Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 µmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 µmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 µmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 µmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Methane-Dependent Extracellular Electron Transfer at the Bioanode by the Anaerobic Archaeal Methanotroph "Candidatus Methanoperedens".

Anaerobic methanotrophic (ANME) archaea have recently been reported to be capable of using insoluble extracellular electron acceptors via extracellular electron transfer (EET). In this study, we investigated EET by a microbial community dominated by "Candidatus Methanoperedens" archaea at the anode of a bioelectrochemical system (BES) poised at 0 V vs. standard hydrogen electrode (SHE), in this way measuring current as a direct proxy of EET by this community. After inoculation of the BES, the maximum current density was 274 mA m-2 (stable current up to 39 mA m-2). Concomitant conversion of 13CH4 into 13CO2 demonstrated that current production was methane-dependent, with 38% of the current attributed directly to methane supply. Based on the current production and methane uptake in a closed system, the Coulombic efficiency was about 17%. Polarization curves demonstrated that the current was limited by microbial activity at potentials above 0 V. The metatranscriptome of the inoculum was mined for the expression of c-type cytochromes potentially used for EET, which led to the identification of several multiheme c-type cytochrome-encoding genes among the most abundant transcripts in "Ca. Methanoperedens." Our study provides strong indications of EET in ANME archaea and describes a system in which ANME-mediated EET can be investigated under laboratory conditions, which provides new research opportunities for mechanistic studies and possibly the generation of axenic ANME cultures.

Hygiene management for long-term ventilated persons in the home health care setting: a scoping review.

BACKGROUND: Evidence and recommendations for hygiene management in home mechanical ventilation (HMV) are rare. In Germany, few regionally limited studies show poor hygiene management or a lack of its implementation. This scoping review of international literature identified the evidence in hygiene management for ventilated patients in the home care setting which has to be implemented for infection prevention and control. METHODS: A review of international literature was conducted in CINAHL, PubMed and Web of Science. The search focused on four key domains: HMV, hygiene management, home care setting, and methicillin-resistant Staphylococcus aureus (MRSA). Data of included studies were extracted using a data charting sheet. Extracted data were assigned to the categories (1) study description, (2) setting and participants, and (3) hygiene management. RESULTS: From 1,718 reviewed articles, n = 8 studies met inclusion criteria. All included studies had a quantitative study design. The approaches were heterogeneous due to different settings, study populations and types of ventilation performed. Regarding aspects of hygiene management, most evidence was found for infectious critical activities (n = 5), quality management for hygiene (n = 4), and training and education (n = 4). This review identified research gaps concerning kitchen hygiene, relatives and visitors of HMV patients, and waste management (n = 0). DISCUSSION: Overall evidence was rather scarce. Consequently, this review could not answer all underlying research questions. No evidence was found for measures in hygiene management relating to ventilated patients' relatives. Evidence for kitchen hygiene, waste management and interaction with relatives is available for inpatient care settings. However, this may not be transferable to outpatient care. Binding legal requirements and audits may help regulate the implementation of HMV hygiene measures. CONCLUSION: Infection control programmes included qualified personnel, hygiene plans, and standards for MRSA and multidrug-resistant organisms (MDRO). The appropriateness of hygiene management measures for outpatient care is the basis for their application in practice.

Methanogenic archaea use a bacteria-like methyltransferase system to demethoxylate aromatic compounds.

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

Investigation of central energy metabolism-related protein complexes of ANME-2d methanotrophic archaea by complexome profiling.

The anaerobic oxidation of methane is important for mitigating emissions of this potent greenhouse gas to the atmosphere and is mediated by anaerobic methanotrophic archaea. In a 'Candidatus Methanoperedens BLZ2' enrichment culture used in this study, methane is oxidized to CO2 with nitrate being the terminal electron acceptor of an anaerobic respiratory chain. Energy conservation mechanisms of anaerobic methanotrophs have mostly been studied at metagenomic level and hardly any protein data is available at this point. To close this gap, we used complexome profiling to investigate the presence and subunit composition of protein complexes involved in energy conservation processes. All enzyme complexes and their subunit composition involved in reverse methanogenesis were identified. The membrane-bound enzymes of the respiratory chain, such as F420H2:quinone oxidoreductase, membrane-bound heterodisulfide reductase, nitrate reductases and Rieske cytochrome bc1 complex were all detected. Additional or putative subunits such as an octaheme subunit as part of the Rieske cytochrome bc1 complex were discovered that will be interesting targets for future studies. Furthermore, several soluble proteins were identified, which are potentially involved in oxidation of reduced ferredoxin produced during reverse methanogenesis leading to formation of small organic molecules. Taken together these findings provide an updated, refined picture of the energy metabolism of the environmentally important group of anaerobic methanotrophic archaea.

Brain Tissue Pulsation in Healthy Volunteers.

It is well known that the brain pulses with each cardiac cycle, but interest in measuring cardiac-induced brain tissue pulsations (BTPs) is relatively recent. This study was aimed at generating BTP reference data from healthy patients for future clinical comparisons and modelling. BTPs were measured through the forehead and temporal positions as a function of age, sex, heart rate, mean arterial pressure and pulse pressure. A multivariate regression model was developed based on transcranial tissue Doppler BTP measurements from 107 healthy adults (56 male) aged from 20-81 y. A subset of 5 participants (aged 20-49 y) underwent a brain magnetic resonance imaging scan to relate the position of the ultrasound beam to anatomy. BTP amplitudes were found to vary widely between patients (from ∼4 to ∼150 µm) and were strongly associated with pulse pressure. Comparison with magnetic resonance images confirmed regional variations in BTP with depth and probe position.

Draft Genome Sequence of a New Methanobacterium Strain Potentially Resistant to Bile Salts, Isolated from Deer Feces.

We present the high-quality draft genome of Methanobacterium subterraneum DF, a hydrogenotrophic methanogen that was isolated from deer feces. This organism has potentially been overlooked in previous studies. Interestingly, its genome encoded bile salt hydrolase, a crucial enzyme for bile salt tolerance that is found in gut organisms.

Severe hydroxymethylbilane synthase deficiency causes depression-like behavior and mitochondrial dysfunction in a mouse model of homozygous dominant acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant inborn error of heme biosynthesis due to a pathogenic mutation in the Hmbs gene, resulting in half-normal activity of hydroxymethylbilane synthase. Factors that induce hepatic heme biosynthesis induce episodic attacks in heterozygous patients. The clinical presentation of acute attacks involves the signature neurovisceral pain and may include psychiatric symptoms. Here we used a knock-in mouse line that is biallelic for the Hmbs c.500G > A (p.R167Q) mutation with ~ 5% of normal hydroxymethylbilane synthase activity to unravel the consequences of severe HMBS deficiency on affective behavior and brain physiology. Hmbs knock-in mice (KI mice) model the rare homozygous dominant form of AIP and were used as tool to elucidate the hitherto unknown pathophysiology of the behavioral manifestations of the disease and its neural underpinnings. Extensive behavioral analyses revealed a selective depression-like phenotype in Hmbs KI mice; transcriptomic and immunohistochemical analyses demonstrated aberrant myelination. The uncovered compromised mitochondrial function in the hippocampus of knock-in mice and its ensuing neurogenic and neuroplastic deficits lead us to propose a mechanistic role for disrupted mitochondrial energy production in the pathogenesis of the behavioral consequences of severe HMBS deficiency and its neuropathological sequelae in the brain.

Effect of Chronic Corticosterone Treatment on Depression-Like Behavior and Sociability in Female and Male C57BL/6N Mice.

Depression is a very common psychiatric disorder affecting approximately 300 million people worldwide with the prevalence being twice as high in women as in men. Despite intense research efforts in recent decades, the neurobiological basis underlying depression remains incompletely understood. However, the exposure to chronic stress is widely accepted to constitute a precipitating factor for the development of this mental disorder. Several animal models for the investigation of the pathogenetic link between chronic stress and depression exist and have yielded important insights. The present study aimed at comparing two published protocols for the induction of depression-like behavior in mice based on chronic oral glucocorticoid application. Given the gender distribution in the prevalence of depression, the second goal of this study was to reveal possible differences in the behavioral responses of female and male mice to corticosterone (CORT) treatment. CORT treatment was found to modulate depression-like behavior in selected behavioral paradigms in a sex- and protocol-specific manner. These data are of relevance for the experimental design and interpretation of future studies in the field and further highlight the relevance of "sex as biological variable" to be considered an important parameter for experimental planning and interpretation of results.

Anaerobic methanotrophic archaea of the ANME-2d clade feature lipid composition that differs from other ANME archaea.

The anaerobic oxidation of methane (AOM) is a microbial process present in marine and freshwater environments. AOM is important for reducing the emission of the second most important greenhouse gas methane. In marine environments anaerobic methanotrophic archaea (ANME) are involved in sulfate-reducing AOM. In contrast, Ca. Methanoperedens of the ANME-2d cluster carries out nitrate AOM in freshwater ecosystems. Despite the importance of those organisms for AOM in non-marine environments little is known about their lipid composition or carbon sources. To close this gap, we analysed the lipid composition of ANME-2d archaea and found that they mainly synthesise archaeol and hydroxyarchaeol as well as different (hydroxy-) glycerol dialkyl glycerol tetraethers, albeit in much lower amounts. Abundant lipid headgroups were dihexose, monomethyl-phosphatidyl ethanolamine and phosphatidyl hexose. Moreover, a monopentose was detected as a lipid headgroup that is rare among microorganisms. Batch incubations with 13C labelled bicarbonate and methane showed that methane is the main carbon source of ANME-2d archaea varying from ANME-1 archaea that primarily assimilate dissolved inorganic carbon (DIC). ANME-2d archaea also assimilate DIC, but to a lower extent than methane. The lipid characterisation and analysis of the carbon source of Ca. Methanoperedens facilitates distinction between ANME-2d and other ANMEs.

Energy conservation in the gut microbe Methanomassiliicoccus luminyensis is based on membrane-bound ferredoxin oxidation coupled to heterodisulfide reduction.

Methanomassiliicoccus luminyensis was originally isolated from human feces and belongs to the seventh order of methanogens, the Methanomassiliicoccales, which are only distantly related to other methanogenic archaea. The organism forms methane from the reduction of methylamines or methanol using molecular hydrogen as reductant. The energy-conserving system in M. luminyensis is unique and the enzymes involved in this process are not found in this combination in members of the other methanogenic orders. In this context our central question was how the organism is able to generate ATP. Energy transduction was dependent on a membrane-bound ferredoxin: heterodisulfide oxidoreductase composed of reduced ferredoxin as an electron donor, at least one protein in the membrane fraction and the heterodisulfide reductase HdrD, which reduced the electron acceptor CoM-S-S-CoB. Electron transfer of this respiratory chain proceeded with a rate of 145 nmol reduced heterodisulfide min-1 ·mg-1 membrane protein. Methanomassiliicoccus luminyensis is the first example of a methanogenic archaeon that does not require Na+ ions for energy conservation. Only protons were used as coupling ions for the generation of the electrochemical ion gradient. The membrane-bound F420 H2 :phenazine oxidoreductase complex (without the electron input module FpoF) probably catalyzed the oxidation of reduced ferredoxin and potentially acted as primary proton pump in this electron transport system. In summary, the energy-conserving system of M. luminyensis possesses features found in the pathways of hydrogenotrophic and methylotrophic/aceticlastic methanogenesis. Consequently, the composition of the enzymes involved in ion translocation across the cytoplasmic membrane is different from all other methanogenic archaea.

Development of a nutritional documentation tool: a Delphi study.

In daily clinical practice, the smooth, timely and comprehensive transfer of information between care settings is important and reflects a cornerstone of high-quality patient care. The integration of nutritional information in the medical information transfer is currently not included in an evidence-based approach. It was, therefore, the aim of this study to develop a nutritional documentation tool (NDoc) on the basis of evidence and test it for its usability in daily clinical practice. Based on the results of a literature review, the authors collected core content using a modified Delphi survey from experts across Europe and included the information collected in a structured, NDoc. The subsequently developed tool included thirty items and was tested for its usability on a daily basis among primary care physicians and clinical physicians. The new NDoc can be introduced for use in any computer-based hospital information system regionally and adapted for worldwide use.

Homozygous hydroxymethylbilane synthase knock-in mice provide pathogenic insights into the severe neurological impairments present in human homozygous dominant acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an inborn error of heme biosynthesis due to the deficiency of hydroxymethylbilane synthase (HMBS) activity. Human AIP heterozygotes have episodic acute neurovisceral attacks that typically start after puberty, whereas patients with homozygous dominant AIP (HD-AIP) have early-onset chronic neurological impairment, including ataxia and psychomotor retardation. To investigate the dramatically different manifestations, knock-in mice with human HD-AIP missense mutations c.500G>A (p.Arg167Glu) or c.518_519GC>AG (p.Arg173Glu), designated R167Q or R173Q mice, respectively, were generated and compared with the previously established T1/T2 mice with ~30% residual HMBS activity and the heterozygous AIP phenotype. Homozygous R173Q mice were embryonic lethal, while R167Q homozygous mice (R167Q+/+) had ~5% of normal HMBS activity, constitutively elevated plasma and urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), profound early-onset ataxia, delayed motor development and markedly impaired rotarod performance. Central nervous system (CNS) histology was grossly intact, but CNS myelination was delayed and overall myelin volume was decreased. Heme concentrations in liver and brain were similar to those of T1/T2 mice. Notably, ALA and PBG concentrations in the cerebral spinal fluid and CNS regions were markedly elevated in R167Q+/+ mice compared with T1/T2 mice. When the T1/T2 mice were administered phenobarbital, ALA and PBG markedly accumulated in their liver and plasma, but not in the CNS, indicating that ALA and PBG do not readily cross the blood-brain barrier. Taken together, these studies suggest that the severe HD-AIP neurological phenotype results from decreased myelination and the accumulation of locally produced neurotoxic porphyrin precursors within the CNS.

Impact of maternal immune activation on maternal care behavior, offspring emotionality and intergenerational transmission in C3H/He mice.

Maternal immune activation (MIA) is a well-established model for the investigation of the deleterious effects of gestational infection on offspring mental health later in life. Hence, MIA represents a critical environmental variable determining brain development and the depending neural and behavioral functions in the progeny. Transgenerational transmission of some of the effects of MIA has been recently reported using the Polyinosinic:polycytidylic acid (Poly (I:C)) MIA model in C57BL/6 (C57) inbred mice. However, little is known about the underlying molecular mechanisms and the possible relevance of the specific genetic make-up of the inbred mouse strain used. Here we set out to characterize the effects of gestational Poly (I:C) treatment in C3H/HeNCrl mice (C3H), focusing on maternal care and offspring depression-like behavior and its intergenerational potential. miRNA expression in the offspring hippocampus in the F1 and F2 generations was examined as possible mechanism contributing to the observed behavioral effects. The impact of MIA on maternal care and its transmission to F1 females was previously observed in C57 mice was also found in C3H mice. Depression-like behavior in the adult offspring in C3H F1 and F2 females differed from reports of the C57 strain in the literature, suggesting a potential modulating role of the genetic background in the Poly(I:C) MIA mouse model. As the pattern of expression of selected candidate miRNAs in the F1 and F2 offspring hippocampus was not conserved between the two generations, it is unlikely to be a direct consequence of altered maternal care, or to be an immediate determinant of offspring emotionality.

High-Quality Draft Genome Sequence of "Candidatus Methanoperedens sp." Strain BLZ2, a Nitrate-Reducing Anaerobic Methane-Oxidizing Archaeon Enriched in an Anoxic Bioreactor.

The high-quality draft genome of "Candidatus Methanoperedens sp." strain BLZ2, a nitrate-reducing archaeon anaerobically oxidizing methane, is presented. The genome was obtained from an enrichment culture and measures 3.74 Mb. It harbors two nitrate reductase gene clusters, an ammonium-forming nitrite reductase, and the complete reverse methanogenesis pathway. Methane that escapes to the atmosphere acts as a potent greenhouse gas. Global methane emissions are mitigated by methanotrophs, which oxidize methane to CO2 "Candidatus Methanoperedens spp." are unique methanotrophic archaea that can perform nitrate-dependent anaerobic oxidation of methane. A high-quality draft genome sequence of only 85 contigs from this archaeon is presented here.

Repeatability and Reproducibility of Intraocular Pressure and Dynamic Corneal Response Parameters Assessed by the Corvis ST.

PURPOSE: To assess the repeatability and reproducibility of dynamic corneal response parameters measured by the Corvis ST (Oculus, Wetzlar, Germany). METHODS: One eye randomly selected from 32 healthy volunteers was examined by the Corvis ST. Three different devices were used in an alternated random order for taking three measurements at each device in each subject. Standard intraocular pressure (IOP), the biomechanical-compensated IOP (bIOP), and DCR parameters were evaluated. The within-subject standard deviation (ζw) and coefficient of variation (CV) were assessed. RESULTS: Regarding pressure indices, the ζw was below 1 mmHg for repeatability (0.98 for IOP and 0.89 for bIOP) and the CV was 6.6% for IOP and 6.1% for bIOP. For reproducibility, the ζw was around 1 mmHg (1.12 for IOP and 1.05 for bIOP) and the CV was 7.6% for IOP and 7.1% for bIOP. Most of DCR indices presented CV for repeatability below 4%. For reproducibility, the CV of most of the indices were below 6%. The deformation amplitude (DA) ratio in 1 mm and integrated radius were below 4% (1.2% and 3.8%, resp.). CONCLUSIONS: The Corvis ST showed good precision (repeatability and reproducibility) for IOP measurements and for DCR in healthy eyes.

BEAP profiles as rapid test system for status analysis and early detection of process incidents in biogas plants.

A method was developed to quantify the performance of microorganisms involved in different digestion levels in biogas plants. The test system was based on the addition of butyrate (BCON), ethanol (ECON), acetate (ACON) or propionate (PCON) to biogas sludge samples and the subsequent analysis of CH4 formation in comparison to control samples. The combination of the four values was referred to as BEAP profile. Determination of BEAP profiles enabled rapid testing of a biogas plant's metabolic state within 24 h and an accurate mapping of all degradation levels in a lab-scale experimental setup. Furthermore, it was possible to distinguish between specific BEAP profiles for standard biogas plants and for biogas reactors with process incidents (beginning of NH4+-N inhibition, start of acidification, insufficient hydrolysis and potential mycotoxin effects). Finally, BEAP profiles also functioned as a warning system for the early prediction of critical NH4+-N concentrations leading to a drop of CH4 formation.

Maternal immune activation transgenerationally modulates maternal care and offspring depression-like behavior.

Gestational infection is increasingly being recognized for its involvement as causative mechanism in severe developmental brain abnormalities and its contribution to the pathogenesis of psychopathologies later in life. First observations in the widely accepted maternal immune activation (MIA) model based upon the systemic administration of the viral mimetic Polyinosinic:polycytidylic acid (poly(I:C)) have recently suggested a transmission of behavioral and transcriptional traits across generations. Although maternal care behavior (MCB) is known as essential mediator of the transgenerational effects of environmental challenges on offspring brain function and behavior, the possible propagation of alterations of MCB resulting from MIA to following generations has not yet been examined. Here we show that poly(I:C) stimulation at embryonic day 12.5 (E12.5) leads to aberrant MCB and that this effect is transmitted to the female F1 offspring. The transgenerational effects on MCB are paralleled by enhanced depression-like behavior in the second generation F2 offspring with contributions of both maternal and paternal heritages. Examination of offspring hippocampal expression of genes known as targets of MCB and relevant for ensuing non-genetic transmission of altered brain function and behavior revealed transgenerationally conserved and modified expressional patterns in the F1 and F2 generation. Collectively these data firstly demonstrate the transgenerational transmission of the impact of gestational immune activation on the reproductive care behavior of the mother. Behavioral and molecular characteristics of first and second generation offspring suggest transgenerationally imprinted consequences of gestational infection on psychopathological traits related to mood disorders which remain to be examined in future cross-fostering experiments.

Maternal immune activation epigenetically regulates hippocampal serotonin transporter levels.

Major depressive disorder (MDD) is one of the most debilitating psychiatric diseases, affecting a large percentage of the population worldwide. Currently, the underlying pathomechanisms remain incompletely understood, hampering the development of critically needed alternative therapeutic strategies, which further largely depends on the availability of suitable model systems. Here we used a mouse model of early life stress - a precipitating factor for the development of MDD - featuring infectious stress through maternal immune activation (MIA) by polyinosinic:polycytidilic acid (Poly(I:C)) to examine epigenetic modulations as potential molecular correlates of the alterations in brain structure, function and behavior. We found that in adult female MIA offspring anhedonic behavior was associated with modulations of the global histone acetylation profile in the hippocampus. Morevoer, specific changes at the promoter and in the expression of the serotonin transporter (SERT), critically involved in the etiology of MDD and pharmacological antidepressant treatment were detected. Furthermore, an accompanying reduction in hippocampal levels of histone deacetylase (HDAC) 1 was observed in MIA as compared to control offspring. Based on these results we propose a model in which the long-lasting impact of MIA on depression-like behavior and associated molecular and cellular aberrations in the offspring is brought about by the modulation of epigenetic processes and consequent enduring changes in gene expression. These data provide additional insights into the principles underlying the impact of early infectious stress on the development of MDD and may contribute to the development of new targets for antidepressant therapy.