New insights into the interaction of emodin with lipid membranes.

Emodin is a natural anthraquinone derivative found in nature, widely known as an herbal medicine. Here, the partition, location, and interaction of emodin with lipid membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are experimentally investigated with different techniques. Our studies have considered the neutral form of emodin (EMH) and its anionic/deprotonated form (EM-), and their interaction with a more and less packed lipid membrane, DMPC at the gel and fluid phases, respectively. Though DSC results indicate that the two species, EMH and EM-, similarly disrupt the packing of DMPC bilayers, spin labels clearly show that EMH causes a stronger bilayer disruption, both in gel and fluid DMPC. Fluorescence spectroscopy shows that both EMH and EM- have a high affinity for DMPC: the binding of EM- to both gel and fluid DMPC bilayers was found to be quite similar, and similar to that of EMH to gel DMPC, Kp = (1.4 ± 0.3)x103. However, EMH was found to bind twice more strongly to fluid DMPC bilayers, Kp = (3.2 ± 0.3)x103. Spin labels and optical absorption spectroscopy indicate that emodin is located close to the lipid bilayer surface, and suggest that EM- is closer to the lipid/water interface than EMH, as expected. The present studies present a relevant contribution to the current understanding of the effect the two species of emodin, EMH and EM-, present on different microregions of an organism, as local pH values can vary significantly, can cause in a neutral lipid membrane, either more or less packed, liked gel and fluid DMPC, respectively, and could be extended to lipid domains of biological membranes.

Inhibition of superoxide and iNOS augment cutaneous nitric oxide-dependent vasodilation in non-Hispanic black young adults.

We assessed the combined effect of superoxide and iNOS inhibition on microvascular function in non-Hispanic Black and non-Hispanic White participants (n = 15 per group). Participants were instrumented with four microdialysis fibers: (1) lactated Ringer's (control), (2) 10 µM tempol (superoxide inhibition), (3) 0.1 mM 1400 W (iNOS inhibition), (4) tempol + 1400 W. Cutaneous vasodilation was induced via local heating and NO-dependent vasodilation was quantified. At control sites, NO-dependent vasodilation was lower in non-Hispanic Black (45 ± 9% NO) relative to non-Hispanic White (79 ± 9% NO; p < 0.01; effect size, d = 3.78) participants. Tempol (62 ± 16% NO), 1400 W (78 ± 12% NO) and tempol +1400 W (80 ± 13% NO) increased NO-dependent vasodilation in non-Hispanic Black participants relative to control sites (all p < 0.01; d = 1.22, 3.05, 3.03, respectively). The effect of 1400 W (p = 0.04, d = 1.11) and tempol +1400 W (p = 0.03, d = 1.22) was greater than tempol in non-Hispanic Black participants. There was no difference between non-Hispanic Black and non-Hispanic White participants at 1400 W or tempol + 1400 W sites. These data suggest iNOS has a greater effect on NO-dependent vasodilation than superoxide in non-Hispanic Black participants.

Dysregulated cerebral blood flow, rather than gray matter Volume, exhibits stronger correlations with blood inflammatory and lipid markers in depression.

Arterial spin labeling (ASL) can be used to detect differences in perfusion for multiple brain regions thought to be important in major depressive disorder (MDD). However, the potential of cerebral blood flow (CBF) to predict MDD and its correlations between the blood lipid levels and immune markers, which are closely related to MDD and brain function change, remain unclear. The 451 individuals - 298 with MDD and 133 healthy controls who underwent MRI at a single time point with arterial spin labelling and a high resolution T1-weighted structural scan. A proportion of MDD also provided blood samples for analysis of lipid and immune markers. We performed CBF case-control comparisons, random forest model construction, and exploratory correlation analyses. Moreover, we investigated the relationship between gray matter volume (GMV), blood lipids, and the immune system within the same sample to assess the differences in CBF and GMV. We found that the left inferior parietal but supramarginal and angular gyrus were significantly different between the MDD patients and HCs (voxel-wise P < 0.001, cluster-wise FWE correction). And bilateral inferior temporal (ITG), right middle temporal gyrus and left precentral gyrus CBF predict MDD (the area under the receiver operating characteristic curve of the random forest model is 0.717) and that CBF is a more sensitive predictor of MDD than GMV. The left ITG showed a positive correlation trend with immunoglobulin G (r = 0.260) and CD4 counts (r = 0.283). The right ITG showed a correlation trend with Total Cholesterol (r = -0.249) and tumour necrosis factor-alpha (r = -0.295). Immunity and lipids were closely related to CBF change, with the immunity relationship potentially playing a greater role. The interactions between CBF, plasma lipids and immune index could therefore represent an MDD pathophysiological mechanism. The current findings provide evidence for targeted regulation of CBF or immune properties in MDD.

Lesion volume and spike frequency on EEG impact perfusion values in focal cortical dysplasia: a pediatric arterial spin labeling study.

Arterial spin labelling (ASL), an MRI sequence non-invasively imaging brain perfusion, has yielded promising results in the presurgical workup of children with focal cortical dysplasia (FCD)-related epilepsy. However, the interpretation of ASL-derived perfusion patterns remains unclear. Hence, we compared ASL qualitative and quantitative findings to their clinical, EEG, and MRI counterparts. We included children with focal structural epilepsy related to an MRI-detectable FCD who underwent single delay pseudo-continuous ASL. ASL perfusion changes were assessed qualitatively by visual inspection and quantitatively by estimating the asymmetry index (AI). We considered 18 scans from 15 children. 16 of 18 (89%) scans showed FCD-related perfusion changes: 10 were hypoperfused, whereas six were hyperperfused. Nine scans had perfusion changes larger than and seven equal to the FCD extent on anatomical images. Hyperperfusion was associated with frequent interictal spikes on EEG (p = 0.047). Perfusion changes in ASL larger than the FCD corresponded to larger lesions (p = 0.017). Higher AI values were determined by frequent interictal spikes on EEG (p = 0.004). ASL showed FCD-related perfusion changes in most cases. Further, higher spike frequency on EEG may increase ASL changes in affected children. These observations may facilitate the interpretation of ASL findings, improving treatment management, counselling, and prognostication in children with FCD-related epilepsy.

Conformational Heterogeneity of ß-Barrel Membrane Proteins Observed In Situ Using Orthogonal Spin Labels and Pulsed ESR Spectroscopy.

Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of ß-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd3+-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.

Developing blood-brain barrier arterial spin labelling as a non-invasive early biomarker of Alzheimer's disease (DEBBIE-AD): a prospective observational multicohort study protocol.

INTRODUCTION: Loss of blood-brain barrier (BBB) integrity is hypothesised to be one of the earliest microvascular signs of Alzheimer's disease (AD). Existing BBB integrity imaging methods involve contrast agents or ionising radiation, and pose limitations in terms of cost and logistics. Arterial spin labelling (ASL) perfusion MRI has been recently adapted to map the BBB permeability non-invasively. The DEveloping BBB-ASL as a non-Invasive Early biomarker (DEBBIE) consortium aims to develop this modified ASL-MRI technique for patient-specific and robust BBB permeability assessments. This article outlines the study design of the DEBBIE cohorts focused on investigating the potential of BBB-ASL as an early biomarker for AD (DEBBIE-AD). METHODS AND ANALYSIS: DEBBIE-AD consists of a multicohort study enrolling participants with subjective cognitive decline, mild cognitive impairment and AD, as well as age-matched healthy controls, from 13 cohorts. The precision and accuracy of BBB-ASL will be evaluated in healthy participants. The clinical value of BBB-ASL will be evaluated by comparing results with both established and novel AD biomarkers. The DEBBIE-AD study aims to provide evidence of the ability of BBB-ASL to measure BBB permeability and demonstrate its utility in AD and AD-related pathologies. ETHICS AND DISSEMINATION: Ethics approval was obtained for 10 cohorts, and is pending for 3 cohorts. The results of the main trial and each of the secondary endpoints will be submitted for publication in a peer-reviewed journal.

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0 nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Identifying delay in glymphatic clearance of labeled protons post-acute head trauma utilizing 3D ASL MRI (arterial spin labeling): a pilot study.

This study correlated mild traumatic brain injury (mTBI) cognitive changes with ASL-MRI glymphatic clearance rates (GCRs) and recovery with GCR improvement. mTBI disrupts the blood brain barrier (BBB), reducing capillary mean transit time and GCRs. mTBI is clinically diagnosed utilizing history/examination findings with no physiologic biomarkers. 3D TGSE (turbo-gradient spin-echo) pulsed arterial spin-labeling 3T MRI with 7 long inversion times (TIs) assessed the signal clearance of labeled protons 2800-4000 ms postlabeling in bifrontal, bitemporal, and biparietal regions within 7 days of mTBI and once clinically cleared to resume activities. The Sport Concussion Assessment Tool Version 5 (SKAT5) and Brief Oculomotor/Vestibular Assessment evaluated injured athletes' cognitive function prior to MRIs. The pilot study demonstrated significant GCRs improvement (95% CI - 0.06 to - 0.03 acute phase; to CI-recovery CI 0.0772 to - 0.0497; P < 0.001 in frontal lobes; and parietal lobes (95% CI - 0.0584 to - 0.0251 acute; CI - 0.0727 to - 0.0392 recovery; P = 0.024) in 9 mTBI athletes (8 female, 1 male). Six age/activity-matched controls (4 females, 2 males) were also compared. mTBI disrupts the BBB, reducing GCR measured using the 3D ASL MRI technique. ASL MRI is a potential noninvasive biomarker of mTBI and subsequent recovery.

Comparison of ASL and DSC perfusion methods in the evaluation of response to treatment in patients with a history of treatment for malignant brain tumor.

OBJECTIVE: Perfusion MRI is of great benefit in the post-treatment evaluation of brain tumors. Interestingly, dynamic susceptibility contrast-enhanced (DSC) perfusion has taken its place in routine examination for this purpose. The use of arterial spin labeling (ASL), a perfusion technique that does not require exogenous contrast material injection, has gained popularity in recent years. The aim of the study was to compare two different perfusion techniques, ASL and DSC, using qualitative and quantitative measurements and to investigate the diagnostic effectiveness of both. The fact that the number of patients is higher than in studies conducted with 3D pseudo-continious ASL (pCASL), the study group is heterogeneous as it consists of patients with both metastases and glial tumors, the use of 3D Turbo Gradient Spin Echo (TGSE), and the inclusion of visual (qualitative) assessment make our study unique. METHODS: Ninety patients, who were treated for malignant brain tumor, were enrolled in the retrospective study. DSC Cerebral Blood Volume (CBV), Cerebral Blood Flow (CBF) and ASL CBF maps of each case were obtained. In qualitative analysis, the lesions of the cases were visually classified as treatment-related changes (TRC) and relapse/residual mass (RRT). In the quantitative analysis, three regions of interest (ROI) measurements were taken from each case. The average of these measurements was compared with the ROI taken from the contralateral white matter and normalized values (n) were obtained. These normalized values were compared across events. RESULTS: Uncorrected DSC normalized CBV (nCBV), DSC normalized CBF (nCBF) and ASL nCBF values of RRT cases were higher than those of TRC cases (p < 0.001). DSC nCBV values were correlated with DSC nCBF (r: 0.94, p < 0.001) and correlated with ASL nCBF (r: 0.75, p < 0.001). Similarly, ASL nCBF was positively correlated with DSC nCBF (r: 0.79 p < 0.01). When the ROC curve parameters were evaluated, the cut-off values were determined as 1.211 for DSC nCBV (AUC: 0.95, 93% sensitivity, 82% specificity), 0.896 for DSC nCBF (AUC; 0.95, 93% sensitivity, 82% specificity), and 0.829 for ASL nCBF (AUC: 0.84, 78% sensitivity, 75% specificity). For qualitative evaluation (visual evaluation), inter-observer agreement was found to be good for ASL CBF (0.714), good for DSC CBF (0.790), and excellent for DSC CBV (0.822). Intra-observer agreement was also evaluated. For the first observer, good agreement was found in ASL CBF (0.626, 70% sensitive, 93% specific), in DSC CBF (0.713, 76% sensitive, 95% specific), and in DSC CBV (0.755, 87% sensitive - 88% specific). In the second observer, moderate agreement was found in ASL CBF (0.584, 61% sensitive, 97% specific) and DSC CBF (0.649, 65% sensitive, 100% specific), and excellent agreement in DSC CBV (0.800, 89% sensitive, 90% specific). CONCLUSION: It was observed that uncorrected DSC nCBV, DSC nCBF and ASL nCBF values were well correlated with each other. In qualitative evaluation, inter-observer and intra-observer agreement was higher in DSC CBV than DSC CBF and ASL CBF. In addition, DSC CBV is found more sensitive, ASL CBF and DSC CBF are found more specific for both observers. From a diagnostic perspective, all three parameters DSC CBV, DSC CBF and ASL CBF can be used, but it was observed that the highest rate belonged to DSC CBV.

Tempol alleviates acute lung injury by affecting glutathione synthesis through Nrf2 and inhibiting ferroptosis in lung epithelial cells.

As a life-threatening disease, acute lung injury (ALI) may progress to chronic pulmonary fibrosis. For the treatment of lung injury, Tempol is a superoxide dismutase mimetic and intracellular redox agent that can be a potential drug. This study investigated the regulatory mechanism of Tempol in the treatment of ALI. A mouse model of ALI was established, and HE staining was used to examine histomorphology. The CCK-8 assay was used to measure cell viability, and oxidative stress was assessed by corresponding kits. Flow cytometry and dichlorodihydrofluorescein diacetate staining assays were used to detect reactive oxygen species (ROS) levels. Protein expression levels were measured by Western blot analysis and ELISA. Pulmonary vascular permeability was used to measure the lung wet/dry weight ratio. The level of oxidative stress was increased in ALI mice, and the level of ferroptosis was upregulated. Tempol inhibited this effect and alleviated ALI. The administration of Tempol alleviated the pathological changes in ALI, inhibited pulmonary vascular permeability, and improved lung injury in ALI mice. The upregulation of genes essential for glutathione (GSH) metabolism induced by lipopolysaccharide (LPS) was inhibited by Tempol. In addition, nuclear factor-related factor 2 (Nrf2) is activated by Tempol therapy to regulate the de novo synthesis pathway of GSH, thereby alleviating LPS-induced lung epithelial cell damage. The results showed that Tempol alleviated ALI by activating the Nrf2 pathway to inhibit oxidative stress and ferroptosis in lung epithelial cells. In conclusion, this study demonstrates that Tempol alleviates ALI by inhibiting ferroptosis in lung epithelial cells through the effect of Nrf2 on GSH synthesis.

Quantifying cerebral blood flow changes using arterial spin labeling: A comparative study of idiopathic rapid eye movement sleep behavior disorder and Parkinson's disease.

INTRODUCTION: Idiopathic rapid eye movement sleep behavior disorder (iRBD) and Parkinson's disease (PD) have been found to have changes in cerebral perfusion and overlap of some of the lesioned brain areas. However, a consensus regarding the specific location and diagnostic significance of these cerebral blood perfusion alternations remains elusive in both iRBD and PD. The present study evaluated the patterns of cerebral blood flow changes in iRBD and PD. MATERIAL AND METHODS: A total of 59 right-handed subjects were enrolled, including 15 patients with iRBD, 20 patients with PD, and 24 healthy controls (HC). They were randomly divided into groups at a ratio of 4 to 1 for training and testing. A PASL sequence was employed to obtain quantitative cerebral blood flow (CBF) maps. The CBF values were calculated from these acquired maps. In addition, AutoGluon was employed to construct a classifier for CBF features selection and classification. An independent t-test was performed for CBF variations, with age and sex as nuisance variables. The performance of the feature was evaluated using receiver operating characteristic (ROC) curves. A significance level of P < 0.05 was considered significant. CBF in several brain regions, including the left median cingulate and paracingulate gyri and the right middle occipital gyrus (MOG), showed significant differences between PD and HC, demonstrating good classification performance. The combined model that integrates all features achieved even higher performance with an AUC of 0.9380. Additionally, CBF values in multiple brain regions, including the right MOG and the left angular gyrus, displayed significant differences between PD and iRBD. Particularly, CBF values in the left angular gyrus exhibited good performance in classifying PD and iRBD. The combined model achieved improved performance, with an AUC of 0.8533. No significant differences were found in brain regions when comparing CBF values between iRBD and HC subjects. CONCLUSIONS: ASL-based quantitative CBF change features can offer reliable biomarkers to assist in the diagnosis of PD. Regarding the characteristic of CBF in the right MOG, it is anticipated to serve as an imaging biomarker for predicting the progression of iRBD to PD.

Arterial Spin-Labeling and DSC Perfusion Metrics Improve Agreement in Neuroradiologists' Clinical Interpretations of Posttreatment High-Grade Glioma Surveillance MR Imaging-An Institutional Experience.

BACKGROUND AND PURPOSE: MR perfusion has shown value in the evaluation of posttreatment high-grade gliomas, but few studies have shown its impact on the consistency and confidence of neuroradiologists' interpretation in routine clinical practice. We evaluated the impact of adding MR perfusion metrics to conventional contrast-enhanced MR imaging in posttreatment high-grade glioma surveillance imaging. MATERIALS AND METHODS: This retrospective study included 45 adults with high-grade gliomas who had posttreatment perfusion MR imaging. Four neuroradiologists assigned Brain Tumor Reporting and Data System scores for each examination on the basis of the interpretation of contrast-enhanced MR imaging and then after the addition of arterial spin-labeling-CBF, DSC-relative CBV, and DSC-fractional tumor burden. Interrater agreement and rater agreement with a multidisciplinary consensus group were assessed with κ statistics. Raters used a 5-point Likert scale to report confidence scores. The frequency of clinically meaningful score changes resulting from the addition of each perfusion metric was determined. RESULTS: Interrater agreement was moderate for contrast-enhanced MR imaging alone (κ = 0.63) and higher with perfusion metrics (arterial spin-labeling-CBF, κ = 0.67; DSC-relative CBV, κ = 0.66; DSC-fractional tumor burden, κ = 0.70). Agreement between raters and consensus was highest with DSC-fractional tumor burden (κ = 0.66-0.80). Confidence scores were highest with DSC-fractional tumor burden. Across all raters, the addition of perfusion resulted in clinically meaningful interpretation changes in 2%-20% of patients compared with contrast-enhanced MR imaging alone. CONCLUSIONS: Adding perfusion to contrast-enhanced MR imaging improved interrater agreement, rater agreement with consensus, and rater confidence in the interpretation of posttreatment high-grade glioma MR imaging, with the highest agreement and confidence scores seen with DSC-fractional tumor burden. Perfusion MR imaging also resulted in interpretation changes that could change therapeutic management in up to 20% of patients.

The effect of arterial spin labeling MR angiography (ASL-MRA) in visualizing the branches of external carotid artery.

This study aimed to assess the performance of arterial-spin labeling MRA (ASL-MRA) for visualizing the external carotid artery (ECA) branches in comparison with time-of-flight MRA (TOF-MRA) and CT angiography (CTA). We retrospectively selected 31 consecutive patients, who underwent both MRAs and CTA, prior to the intra-arterial chemoradiotherapy (IACRT) for head and neck cancer. Four patients underwent IACRT bilaterally, so we analyzed 35 ECAs. Pseudo-continuous, three-dimensional ASL using a turbo field echo sequence was acquired. For the TOF-MRA and CTA, clinically used parameters were applied. Two observers evaluated each ECA branch with reference to the angiogram at the IACRT, using five-point scale, in consensus. Friedman test for multiple comparisons was applied. ASL-MRA and CTA better visualized the superior thyroid, lingual, facial, submental, transverse facial, and internal maxillary arteries (IMAs) better than TOF-MRA (p < 0.05). In addition, CTA was superior to ASL-MRA in visualizing only submental artery among these arteries (p = 0.0005). Alternatively, the ASL-MRA was superior for visualizing the middle meningeal artery (MMA) and IMA, compared to the CTA (p = 0.0001 and 0.0007, respectively). ASL-MRA was superior to the TOF-MRA and similar to the CTA in visualizing most of ECA branches. Furthermore, ASL-MRA can better visualize the periphery of MMA and IMA than CTA.

Insights into single-timepoint ASL hemodynamics: what visual assessment and spatial coefficient of variation can tell.

PURPOSE: Arterial spin labeling (ASL) represents a noninvasive perfusion biomarker, and, in the study of nonvascular disease, the use of the single-timepoint ASL technique is recommended. However, the obtained cerebral blood flow (CBF) maps may be highly influenced by delayed arterial transit time (ATT). Our aim was to assess the complexity of hemodynamic information of single-timepoint CBF maps using a new visual scale and comparing it with an ATT proxy, the "coefficient of spatial variation" (sCoV). MATERIAL AND METHODS: Individual CBF maps were estimated in a memory clinic population (mild cognitive impairment, dementia and cognitively unimpaired controls) and classified into four levels of delayed perfusion based on a visual rating scale. Calculated measures included global/regional sCoVs and common CBF statistics, as mean, median and standard deviation. One-way ANOVA was performed to compare these measures across the four groups of delayed perfusion. Spearman correlation was used to study the association of global sCoV with clinical data and CBF statistics. RESULTS: One hundred and forty-four participants (72 ± 7 years, 53% women) were included in the study. The proportion of maps with none, mild, moderate, and severe delayed perfusion was 15, 20, 37, and 28%, respectively. SCoV demonstrated a significant increase (p < 0.05) across the four groups, except when comparing none vs mild delayed perfusion groups (pBonf > 0.05). Global sCoV values, as an ATT proxy, ranged from 67 ± 4% (none) to 121 ± 24% (severe delayed) and were significantly associated with age and CBF statistics (p < 0.05). CONCLUSION: The impact of ATT delay in single-time CBF maps requires the use of a visual scale or sCoV in clinical or research settings.

Designing surface exposed sites on Bacillus subtilis lipase A for spin-labeling and hydration studies.

Spin-labeling with electron paramagnetic resonance spectroscopy (EPR) is a facile method for interrogating macromolecular flexibility, conformational changes, accessibility, and hydration. Within we present a computationally based approach for the rational selection of reporter sites in Bacillus subtilis lipase A (BSLA) for substitution to cysteine residues with subsequent modification with a spin-label that are expected to not significantly perturb the wild-type structure, dynamics, or enzymatic function. Experimental circular dichroism spectroscopy, Michaelis-Menten kinetic parameters and EPR spectroscopy data validate the success of this approach to computationally select reporter sites for future magnetic resonance investigations of hydration and hydration changes induced by polymer conjugation, tethering, immobilization, or amino acid substitution in BSLA. Analysis of molecular dynamic simulations of the impact of substitutions on the secondary structure agree well with experimental findings. We propose that this computationally guided approach for choosing spin-labeled EPR reporter sites, which evaluates relative surface accessibility coupled with hydrogen bonding occupancy of amino acids to the catalytic pocket via atomistic simulations, should be readily transferable to other macromolecular systems of interest including selecting sites for paramagnetic relaxation enhancement NMR studies, other spin-labeling EPR studies or any method requiring a tagging method where it is desirable to not alter enzyme stability or activity.

Assessing the effects of tempol on renal fibrosis, inflammation, and oxidative stress in a high-salt diet combined with 5/6 nephrectomy rat model: utilizing oxidized albumin as a biomarker.

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of chronic kidney disease (CKD), prompting the exploration of antioxidants as a potential therapeutic avenue for mitigating disease progression. This study aims to investigate the beneficial impact of Tempol on the progression of CKD in a rat model utilizing oxidized albumin as a biomarker. METHODS: After four weeks of treatment, metabolic parameters, including body weight, left ventricle residual weight, kidney weight, urine volume, and water and food intake, were measured. Systolic blood pressure, urinary protein, oxidized albumin level, serum creatinine (Scr), blood urea nitrogen (BUN), 8-OHdG, TGF-ß1, and micro-albumin were also assessed. Renal fibrosis was evaluated through histological and biochemical assays. P65-NF-κB was quantified using an immunofluorescence test, while Smad3, P65-NF-κB, and Collagen I were measured using western blot. TNF-α, IL-6, MCP-1, TGF-ß1, Smad3, and P65-NF-κB were analyzed by RT-qPCR. RESULTS: Rats in the high-salt diet group exhibited impaired renal function, characterized by elevated levels of blood urea nitrogen, serum creatinine, 8-OHdG, urine albumin, and tubulointerstitial damage, along with reduced body weight. However, these effects were significantly ameliorated by Tempol administration. In the high-salt diet group, blood pressure, urinary protein, and oxidized albumin levels were notably higher compared to the normal diet group, but Tempol administration in the treatment group reversed these effects. Rats in the high-salt diet group also displayed increased levels of proinflammatory factors (TNF-α, IL-6, MCP1) and profibrotic factors (NF-κB activation, Collagen I), elevated expression of NADPH oxidation-related subunits (P65), and activation of the TGF-ß1/Smad3 signaling pathway. Tempol treatment inhibited NF-κB-mediated inflammation and TGF-ß1/Smad3-induced renal fibrosis signaling pathway activation. CONCLUSION: These findings suggest that Tempol may hold therapeutic potential for preventing and treating rats undergoing 5/6 nephrectomy. Further research is warranted to elucidate the mechanisms underlying Tempol's protective effects and its potential clinical applications. Besides, there is a discernible positive relationship between oxidized albumin and other biomarkers, such as 8-OHG, urinary protein levels, mALB, Scr, BUN, and TGF-ß1 in a High-salt diet combined with 5/6 nephrectomy rat model. These findings suggest the potential utility of oxidized albumin as a sensitive indicator for oxidative stress assessment.

Extending the Range of Distances Accessible by 19F Electron-Nuclear Double Resonance in Proteins Using High-Spin Gd(III) Labels.

Fluorine electron-nuclear double resonance (19F ENDOR) has recently emerged as a valuable tool in structural biology for distance determination between F atoms and a paramagnetic center, either intrinsic or conjugated to a biomolecule via spin labeling. Such measurements allow access to distances too short to be measured by double electron-electron resonance (DEER). To further extend the accessible distance range, we exploit the high-spin properties of Gd(III) and focus on transitions other than the central transition (|-1/2⟩ ↔ |+1/2⟩), that become more populated at high magnetic fields and low temperatures. This increases the spectral resolution up to ca. 7 times, thus raising the long-distance limit of 19F ENDOR almost 2-fold. We first demonstrate this on a model fluorine-containing Gd(III) complex with a well-resolved 19F spectrum in conventional central transition measurements and show quantitative agreement between the experimental spectra and theoretical predictions. We then validate our approach on two proteins labeled with 19F and Gd(III), in which the Gd-F distance is too long to produce a well-resolved 19F ENDOR doublet when measured at the central transition. By focusing on the |-5/2⟩ ↔ |-3/2⟩ and |-7/2⟩ ↔ |-5/2⟩ EPR transitions, a resolution enhancement of 4.5- and 7-fold was obtained, respectively. We also present data analysis strategies to handle contributions of different electron spin manifolds to the ENDOR spectrum. Our new extended 19F ENDOR approach may be applicable to Gd-F distances as large as 20 Å, widening the current ENDOR distance window.

Exploring the dynamics and structure of PpiB in living Escherichia coli cells using electron paramagnetic resonance spectroscopy.

The combined effects of the cellular environment on proteins led to the definition of a fifth level of protein structural organization termed quinary structure. To explore the implication of potential quinary structure for globular proteins, we studied the dynamics and conformations of Escherichia coli (E. coli) peptidyl-prolyl cis/trans isomerase B (PpiB) in E. coli cells. PpiB plays a major role in maturation and regulation of folded proteins by catalyzing the cis/trans isomerization of the proline imidic peptide bond. We applied electron paramagnetic resonance (EPR) techniques, utilizing both Gadolinium (Gd(III)) and nitroxide spin labels. In addition to using standard spin labeling approaches with genetically engineered cysteines, we incorporated an unnatural amino acid to achieve Gd(III)-nitroxide orthogonal labeling. We probed PpiB's residue-specific dynamics by X-band continuous wave EPR at ambient temperatures and its structure by double electron-electron resonance (DEER) on frozen samples. PpiB was delivered to E. coli cells by electroporation. We report a significant decrease in the dynamics induced by the cellular environment for two chosen labeling positions. These changes could not be reproduced by adding crowding agents and cell extracts. Concomitantly, we report a broadening of the distance distribution in E. coli, determined by Gd(III)-Gd(III) DEER measurements, as compared with solution and human HeLa cells. This suggests an increase in the number of PpiB conformations present in E. coli cells, possibly due to interactions with other cell components, which also contributes to the reduction in mobility and suggests the presence of a quinary structure.

Effect of Selected Antioxidants on the In Vitro Aging of Human Fibroblasts.

The modification of the replicative lifespan (RLS) of fibroblasts is of interest both from a knowledge point of view and for the attenuation of skin aging. The effect of six antioxidants at a concentration of 1 µM on the replicative lifespan of human dermal fibroblasts was studied. The nitroxide 4-hydroxy-TEMPO (TEMPOL), ergothioneine, and Trolox extended the replicative lifespan (RLS) (40 ± 1 population doublings (PD)) by 7 ± 2, 4 ± 1, and 3 ± 1 PD and lowered the expression of p21 at late passages. Coumaric acid, curcumin and resveratrol did not affect the RLS . The level of reactive oxygen species (ROS) was decreased or not affected by the antioxidants although TEMPOL and coumaric acid decreased the level of glutathione. Only ergothioneine and resveratrol decreased the level of protein carbonylation. The antioxidants that could prolong the RLS elevated the mitochondrial membrane potential. Protecting the activity of mitochondria seems to be important for maintaining the replicative capacity of fibroblasts.

Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl.

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT.