Giant supermagnonic Bloch point velocities in cylindrical ferromagnetic nanowires.

Achieving high velocities of magnetic domain walls is a crucial factor for their use as information carriers in modern nanoelectronic applications. In nanomagnetism and spintronics, these velocities are often limited either by internal domain wall instabilities, known as the Walker breakdown phenomenon, or by spin wave emission, known as the magnonic regime. In the rigid domain wall model, the maximum magnon velocity acts as an effective "speed of light", providing a relativistic analogy for the domain wall speed limitation. Cylindrical magnetic nanowires are an example of systems without the Walker breakdown phenomenon. Here we demonstrate that the magnonic limit could be outstandingly surpassed in cylindrical nanowires with high magnetization, such as iron. Our numerical modeling shows the Bloch point domain wall velocities as high as 14 km s-1, well above the magnonic limit estimated in the interval 1.7-2.0 km s-1. The key ingredient is the three-dimensional conical shape of the domain wall, which elongates and breaks during the dynamics, expelling backwards pairs of Bloch points. This leads to domain wall acceleration, the effect, which resembles the "jet propulsion". This effect will be very important for three-dimensional networks based on cylindrical magnetic nanowires.

Renal Proximal Tubule Cell-specific Megalin Deletion Does Not Affect Atherosclerosis But Induces Tubulointerstitial Nephritis in Mice Fed Western Diet.

Background: Pharmacological inhibition of megalin (also known as low-density lipoprotein receptor-related protein 2: LRP2) attenuates atherosclerosis in hypercholesterolemic mice. Since megalin is abundant in renal proximal tubule cells (PTCs), the purpose of this study was to determine whether PTC-specific deletion of megalin reduces hypercholesterolemia-induced atherosclerosis in mice. Methods: Female Lrp2 f/f mice were bred with male Ndrg1-Cre ERT2 +/0 mice to develop PTC-LRP2 +/+ and -/- littermates. To study atherosclerosis, all mice were to bred to an LDL receptor -/- background and fed a Western diet to induce atherosclerosis. Results: PTC-specific megalin deletion did not attenuate atherosclerosis in LDL receptor -/- mice in either sex. Serendipitously, we discovered that PTC-specific megalin deletion led to interstitial infiltration of CD68+ cells and tubular atrophy. The pathology was only evident in male PTC-LRP2 -/- mice fed the Western diet, but not in mice fed a normal laboratory diet. Renal pathologies were also observed in male PTC-LRP2 -/- mice in an LDL receptor +/+ background fed the same Western diet, demonstrating that the renal pathologies were dependent on diet and not hypercholesterolemia. By contrast, female PTC-LRP2 -/- mice had no apparent renal pathologies. In vivo multiphoton microscopy demonstrated that PTC-specific megalin deletion dramatically diminished albumin accumulation in PTCs within 10 days of Western diet feeding. RNA sequencing analyses demonstrated the upregulation of inflammation-related pathways in kidney. Conclusions: PTC-specific megalin deletion does not affect atherosclerosis, but leads to tubulointerstitial nephritis in mice fed Western diet, with severe pathologies in male mice.

Therapeutic α-1-microglobulin ameliorates kidney ischemia-reperfusion injury.

α-1-Microglobulin (A1M) is a circulating glycoprotein with antioxidant, heme-binding, and mitochondrial protection properties. The investigational drug RMC-035, a modified therapeutic A1M protein, was assessed for biodistribution and pharmacological activity in a broad set of in vitro and in vivo experiments, supporting its clinical development. Efficacy and treatment posology were assessed in various models of kidney ischemia and reperfusion injury (IRI). Real-time glomerular filtration rate (GFR), functional renal biomarkers, tubular injury biomarkers (NGAL and KIM-1), and histopathology were evaluated. Fluorescently labeled RMC-035 was used to assess biodistribution. RMC-035 demonstrated consistent and reproducible kidney protection in rat IRI models as well as in a model of IRI imposed on renal impairment and in a mouse IRI model, where it reduced mortality. Its pharmacological activity was most pronounced with combined dosing pre- and post-ischemia and weaker with either pre- or post-ischemia dosing alone. RMC-035 rapidly distributed to the kidneys via glomerular filtration and selective luminal uptake by proximal tubular cells. IRI-induced expression of kidney heme oxygenase-1 was inhibited by RMC-035, consistent with its antioxidative properties. RMC-035 also dampened IRI-associated inflammation and improved mitochondrial function, as shown by tubular autofluorescence. Taken together, the efficacy of RMC-035 is congruent with its targeted mechanism(s) and biodistribution profile, supporting its further clinical evaluation as a novel kidney-protective therapy.NEW & NOTEWORTHY A therapeutic A1M protein variant (RMC-035) is currently in phase 2 clinical development for renal protection in patients undergoing open-chest cardiac surgery. It targets several key pathways underlying kidney injury in this patient group, including oxidative stress, heme toxicity, and mitochondrial dysfunction. RMC-035 is rapidly eliminated from plasma, distributing to kidney proximal tubules, and demonstrates dose-dependent efficacy in numerous models of ischemia-reperfusion injury, particularly when administered before ischemia. These results support its continued clinical evaluation.

Cryopreservation and Rapid Recovery of Differentiated Intestinal Epithelial Barrier Cells at Complex Transwell Interfaces Is Enabled by Chemically Induced Ice Nucleation.

Cell-based models, such as organ-on-chips, can replace and inform in vivo (animal) studies for drug discovery, toxicology, and biomedical science, but most cannot be banked "ready to use" as they do not survive conventional cryopreservation with DMSO alone. Here, we demonstrate how macromolecular ice nucleators enable the successful cryopreservation of epithelial intestinal models supported upon the interface of transwells, allowing recovery of function in just 7 days post-thaw directly from the freezer, compared to 21 days from conventional suspension cryopreservation. Caco-2 cells and Caco-2/HT29-MTX cocultures are cryopreserved on transwell inserts, with chemically induced ice nucleation at warmer temperatures resulting in increased cell viability but crucially retaining the complex cellular adhesion on the transwell insert interfaces, which other cryoprotectants do not. Trans-epithelial electrical resistance measurements, confocal microscopy, histology, and whole-cell proteomics demonstrated the rapid recovery of differentiated cell function, including the formation of tight junctions. Lucifer yellow permeability assays confirmed that the barrier functions of the cells were intact. This work will help solve the long-standing problem of transwell tissue barrier model storage, facilitating access to advanced predictive cellular models. This is underpinned by precise control of the nucleation temperature, addressing a crucial biophysical mode of damage.

Clinical effectiveness, cost-effectiveness and process evaluation of group schema therapy for eating disorders: study protocol for a multicenter randomized controlled trial.

BACKGROUND: Eating disorders (EDs), such as (atypical) Anorexia (AN) and Bulimia Nervosa (BN), are difficult to treat, causing socioeconomic impediments. Although enhanced cognitive behavioral therapy (CBT-E) is widely considered clinically effective, it may not be the most beneficial treatment for (atypical) AN and BN patients who do not show a rapid response after the first 4 weeks (8 sessions) of a CBT-E treatment. Alternatively, group schema therapy (GST) may be a valuable treatment for this ED population. Even though GST for EDs has yielded promising preliminary findings, the current body of evidence requires expansion. On top of that, data on cost-effectiveness is lacking. In light of these gaps, we aim to describe a protocol to examine whether GST is more (1) clinically effective and (2) cost-effective than CBT-E for (atypical) AN and BN patients, who do not show a rapid response after the first 4 weeks of treatment. Additionally, we will conduct (3) process evaluations for both treatments. METHODS: Using a multicenter RCT design, 232 Dutch (atypical) AN and BN patients with a CBT-E referral will be recruited from five treatment centers. Clinical effectiveness and cost-effectiveness will be measured before treatment, directly after treatment, at 6 and at 12 months follow-up. In order to rate process evaluation, patient experiences and the degree to which treatments are implemented according to protocol will be measured. In order to assess the quality of life and the achievement of personalized goals, interviews will be conducted at the end of treatment. Data will be analyzed, using a regression-based approach to mixed modelling, multivariate sensitivity analyses and coding trees for qualitative data. We hypothesize GST to be superior to CBT-E in terms of clinical effectiveness and cost-effectiveness for patients who do not show a rapid response to the first 4 weeks of a CBT-E treatment. DISCUSSION: To our knowledge, this is the first study protocol describing a multicenter RCT to explore the three aforementioned objectives. Related risks in performing the study protocol have been outlined. The expected findings may serve as a guide for healthcare stakeholders to optimize ED care trajectories. TRIAL REGISTRATION: clinicaltrials.gov (NCT05812950).

Sex Differences in Prehospital Identification of Large Vessel Occlusion in Patients With Suspected Stroke.

BACKGROUND: Differences in clinical presentation of acute ischemic stroke between men and women may affect prehospital identification of anterior circulation large vessel occlusion (aLVO). We assessed sex differences in diagnostic performance of 8 prehospital scales to detect aLVO. METHODS: We analyzed pooled individual patient data from 2 prospective cohort studies (LPSS [Leiden Prehospital Stroke Study] and PRESTO [Prehospital Triage of Patients With Suspected Stroke Study]) conducted in the Netherlands between 2018 and 2019, including consecutive patients ≥18 years suspected of acute stroke who presented within 6 hours after symptom onset. Ambulance paramedics assessed clinical items from 8 prehospital aLVO detection scales: Los Angeles Motor Scale, Rapid Arterial Occlusion Evaluation, Cincinnati Stroke Triage Assessment Tool, Cincinnati Prehospital Stroke Scale, Prehospital Acute Stroke Severity, gaze-face-arm-speech-time, Conveniently Grasped Field Assessment Stroke Triage, and Face-Arm-Speech-Time Plus Severe Arm or Leg Motor Deficit. We assessed the diagnostic performance of these scales for identifying aLVO at prespecified cut points for men and women. RESULTS: Of 2358 patients with suspected stroke (median age, 73 years; 47% women), 231 (10%) had aLVO (100/1114 [9%] women and 131/1244 [11%] men). The area under the curve of the scales ranged from 0.70 (95% CI, 0.65-0.75) to 0.77 (95% CI, 0.73-0.82) in women versus 0.69 (95% CI, 0.64-0.73) to 0.75 (95% CI, 0.71-0.79) in men. Positive predictive values ranged from 0.23 (95% CI, 0.20-0.27) to 0.29 (95% CI, 0.26-0.31) in women versus 0.29 (95% CI, 0.24-0.33) to 0.37 (95% CI, 0.32-0.43) in men. Negative predictive values were similar (0.95 [95% CI, 0.94-0.96] to 0.98 [95% CI, 0.97-0.98] in women versus 0.94 [95% CI, 0.93-0.95] to 0.96 [95% CI, 0.94-0.97] in men). Sensitivity of the scales was slightly higher in women than in men (0.53 [95% CI, 0.43-0.63] to 0.76 [95% CI, 0.68-0.84] versus 0.49 [95% CI, 0.40-0.57] to 0.63 [95% CI, 0.55-0.73]), whereas specificity was lower (0.79 [95% CI, 0.76-0.81] to 0.87 [95% CI, 0.84-0.89] versus 0.82 [95% CI, 0.79-0.84] to 0.90 [95% CI, 0.88-0.91]). Rapid arterial occlusion evaluation showed the highest positive predictive values in both sexes (0.29 in women and 0.37 in men), reflecting the different event rates. CONCLUSIONS: aLVO scales show similar diagnostic performance in both sexes. The rapid arterial occlusion evaluation scale may help optimize prehospital transport decision-making in men as well as in women with suspected stroke.

Light-sensitive phosphorylation regulates retinal IMPDH1 activity and filament assembly.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.

Long-Term Results of the Linear Incision Technique With Tissue Reduction Versus Tissue Preservation for Inserting Bone-Anchored Hearing Implants: The Ongoing Optimization in Bone Implant Surgery.

OBJECTIVE: To compare the long-term outcomes of the linear incision technique with tissue reduction (LIT-TR) and the linear incision technique with tissue preservation (LIT-TP) for inserting bone-anchored hearing implants (BAHIs). STUDY DESIGN: Single-center retrospective cohort study. SETTING: Large general teaching hospital. PATIENTS: A total of 231 adult patients were included between August 2005 and October 2020, with a minimum follow-up time of 6 months. INTERVENTION: The test group received a BAHI using the LIT-TP (N = 147). The control group underwent surgery using the LIT-TR (N = 84). MAIN OUTCOME MEASURES: Soft tissue reactions, skin thickening, postoperative complications (e.g., wound dehiscence), and implant loss were compared between the test and control group. Furthermore, Cochlear Bone Anchored Solutions AB (Mölnlycke, Sweden) and Oticon Medical AB (Askim, Sweden) implants/abutments within the LIT-TP cohort were compared. Validated questionnaires were used to quantify patients' health-related quality of life (HRQoL). RESULTS: Significantly more cases with wound dehiscence and adverse soft tissue reactions (Holgers ≥2) were observed in the LIT-TR cohort (p < 0.001). However, the LIT-TP cohort showed significantly more cases with skin thickening (requiring treatment) within the first 2 years after implantation. There were no differences in implant loss rates, overall soft tissue reactions (Holgers >1), and overall HRQoL between the two patient groups. Significant improvement in the patients' HRQoL after implementation of a BAHI was found in both techniques. The Ponto Wide implant/abutment showed less frequent skin thickening (requiring treatment) and fewer soft tissue reactions compared with the BIA400 implant/abutment. CONCLUSION: This large-scale study demonstrates that the LIT-TP shows excellent long-term outcomes, including a low incidence of implant failure.

HSP70 Is a Critical Regulator of HSP90 Inhibitor's Effectiveness in Preventing HCl-Induced Chronic Lung Injury and Pulmonary Fibrosis.

Exposure to hydrochloric acid (HCl) can provoke acute and chronic lung injury. Because of its extensive production for industrial use, frequent accidental exposures occur, making HCl one of the top five chemicals causing inhalation injuries. There are no Food and Drug Administration (FDA)-approved treatments for HCl exposure. Heat shock protein 90 (HSP90) inhibitors modulate transforming growth factor-ß (TGF-ß) signaling and the development of chemical-induced pulmonary fibrosis. However, little is known on the role of Heat Shock Protein 70 (HSP70) during injury and treatment with HSP90 inhibitors. We hypothesized that administration of geranylgeranyl-acetone (GGA), an HSP70 inducer, or gefitinib (GFT), an HSP70 suppressant, alone or in combination with the HSP90 inhibitor, TAS-116, would improve or worsen, respectively, HCl-induced chronic lung injury in vivo and endothelial barrier dysfunction in vitro. GGA, alone, improved HCl-induced human lung microvascular endothelial cells (HLMVEC) barrier dysfunction and, in combination with TAS-116, improved the protective effect of TAS-116. In mice, GGA reduced HCl toxicity and while TAS-116 alone blocked HCl-induced chronic lung injury, co-administration with GGA, resulted in further improvement. Conversely, GFT potentiated HCl-induced barrier dysfunction and impaired the antidotal effects of TAS-116. We conclude that combined treatments with HSP90 inhibitors and HSP70 inducers may represent a novel therapeutic approach to manage HCl-induced chronic lung injury and pulmonary fibrosis.

Synergistic effects of nanosecond pulsed plasma and electric field on inactivation of pancreatic cancer cells in vitro.

Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on the metabolic activities of cells and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for > 2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, > 20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-fivefold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.

Extracellular vesicles in acute respiratory distress syndrome: Understanding protective and harmful signaling for the development of new therapeutics.

Acute respiratory distress syndrome (ARDS) is a severe respiratory condition characterized by increased lung permeability, hyper-inflammatory state, and fluid leak into the alveolar spaces. ARDS is a heterogeneous disease, with multiple direct and indirect causes that result in a mortality of up to 40%. Due to the ongoing Covid-19 pandemic, its incidence has increased up to ten-fold. Extracellular vesicles (EVs) are small liposome-like particles that mediate intercellular communication and play a major role in ARDS pathophysiology. Indeed, they participate in endothelial barrier dysfunction and permeability, neutrophil, and macrophage activation, and also in the development of a hypercoagulable state. A more thorough understanding of the variegated and cell-specific functions of EVs may lead to the development of safe and effective therapeutics. In this review, we have collected evidence of EVs role in ARDS, revise the main mechanisms of production and internalization and summarize the current therapeutical approaches that have shown the ability to modulate EV signaling.

Assessing Polymorphic Purity of Rifampicin in Double and Triple-Drug Fixed-Dose Combination Products.

First-line tuberculostatic agents, Rifampicin (RIF), Isoniazid (ISH), Ethambutol (ETB), and Pyrazinamide (PZA) are generally administered as a fixed-dose combination (FDC) for improving patient adherence. The major quality challenge of these FDC products is their variable bioavailability, where RIF and its solid state are key factors. In this work, the analysis of the impact of the polymorphism in the performance of RIF in RIF-ISH and PZA-RIF-ISH combined products was carried out by an overall approach that included the development and validation of two methodologies combining near-infrared (NIR) spectroscopy and partial least squares (PLS) to the further evaluation of commercial products. For NIR-PLS methods, training and validation sets were prepared with mixtures of Form I/Form II of RIF, and the appropriate amount of ISH (for double associations) or ISH-PZA (for triple associations). The corresponding matrix of the excipients was added to the mixture of APIs to simulate the environment of each FDC product. Four PLS factors, reduced spectral range, and the combination of standard normal variate and Savitzky-Golay 1st derivative (SNV-D') were selected as optimum data pre-treatment for both methods, yielding satisfactory recoveries during the analysis of validation sets (98.5±2.0%, and 98.7±1.8% for double- and triple-FDC products, respectively). The NIR-PLS model for RIF-ISH successfully estimated the polymorphic purity of Form II in double-FDC capsules (1.02 ± 0.02w/w). On the other hand, the NIR-PLS model for RIF-ISH-PZA detected a low purity of Form II in triple FDC tablets (0.800 ± 0.021w/w), these results were confirmed by X-ray powder diffraction. Nevertheless, the triple-FDC tablets showed good performance in the dissolution test (Q=99-102%), implying a Form II purity about of 80% is not low enough to affect the safety and efficacy of the product.

Regioselective Glycosylation of Polyphenols by Family 1 Glycosyltransferases: Experiments and Simulations.

Family 1 glycosyltransferases (GT1s, UGTs) form natural product glycosides with exquisite control over regio- and stereoselectivity, representing attractive biotechnological targets. However, regioselectivity cannot be predicted and large-scale activity assessment efforts of UGTs are commonly performed via mass spectrometry or indirect assays that are blind to regioselectivity. Here, we present a large high performance liquid chromatography screening discriminating between regioisomeric products of 40 diverse UGTs (28.6% average pairwise sequence identity) against 32 polyphenols, identifying enzymes able to reach high glycosylation yields (≥90% in 24 h) in 26/32 cases. In reactions with >50% yield, we observed perfect regioselectivity for 47% (75/158) on polyphenols presenting two hydroxyl groups and for 30% (43/143) on polyphenols presenting ≥3 hydroxyl groups. Moreover, we developed a nuclear magnetic resonance-based procedure to identify the site of glycosylation directly on enzymatic mixtures. We further selected seven regiospecific reactions catalyzed by four enzymes on five dihydroxycoumarins. We characterized the four enzymes, showing that temperature optima are functions of the acceptor substrate, varying by up to 20 °C for the same enzyme. Furthermore, we performed short molecular dynamics simulations of 311 ternary complexes (UGT, UDP-Glc, and glycosyl acceptor) to investigate the molecular basis for regioselectivity. Interestingly, it appeared that most UGTs can accommodate acceptors in configurations favorable to the glycosylation of either hydroxyl. In contrast, evaluation of hydroxyl nucleophilicity appeared to be a strong predictor of the hydroxyl predominantly glycosylated by most enzymes.

Small protein mediates inhibition of ammonium transport in Methanosarcina mazei-an ancient mechanism?

IMPORTANCE: Small proteins containing fewer than 70 amino acids, which were previously disregarded due to computational prediction and biochemical detection challenges, have gained increased attention in the scientific community in recent years. However, the number of functionally characterized small proteins, especially in archaea, is still limited. Here, by using biochemical and genetic approaches, we demonstrate a crucial role of the small protein sP36 in the nitrogen metabolism of M. mazei, which modulates the ammonium transporter AmtB1 according to nitrogen availability. This modulation might represent an ancient archaeal mechanism of AmtB1 inhibition, in contrast to the well-studied uridylylation-dependent regulation in bacteria.

Promiscuous Yet Specific: A Methionine-Aromatic Interaction Drives the Reaction Scope of the Family 1 Glycosyltransferase GmUGT88E3 from Soybean.

Family 1 glycosyltransferases (GT1s, UGTs) catalyze the regioselective glycosylation of natural products in a single step. We identified GmUGT88E3 as a particularly promising biocatalyst able to produce a variety of pure, single glycosidic products from polyphenols with high chemical yields. We investigated this particularly desirable duality toward specificity, i.e., promiscuous toward acceptors while regiospecific. Using high-field NMR, kinetic characterization, molecular dynamics simulations, and mutagenesis studies, we uncovered that the main molecular determinant of GmUGT88E3 specificity is a methionine-aromatic bridge, an interaction often present in protein structures but never reported for enzyme-substrate interactions. Here, mutating Met127 led to inactive proteins or 100-fold reduced activity.

Tailoring elastic and inelastic collisions of relativistic antiferromagnetic domain walls.

Soliton-based computing relies on their unique properties for transporting energy and emerging intact from head-on collisions. Magnetic domain walls are often referred to as solitons disregarding the strict mathematical definition requiring the above scattering property. Here we demonstrate the conditions of elastic and inelastic scattering for spin-orbit torque-induced dynamics of relativistic domain walls on the technologically relevant Mn[Formula: see text]Au antiferromagnetic material. We show that even domain walls with opposite winding numbers can experience elastic scattering and we present the corresponding phase diagram as a function of the spin-orbit field strength and duration. The elastic collision requires minimum domain walls speed, which we explain assuming an attractive potential created by domain wall pair. On the contrary, when the domain walls move at lower speeds, their collision is inelastic and results in a dispersing breather. Our findings will be important for the development of soliton-based computing using antiferromagnetic spintronics and we discuss their prospects for building NOT and XOR gates.

Molecular orbital and topological electron density study of n → π* interactions: amides and thioamides cases.

The n → π* interactions were studied in amides and thioamides systems models, through the analysis of the electron density topology along with the Natural Bonding Orbital (NBO) approach. The effect of the dispersion terms was assessed using different DFT functionals. The NBO, independent gradient model (IGM), and the analysis of the reduced density gradient outcomes show that dispersion forces play a significant role in the strength of n → π* interactions. The IGM results indicate that δg height values for n → π* interactions do not extend beyond 0.025. All the methods used in this work predict that n → π* interaction between pairs of thioamides is stronger than those between amides. However, the electron density topology-based methods were not able to replicate the trends in the relative force of this interaction found in the experimental and NBO results.

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants.

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands.