A single hydrogen bond that tunes flavin redox reactivity and activates it for modification.

Electron bifurcation produces high-energy products based on less energetic reagents. This feat enables biological systems to exploit abundant mediocre fuel to drive vital but demanding reactions, including nitrogen fixation and CO2 capture. Thus, there is great interest in understanding principles that can be portable to man-made devices. Bifurcating electron transfer flavoproteins (Bf ETFs) employ two flavins with contrasting reactivities to acquire pairs of electrons from a modest reductant, NADH. The bifurcating flavin then dispatches the electrons individually to a high and a low reduction midpoint potential (E°) acceptor, the latter of which captures most of the energy. Maximum efficiency requires that only one electron accesses the exergonic path that will 'pay for' the production of the low-E° product. It is therefore critical that one of the flavins, the 'electron transfer' (ET) flavin, is tuned to execute single-electron (1e-) chemistry only. To learn how, and extract fundamental principles, we systematically altered interactions with the ET-flavin O2 position. Removal of a single hydrogen bond (H-bond) disfavored the formation of the flavin anionic semiquinone (ASQ) relative to the oxidized (OX) state, lowering by 150 mV and retuning the flavin's tendency for 1e-vs. 2e- reactivity. This was achieved by replacing conserved His 290 with Phe, while also replacing the supporting Tyr 279 with Ile. Although this variant binds oxidized FADs at 90% the WT level, the ASQ state of the ET-flavin is not stable in the absence of H290's H-bond, and dissociates, in contrast to the WT. Removal of this H-bond also altered the ET-flavin's covalent chemistry. While the WT ETF accumulates modified flavins whose formation is believed to rely on an anionic paraquinone methide intermediate, the FADs of the H-bond lacking variant remain unchanged over weeks. Hence the variant that destabilizes the anionic semiquinone also suppresses the anionic intermediate in flavin modification, verifying electronic similarities between these two species. These correlations suggest that the H-bond that stabilizes the crucial flavin ASQ also promotes flavin modification. The two effects may indeed be inseparable, as a Jekyll and Hydrogen bond.

Prolonged cytopenias after immune effector cell therapy and lymphodepletion in patients with leukemia, lymphoma and solid tumors.

BACKGROUND AIMS: The success of chimeric antigen receptor (CAR) T-cell therapy in treating B-cell malignancies has led to the evaluation of CAR T-cells targeting a variety of other malignancies. Although the efficacy of CAR T-cells is enhanced when administered post-lymphodepleting chemotherapy, this can trigger bone marrow suppression and sustained cytopenia after CD19.CAR T-cell therapy. Additionally, systemic inflammation associated with CAR T-cell activity may contribute to myelosuppression. Cytopenias, such as neutropenia and thrombocytopenia, elevate the risk of severe infections and bleeding, respectively. However, data on the incidence of prolonged cytopenias after immune effector therapy in the solid tumor context remain limited. OBJECTIVE: We compared the incidence of prolonged cytopenias after immune effector therapy including genetically modified T-cells, virus-specific T-cells (VSTs) and NKT-cells, as well non-gene-modified VSTs for leukemia, lymphoma, and solid tumors (ST) to identify associated risk factors. METHODS: A retrospective analysis was conducted of 112 pediatric and adult patients with relapsed and/or refractory cancers who received lymphodepleting chemotherapy followed by immune effector therapy. Patients treated with 13 distinct immune effector cell therapies through 11 single-center clinical trials and 2 commercial products over a 6-year period were categorized into 3 types of malignancies: leukemia, lymphoma and ST. We obtained baseline patient characteristics and adverse events data for each participant, and tracked neutrophil and platelet counts following lymphodepletion. RESULTS: Of 112 patients, 104 (92.9%) experienced cytopenias and 88 (79%) experienced severe cytopenias. Patients with leukemia experienced significantly longer durations of severe neutropenia (median duration of 14 days) compared with patients with lymphoma (7 days) or ST (11 days) (P = 0.002). Patients with leukemia also had a higher incidence of severe thrombocytopenia (74.1%), compared with lymphoma (46%, P = 0.03) and ST (14.3%, P < 0.0001). Prolonged cytopenias were significantly associated with disease type (63% of patients with leukemia, 44% of patients with lymphoma, and 22.9% of patients with ST, P = 0.006), prior hematopoietic stem cell transplant (HSCT) (66.7% with prior HSCT versus 38.3% without prior HSCT, P = 0.039), and development of immune effector cell-associated neurotoxicity syndrome (ICANS) (75% with ICANS versus 38% without ICANS, P = 0.027). There was no significant association between prolonged cytopenias and cytokine release syndrome. CONCLUSIONS: Immune effector recipients often experience significant cytopenias due to marrow suppression following lymphodepletion regardless of disease, but prolonged severe cytopenias are significantly less common after treatment of patients with lymphoma and solid tumors.

Clinicians Address Gestational Weight Gain and Nutrition Using the International Federation of Gynecology and Obstetrics (FIGO) Nutrition Checklist.

The negative effects of excessive gestational weight gain (GWG) and obesity during pregnancy are well documented in the literature. However, lack of time, education, comfort, and confidence among health care providers often make it difficult to provide proper nutrition and weight gain guidance for pregnant persons. In response, the International Federation of Gynecology and Obstetrics (FIGO) has developed a nutrition checklist that can standardize recommendations for GWG, facilitate discussions with pregnant persons, and aid providers with nutrition education. The checklist is an innovative tool that can help reduce complications associated with excessive GWG. This article discusses the benefits of FIGO Nutrition Checklist and its implementation at a midwifery clinic that primarily serves Native American women. By using this quick, simple, guided, time-efficient tool, clinics can be successful in facilitating important conversations and education about nutrition and GWG during pregnancy.

Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects.

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcating electron transfer flavoprotein (SulfETF), tryptophan synthases from Salmonella typhimurium (StTS) and the dimeric ß-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from µM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20-30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Structure, dynamics, and redox reactivity of an all-purpose flavodoxin.

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-19F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.

Sensing in Sacral Neuromodulation: A Feasibility Study in Subjects With Urinary Incontinence and Retention.

OBJECTIVES: Sacral neuromodulation (SNM) therapy standard of care relies on visual-motor responses and patient-reported sensory responses in deciding optimized lead placement and programming. Automatic detection of stimulation responses could offer a simple, consistent indicator for optimizing SNM. The purpose of this study was to measure and characterize sacral evoked responses (SERs) resulting from sacral nerve stimulation using a commercial, tined SNM lead. MATERIALS AND METHODS: A custom external research system with stimulation and sensing hardware was connected to the percutaneous extension of an implanted lead during a staged (tined lead) evaluation for SNM. The system collected SER recordings across a range of prespecified stimulation settings (electrode configuration combinations for bipolar stimulation and bipolar sensing) during intraoperative and postoperative sessions in 21 subjects with overactive bladder (OAB) and nonobstructive urinary retention (NOUR). Motor and sensory thresholds were collected during the same sessions. RESULTS: SERs were detected in all 21 subjects. SER morphology (number of peaks, magnitude, and timing) varied across electrode configurations within and across subjects. Among subjects and electrode configurations tested, recordings contained SERs at motor threshold and/or sensory threshold in 75% to 80% of subjects. CONCLUSIONS: This study confirmed that implanted SNM leads can be used to directly record SERs elicited by stimulation in subjects with OAB and NOUR. SERs were readily detectable at typical SNM stimulation settings and procedural time points. Using these SERs as possible objective measures of SNM response has the capability to automate patient-specific SNM therapy, potentially providing consistent lead placement, programming, and/or closed-loop therapy.

The dentate gyrus differentially metabolizes glucose and alternative fuels during rest and stimulation.

The metabolic demands of neuronal activity are both temporally and spatially dynamic, and neurons are particularly sensitive to disruptions in fuel and oxygen supply. Glucose is considered an obligate fuel for supporting brain metabolism. Although alternative fuels are often available, the extent of their contribution to central carbon metabolism remains debated. Differential fuel metabolism likely depends on cell type, location, and activity state, complicating its study. While biosensors provide excellent spatial and temporal information, they are limited to observations of only a few metabolites. On the other hand, mass spectrometry is rich in chemical information, but traditionally relies on cell culture or homogenized tissue samples. Here, we use mass spectrometry imaging (MALDI-MSI) to focus on the fuel metabolism of the dentate granule cell (DGC) layer in murine hippocampal slices. Using stable isotopes, we explore labeling dynamics at baseline, as well as in response to brief stimulation or fuel competition. We find that at rest, glucose is the predominant fuel metabolized through glycolysis, with little to no measurable contribution from glycerol or fructose. However, lactate/pyruvate, ß-hydroxybutyrate (ßHB), octanoate, and glutamine can contribute to TCA metabolism to varying degrees. In response to brief depolarization with 50 mM KCl, glucose metabolism was preferentially increased relative to the metabolism of alternative fuels. With an increased supply of alternative fuels, both lactate/pyruvate and ßHB can outcompete glucose for TCA cycle entry. While lactate/pyruvate modestly reduced glucose contribution to glycolysis, ßHB caused little change in glycolysis. This approach achieves broad metabolite coverage from a spatially defined region of physiological tissue, in which metabolic states are rapidly preserved following experimental manipulation. Using this powerful methodology, we investigated metabolism within the dentate gyrus not only at rest, but also in response to the energetic demand of activation, and in states of fuel competition.

A fingerprint of 2-[18F]FDG radiometabolites - How tissue-specific metabolism beyond 2-[18F]FDG-6-P could affect tracer accumulation.

Studies indicate that the radiotracer 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) can be metabolized beyond 2-[18F]FDG-6-phosphate (2-[18F]FDG-6-P), but its metabolism is incompletely understood. Most importantly, it remains unclear whether downstream metabolism affects tracer accumulation in vivo. Here we present a fingerprint of 2-[18F]FDG radiometabolites over time in cancer cells, corresponding tumor xenografts and murine organs. Strikingly, radiometabolites representing glycogen metabolism or the oxPPP correlated inversely with tracer accumulation across all examined tissues. Recent studies suggest that not only hexokinase, but also hexose-6-phosphate dehydrogenase (H6PD), an enzyme of the oxidative pentose phosphate pathway (oxPPP), determines 2-[18F]FDG accumulation. However, little is known about the corresponding enzyme glucose-6-phosphate dehydrogenase (G6PD). Our mechanistic in vitro experiments on the role of the oxPPP propose that 2-[18F]FDG can be metabolized via both G6PD and H6PD, but data from separate enzyme knockdown suggest diverging roles in downstream tracer metabolism. Overall, we propose that tissue-specific metabolism beyond 2-[18F]FDG-6-P could matter for imaging.

Spatially resolved metabolomics and isotope tracing reveal dynamic metabolic responses of dentate granule neurons with acute stimulation.

Neuronal activity creates an intense energy demand that must be met by rapid metabolic responses. To investigate metabolic adaptations in the neuron-enriched dentate granule cell (DGC) layer within its native tissue environment, we employed murine acute hippocampal brain slices, coupled with fast metabolite preservation and followed by mass spectrometry (MS) imaging, to generate spatially resolved metabolomics and isotope-tracing data. Here we show that membrane depolarization induces broad metabolic changes, including increased glycolytic activity in DGCs. Increased glucose metabolism in response to stimulation is accompanied by mobilization of endogenous inosine into pentose phosphates via the action of purine nucleotide phosphorylase (PNP). The PNP reaction is an integral part of the neuronal response to stimulation, because inhibition of PNP leaves DGCs energetically impaired during recovery from strong activation. Performing MS imaging on brain slices bridges the gap between live-cell physiology and the deep chemical analysis enabled by MS.

Rationale and design of an implant procedure and pivotal study to evaluate safety and effectiveness of Medtronic's tibial neuromodulation device.

Percutaneous tibial neuromodulation is a medical guideline recommended therapy for treating symptoms of overactive bladder. Stimulation is delivered to the tibial nerve via a thin needle placed percutaneously for 30 min once a week for 12-weeks, and monthly thereafter. Studies have shown that this therapy can effectively relieve symptoms of overactive bladder; however, the frequent office visits present a barrier to patients and can impact therapy effectiveness. To mitigate the burden of frequent clinic visits, small implantable devices are being developed to deliver tibial neuromodulation. These devices are implanted during a single minimally invasive procedure and deliver stimulation intermittently, similar to percutaneous tibial neuromodulation. Here, we describe the implant procedure and design of a pivotal study evaluating the safety and effectiveness for an implantable tibial neuromodulation device. The Evaluation of Implantable Tibial Neuromodulation (TITAN 2) pivotal study is a prospective, multicenter, investigational device exemption study being conducted at up to 30 sites in the United States and enrolling subjects with symptoms of overactive bladder.

Spatially resolved metabolomics and isotope tracing reveal dynamic metabolic responses of dentate granule neurons with acute stimulation.

Neuronal activity creates an intense energy demand that must be met by rapid metabolic responses. To investigate metabolic adaptations in the neuron-enriched dentate granule cell (DGC) layer within its native tissue environment, we employed murine acute hippocampal brain slices coupled with fast metabolite preservation, followed by mass spectrometry imaging (MALDI-MSI) to generate spatially resolved metabolomics and isotope tracing data. Here we show that membrane depolarization induces broad metabolic changes, including increased glycolytic activity in DGCs. Increased glucose metabolism in response to stimulation is accompanied by mobilization of endogenous inosine into pentose phosphates, via the action of purine nucleotide phosphorylase (PNP). The PNP reaction is an integral part of the neuronal response to stimulation, as inhibiting PNP leaves DGCs energetically impaired during recovery from strong activation. Performing MSI on brain slices bridges the gap between live cell physiology and the deep chemical analysis enabled by mass spectrometry.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

Unusual reactivity of a flavin in a bifurcating electron-transferring flavoprotein leads to flavin modification and a charge-transfer complex.

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.

Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins.

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.

Tuning the Quantum Chemical Properties of Flavins via Modification at C8.

Flavins are central to countless enzymes but display different reactivities depending on their environments. This is understood to reflect modulation of the flavin electronic structure. To understand changes in orbital natures, energies, and correlation over the ring system, we begin by comparing seven flavin variants differing at C8, exploiting their different electronic spectra to validate quantum chemical calculations. Ground state calculations replicate a Hammett trend and reveal the significance of the flavin π-system. Comparison of higher-level theories establishes CC2 and ACD(2) as methods of choice for characterization of electronic transitions. Charge transfer character and electron correlation prove responsive to the identity of the substituent at C8. Indeed, bond length alternation analysis demonstrates extensive conjugation and delocalization from the C8 position throughout the ring system. Moreover, we succeed in replicating a particularly challenging UV/Vis spectrum by implementing hybrid QM/MM in explicit solvents. Our calculations reveal that the presence of nonbonding lone pairs correlates with the change in the UV/Vis spectrum observed when the 8-methyl is replaced by NH2, OH, or SH. Thus, our computations offer routes to understanding the spectra of flavins with different modifications. This is a first step toward understanding how the same is accomplished by different binding environments.

If It Works, Don't Touch It? A Cell-Based Approach to Studying 2-[18F]FDG Metabolism.

The glucose derivative 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG) is still the most used radiotracer for positron emission tomography, as it visualizes glucose utilization and energy demand. In general, 2-[18F]FDG is said to be trapped intracellularly as 2-[18F]FDG-6-phosphate, which cannot be further metabolized. However, increasingly, this dogma is being questioned because of publications showing metabolism beyond 2-[18F]FDG-6-phosphate and even postulating 2-[18F]FDG imaging to depend on the enzyme hexose-6-phosphate dehydrogenase in the endoplasmic reticulum. Therefore, we aimed to study 2-[18F]FDG metabolism in the human cancer cell lines HT1080, HT29 and Huh7 applying HPLC. We then compared 2-[18F]FDG metabolism with intracellular tracer accumulation, efflux and the cells' metabolic state and used a graphical Gaussian model to visualize metabolic patterns. The extent of 2-[18F]FDG metabolism varied considerably, dependent on the cell line, and was significantly enhanced by glucose withdrawal. However, the metabolic pattern was quite conserved. The most important radiometabolites beyond 2-[18F]FDG-6-phosphate were 2-[18F]FDMannose-6-phosphate, 2-[18F]FDG-1,6-bisphosphate and 2-[18F]FD-phosphogluconolactone. Enhanced radiometabolite formation under glucose reduction was accompanied by reduced efflux and mirrored the cells' metabolic switch as assessed via extracellular lactate levels. We conclude that there can be considerable metabolism beyond 2-[18F]FDG-6-phosphate in cancer cell lines and a comprehensive understanding of 2-[18F]FDG metabolism might help to improve cancer research and tumor diagnosis.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Clinical characteristics and outcomes of COVID-19 in a low-prevalence, well resourced setting, Sydney, Australia.

BACKGROUND: The Northern Sydney Local Health District was one of the first health regions to be affected by COVID-19 in Australia. AIMS: To describe the clinical characteristics, risk factors and outcomes in our low-prevalence Australian population. METHODS: This is a retrospective analysis of 517 laboratory-confirmed COVID-19 cases between January and June 2020. Patient information was collected as part of routine care within the COVID-19 Virtual Hospital system. Outcomes examined were death, recovery at 30 days and intensive care unit (ICU) admission. RESULTS: The case fatality rate was 1.8%. Multivariate analysis showed factors independently associated with death, composite outcome of death/ICU admission or incomplete recovery at 30 days were age >80 years and presence of two or more comorbidities. Most cases acquired COVID-19 through international (50.9%) or cruise ship travel (9.1%). Healthcare workers comprised 12.8% of the cohort and represented a disproportionately high percentage of the 'unknown' source group (27.6%). The median incubation period was 5 days (interquartile range 3-8); one patient had an incubation period of 15 days. Hospitalisation was required in 11.8%, ICU admission in 2.1% and ventilation in 1.4%. A Radiographic Assessment of Lung Oedema score on chest X-ray of >10 was independently associated with death. CONCLUSIONS: In this low prevalence, well resourced Australian setting, we report an overall low mortality. Factors associated with adverse patient outcomes on multivariate analysis were age greater than 80 and the presence of two or more comorbidities. These data can assist in early risk stratification of COVID-19 patients, and in surge capacity planning for hospitals.

Assignments of 19F NMR resonances and exploration of dynamics in a long-chain flavodoxin.

Flavodoxin is a small protein that employs a non-covalently bound flavin to mediate single-electron transfer at low potentials. The long-chain flavodoxins possess a long surface loop that is proposed to interact with partner proteins. We have incorporated 19F-labeled tyrosine in long-chain flavodoxin from Rhodopseudomonas palustris to gain a probe of possible loop dynamics, exploiting the presence of a Tyr in the long loop in addition to Tyr residues near the flavin. We report 19F resonance assignments for all four Tyrs, and demonstration of a pair of resonances in slow exchange, both corresponding to a Tyr adjacent to the flavin. We also provide evidence for dynamics affecting the Tyr in the long loop. Thus, we show that 19F NMR of 19F-Tyr labeled flavodoxin holds promise for monitoring possible changes in conformation upon binding to partner proteins.

Photogeneration and reactivity of flavin anionic semiquinone in a bifurcating electron transfer flavoprotein.

Electron transfer bifurcation allows production of a strongly reducing carrier at the expense of a weaker one, by redistributing energy among a pair of electrons. Thus, two weakly-reducing electrons from NADH are consumed to produce a strongly reducing ferredoxin or flavodoxin, paid for by reduction of an oxidizing acceptor. The prevailing mechanism calls for participation of a strongly reducing flavin semiquinone which has been difficult to observe with site-certainly in multi-flavin systems. Using blue light (450 nm) to photoexcite the flavins of bifurcating electron transfer flavoprotein (ETF), we demonstrate accumulation of anionic flavin semiquinone in excess of what is observed in equilibrium titrations, and establish its ability to reduce the low-potential electron acceptor benzyl viologen. This must occur at the bifurcating flavin because the midpoint potentials of the electron transfer (ET) flavin are not sufficiently negative. We show that bis-tris propane buffer is an effective electron donor to the flavin photoreduction, but that if the system is prepared with the ET flavin chemically reduced, so that only the bifurcating flavin is oxidized and photochemically active, flavin anionic semiquinone is formed more rapidly. Thus, excited bifurcating flavin is able to draw on an electron stored at the ET flavin. Flavin semiquinone photogenerated at the bifurcation site must therefore be accompanied by additional semiquinone formation by oxidation of the ET flavin. Consistent with the expected instability of bifurcating flavin semiquinone, it subsides immediately upon cessation of illumination. However comparison with yields of semiquinone in equilibrium titrations suggest that during continuous illumination at pH 9 a steady state population of 0.3 equivalents of bifurcating flavin semiquinone accumulates, and then undergoes further photoreduction to the hydroquinone. Although transient, the population of bifurcating flavin semiquinone explains the system's ability to conduct light-driven electron transfer from bis-tris propane to benzyl viologen, in effect trapping energy from light.