Modeling the Fluorescence Quantum Yields of Aromatic Compounds: Benchmarking the Machinery to Compute Intersystem Crossing Rates.

The from-first-principles calculation of fluorescence quantum yields (FQYs) and lifetimes of organic dyes remains very challenging. In this article, we extensively test the machinery to calculate FQYs. Specifically, we perform an extensive analysis on the parameters influencing the intersystem crossing (ISC), internal conversion (IC), and fluorescence rate constants calculations. The impact of (i) the electronic structure (chosen exchange-correlation functional and spin-orbit Hamiltonian), (ii) the vibronic parameters (coordinate system, broadening function, and dipole expansion), and (iii) the excited-state kinetic models are systematically assessed for a series of seven rigid aromatic molecules. Our studies provide more insights into the choice of parameters and the expected accuracy for the computational protocols aiming to deliver FQY values. Some challenges are highlighted, such as, on the one hand, the difficulty to benchmark against the experimental nonradiative rate constants, for which the separation between the IC and ISC contributions is often not provided in the literature and, on the other hand, the need to go beyond the harmonic approximation for the calculation of the IC rates.

Detailed protocol for a corneal thermal cauterization-based (lymph-)angiogenesis assay in mice.

Angiogenesis and lymphangiogenesis, the formation of new blood or lymphatic vessels, respectively, from preexisting vasculature is essential during embryonic development, but also occurs during tissue repair and in pathological conditions (cancer; ocular disease; ischemic, infectious and inflammatory disorders), which are all characterized to a certain extent by inflammatory conditions. Hence, a rapid, inexpensive, feasible / technically easy, reliable assay of inflammation-induced (lymph-)angiogenesis is highly valuable. In this context, the corneal thermal cauterization assay in mice is a simple, low-cost, reproducible, insightful and labor-saving assay to gauge the role of inflammation in angiogenesis and lymphangiogenesis. However, to the best of our knowledge, there is no standardized protocol to perform this assay. Here, we provide a step-by-step description of the model's procedures, which include:•The thermal cauterization of the corneas,•Enucleation and dissection of the corneas,•Subsequent immunofluorescence staining of the neovasculature, and morphometric analysis. We also discuss ethical considerations and aspects related to animal welfare guidelines. Altogether, this paper will help to increase the reproducibility of the corneal thermal cauterization model and facilitate its use for angiogenesis and lymphangiogenesis research.

First-Principles Calculations of Excited-State Decay Rate Constants in Organic Fluorophores.

In this Perspective, we discuss recent advances made to evaluate from first-principles the excited-state decay rate constants of organic fluorophores, focusing on the so-called static strategy. In this strategy, one essentially takes advantage of Fermi's golden rule (FGR) to evaluate rate constants at key points of the potential energy surfaces, a procedure that can be refined in a variety of ways. In this way, the radiative rate constant can be straightforwardly obtained by integrating the fluorescence line shape, itself determined from vibronic calculations. Likewise, FGR allows for a consistent calculation of the internal conversion (related to the non-adiabatic couplings) in the weak-coupling regime and intersystem crossing rates, therefore giving access to estimates of the emission yields when no complex photophysical phenomenon is at play. Beyond outlining the underlying theories, we summarize here the results of benchmarks performed for various types of rates, highlighting that both the quality of the vibronic calculations and the accuracy of the relative energies are crucial to reaching semiquantitative estimates. Finally, we illustrate the successes and challenges in determining the fluorescence quantum yields using a series of organic fluorophores.

Extensive Analysis of the Parameters Influencing Radiative Rates Obtained through Vibronic Calculations.

Defining a theoretical model systematically delivering accurate ab initio predictions of the fluorescence quantum yields of organic dyes is highly desirable for designing improved fluorophores in a systematic rather than trial-and-error way. To this end, the first required step is to obtain reliable radiative rates (kr), as low kr typically precludes effective emission. In the present contribution, using a series of 10 substituted phenyls with known experimental kr, we analyze the impact of the computational protocol on the kr determined through the thermal vibration correlation function (TVCF) approach on the basis of time-dependent density functional theory (TD-DFT) calculations of the energies, structures, and vibrational parameters. Both the electronic structure (selected exchange-correlation functional, application or not of the Tamm-Dancoff approximation) and the vibronic parameters (line-shape formalism, coordinate system, potential energy surface model, and dipole expansion) are tackled. Considering all possible combinations yields more than 3500 cases, allowing to extract statistically-relevant information regarding the impact of each computational parameter on the magnitude of the estimated kr. It turns out that the selected vibronic model can have a significant impact on the computed kr, especially the potential energy surface model. This effect is of the same order of magnitude as the difference noted between B3LYP and CAM-B3LYP estimates. For the treated compounds, all evaluated functionals do deliver reasonable trends, fitting the experimental values.

Systematic reviews on platelet transfusions: Is there unnecessary duplication of effort? A scoping review.

BACKGROUND AND OBJECTIVES: Platelet transfusions are used across multiple patient populations to prevent and correct bleeding. This scoping review aimed to map the currently available systematic reviews (SRs) and evidence-based guidelines in the field of platelet transfusion. MATERIALS AND METHODS: A systematic literature search was conducted in seven databases for SRs on effectiveness (including dose and timing, transfusion trigger and ratio to other blood products), production modalities and decision support related to platelet transfusion. The following data were charted: methodological features of the SR, population, concept and context features, outcomes reported, study design and number of studies included. Results were synthesized in interactive evidence maps. RESULTS: We identified 110 SRs. The majority focused on clinical effectiveness, including prophylactic or therapeutic transfusions compared to no platelet transfusion (34 SRs), prophylactic compared to therapeutic-only transfusion (8 SRs), dose, timing (11 SRs) and threshold for platelet transfusion (15 SRs) and the ratio of platelet transfusion to other blood products in massive transfusion (14 SRs). Furthermore, we included 34 SRs on decision support, of which 26 evaluated viscoelastic testing. Finally, we identified 22 SRs on platelet production modalities, including derivation (4 SRs), pathogen inactivation (6 SRs), leucodepletion (4 SRs) and ABO/human leucocyte antigen matching (5 SRs). The SRs were mapped according to concept and clinical context. CONCLUSION: An interactive evidence map of SRs and evidence-based guidelines in the field of platelet transfusion has been developed and identified multiple reviews. This work serves as a tool for researchers looking for evidence gaps, thereby both supporting research and avoiding unnecessary duplication.

Unusually high energy barriers for internal conversion in a {Ru(bpy)} chromophore.

Internal conversion (IC) coupled to vibrational relaxation (VR) in molecular chromophores is a source of major energy losses in natural and artificial solar-to-chemical energy conversion schemes. The development of anti-Kasha chromophores, where dissipative IC channels are blocked, is a promising strategy to boost energy conversion efficiencies. In this contribution, we demonstrate the presence of an unusually high kinetic barrier for IC in [Ru(tpm)(bpy)(NCS)]+ (RuNCS), where tpm is tris(1-pyrazolyl)methane and bpy is 2,2'-bipyridine, by means of an arsenal of temperature-dependent spectroscopic methods including nanosecond and femtosecond transient absorption spectroscopies. These studies are complemented with theoretical investigations, that provide a detailed atomistic description of the dissipation process, including the electronic structures of the excited states involved. The observed IC is mainly a hole reconfiguration within the octahedral t2g set of the Ru ion, with contributions from a Ru to NCS charge transfer. Thus, in a Marcus picture, inner and outer reorganizations contribute to the observed barrier. The results presented here show that wavefunction symmetry within a molecular chromophore can be exploited to inhibit dissipative IC. Finally, guidelines for the design of anti-Kasha chromophores that prevent dissipation in energy conversion schemes, based on minimum energy conical intersection calculations, are provided.

Anti-Kasha Fluorescence in Molecular Entities: Central Role of Electron-Vibrational Coupling.

According to Kasha's rule, the emission of a photon in a molecular system always comes from the lowest excited state. A corollary of this rule (i.e., the Kasha-Vavilov rule) states that the emission spectra are independent of the excitation wavelength. Although these rules apply for most of the molecular systems, violations of these rules are often reported. The prototypical case of a Kasha's rule violation is the fluorescence observed from S2 in azulene. Thanks to the advances in both theoretical and experimental research, other types of anomalous fluorescence (e.g., excitation energy transfer (EET)-based dual emissions, thermally activated fluorescence, etc.) are more recurrently reported in the literature. Sometimes, these anomalous processes involve higher-lying excited states but are mechanistically different from the azulene-like anomalous fluorescence. However, the underlying mechanisms leading to these anomalous emissions can be numerous and are not yet well understood.In order to shed some light on the above phenomena, this Account provides a comprehensive review of this topic. We herein report quantum chemical investigations in target molecular systems breaking Kasha's rule. The latter molecules were chosen because they were unambiguously reported to display anti-Kasha fluorescence. Our studies highlight three different types of anti-Kasha scenarios. Specifically, (i) the strong electronic, weak vibrational nonadiabatic coupling (NAC) regime (here named the type I case, i.e., azulene-like); (ii) the strong electronic, strong vibrational NAC regime (type II case, i.e., thermally activated S2 fluorescence); and the (iii) very weak electronic NAC regime (type III case, i.e., EET dyads). In addition, by combining state-of-the-art quantum chemical calculations with excited-state decay rate theories and appropriate excited-state kinetic models, we provide semiquantitative estimations of photoluminescence quantum yields for the most rigid molecular entities. Finally, we propose the use of simple theoretical descriptors relying on calculations of the excited-state density difference and the electron-vibrational coupling to classify anomalous emissions according to their coupling scenario.Besides the fundamental interest of the above investigations, the herein developed computational protocols and descriptors will be useful for the tailored design of dyes with tunable and unconventional fluorescence properties and their exploitation in a wide range of areas (i.e., from organic light-emitting diodes (OLEDs) to bioimaging, small-molecule fluorescent probes, and photocatalysis). Finally, our theoretical framework enables the attainment of a holistic understanding of the interconversion processes between excited states, where the electron-vibrational coupling is shown to play a central role in determining the efficacy.

Protocols for endothelial cell isolation from mouse tissues: brain, choroid, lung, and muscle.

Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).

The effect of hand hygiene promotion programs during epidemics and pandemics of respiratory droplet-transmissible infections on health outcomes: a rapid systematic review.

BACKGROUND: Public health strategies in the context of respiratory droplet-transmissible diseases (such as influenza or COVID-19) include intensified hand hygiene promotion, but a review on the effectiveness of different ways of promoting hand hygiene in the community, specifically for this type of infections, has not been performed. This rapid systematic review aims to summarize the effectiveness of community-based hand hygiene promotion programs on infection transmission, health outcomes and behavioral outcomes during epidemic periods in the context of respiratory droplet-transmissible diseases. We also included laboratory-confirmed health outcomes for epidemic-prone disease during interepidemic periods. METHODS: We searched for controlled experimental studies. A rapid systematic review was performed in three databases and a COVID-19 resource. Following study selection (in which studies performed in the (pre-)hospital/health care setting were excluded), study characteristics and effect measures were synthesized, using meta-analyses of cluster-RCTs where possible. Risk of bias of each study was assessed and the certainty of evidence was appraised according to the GRADE methodology. RESULTS: Out of 2050 unique references, 12 cluster-RCTs, all in the context of influenza, were selected. There were no controlled experimental studies evaluating the effectiveness of hand hygiene promotion programs in the context of COVID-19 that met the in-/exclusion criteria. There was evidence that preventive hand hygiene promotion interventions in interepidemic periods significantly decreased influenza positive cases in the school setting. However, no improvement could be demonstrated for programs implemented in households to prevent secondary influenza transmission from previously identified cases (epidemic and interepidemic periods). CONCLUSIONS: The data suggest that proactive hand hygiene promotion interventions, i.e. regardless of the identification of infected cases, can improve health outcomes upon implementation of such a program, in contrast to reactive interventions in which the program is implemented after (household) index cases are identified.

Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis.

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Protocols for endothelial cell isolation from mouse tissues: small intestine, colon, heart, and liver.

Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).

Day care as a strategy for drowning prevention in children under 6 years of age in low- and middle-income countries.

BACKGROUND: Drowning is responsible for an estimated 320,000 deaths a year, and over 90% of drowning mortality occurs in low- to middle-income countries (LMICs), with peak drowning rates among children aged 1 to 4 years. In this age group, mortality due to drowning is particularly common in rural settings and about 75% of drowning accidents happen in natural bodies of water close to the home. Providing adequate child supervision can protect children from drowning, and organized formal day care programs could offer a way to achieve this. OBJECTIVES: Primary objective • To assess the effects of day care programs for children under 6 years of age on drowning-related mortality or morbidity, or on total drowning accidents (fatal and non-fatal), in LMICs, compared to no day care programs or other drowning prevention interventions Secondary objectives • To assess the effects of day care programs in LMICs for children under 6 years of age on unsafe water exposure • To assess safety within these programs (e.g. transmission of infection within day care, physical or sexual abuse of children within day care) • To assess the incidence of unintentional injury within these programs • To describe the cost-effectiveness of such programs, in relation to averted drowning-related mortality or morbidity SEARCH METHODS: On November 23, 2019, and for an update on August 18, 2020, we searched MEDLINE (PubMed), Embase, CENTRAL, ERIC, and CINAHL, as well as two trial registries. On December 16, 2019, and for an update on February 9, 2021, we searched 12 other resources, including websites of organizations that develop programs targeted to children. SELECTION CRITERIA: We included randomized, quasi-randomized, and non-randomized controlled studies (with explicitly listed specific study design features) that implemented formal day care programs as a single program or combined with additional out-of-day care components (such as educational activities aimed at preventing injury or drowning or early childhood development activities) for children of preschool age (below 6 years of age) in LMICs for comparison with no such programs or with other drowning prevention interventions. Studies had to report at least one outcome related to drowning or injury prevention for the children enrolled. DATA COLLECTION AND ANALYSIS: Two review authors independently performed study selection and data extraction, as well as risk of bias and GRADE assessment. MAIN RESULTS: Two non-randomized observational studies, conducted in rural Bangladesh, involving a total of 252,631 participants, met the inclusion criteria for this review. One of these studies compared a formal day care program combined with parent education, playpens provided to parents, and community-based activities as additional out-of-day care components versus no such program. Overall we assessed this study to be at moderate risk of bias (moderate risk of bias due to confounding, low risk of bias for other domains). This study showed that implementation of a formal day care program combined with parent education, provision of playpens to parents, and community-based activities, in a rural area with a high drowning incidence, likely reduces the risk of death from drowning over the study period of 4 years and 8 months compared to no day care program (hazard ratio 0.18, 95% confidence interval [CI] 0.06 to 0.58; 1 study, 136,577 participants; moderate-certainty evidence). Drowning morbidity (non-fatal drowning resulting in complications), total drowning (fatal and non-fatal), unsafe water exposure, and program safety (e.g. transmission of infection within day care, physical or sexual abuse of children within day care) were not reported, nor was the incidence of other unintentional injuries. Cost-effectiveness was reported as 812 USD (95% CI 589 to 1777) per disability-adjusted life-year averted as a consequence of drowning (moderate-certainty evidence). The second study compared day care programs with or without playpens provided to parents as an additional component versus only playpens provided to parents as an alternative drowning prevention intervention. Overall we assessed the study to be at critical risk of bias because we judged bias due to confounding to be at critical risk. As the certainty of evidence was very low, we are uncertain about the effects on drowning mortality rate of implementing a day care program compared to providing playpens (rate ratio 0.25, 95% CI 0.15 to 0.41; 1 study; 76,575 participants; very low-certainty evidence). Likewise, we are uncertain about the effects of a day care program with playpens provided as an additional component versus playpens provided alone (rate ratio 0.06, 95% CI 0.02 to 0.12; 1 study, 45,460 participants; very low-certainty evidence). The other outcomes of interest - drowning morbidity, total drowning, unsafe water exposure, program safety, incidence of other unintentional injuries, and cost-effectiveness - were not reported. AUTHORS' CONCLUSIONS: This review provides evidence suggesting that a day care program with additional out-of-day care components such as community-based education, parent education, and playpens provided to parents likely reduces the drowning mortality risk in regions with a high burden of drowning compared to no intervention.

Fat Induces Glucose Metabolism in Nontransformed Liver Cells and Promotes Liver Tumorigenesis.

Hepatic fat accumulation is associated with diabetes and hepatocellular carcinoma (HCC). Here, we characterize the metabolic response that high-fat availability elicits in livers before disease development. After a short term on a high-fat diet (HFD), otherwise healthy mice showed elevated hepatic glucose uptake and increased glucose contribution to serine and pyruvate carboxylase activity compared with control diet (CD) mice. This glucose phenotype occurred independently from transcriptional or proteomic programming, which identifies increased peroxisomal and lipid metabolism pathways. HFD-fed mice exhibited increased lactate production when challenged with glucose. Consistently, administration of an oral glucose bolus to healthy individuals revealed a correlation between waist circumference and lactate secretion in a human cohort. In vitro, palmitate exposure stimulated production of reactive oxygen species and subsequent glucose uptake and lactate secretion in hepatocytes and liver cancer cells. Furthermore, HFD enhanced the formation of HCC compared with CD in mice exposed to a hepatic carcinogen. Regardless of the dietary background, all murine tumors showed similar alterations in glucose metabolism to those identified in fat exposed nontransformed mouse livers, however, particular lipid species were elevated in HFD tumor and nontumor-bearing HFD liver tissue. These findings suggest that fat can induce glucose-mediated metabolic changes in nontransformed liver cells similar to those found in HCC. SIGNIFICANCE: With obesity-induced hepatocellular carcinoma on a rising trend, this study shows in normal, nontransformed livers that fat induces glucose metabolism similar to an oncogenic transformation.

Computational Protocol To Predict Anti-Kasha Emissions: The Case of Azulene Derivatives.

In this contribution, we present a computational protocol to predict anti-Kasha photoluminescence. The herein developed protocol is based on state-of-the-art quantum chemical calculations and excited-state decay rate theories (i.e., thermal vibration correlation function formalism), along with appropriate kinetic models which include all relevant electronic states. This protocol is validated for a series of azulene derivatives. For this series, we have computed absorption and emission spectra for both their first and second excited states, their radiative and nonradiative rates, as well as fluorescence yields from the two different excited states. All the studied azulene derivatives are predicted to exclusively display anomalous anti-Kasha S2 emission. A quantitative agreement for the herein computed excited-state spectra, lifetimes, and fluorescence quantum yields is obtained with respect to the experimental values. Given the increasing interest in anti-Kasha emitters, we foresee that the herein developed computational protocol can be used to prescreen dyes with the desired aforementioned anomalous photoluminescence properties.

Role of the GLUT1 Glucose Transporter in Postnatal CNS Angiogenesis and Blood-Brain Barrier Integrity.

RATIONALE: Endothelial cells (ECs) are highly glycolytic and generate the majority of their energy via the breakdown of glucose to lactate. At the same time, a main role of ECs is to allow the transport of glucose to the surrounding tissues. GLUT1 (glucose transporter isoform 1/Slc2a1) is highly expressed in ECs of the central nervous system (CNS) and is often implicated in blood-brain barrier (BBB) dysfunction, but whether and how GLUT1 controls EC metabolism and function is poorly understood. OBJECTIVE: We evaluated the role of GLUT1 in endothelial metabolism and function during postnatal CNS development as well as at the adult BBB. METHODS AND RESULTS: Inhibition of GLUT1 decreases EC glucose uptake and glycolysis, leading to energy depletion and the activation of the cellular energy sensor AMPK (AMP-activated protein kinase), and decreases EC proliferation without affecting migration. Deletion of GLUT1 from the developing postnatal retinal endothelium reduces retinal EC proliferation and lowers vascular outgrowth, without affecting the number of tip cells. In contrast, in the brain, we observed a lower number of tip cells in addition to reduced brain EC proliferation, indicating that within the CNS, organotypic differences in EC metabolism exist. Interestingly, when ECs become quiescent, endothelial glycolysis is repressed, and GLUT1 expression increases in a Notch-dependent fashion. GLUT1 deletion from quiescent adult ECs leads to severe seizures, accompanied by neuronal loss and CNS inflammation. Strikingly, this does not coincide with BBB leakiness, altered expression of genes crucial for BBB barrier functioning nor reduced vascular function. Instead, we found a selective activation of inflammatory and extracellular matrix related gene sets. CONCLUSIONS: GLUT1 is the main glucose transporter in ECs and becomes uncoupled from glycolysis during quiescence in a Notch-dependent manner. It is crucial for developmental CNS angiogenesis and adult CNS homeostasis but does not affect BBB barrier function.

BOPAHY: a doubly chelated highly fluorescent pyrrole-acyl hydrazone -BF2 chromophore.

New easily functionalisable and highly fluorescent BOPAHY chromophores are synthesised via a one-pot two-step reaction starting from commercially available pyrrole-2-carbaldehydes and respective acyl hydrazides in the presence of BF3·OEt2. Most importantly, all BOPAHY dyes show excellent photophysical properties with quantum yields up to 0.92. Steady-state spectroscopy and quantum chemical calculations provide a first insight into these promising properties.

Reactivity of Cobalt-Fullerene Complexes towards Deuterium.

The adsorption of molecular deuterium (D2 ) onto charged cobalt-fullerene-complexes Con C60 + (n=1-8) is measured experimentally in a few-collision reaction cell. The reactivity is strongly size-dependent, hinting at clustering of the transition metal atoms on the fullerenes. Formation and desorption rate constants are obtained from the pressure-dependent deuterogenation curves. DFT calculations indeed find that this transition metal clustering is energetically more favorable than decorating the fullerene. For n=1, D2 is predicted to bind molecularly and for n=2 dissociative and molecular configurations are quasi-isoenergetic. For n=3-8, dissociation of D2 is thermodynamically preferred. However, reaching the ground state configuration with dissociated deuterium on the timescale of the experiment may be hindered by dissociation barriers.

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase.

mTORC1 is an important regulator of muscle mass but how it is modulated by oxygen and nutrients is not completely understood. We show that loss of the prolyl hydroxylase domain isoform 1 oxygen sensor in mice (PHD1KO) reduces muscle mass. PHD1KO muscles show impaired mTORC1 activation in response to leucine whereas mTORC1 activation by growth factors or eccentric contractions was preserved. The ability of PHD1 to promote mTORC1 activity is independent of its hydroxylation activity but is caused by decreased protein content of the leucyl tRNA synthetase (LRS) leucine sensor. Mechanistically, PHD1 interacts with and stabilizes LRS. This interaction is promoted during oxygen and amino acid depletion and protects LRS from degradation. Finally, elderly subjects have lower PHD1 levels and LRS activity in muscle from aged versus young human subjects. In conclusion, PHD1 ensures an optimal mTORC1 response to leucine after episodes of metabolic scarcity.

Differentiation but not ALS mutations in FUS rewires motor neuron metabolism.

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.