In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

Interaction of ceramides and tear lipocalin

The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.     

Human tears reveal insights into corneal neovascularization

Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization.     

Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmer''s I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.     

Borna disease virus infection perturbs energy metabolites and amino acids in cultured human oligodendroglia cells

Background

Borna disease virus is a neurotropic, non-cytolytic virus that has been widely employed in neuroscientific research. Previous studies have revealed that metabolic perturbations are associated with Borna disease viral infection. However, the pathophysiological mechanism underlying its mode of action remains unclear.

Methodology

Human oligodendroglia cells infected with the human strain Borna disease virus Hu-H1 and non-infected matched control cells were cultured in vitro. At day 14 post-infection, a proton nuclear magnetic resonance-based metabonomic approach was used to differentiate the metabonomic profiles of 28 independent intracellular samples from Borna disease virus-infected cells (n = 14) and matched control cells (n = 14). Partial least squares discriminant analysis was performed to demonstrate that the whole metabonomic patterns enabled discrimination between the two groups, and further statistical testing was applied to determine which individual metabolites displayed significant differences between the two groups.

Findings

Metabonomic profiling revealed perturbations in 23 metabolites, 19 of which were deemed individually significant: nine energy metabolites (α-glucose, acetate, choline, creatine, formate, myo-inositol, nicotinamide adenine dinucleotide, pyruvate, succinate) and ten amino acids (aspartate, glutamate, glutamine, glycine, histidine, isoleucine, phenylalanine, threonine, tyrosine, valine). Partial least squares discriminant analysis demonstrated that the whole metabolic patterns enabled statistical discrimination between the two groups.

Conclusion

Borna disease viral infection perturbs the metabonomic profiles of several metabolites in human oligodendroglia cells cultured in vitro. The findings suggest that Borna disease virus manipulates the host cell’s metabolic network to support viral replication and proliferation.     

Characterization of human reflex tear proteome reveals high expression of lacrimal proline‐rich protein 4 (PRR4)

In‐depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC‐ESI‐MS/MS strategy for label‐free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC‐ESI‐MS/MS and targeted‐MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline‐rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland‐specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha‐1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.     

Third degree obstetric anal sphincter tears: risk factors and outcome of primary repair.

OBJECTIVES--To determine (i) risk factors in the development of third degree obstetric tears and (ii) the success of primary sphincter repair. DESIGN--(i) Retrospective analysis of obstetric variables in 50 women who had sustained a third degree tear, compared with the remaining 8553 vaginal deliveries during the same period. (ii) Women who had sustained a third degree tear and had primary sphincter repair and control subjects were interviewed and investigated with anal endosonography, anal manometry, and pudendal nerve terminal motor latency measurements. SETTING--Antenatal clinic in teaching hospital in inner London. SUBJECTS--(i) All women (n = 8603) who delivered vaginally over a 31 month period. (ii) 34 women who sustained a third degree tear and 88 matched controls. MAIN OUTCOME MEASURES--Obstetric risk factors, defecatory symptoms, sonographic sphincter defects, and pudendal nerve damage. RESULTS--(i) Factors significantly associated with development of a third degree tear were: forceps delivery (50% v 7% in controls; P = 0.00001), primiparous delivery (85% v 43%; P = 0.00001), birth weight > 4 kg (P = 0.00002), and occipito-posterior position at delivery (P = 0.003). No third degree tear occurred during 351 vacuum extractions. Eleven of 25 (44%) women who were delivered without instruments and had a third degree tear did so despite a posterolateral episiotomy. (ii) Anal incontinence or faecal urgency was present in 16 women with tears and 11 controls (47% v 13%; P = 0.00001). Sonographic sphincter defects were identified in 29 with tears and 29 controls (85% v 33%; P = 0.00001). Every symptomatic patient had persistent combined internal and external sphincter defects, and these were associated with significantly lower anal pressures. Pudendal nerve terminal motor latency measurements were not significantly different. CONCLUSIONS--Vacuum extraction is associated with fewer third degree tears than forceps delivery. An episiotomy does not always prevent a third degree tear. Primary repair is inadequate in most women who sustain third degree tears, most having residual sphincter defects and about half experiencing anal incontinence, which is caused by persistent mechanical sphincter disruption rather than pudendal nerve damage. Attention should be directed towards preventive obstetric practice and surgical techniques of repair.     

Sex Difference in Contusion of the Medial Femoral Condyle Rim-Association with MRI Occult Meniscal Tears in the ACL Deficient Knee

BackgroundMeniscal tears, specifically lateral meniscal tears, have a larger than expected un-derdiagnosis rate in the presence of an ACL tear. The purpose of our study was to search for an MRI bone contusion pattern associated with MRI occult meniscal tears in patients with an ACL tear, specifically a contusion of the rim of the medial femoral condyle (RMFC). Our hypothesis was that there would be a significant association between RMFC contusions and MRI occult meniscal tears in patients with an ACL tear. We also searched for a difference between sexes with respect to the presence of the RMFC contusion in the setting of an occult meniscal tear. We also categorized the type, size, and location of these occult meniscal tears in the setting of an ACL tear.Methods This was a retrospective study that examined characteristics of occult meniscal tears and their association with a RMFC bone contusion. IRB approval was obtained. The date range of the study was June 2009 through December 2015. 6392 consecutive knee MRI reports in patients with an ACL deficient knee were reviewed. The study group included 22 patients with MRI occult meniscal tears, the control group included 110 patients. Relevant statistical values were calculated.ResultsThe most common type of occult meniscal tears were small radial and small longitudinal tears of the lateral meniscus. Occult meniscal tears were associated with an RMFC contusion in the study group (p=0.0457), particularly in males (p = 0.0003). In males with a torn ACL, the sensitivity of an RMFC contusion for an occult meniscal tear was 80%.ConclusionIn males with an ACL tear, there was a significant association between a contusion of the RMFC and an occult meniscal tear (commonly small radial or small peripheral partial-thickness longitudinal tears). RMFC contusions were reliably identified by radiologists in this study.Level of Evidence: II     

Role of lactoferrin in the tear film

The surface of the eye provides an inert barrier against infection. Through its unique combination of antimicrobial action and anti-inflammatory activities lactoferrin (Lf) in the tear film plays an important role in the maintenance of ocular health. In order to maintain clarity the eye must provide immunological defense without immunopathology. Along with physical barriers, soluble plasma factors and other proteins such as lysozyme, Lf produced by the acinar cells of the lacrimal gland serves a number of roles in defense for this purpose. Lf in tears provides antimicrobial efficacy by binding free iron thus reducing the availability of iron necessary for microbial growth and survival as well as pathogenesis. Lf has been shown to inhibit biofilm formation and thus may play a role in protecting contact lens surfaces from colonization. Virus particles' entry into epithelial cells is inhibited by Lf while an excess of Lf in tear film is thought to limit the opportunistic Lf-mediated bridging of adenovirus and host cell that occurs in other tissues. Lf dampens the classical complement activation pathway by binding to markers of inflammation and immune activation while pathogen-associated molecular patterns such as lipopolysaccharide (LPS) are targeted by Lf for removal through tears and hydrodynamic flushing. This review focuses on the role of Lf in human tear film and its contribution to ocular health during contact lens wear.     

Proteomic analysis of human tears: defensin expression after ocular surface surgery

Human tear protein profiles were monitored by surface enhanced laser desorption/ionization-time-of-flight mass spectrometry ProteinChip technology (SELDI-TOF ProteinChip) and liquid chromatography-mass spectrometry (LC-MS). Tears were collected from 21 patients scheduled for surgery to remove an ocular surface neoplasm prior to surgery (day 0) and on days 1, 3, and 30 postoperatively. Using this proteomic approach, we verified that three human alpha-defensins (HNP-1, HNP-2, and HNP-3) were significantly up-regulated in their expression after surgery and that their levels decreased to approximately normal by day 30 by which time healing was complete. Further confirmation of the identity of the alpha-defensins in human tears was made by LC purification, trypsin digestion, and ESI-MS/MS analysis of their tryptic digests. The concentrations of HNP-1 and HNP-2 were determined and shown to be markedly increased after ocular surface surgery. The results of the study suggest that human alpha-defensins HNP-1, HNP-2, and HNP-3 are up-regulated after surgery, and may in addition to their antimicrobial properties have an important role in wound healing.     

Proteomic analysis of rabbit tear fluid: Defensin levels after an experimental corneal wound are correlated to wound closure

The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1-5) following a standard unilateral 6 mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13 peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1- 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability.     

Serum metabolic profiling of abnormal savda by liquid chromatography/mass spectrometry

Abnormal savda is a special symptom in Uigur medicine. The understanding of its metabolic origins is of great importance for the subsequent treatment. Here, a metabonomic study of this symptom was carried out using LC-MS based human serum metabolic profiling. Orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA) was used for the classification and prediction of abnormal savda. Potential biomarkers from metabonomics were also identified for a metabolic understanding of abnormal savda. As a result, our OSC-PLS-DA model had a satisfactory ability for separation and prediction of abnormal savda. The potential biomarkers including bilirubin, bile acids, tryptophan, phenylalanine and lyso-phosphatidylcholines indicated that abnormal savda could be related to some abnormal metabolisms within the body, including energy metabolism, absorption of nutrition, metabolism of lecithin on cell membrane, etc. To the best of our knowledge, this is the first study of abnormal savda based on serum metabolic profiling. The LC/MS-based metabonomic platform could be a powerful tool for the classification of symptoms and for the development of this traditional medicine into an evidence-based one.     

Quantitative determination of besifloxacin, a novel fluoroquinolone antimicrobial agent, in human tears by liquid chromatography-tandem mass spectrometry

A rapid and sensitive method was developed using high-performance liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the quantification of besifloxacin in human tears using sparfloxacin as the internal standard (IS). Besifloxacin was extracted from human tear samples using an ammonium formate buffer at pH 3.25. The method was validated over a concentration range of 2-2000 ng/mL, with a total run time of less than 4 min. The overall intra- and inter-day precision for this method was less than 6%. The method was used to measure besifloxacin concentrations in tear samples collected after topical ocular administration to humans; besifloxacin concentrations were 610+/-540 microg/g (15 min) and 1.60+/-2.28 microg/g (24h).     

Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.     

Proteomic analysis revealed the altered tear protein profile in a rabbit model of Sjögren's syndrome‐associated dry eye

Sjögren's syndrome (SS) is an autoimmune disease that results in pathological dryness of mouth and eye. The diagnosis of SS depends on both clinical evaluation and specific antibodies. The goal of this study was to use quantitative proteomics to investigate changes in tear proteins in a rabbit model of SS‐associated dry eye, induced autoimmune dacryoadenitis (IAD). Proteomic analysis was performed by iTRAQ and nano LC‐MS/MS on tears collected from the ocular surface, and specific proteins were verified by high resolution MRM. It was found that in the tears of IAD rabbits at 2 and 4 weeks after induction, S100 A6, S100 A9, and serum albumin were upregulated, whereas serotransferrin (TF), prolactin‐inducible protein (PIP), polymeric immunoglobulin receptor (pIgR), and Ig gamma chain C region were downregulated. High resolution MRM with mTRAQ labeling verified the changes in S100 A6, TF, PIP, and pIgR. Our results indicated significant changes of tear proteins in IAD rabbits, suggesting these proteins could potentially be used as biomarkers for the diagnosis and prognosis of dry eye. Several of these proteins were also found in the tears of non‐SS dry eye patients indicating a common basis of ocular surface pathology, however, pIgR appears to be unique to SS.     

Human tear fluid lipidome: from composition to function

We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 μM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.     

GC/MS analysis of the rat urine for metabonomic research

In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine+water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.     

THE OCULAR SECRETIONS OF THE BOTTLENOSE DOLPHIN TURSIOPS TRUNCATUS

A viscous ocular secretion streams from the eyes of bottlenose dolphins, Tursiops truncatuss , removed from the water. The chemical composition and environmentally adaptive implications of this substance continue to perplex curators and scientists. Secretions from three dolphins were examined for deviations from literature values for normal human and terrestrial mammalian tear film components. Osmolality (= 470 mOsm/kg), pH (= 8.44) and glucose (= 26.58 mg/100 ml) were significantly greater than human and terrestrial mammalian tear film values; while lysozyme (= 0.005 mg/ml) and total cholesterol (= 0.17 μg/μ1) were significantly less. Refractive index, sodium ion concentration, and potassium tear to plasma ratios in the dolphin ocular secretion did not deviate significantly from human or terrestrial mammalian tears. Electrophoresis of dolphin tears yielded four major protein bands (probably lactoferrin, serum albumin, teat specific prealbumin, and lysozyme) and high molecular weight glycoproteins similar to human tears. The chemical composition of the T. truncatus ocular secretion suggests that the circumorbital conjunctival gland may be analogous (relative to refractive index, electrolytes, and component protein) to the terrestrial lachrymal gland. At the functional level the T. truncatus ocular secretion may reduce hydrodynamic resistance on the cornea and protect it from bacterial invasion.     

An 1H NMR and UPLC�CMS-based plasma metabonomic study to investigate the biochemical changes in chronic unpredictable mild stress model of depression

Depression is a common and highly debilitating psychiatric illness. However, the pathophysiology of depression is not fully understood. In this study Sprague-Dawley rats were exposed to chronic unpredictable mild stress (CUMS) to induce depression. A metabonomic study on plasma of CUMS-induced depressive rats was performed to research the pathologic mechanism of depression by using ¹H nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography coupled to mass spectrometry (UPLC–MS). Clear separations between depressive rats and control rats were observed by principal component analysis (PCA) based on the data obtained using both analytical techniques and 18 significantly changed metabolites were identified as potential biomarkers of depression. Depressive rats were characterized by altered levels of plasma lysophosphatidylcholines, amino acids, cholic acid, choline, lactate, glycoproteins, glucose, ketone bodies, nucleosides and gut microflora metabolites, which were related to multiple perturbed metabolic pathways and contributed to the elucidation of the complex mechanism of depression. To the best of our knowledge, this is the first plasma metabonomic study on CUMS-induced depressive rats by using two complementary analytical technologies. Our results showed that metabonomic approach offers a useful tool to identify depression-specific biomarkers and provide new insights into the pathophysiology of depression.