Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity.

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD+ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD+ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K+ , typical correlates of neuronal activity, induced a more reduced NADH/NAD+ redox ratio. While application of glutamate decreased [ATP], K+ as well as the combination of glutamate and K+ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K+ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg2+ caused a shift of the NADH/NAD+ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.

Structural myelin defects are associated with low axonal ATP levels but rapid recovery from energy deprivation in a mouse model of spastic paraplegia.

In several neurodegenerative disorders, axonal pathology may originate from impaired oligodendrocyte-to-axon support of energy substrates. We previously established transgenic mice that allow measuring axonal ATP levels in electrically active optic nerves. Here, we utilize this technique to explore axonal ATP dynamics in the Plpnull/y mouse model of spastic paraplegia. Optic nerves from Plpnull/y mice exhibited lower and more variable basal axonal ATP levels and reduced compound action potential (CAP) amplitudes, providing a missing link between axonal pathology and a role of oligodendrocytes in brain energy metabolism. Surprisingly, when Plpnull/y optic nerves are challenged with transient glucose deprivation, both ATP levels and CAP decline slower, but recover faster upon reperfusion of glucose. Structurally, myelin sheaths display an increased frequency of cytosolic channels comprising glucose and monocarboxylate transporters, possibly facilitating accessibility of energy substrates to the axon. These data imply that complex metabolic alterations of the axon-myelin unit contribute to the phenotype of Plpnull/y mice.

Requirements analysis for an AI-based clinical decision support system for general practitioners: a user-centered design process.

BACKGROUND: As the first point of contact for patients with health issues, general practitioners (GPs) are frequently confronted with patients presenting with non-specific symptoms of unclear origin. This can result in delayed, prolonged or false diagnoses. To accelerate and improve the diagnosis of diseases, clinical decision support systems would appear to be an appropriate tool. The objective of the project 'Smart physician portal for patients with unclear disease' (SATURN) is to employ a user-centered design process based on the requirements analysis presented in this paper to develop an artificial Intelligence (AI)-based diagnosis support system that specifically addresses the needs of German GPs. METHODS: Requirements analysis for a GP-specific diagnosis support system was conducted in an iterative process with five GPs. First, interviews were conducted to analyze current workflows and the use of digital applications in cases of diagnostic uncertainty (as-is situation). Second, we focused on collecting and prioritizing tasks to be performed by an ideal smart physician portal (to-be situation) in a workshop. We then developed a task model with corresponding user requirements. RESULTS: Numerous GP-specific user requirements were identified concerning the tasks and subtasks: performing data entry (open system, enter patient data), reviewing results (receiving and evaluating results), discussing results (with patients and colleagues), scheduling further diagnostic procedures, referring to specialists (select, contact, make appointments), and case closure. Suggested features particularly concerned the process of screening and assessing results: e.g., the system should focus more on atypical patterns of common diseases than on rare diseases only, display probabilities of differential diagnoses, ensure sources and results are transparent, and mark diagnoses that have already been ruled out. Moreover, establishing a means of using the platform to communicate with colleagues and transferring patient data directly from electronic patient records to the system was strongly recommended. CONCLUSIONS: Essential user requirements to be considered in the development and design of a diagnosis system for primary care could be derived from the analysis. They form the basis for mockup-development and system engineering.

Risk Disclosure in Prodromal Parkinson's Disease.

BACKGROUND: Impressive progress in the understanding of the prodromal phase of Parkinson's disease (PD) in recent years has enabled the generation of disease prediction models. However, a remaining diagnostic uncertainty and lack of therapeutic options for affected individuals has resulted in a variety of ethical issues that have not to date been addressed sufficiently. Moreover, differences in the specificity of prodromal symptoms and possible subtypes of PD, especially the presence of rapid eye movement (REM) sleep behavior disorder (RBD), may have an important impact on prognostic counseling. OBJECTIVES: To derive a guideline for risk disclosure in prodromal PD based on the current literature and expert opinion. METHODS: We performed (1) a literature review on prognostic counseling in PD and (2) consulted with international experts on prodromal PD using a semi-structured questionnaire based on a Delphi approach to evaluate recommendations for risk disclosure in PD. RESULTS: The literature research revealed only 11 publications addressing prognostic counseling, with only two studies directly addressing affected individuals and most studies focusing on risk disclosure in RBD. The expert survey revealed the importance of distinguishing between individuals with and without RBD in prognostic counseling. CONCLUSIONS: Based on the current literature and expert recommendations, a guideline for risk disclosure in prodromal PD for clinical care and research could be elaborated. Prognostic counseling should include differentiation between individuals with and without RBD, taking into account the high uncertainty of risk calculation in RBD-negative prodromal PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Heterogeneity of Astrocytes in Grey and White Matter.

Astrocytes are a diverse and heterogeneous type of glial cells. The major task of grey and white matter areas in the brain are computation of information at neuronal synapses and propagation of action potentials along axons, respectively, resulting in diverse demands for astrocytes. Adapting their function to the requirements in the local environment, astrocytes differ in morphology, gene expression, metabolism, and many other properties. Here we review the differential properties of protoplasmic astrocytes of grey matter and fibrous astrocytes located in white matter in respect to glutamate and energy metabolism, to their function at the blood-brain interface and to coupling via gap junctions. Finally, we discuss how this astrocytic heterogeneity might contribute to the different susceptibility of grey and white matter to ischemic insults.

Life Cycle Assessment of Bitcoin Mining.

This study estimates the environmental impact of mining Bitcoin, the most well-known blockchain-based cryptocurrency, and contributes to the discussion on the technology's supposedly large energy consumption and carbon footprint. The lack of a robust methodological framework and of accurate data on key factors determining Bitcoin's impact have so far been the main obstacles in such an assessment. This study applied the well-established Life Cycle Assessment methodology to an in-depth analysis of drivers of past and future environmental impacts of the Bitcoin mining network. It was found that, in 2018, the Bitcoin network consumed 31.29 TWh with a carbon footprint of 17.29 MtCO2-eq, an estimate that is in the lower end of the range of results from previous studies. The main drivers of such impact were found to be the geographical distribution of miners and the efficiency of the mining equipment. In contrast to previous studies, it was found that the service life, production, and end-of-life of such equipment had only a minor contribution to the total impact, and that while the overall hashrate is expected to increase, the energy consumption and environmental footprint per TH mined is expected to decrease.

NBCe1 mediates the regulation of the NADH/NAD+ redox state in cortical astrocytes by neuronal signals.

Astrocytes are a glial cell type, which is indispensable for brain energy metabolism. Within cells, the NADH/NAD+ redox state is a crucial node in metabolism connecting catabolic pathways to oxidative phosphorylation and ATP production in mitochondria. To characterize the dynamics of the intracellular NADH/NAD+ redox state in cortical astrocytes Peredox, a genetically encoded sensor for the NADH/NAD+ redox state, was expressed in cultured cortical astrocytes as well as in cortical astrocytes in acutely isolated brain slices. Calibration of the sensor in cultured astrocytes revealed a mean basal cytosolic NADH/NAD+ redox ratio of about 0.01; however, with a broad distribution and heterogeneity in the cell population, which was mirrored by a heterogeneous basal cellular concentration of lactate. Inhibition of glucose uptake decreased the NADH/NAD+ redox state while inhibition of lactate dehydrogenase or of lactate release resulted in an increase in the NADH/NAD+ redox ratio. Furthermore, the NADH/NAD+ redox state was regulated by the extracellular concentration of K+ , and application of the neurotransmitters ATP or glutamate increased the NADH/NAD+ redox state dependent on purinergic receptors and glutamate uptake, respectively. This regulation by K+ , ATP, and glutamate involved NBCe1 mediated sodium-bicarbonate transport. These results demonstrate that the NADH/NAD+ redox state in astrocytes is a metabolic node regulated by neuronal signals reflecting physiological activity, most likely contributing to adjust astrocytic metabolism to energy demand of the brain.

Activity-dependent modulation of intracellular ATP in cultured cortical astrocytes.

Brain function is absolutely dependent on an appropriate supply of energy. A shortfall in supply-as occurs, for instance, following stroke-can lead rapidly to irreversible damage to this vital organ. While the consequences of pathophysiological energy depletion have been well documented, much less is known about the physiological energy dynamics of brain cells, although changes in the intracellular concentration of adenosine triphosphate (ATP), the major energy carrier of cells, have been postulated to contribute to cellular signaling. To address this issue more closely, we have investigated intracellular ATP in cultured primary cortical astrocytes by time-lapse microscopy using a genetically encoded fluorescent sensor for ATP. The cytosolic ATP sensor signal decreased after application of the neurotransmitter glutamate in a manner dependent on both glutamate concentration and glutamate transporter activity, but independent of glutamate receptors. The application of dopamine did not affect ATP levels within astrocytes. These results confirm that intracellular ATP levels in astrocytes do indeed respond to changes in physiological activity and pave the way for further studies addressing factors that affect regulation of ATP. © 2017 Wiley Periodicals, Inc.

Exploring patient-centered design solutions of a telehealth app for HIV - A qualitative study.

BACKGROUND: The Communication and Tracing App HIV (COMTRAC-HIV) project is developing a mobile health (mHealth) app for integrated care of HIV patients in Germany. The complexity of HIV treatment and continuous care necessitates the need for tailored mHealth solutions. This qualitative study explores design solutions and a prototype to enhance the app's functionality and effectiveness. METHODS: A total of eight HIV patients and pre-exposure prophylaxis (PrEP) users, recruited at the HIV Center Frankfurt, participated in focus groups and thinking-aloud tests (TA test). In the focus groups, design solutions were discussed for user-interface clarity, leading to the development of an interactive prototype, the usability of which was evaluated with a TA test. Data collection involved video/audio recordings. Qualitative analysis was conducted using a deductive category system, and focused on app design and usage in focus groups, and layout, navigation, interaction, terminology, comprehension, feedback, and level of satisfaction in TA tests. RESULTS: The app was commended for its simple, clear design, especially its medication reminders and health tracking features. Opinions on the symptom diary varied however, respondents noting it more suitable for HIV users than PrEP users. Privacy concerns suggest avoiding display of HIV-specific information. Suggested improvements include e.g. image uploads, drug interaction checks and prescription tracking. A total of 25 usability issues were identified in the TA test, with most found in the layout (n = 6), navigation (n = 5), interaction (n = 5), and terminology (n = 5) categories. Two examples are non-intuitive controls and illogical button placement. Despite these disadvantages, participants noted positive impressions (n = 5) in the satisfaction category. CONCLUSION: The study emphasizes the need for patient-centered design in mobile HIV care solutions, highlighting to the app's user-friendliness and potential to enhance care. Further research is necessary to refine the app's functionality and to align it with clinical and patients' privacy needs.

Initial User-Centred Design of an AI-Based Clinical Decision Support System for Primary Care.

A clinical decision support system based on different methods of artificial intelligence (AI) can support the diagnosis of patients with unclear diseases by providing tentative diagnoses as well as proposals for further steps. In a user-centred-design process, we aim to find out how general practitioners envision the user interface of an AI-based clinical decision support system for primary care. A first user-interface prototype was developed using the task model based on user requirements from preliminary work. Five general practitioners evaluated the prototype in two workshops. The discussion of the prototype resulted in categorized suggestions with key messages for further development of the AI-based clinical decision support system, such as the integration of intelligent parameter requests. The early inclusion of different user feedback facilitated the implementation of a user interface for a user-friendly decision support system.

Interviews with HIV Experts for Development of a Mobile Health Application in HIV Care-A Qualitative Study.

The Communication and Tracing App HIV (COMTRAC-HIV) project aims to develop a mobile health application for integrated care of HIV patients due to the low availability of those apps in Germany. This study addressed organizational conditions and necessary app functionalities, especially for the care of late diagnosed individuals (late presenters) and those using pre-exposure prophylaxis. We followed a human-centered design approach and interviewed HIV experts in Germany to describe the context of use of the app. The interviews were paraphrased and analyzed with a qualitative content analysis. To define the context of use, user group profiles were defined and tasks derived, which will represent the functionalities of the app. A total of eight experts were included in the study. The results show that the app should include a symptom diary for entering symptoms, side effects, and their intensity. It offers chat/video call functionality for communication with an HIV expert, appointment organization, and sharing findings. The app should also provide medication overview and reminders for medications and appointments. This qualitative study is a first step towards the development of an app for HIV individuals in Germany. Further research includes involving patients in the initial app design and test design usability.

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Synthesis and immunochemical evaluation of a non-methylated disaccharide analogue of the anthrax tetrasaccharide.

Anthrax tetrasaccharide is an oligosaccharide expressed at the outermost surface of the Bacillus anthracis spores, featuring three rhamnoses and a rare sugar called anthrose. This motif has now been identified as a plausible component of future human vaccines against anthrax. We report herein the synthesis of a 2-O-demethylated-ß-D-anthropyranosyl-(1→3)-α-L-rhamnopyranose disaccharide analogue of this tetrasaccharide from a cyclic sulfate intermediate. This disaccharide conjugated to BSA induces an anti-native tetrasaccharide IgG antibody response when administered in BALB/c mice. Moreover, induced sera bound to native B. anthracis endospores. These results suggest that the disaccharide analogue, easily amenable for a synthetic scale-up, could be used in a glycoconjugate antigen formulation.

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines.

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type-specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

A Dual Nanosensor Approach to Determine the Cytosolic Concentration of ATP in Astrocytes.

Adenosine triphosphate (ATP) is the central energy carrier of all cells and knowledge on the dynamics of the concentration of ATP ([ATP]) provides important insights into the energetic state of a cell. Several genetically encoded fluorescent nanosensors for ATP were developed, which allow following the cytosolic [ATP] at high spatial and temporal resolution using fluorescence microscopy. However, to calibrate the fluorescent signal to [ATP] has remained challenging. To estimate basal cytosolic [ATP] ([ATP]0) in astrocytes, we here took advantage of two ATP nanosensors of the ATeam-family (ATeam1.03; ATeam1.03YEMK) with different affinities for ATP. Altering [ATP] by external stimuli resulted in characteristic pairs of signal changes of both nanosensors, which depend on [ATP]0. Using this dual nanosensor strategy and epifluorescence microscopy, [ATP]0 was estimated to be around 1.5 mM in primary cultures of cortical astrocytes from mice. Furthermore, in astrocytes in acutely isolated cortical slices from mice expressing both nanosensors after stereotactic injection of AAV-vectors, 2-photon microscopy revealed [ATP]0 of 0.7 mM to 1.3 mM. Finally, the change in [ATP] induced in the cytosol of cultured cortical astrocytes by application of azide, glutamate, and an increased extracellular concentration of K+ were calculated as -0.50 mM, -0.16 mM, and 0.07 mM, respectively. In summary, the dual nanosensor approach adds another option for determining the concentration of [ATP] to the increasing toolbox of fluorescent nanosensors for metabolites. This approach can also be applied to other metabolites when two sensors with different binding properties are available.

GABA-Glycine Cotransmitting Neurons in the Ventrolateral Medulla: Development and Functional Relevance for Breathing.

Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.

Novel Mutations in the Asparagine Synthetase Gene (ASNS) Associated With Microcephaly.

Microcephaly is a devastating condition defined by a small head and small brain compared to the age- and sex-matched population. Mutations in a number of different genes causative for microcephaly have been identified, e.g., MCPH1, WDR62, and ASPM. Recently, mutations in the gene encoding the enzyme asparagine synthetase (ASNS) were associated to microcephaly and so far 24 different mutations in ASNS causing microcephaly have been described. In a family with two affected girls, we identified novel compound heterozygous variants in ASNS (c.1165G > C, p.E389Q and c.601delA, p.M201Wfs∗28). The first mutation (E389Q) is a missense mutation resulting in the replacement of a glutamate residue evolutionary conserved from Escherichia coli to Homo sapiens by glutamine. Protein modeling based on the known crystal structure of ASNS of E. coli predicted a destabilization of the protein by E389Q. The second mutation (p.M201Wfs∗28) results in a premature stop codon after amino acid 227, thereby truncating more than half of the protein. The novel variants expand the growing list of microcephaly causing mutations in ASNS.