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.

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.

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.

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.

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.

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.

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.

Anhedonic behavior in cryptochrome 2-deficient mice is paralleled by altered diurnal patterns of amygdala gene expression.

Mood disorders are frequently paralleled by disturbances in circadian rhythm-related physiological and behavioral states and genetic variants of clock genes have been associated with depression. Cryptochrome 2 (Cry2) is one of the core components of the molecular circadian machinery which has been linked to depression, both, in patients suffering from the disease and animal models of the disorder. Despite this circumstantial evidence, a direct causal relationship between Cry2 expression and depression has not been established. Here, a genetic mouse model of Cry2 deficiency (Cry2 (-/-) mice) was employed to test the direct relevance of Cry2 for depression-like behavior. Augmented anhedonic behavior in the sucrose preference test, without alterations in behavioral despair, was observed in Cry2 (-/-) mice. The novelty suppressed feeding paradigm revealed reduced hyponeophagia in Cry2 (-/-) mice compared to wild-type littermates. Given the importance of the amygdala in the regulation of emotion and their relevance for the pathophysiology of depression, potential alterations in diurnal patterns of basolateral amygdala gene expression in Cry2 (-/-) mice were investigated focusing on core clock genes and neurotrophic factor systems implicated in the pathophysiology of depression. Differential expression of the clock gene Bhlhe40 and the neurotrophic factor Vegfb were found in the beginning of the active (dark) phase in Cry2 (-/-) compared to wild-type animals. Furthermore, amygdala tissue of Cry2 (-/-) mice contained lower levels of Bdnf-III. Collectively, these results indicate that Cry2 exerts a critical role in the control of depression-related emotional states and modulates the chronobiological gene expression profile in the mouse amygdala.

Increase of methane formation by ethanol addition during continuous fermentation of biogas sludge.

Very recently, it was shown that the addition of acetate or ethanol led to enhanced biogas formation rates during an observation period of 24 h. To determine if increased methane production rates due to ethanol addition can be maintained over longer time periods, continuous reactors filled with biogas sludge were developed which were fed with the same substrates as the full-scale reactor from which the sludge was derived. These reactors are well reflected conditions of a full-scale biogas plant during a period of 14 days. When the fermenters were pulsed with 50-100 mM ethanol, biomethanation increased by 50-150 %, depending on the composition of the biogas sludge. It was also possible to increase methane formation significantly when 10-20 mM pure ethanol or ethanolic solutions (e.g. beer) were added daily. In summary, the experiments revealed that "normal" methane production continued to take place, but ethanol led to production of additional methane.

Acetate activation in Methanosaeta thermophila: characterization of the key enzymes pyrophosphatase and acetyl-CoA synthetase.

The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PP(i)) and was only moderately inhibited by PP(i). The breakdown of PP(i) was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PP(i) (K(M) = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PP(i). However, it cannot be excluded that other PP(i)-dependent enzymes take advantage of the remaining PP(i) and contribute to the energy balance of the cell.

Ruthenium-catalyzed allylation-cyclization reactions of cyclic 1,3-dicarbonyl compounds with 1-vinyl propargyl alcohols.

Ruthenium-catalyzed allylation-cyclization reactions of cyclic 1,3-dicarbonyl compounds with 1-vinyl propargyl alcohols that lead to diverse carbo- or heterocyclic products in a one-pot cascade process are reported. These mechanistically distinct reactions are catalyzed by a single ruthenium(0) complex that contains a redox-coupled dienone ligand. The reaction pathway strongly depends on the substrate substitution pattern, which determines the mode of activation of the 1-vinyl propargyl alcohol. The environmentally benign process, which generates water as the only waste product, is of wide scope and allows the atom-economic synthesis of highly functionalized furans, pyrans, and spirocarbocycles.

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.

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.

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.

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.

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.

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.

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.