDE1-knockdown-impaired oligodendrocyte process formation was rescued by siRNA-resistant wild-type NDE1 but not by DIC-binding region-deficient NDE1 overexpression. These results suggest that NDE1 plays a crucial role in oligodendrocyte morphological differentiation via interaction with dynein.

MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors.

Abnormalities in limbic neural circuits have been implicated in the onset of anxiety disorders. However, the molecular pathogenesis underlying anxiety disorders remains poorly elucidated. Here, we demonstrate that myristoylated alanine-rich C-kinase substrate like 1 (MARCKSL1) regulates amygdala circuitry to control the activity of the hypothalamic-pituitary-adrenal (HPA) axis, as well as induces anxiety-like behaviors in mice. MARCKSL1 expression was predominantly localized in the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala of the adult mouse brain. MARCKSL1 transgenic (Tg) mice exhibited anxiety-like behaviors dependent on corticotropin-releasing hormone. MARCKSL1 increased spine formation in the central amygdala, and downregulation of MARCKSL1 in the amygdala normalized both increased HPA axis activity and elevated anxiety-like behaviors in Tg mice. Furthermore, MARCKSL1 expression was increased in the PFC and amygdala in a brain injury model associated with anxiety-like behaviors. Our findings suggest that MARCKSL1 expression in the amygdala plays an important role in anxiety-like behaviors.

Effects of task-specific paretic ankle plantar flexor training on walking in a stroke patient: a single-case study.

[Purpose] The purpose of this study was to examine the effects of task-specific plantar flexor training on walking ability indices in a patient with a paretic ankle. [Subject and Methods] The subject was a 65-year-old male patient with right hemiplegia due to a left medullary ventral infarction. An ABA' single-subject design was adopted. The independent variable was a task-specific plantar flexor training exercise, similar to that during walking, targeting the paretic ankle. The dependent variables were the isometric ankle plantar flexor strength, maximum walking speed, step length, and trailing limb angle in the paretic terminal stance phase. The B study phase was divided into B1 and B2 phases. A two standard-deviation-band method was used to evaluate improvement. [Results] Improvements in the paretic plantar flexor strength, maximum walking speed, step length, and trailing limb angle in the B2 phase were observed. The improvements in the maximum walking speed, step length, and trailing limb angle were sustained in the A' study phase. [Conclusion] These results suggest that task-specific plantar flexor training exercise is efficacious in improving the walking ability index of a paretic ankle.

Kaempferol stimulates gene expression of low-density lipoprotein receptor through activation of Sp1 in cultured hepatocytes.

A high level of plasma low-density lipoprotein (LDL) cholesterol is considered a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) is essential for clearing plasma LDL cholesterol, activation of LDLR is a promising therapeutic target for patients with atherosclerotic disease. Here we demonstrated how the flavonoid kaempferol stimulated the gene expression and activity of LDLR in HepG2 cells. The kaempferol-mediated stimulation of LDLR gene expression was completely inhibited by knockdown of Sp1 gene expression. Treatment of HepG2 cells with kaempferol stimulated the recruitment of Sp1 to the promoter region of the LDLR gene, as well as the phosphorylation of Sp1 on Thr-453 and Thr-739. Moreover, these kaempferol-mediated processes were inhibited in the presence of U0126, an ERK pathway inhibitor. These results suggest that kaempferol may increase the activity of Sp1 through stimulation of Sp1 phosphorylation by ERK1/2 and subsequent induction of LDLR expression and activity.

Association between chronic stress-induced structural abnormalities in Ranvier nodes and reduced oligodendrocyte activity in major depression.

Repeated stressful events are associated with the onset of major depressive disorder (MDD). We previously showed oligodendrocyte (OL)-specific activation of the serum/glucocorticoid-regulated kinase (SGK)1 cascade, increased expression of axon-myelin adhesion molecules, and elaboration of the oligodendrocytic arbor in the corpus callosum of chronically stressed mice. In the current study, we demonstrate that the nodes and paranodes of Ranvier in the corpus callosum were narrower in these mice. Chronic stress also led to diffuse redistribution of Caspr and Kv 1.1 and decreased the activity in white matter, suggesting a link between morphological changes in OLs and inhibition of axonal activity. OL primary cultures subjected to chronic stress resulted in SGK1 activation and translocation to the nucleus, where it inhibited the transcription of metabotropic glutamate receptors (mGluRs). Furthermore, the cAMP level and membrane potential of OLs were reduced by chronic stress exposure. We showed by diffusion tensor imaging that the corpus callosum of patients with MDD exhibited reduced fractional anisotropy, reflecting compromised white matter integrity possibly caused by axonal damage. Our findings suggest that chronic stress disrupts the organization of the nodes of Ranvier by suppressing mGluR activation in OLs, and that specific white matter abnormalities are closely associated with MDD onset.

Allyl isothiocyanate suppresses the proteolytic activation of sterol regulatory element-binding proteins and de novo fatty acid and cholesterol synthesis.

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of genes involved in fatty acid and cholesterol synthesis. In this study, we used a stable cell line that expresses a luciferase reporter gene driven by an SRE-containing fatty acid synthase promoter to identify allyl isothiocyanate (AITC), one of the major isothiocyanates in cruciferous vegetables, as a novel SREBP inactivator. We found that AITC downregulated the proteolytic processing of SREBPs and the expression of their target genes in human hepatoma Huh-7 cells. Furthermore, AITC reduced the de novo synthesis of both fatty acids and cholesterol. Our results indicate a novel physiological function of AITC in lipid metabolism regulation.

Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins.

Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD-15 cells, which lack insulin-induced gene-1 (Insig-1) and Insig-2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.

Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation.

Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome.

The Kampo Medicine Yokukansan Decreases MicroRNA-18 Expression and Recovers Glucocorticoid Receptors Protein Expression in the Hypothalamus of Stressed Mice.

It is well known that glucocorticoid receptor (GR) signaling regulates the hypothalamic-pituitary-adrenal (HPA) axis, and GR expression level is associated with HPA axis activity. Recent studies revealed that microRNA- (miR-) 18 and/or 124a are candidate negative regulators of GR in the brain. The Kampo medicine Yokukansan (YKS) can affect psychological symptoms such as depression and anxiety that are associated with stress responses. In this study, we evaluated the effect of YKS on miR-18 and 124a and GR levels in mice exposed to stress. We found that YKS pretreatment normalized elevated plasma corticosterone levels in stress-exposed mice. In addition, GR mRNA levels were downregulated in the brain following stress exposure. While miR-124a expression levels were not altered in the hypothalamus of stress-exposed mice, miR-18 levels decreased in the hypothalamus of YKS-pretreated mice after stress exposure. Finally, GR protein levels in the paraventricular nucleus (PVN) of the hypothalamus after stress exposure recovered in YKS-pretreated mice. Collectively, these data suggest that YKS normalizes GR protein levels by regulating miR-18 expression in the hypothalamus, thus normalizing HPA axis activity following stress exposure.

Sgk1 regulates desmoglein 1 expression levels in oligodendrocytes in the mouse corpus callosum after chronic stress exposure.

Major depression, one of the most prevalent mental illnesses, is thought to be a multifactorial disease related to both genetic and environmental factors. However, the genes responsible for and the pathogenesis of major depression at the molecular level remain unclear. Recently, we reported that stressed mice with elevated plasma corticosterone levels show upregulation and activation of serum glucocorticoid-regulated kinase (Sgk1) in oligodendrocytes. Active Sgk1 causes phosphorylation of N-myc downstream-regulated gene 1 (Ndrg1), and phospho-Ndrg1 increases the expression of N-cadherin, α-catenin, and ß-catenin in oligodendrocytes. This activation of the Sgk1 cascade results in morphological changes in the oligodendrocytes of nerve fiber bundles, such as those present in the corpus callosum. However, little is known about the molecular functions of the traditional and/or desmosomal cadherin superfamily in oligodendrocytes. Therefore, in this study, we aimed to elucidate the functions of the desmosomal cadherin superfamily in oligodendrocytes. Desmoglein (Dsg) 1, Dsg2, and desmocollin 1 (Dsc1) were found to be expressed in the corpus callosum of mouse brain, and the expression of a subtype of Dsg1, Dsg1c, was upregulated in oligodendrocytes after chronic stress exposure. Furthermore, Dsg1 proteins were localized around the plasma membrane regions of oligodendrocytes. A study in primary oligodendrocyte cultures also revealed that chronic upregulation of Sgk1 by dexamethasone administration is involved in upregulation of Dsg1c mRNA. These results may indicate that chronic stress induced Sgk1 activation in oligodendrocytes, which increases Dsg1 expression near the plasma membrane. Thus, Dsg1 upregulation may be implicated in the molecular mechanisms underlying the morphological changes in oligodendrocytes in response to chronic stress exposure.

Yokukansan normalizes glucocorticoid receptor protein expression in oligodendrocytes of the corpus callosum by regulating microRNA-124a expression after stress exposure.

Stressful events are known to down-regulate expression levels of glucocorticoid receptors (GRs) in the brain. Recently, we reported that stressed mice with elevated plasma levels of corticosterone exhibit morphological changes in the oligodendrocytes of nerve fiber bundles, such as those in the corpus callosum. However, little is known about the molecular mechanism of GR expression regulation in oligodendrocytes after stress exposure. A previous report has suggested that GR protein levels might be regulated by microRNA (miR)-18 and/or -124a in the brain. In this study, we aimed to elucidate the GR regulation mechanism in oligodendrocytes and evaluate the effects of yokukansan (YKS), a Kampo medicine, on GR protein regulation. Acute exposure to stress increased plasma corticosterone levels, decreased GR protein expression, and increased miR-124a expression in the corpus callosum of adult male mice, though the GR mRNA and miR-18 expression levels were not significant changes. YKS normalized the stress-induced changes in the plasma corticosterone, GR protein, and miR124a expression levels. An oligodendrocyte primary culture study also showed that YKS down-regulated miR-124a, but not miR-18, expression levels in dexamethasone-treated cells. These results suggest that the down-regulation of miR124a expression might be involved in the normalization of stress-induced decreases in GR protein in oligodendrocytes by YKS. This effect may imply the molecular mechanisms underlying the ameliorative effects of YKS on psychological symptoms and stress-related behaviors.