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Alzheimer’s disease (AD) develops into dementia over a period of several years, during which subjective cognitive impairment (SCI) and mild cognitive impairment (MCI) can be used as intermediary diagnoses of increasing severity. Chronic neuroinflammation resulting from insufficient resolution is involved in the pathogenesis of AD and is associated with cognitive impairment. Specialized pro-resolving lipid mediators (LMs) that promote the resolution of inflammation may be valuable markers in AD diagnosis and as therapeutic targets. Liquid chromatography–tandem mass spectrometry was used to analyze pro-resolving and pro-inflammatory LMs in cerebrospinal fluid (CSF) from patients with cognitive impairment ranging from subjective impairment to a diagnosis of AD and correlated to cognition, CSF tau, and β-amyloid. Resolvin (Rv) D4, RvD1, neuroprotectin D1 (NPD1), maresin 1 (MaR1), and RvE4 were lower in AD and/or MCI compared to SCI. The pro-inflammatory LTB4 and 15-HETE were higher in AD and MCI, respectively, while PGD2, PGE2, and PGF2a were decreased in AD, compared to SCI. RvD4 was also negatively correlated to AD tangle biomarkers, and positive correlations to cognitive test scores were observed for both pro-resolving LMs and their precursor fatty acids. In this exploratory study of the lipidome in CSF of AD, MCI, and SCI, the results indicate a shift in the LM profile from pro-resolving to pro-inflammatory in progression to AD, suggesting that it may be of use as a biomarker when followed by confirmation by replication studies. Graphical Abstract

The safe and effective management of pain is a critical healthcare and societal need. The potential for misuse and addiction associated with opioids, nephrotoxicity, and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use, as well as acute liver injury from paracetamol (ApAP) overdose, are unresolved challenges. To address them, we developed a non-opioid and non-hepatotoxic small molecule, SRP-001. Compared to ApAP, SRP-001 is not hepatotoxic as it does not produce N-acetyl-p-benzoquinone-imine (NAPQI) and maintains hepatic tight junction integrity at high doses. SRP-001 has comparable analgesia in pain models, including the complete Freund’s adjuvant (CFA) inflammatory von Frey. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception area, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways, including the endocannabinoid, mechanical nociception, and fatty acid amide hydrolase (FAAH) pathways. Both regulate the expression of key genes encoding FAAH, 2-AG, CNR1, CNR2, TRPV4, and voltage-gated Ca2+ channel. Interim Phase 1 trial results demonstrate SRP-001’s safety, tolerability, and favorable pharmacokinetics (NCT05484414). Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.

Little is known of the lipid anti-inflammatory mediators, docosanoids, in intracerebral hemorrhage (ICH). We aim to characterize the abundance of the docosanoid, Neuroprotectin D1 (NPD1), in ICH patients. Blood samples (whole blood in PAXgene-blood-RNA tubes and plasma) were collected from consecutive patients with acute spontaneous ICH within 48 h of admission. A liquid-liquid lipid extraction was used for liquid chromatography-mass spectrometry (LC-MS/MS) and analyzed using MassLynx Mass Spectrometry Software with results normalized to internal standards. RNA was extracted from PAXgene-blood-RNA tubes for 15-LOX-1 gene expression, a critical enzyme in NPD1 synthesis. Demographic and clinical data were collected. Outcome measures included 90-day modified-rankin-score. Sixteen patients were included in the study with a mean age of 62.5years (SD13.5). Three abundant isomers were detected and analyzed - NPD1, PDX, and an uncharacterized isomer designated as NPD1-C. NPD1 levels were higher in patients with 90-day MRS 0-3 (49.63pg/mL SD43.78 vs. 1.88pg/mL SD1.7 p = 0.0012). ROC-AUC analysis showed an NPD1 cutoff of 2.9pg/mL differentiated 90-day MRS 0-3 (sensitivity 100%, specificity 88.89%, AUC 0.98 p = 0.0002). A Spearman correlation demonstrated an inverse relationship with NPD1 and 90-day MRS (rho -7.392 p = 0.0011). 15-LOX-1 gene was almost undetectable in patients with MRS 4-6. Though not significant, NPD1 levels were higher in patients65 years, ICH volume30 ml, and non-whites. NPD1 was abundant and significantly higher in ICH patients with MRS 0-3.15-LOX-1 was significantly under-expressed in patients with MRS 4-6. Early synthesis and abundance of NPD1 is likely an important protective mediator in ICH pathophysiology.

Sustained brain chronic inflammation in Alzheimer’s disease (AD) includes glial cell activation, an increase in cytokines and chemokines, and lipid mediators (LMs), concomitant with decreased pro-homeostatic mediators. The inflammatory response at the onset of pathology engages activation of pro-resolving, pro-homeostatic LMs followed by a gradual decrease. We used an APP knock-in (App KI) AD mouse that accumulates β-amyloid (Aβ) and presents cognitive deficits (at 2 and 6 months of age, respectively) to investigate LMs, their precursors, biosynthetic enzymes and receptors, glial activation, and inflammatory proteins in the cerebral cortex and hippocampus at 2-, 4-, 8- and 18-month-old in comparison with wild-type (WT) mice. We used LC-mass-spectrometry and MALDI molecular imaging to analyze LMs and phospholipids, and immunochemistry for proteins. Our results revealed an age-specific lipid and cytokine profile, and glial activation in the App KI mice. Despite an early onset of Aβ pathology, pro-inflammatory and pro-resolving LMs were prominently increased only in the oldest age group. Furthermore, the LM biosynthetic enzymes increased, and their receptor expression decreased in the aged App KI mice. Arachidonic acid (AA)-containing phospholipid molecular species were elevated, correlating with decreased cPLA2 activity. MALDI molecular imaging depicted differential distribution of phospholipids according to genotype in hippocampal layers. Brain histology disclosed increased microglia proliferation starting from young age in the App KI mice, while astrocyte numbers were enhanced in older ages. Our results demonstrate that the brain lipidome is modified preferentially during aging as compared to amyloid pathology in the model studied here. However, alterations in phospholipids signal early pathological changes in membrane composition.

International audience; Spatial changes of fatty acids in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids, EPA-containing triglycerides, DHA-containing phospholipids, or DHA-containing triglycerides. Qualitative changes in fatty acid composition of plasma, erythrocytes and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in retina were determined by liquid chromatography coupled to tandem mass spectrometry and MALDI (Matrix Assisted Laser Desorption Ionization) coupled to mass spectrometry imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that phospholipids as well as triglycerides are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain polyunsaturated fatty acids (VLC-PUFAs, more than 28 carbons) following phospholipid-EPA and triglyceride-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of fatty acids in the rat retina.

Objective: Ischemic stroke induces multiple pathophysiological processes involving oxidative stress, loss of homeostasis, excitotoxicity, disruption of the blood-brain barrier and neurovascular unit, contributing to neuronal death. In the early phases following the onset of ischemic damage, neuroinflammation contributes to tissue damage and enlargement of the infarct area. Elovanoids (ELVs) are a class of pro-homeostatic lipid mediators that are derivatives of very long-chain polyunsaturated fatty acids (VLC-PUFAs). VLC-PUFAs are synthesized by ELOVL4 an enzyme primarily expressed in neurons. ELVs have been shown to reduce infarct volume and promote cell survival when administered IV following 2h of middle cerebral artery occlusion (MCAo). This study aimed to test the neuroprotective efficacy of these VLC-PUFA ELV precursors when administered intranasally (IN) in a model of experimental ischemic stroke. Methods: Male Sprague-Dawley rats received 2h of the middle cerebral artery occlusion (MCAo). Treatment with C-36:6, C-38:6 (1μg/μl, 20 μg) or vehicle (saline + ethanol) was administered at 1, 24, and 48h after onset MCAo. Behavior was evaluated using a composite neurological battery; a grading scale of 0-12 was employed (normal score=0, maximal deficit=12). Behavior testing was performed on days 1, 2, 3, and 7, followed by ex vivo MRI for lesion volumes and edema (T2WI) and immunohistochemistry on day 7. Results: Following drug administration, no behavioral side effects were observed. Neurologic function was improved on days 1, 3, and 7 by 24%, 37%, and 28% in animals receiving C-36:6 and by 26%, 26%, and 29% in the group receiving C-38:6. Total, core, and penumbra lesion volumes, computed using T2WI, were reduced with C-36:6 by 60%, 68%, and 53%, and C-38:6 by 35%, 43% 25% vs vehicle. Total, cortical and subcortical infarct areas and volumes were reduced by treatments with C-36:6 (by 42% 48% and 34%) and C-38:6 (by 28% 31% and 20%) vs vehicle. Conclusion: IN delivered ELV precursors demonstrated neuroprotective efficacy improving behavior and reducing infarct volume compared to vehicle. We are exploring neuroprotective molecular mechanisms exerted by intranasally delivered VLC-PUFAs as an experimental therapeutic approach.

Background: Alzheimer's disease (AD) develops into dementia over a period of several years, during which subjective cognitive impairment (SCI) and mild cognitive impairment (MCI) are used as intermediary diagnoses of increasing severity. Chronic neuroinflammation resulting from insufficient resolution is involved in the pathogenesis of AD and is associated with cognitive impairment. Specialized pro-resolving lipid mediators (LMs) that promote the resolution of inflammation may be valuable markers in AD diagnosis and as therapeutic targets.Methods: Liquid chromatography-tandem mass spectrometry was used to analyze pro-resolving and pro-inflammatory LMs in cerebrospinal fluid (CSF) from patients with cognitive impairment ranging from subjective impairment to a diagnosis of AD, and correlated to cognition, CSF tau and β-amyloid (Aβ), and an inflammation biomarker (YKL-40). Results: RvD4, neuroprotectin D1, MaR1, and RvE4 were lower in AD and/or MCI compared to SCI. The pro-inflammatory LTB4 and 15-HETE were higher in AD and MCI, respectively, while PGD2 and PGE2 were decreased in AD, compared to SCI. RvD4 was also negatively correlated to AD tangle biomarkers. Many differences were dependent on gender.Conclusion: In this exploratory study of the lipidome in CSF of AD, MCI and SCI, the results indicate a gender-dependent shift in the LM profile from pro-resolving to pro-inflammatory in progression to AD, suggesting that it may be of use as a biomarker when followed by confirmation by replication studies.

Sustained microglial activation and increased pro-inflammatory signalling cause chronic inflammation and neuronal damage in Alzheimer's disease (AD). Resolution of inflammation follows neutralization of pathogens and is a response to limit damage and promote healing, mediated by pro-resolving lipid mediators (LMs). Since resolution is impaired in AD brains, we decided to test if intranasal administration of pro-resolving LMs in the App

Significance This study uncovers biosynthetic pathway insufficiencies of prohomeostatic/neuroprotective mediators neuroprotectin D1 and elovanoids in the retina during early pathogenesis in transgenic Alzheimer’s disease 5xFAD mouse. These changes correlate with photoreceptor cell functional impairments preceding their loss. Amyloid beta (Aβ) peptide accumulates in drusen in AMD. Thus, injecting oligomeric Aβ in wild-type mice behind the retina leads to photoreceptor cell degeneration and transcriptional disruptions including upregulation of a senescence program and of senescence-associated secretory phenotype (SASP). Similar changes take place in human retinal pigment epithelium cells in culture. Novel lipid mediators, the elovanoids, restore Aβ-peptide-induced gene expression changes and SASP secretome and, in turn, protect these cells. This study opens avenues of potential therapeutic exploration of elovanoids for AMD., The onset of neurodegenerative diseases activates inflammation that leads to progressive neuronal cell death and impairments in cognition (Alzheimer’s disease) and sight (age-related macular degeneration [AMD]). How neuroinflammation can be counteracted is not known. In AMD, amyloid β-peptide (Aβ) accumulates in subretinal drusen. In the 5xFAD retina, we found early functional deficiencies (ERG) without photoreceptor cell (PRC) death and identified early insufficiency in biosynthetic pathways of prohomeostatic/neuroprotective mediators neuroprotectin D1 (NPD1) and elovanoids (ELVs). To mimic an inflammatory milieu in wild-type mouse, we triggered retinal pigment epithelium (RPE) damage/PRC death by subretinally injected oligomeric β-amyloid (OAβ) and observed that ELVs administration counteracted their effects, protecting these cells. In addition, ELVs prevented OAβ-induced changes in gene expression engaged in senescence, inflammation, autophagy, extracellular matrix remodeling, and AMD. Moreover, as OAβ targets the RPE, we used primary human RPE cell cultures and demonstrated that OAβ caused cell damage, while ELVs protected and restored gene expression as in mouse. Our data show OAβ activates senescence as reflected by enhanced expression of p16INK4a, MMP1, p53, p21, p27, and Il-6, and of senescence-associated phenotype secretome, followed by RPE and PRC demise, and that ELVs 32 and 34 blunt these events and elicit protection. In addition, ELVs counteracted OAβ-induced expression of genes engaged in AMD, autophagy, and extracellular matrix remodeling. Overall, our data uncovered that ELVs downplay OAβ-senescence program induction and inflammatory transcriptional events and protect RPE cells and PRC, and therefore have potential as a possible therapeutic avenue for AMD.

Innervation sustains cornea integrity. Pigment epithelium‐derived factor (PEDF) plus docosahexaenoic acid (DHA) regenerated damaged nerves by stimulating the synthesis of a new stereoisomer of Resolvin D6 (RvD6si). Here, we resolved the structure of this lipid isolated from mouse tears after injured corneas were treated with PEDF + DHA. RvD6si synthesis was inhibited by fluvoxamine, a cytochrome P450 inhibitor, but not by 15‐ or 5‐LOX inhibitors, suggesting that the 4‐ and 17‐hydroxy of DHA have an R R‐ or S R‐configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si1‐4 from tears were resolved. The R R‐RvD6 standard eluted as a single peak with RvD61 while pure S R‐RvD6 eluted with RvD63. The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that R R‐RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. R R‐RvD6 stimulates Rictor and hepatocyte growth factor (hgf) genes specifically as upstream regulators and a gene network involved in axon growth and suppression of neuropathic pain, indicating a novel function of this lipid mediator to maintain cornea integrity and homeostasis after injury.

Cancer cells exhibit dysregulation of critical genes including those involved in lipid biosynthesis, with subsequent defects in metabolism. Here, we show that ELOngation of Very Long chain fatty acids protein 4 (ELOVL4), a rate-limiting enzyme in the biosynthesis of very-long polyunsaturated fatty acids (n-3, ≥28 C), is expressed and transcriptionally repressed by the oncogene MYCN in neuroblastoma cells. In keeping, ELOVL4 positively regulates neuronal differentiation and lipids droplets accumulation in neuroblastoma cells. At the molecular level we found that MYCN binds to the promoter of ELOVL4 in close proximity to the histone deacetylases HDAC1, HDAC2, and the transcription factor Sp1 that can cooperate in the repression of ELOVL4 expression. Accordingly, in vitro differentiation results in an increase of fatty acid with 34 carbons with 6 double bonds (FA34:6); and when MYCN is silenced, FA34:6 metabolite is increased compared with the scrambled. In addition, analysis of large neuroblastoma datasets revealed that ELOVL4 expression is highly expressed in localized clinical stages 1 and 2, and low in high-risk stages 3 and 4. More importantly, high expression of ELOVL4 stratifies a subsets of neuroblastoma patients with good prognosis. Indeed, ELOVL4 expression is a marker of better overall clinical survival also in MYCN not amplified patients and in those with neuroblastoma-associated mutations. In summary, our findings indicate that MYCN, by repressing the expression of ELOVL4 and lipid metabolism, contributes to the progression of neuroblastoma.

The main risk factor for primary open-angle glaucoma (POAG) is increased intraocular pressure (IOP). It is of interest that about half of the patients have an IOP within the normal range (normal-tension glaucoma, NTG). Additionally, there is a group of patients with a high IOP but no glaucomatous neurodegeneration (ocular hypertension, OHT). Therefore, risk factors other than IOP are involved in the pathogenesis of glaucoma. Since the retina has a very high oxygen-demand, decreased autoregulation and a fluctuating oxygen supply to the retina have been linked to glaucomatous neurodegeneration. To assess the significance of these mechanisms, we have utilized a human experimental model, in which we stress participants with a fluctuating oxygen supply. Levels of oxidative stress molecules, antioxidants, and lipid mediators were measured in the plasma. Patients with NTG, OHT, and control subjects were found to have similar levels of oxidative stress markers. In contrast, patients with OHT had a higher level of total antioxidant capacity (TAC) and pro-homeostatic lipid mediators. Thus, we suggest that OHT patients manage fluctuating oxygen levels more efficiently and, thus, are less susceptible to glaucomatous neurodegenerations, due to enhanced systemic antioxidant protection.

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA,n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

14S,22-Dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (maresin-L1) and 14R,22-dihydroxy-docosa-4Z,7Z,10Z, 12E,16Z,19Z-hexaenoic acid (maresin-L2) were chemically synthesized. They were identical to activated macrophage-produced counterparts and their total synthesis was highly stereoselective, as revealed by chiral LC-UV-MS/MS analysis. The synthesis involved the following steps: (1) kinetic resolution of a racemic allylic alcohol by the asymmetric epoxidation; (2) transformation of the epoxy alcohol to γ-hydroxyenal derivative; and (3) the Wittig reaction to furnish the Z-olefin.

Although acetaminophen (ApAP) is one of the most commonly used medicines worldwide, hepatotoxicity is a risk with overdose or in patients with compromised liver function. ApAP overdose is the most common cause of acute fulminant hepatic failure. Oxidation of ApAP to N-acetyl-p-benzoquinone imine (NAPQI) is the mechanism for hepatotoxicity. 1 is a non-hepatotoxic, metabolically unstable lipophilic ApAP analog that is not antipyretic. The newly synthesized 3 is a non-hepatotoxic ApAP analog that is stable, lipophilic, and retains analgesia and antipyresis. Intraperitoneal or po administration of the new chemical entities (NCEs), 3b and 3r, in concentrations equal to a toxic dose of ApAP did not result in the formation of NAPQI. Unlike livers from NCE-treated mice, the livers from ApAP-treated mice demonstrated large amounts of nitrotyrosine, a marker of mitochondrial free radical formation, and loss of hepatic tight junction integrity. Given the widespread use of ApAP, hepatotoxicity risk with overuse, and the ongoing opioid epidemic, these NCEs represent a novel, non-narcotic therapeutic pipeline., Graphical abstract Image 1, Highlights • We describe a novel class of diethylaminocarbonyl-2-benzenesulfamide analogs to acetaminophen/paracetamol. • Compounds, 3b and 3r, lack the hepatotoxicity present in acetaminophen/paracetamol. • 3b and 3r maintain analgesia and antipyresis in in vivo models.

The high-density corneal innervation plays a pivotal role in sustaining the integrity of the ocular surface. We have previously demonstrated that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) promotes corneal nerve regeneration; here, we report the mechanism involved and the discovery of a stereospecific Resolvin D6-isomer (RvD6si) that drives the process. RvD6si promotes corneal wound healing and functional recovery by restoring corneal innervation after injury. RvD6si applied to the eye surface elicits a specific transcriptome signature in the trigeminal ganglion (TG) that includes Rictor, the rapamycin-insensitive complex-2 of mTOR (mTORC2), and genes involved in axon growth, whereas genes related to neuropathic pain are decreased. As a result, attenuation of ocular neuropathic pain and dry eye will take place. Thus, RvD6si opens up new therapeutic avenues for pathologies that affect corneal innervation.

Purpose Low levels of the long chain polyunsaturated fatty acid (LCPUFA) docosahexaenoic acid (DHA) have been implicated in retinopathy of prematurity (ROP). However, oral DHA suffers from poor palatability and is associated with increased bleeding in premature infants. We asked whether oral administration of the neutraceutical arginine-glutamine (Arg-Glu) could increase retinal DHA and improve outcomes in a mouse model of oxygen-induced retinopathy (OIR). Methods Postnatal day 7 (P7) pups were maintained at 75% oxygen for 5 days and then returned to room air on P12. Pups were gavaged twice daily with Arg-Gln or vehicle from P12 to P17 and eyes were harvested for analysis on P17. Vaso-obliteration and vascular density were assessed on retinal flat mounts and preretinal neovascularization was assessed on retinal cross sections. Retinas were used for measurement of DHA and 10,17S-docosatriene (neuroprotectin D1, NPD1), a key DHA-derived lipid, and for analysis by reverse-phase protein array (RPPA). Results With Arg-Gln treatment, retinal DHA and NPD1 levels were increased in OIR pups. Arg-Gln reduced preretinal neovascularization by 39 ± 6% (P < 0.05) relative to vehicle control. This was accompanied by a restoration of vascular density of the retina in the pups treated with Arg-Gln (73.0 ± 3.0%) compared to vehicle (53.1 ± 3.4%; P < 0.05). Arg-Gln dipeptide restored OIR-induced signaling changes toward normoxia and was associated with normalization of insulin-like growth factor receptor 1 signaling and reduction of apoptosis and an increase in anti-apoptosis proteins. Conclusions Arg-Gln may serve as a safer and easily tolerated nutraceutical agent for prevention or treatment of ROP.

Synthesis of 14S,21R- and 14S,21S-dihydroxy-DHA (diHDHA) among the four possible stereoisomers of 14,21-diHDHA was studied. Methyl (R)-lactate (>97% ee), selected as a C20–C22 fragment (DHA numbering), was converted to the C17–C22 phosphonium salt, which was subjected to a Wittig reaction with racemic C16-aldehyde of the C12–C16 part with the TMS and TBS-oxy groups at C12 and C14, yielding the C12–C22 derivative with 14R/S and 21R chirality. Kinetic resolution using Sharpless asymmetric epoxidation of the TBS-deprotected allylic alcohol with l-(+)-DIPT/Ti(O-i-Pr)(4) afforded 14S-epoxy alcohol and 14R-allylic alcohol with >99% diastereomeric excess (de) for both. The CN group was introduced to the epoxy alcohol by reaction with Et(2)AlCN. The 14R-allylic alcohol was also converted to the nitrile via Mitsunobu inversion. Reduction of the nitrile with DIBAL afforded the key aldehyde corresponding to the C11–C22 moiety. The Wittig reaction of this aldehyde with a phosphonium salt of the remaining C1–C10 part followed by functional group manipulation gave 14S,21R-diHDHA. Similarly, ethyl (S)-lactate (>99% ee) was converted to 14S,21S-diHDHA. The chiral LC-UV-MS/MS analysis demonstrated that each of these two 14,21-diHDHAs synthesized using the presented total organic synthesis was highly stereoselective and identical to the macrophage-produced counterpart.

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are necessary for functional cell integrity. Preconditioning (PC), as we define it, is an acquired protection or resilience by a cell, tissue, or organ to a lethal stimulus enabled by a previous sublethal stressor or stimulus. In this study, we provide evidence that the omega-3 fatty acid docosahexaenoic acid (DHA) and its derivatives, the docosanoids 17-hydroxy docosahexaenoic acid (17-HDHA) and neuroprotectin D1 (NPD1), facilitate cell survival in both in vitro and in vivo models of retinal PC. We also demonstrate that PC requires the enzyme 15-lipoxygenase-1 (15-LOX-1), which synthesizes 17-HDHA and NPD1, and that this is specific to docosanoid signaling despite the concomitant release of the omega-6 arachidonic acid and eicosanoid synthesis. These findings advocate that DHA and docosanoids are protective enablers of PC in photoreceptor and retinal pigment epithelial cells. Electronic supplementary material The online version of this article (10.1007/s10571-017-0565-2) contains supplementary material, which is available to authorized users.

Objectives: Ischemic stroke triggers a pattern of cellular and molecular disturbances that include lipid peroxidation, uncompensated oxidative stress, and neuronal injury. Recently, we have uncovered and characterized a novel neuroprotective signaling mechanism, which involves the activation of the biosynthesis of a family of lipid mediators in the brain made from omega-3 very-long-chain polyunsaturated fatty acids, that we named Elovanoids (ELVs). The present study evaluated ELVs, made of 32 and 34 C atoms in length (ELV-N32 and ELV-N34) and their potential mechanisms of action in cerebral ischemia. Methods: Male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo). Sodium salts (Na) or methyl esters (Me) ELVs were dissolved in artificial CSF and administered into right lateral ventricle at 3 h after onset of stroke. There were five groups: ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, ELV-N34-Me (5μg/50μl), and CSF (50μl). Neurological function was evaluated on days 1, 3, and 7 after MCAo. Ex vivo MRI and immunohistochemistry were conducted on day 7. Results: All ELV treatments greatly improved neurologic scores in a sustained fashion up to the 7-day survival period. Ischemic core and penumbra volumes (computed from T2WI) were significantly reduced by all ELV treatments, and total lesion volumes were significantly reduced by ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, and ELV-N34-Me compared to CSF-treated group (by 60%, 56%, 99%, and 91%, respectively). ELV-treated rats showed less infarction with an increased number of NeuN- and GFAP-positive cells as well as SMI-71-positive vessels in the cortex and less IgG staining in the cortex. ELV-mediated protection was extensive in the frontal-parietal cortex (tissue was salvaged by 57-96%), subcortex (73-75%), and total infarct volume, correction for brain swelling was dramatically reduced in all ELV-treated groups by 55-91%. Conclusion: We have shown that the administration of ELVs provides high-grade neurobehavioral recovery, decreases ischemic core and penumbra volumes, as well as attenuates cellular damage, blood vessel integrity, and BBB disruption.

Objective: Ischemic injury induces neurogenesis in the subventricular zone (SVZ) of the lateral ventricles and subgranular zone (SGZ) of dentate gyrus (DG) promotes the migration of neuroblasts, guided by blood vessels, into the ischemic damaged area. Neurogenesis leads the functional and histological recovery in the ischemic-damaged brain through the proliferation, migration, differentiation and integration of newly generated neural cells into the existing neural circuit. Recently, we have shown that Docosahexaenoic acid (DHA) and Neuroprotectin D1 (NPD1) therapy improves functional and histological outcomes following experimental stroke. It has also been shown that DHA promotes neurite outgrowth of immature neurons, such as embryonic hippocampal cells and embryonic cortical cells. The objective of this study was to explore underlying mechanisms of docosanoid neuroprotection after cerebral ischemia. Methods: Male Sprague-Dawley rats were anesthetized with isoflurane/nitrous oxide, underwent 2h of middle cerebral artery occlusion (MCAo) and treated with DHA (5mg/kg, IV) or NPD1 (5μg/per rat, ICV) and vehicles 1h after. Neuro-behavioral assessments were conducted on days 1, 2, 3 and on weeks 1, 2, 3 or 4. BrdU was injected on days 4, 5 and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7 and lipidomic analysis at 4 and 5h after onset of stroke. Results: DHA improved short- and long-term behavioral functions and reduced cortical, subcortical and total infarct volumes (by 42%, 47% and 31%, respectively) after 2 weeks and reduced tissue loss by 50% after 4 weeks. DHA increased the number of BrdU + /Ki-67 + , BrdU + /DCX + and BrdU + /NeuN + cells in the cortex, subventricular zone and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71 positive cells and vessels, axonal regeneration in the penumbra and attenuated blood-brain barrier (BBB) after MCAo. Conclusion: We conclude that docosanoid administration increases neurogenesis, angiogenesis, activates NPD1 synthesis in the penumbra and diminishes BBB, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.

Thrombotic microangiopathy (TMA) is a disorder characterized by microvascular occlusion that can lead to thrombocytopenia, hemolytic anemia, and glomerular damage. Complement activation is the central event in most cases of TMA. Primary forms of TMA are caused by mutations in genes encoding components of the complement or regulators of the complement cascade. Recently, we and others have described a genetic form of TMA caused by mutations in the gene diacylglycerol kinase-ε ( DGKE) that encodes the lipid kinase DGKε(Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi MR, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji WZ, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler MC, Hwa J, Loirat C, Lifton RP. Nat Genet 45: 531–536, 2013; Ozaltin F, Li BH, Rauhauser A, An SW, Soylemezoglu O, Gonul II, Taskiran EZ, Ibsirlioglu T, Korkmaz E, Bilginer Y, Duzova A, Ozen S, Topaloglu R, Besbas N, Ashraf S, Du Y, Liang CY, Chen P, Lu DM, Vadnagara K, Arbuckle S, Lewis D, Wakeland B, Quigg RJ, Ransom RF, Wakeland EK, Topham MK, Bazan NG, Mohan C, Hildebrandt F, Bakkaloglu A, Huang CL, Attanasio M. J Am Soc Nephrol 24: 377–384, 2013). DGKεis unrelated to the complement pathway, which suggests that unidentified pathogenic mechanisms independent of complement dysregulation may result in TMA. Studying Dgke knockout mice may help to understand the pathogenesis of this disease, but no glomerular phenotype has been described in these animals so far. Here we report that Dgke null mice present subclinical microscopic anomalies of the glomerular endothelium and basal membrane that worsen with age and develop glomerular capillary occlusion when exposed to nephrotoxic serum. We found that induction of cyclooxygenase-2 and of the proangiogenic prostaglandin E2are impaired in Dgke null kidneys and are associated with reduced expression of the antithrombotic cell adhesion molecule platelet endothelial cell adhesion molecule-1/CD31 in the glomerular endothelium. Notably, prostaglandin E2supplementation was able to rescue motility defects of Dgke knockdown cells in vitro and to restore angiogenesis in a test in vivo. Our results unveil an unexpected role of Dgke in the induction of cyclooxygenase-2 and in the regulation of glomerular prostanoids synthesis under stress.

Docosahexaenoic acid (DHA) and neuroprotectin D1 (NPD1) are neuroprotective after experimental ischemic stroke. To explore underlying mechanisms, SD rats underwent 2h of middle cerebral artery occlusion (MCAo) and treated with DHA (5mg/kg, IV) or NPD1 (5μg/per rat, ICV) and vehicles 1h after. Neuro-behavioral assessments was conducted on days 1, 2, and 3, and on week 1, 2, 3, or 4. BrdU was injected on days 4, 5, and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7, and lipidomic analysis at 4 and 5h after onset of stroke. DHA improved short- and long-term behavioral functions and reduced cortical, subcortical, and total infarct volumes (by 42, 47, and 31%, respectively) after 2weeks and reduced tissue loss by 50% after 4weeks. DHA increased the number of BrdU+/Ki-67+, BrdU+/DCX+, and BrdU+/NeuN+ cells in the cortex, subventricular zone, and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71-positive cells and vessels, axonal regeneration in the penumbra, and attenuated blood-brain barrier (BBB) after MCAo. We conclude that docosanoid administration increases neurogenesis and angiogenesis, activates NPD1 synthesis in the penumbra, and diminishes BBB permeability, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.

Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived mediator, induces cell survival in uncompensated oxidative stress (OS), neurodegenerations or ischemic stroke. The molecular principles underlying this protection remain unresolved. We report here that, in retinal pigment epithelial cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways. RelB expression follows a similar pattern as BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. BIRC3 silencing prevents NPD1 induction of survival against OS. Moreover, brain NPD1 biosynthesis and selective neuronal BIRC3 abundance are increased by DHA after experimental ischemic stroke followed by remarkable neurological recovery. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

Introduction A major clinical challenge in treating allergic contact dermatitis (ACD) is that the first line of treatment is based on the use of corticosteroids. In this study, we aimed to develop a formulation that is devoid of steroids. Methods We used mouse ears treated with dinitrofluorobenzene (DNFB) to induce ACD. The efficacy of the test formulation to ameliorate and to prevent induced ACD was determined. Results To treat this experimentally induced ACD, we developed a formulation containing BIPxine (a mixture of Rosa moschata and Croton lechleri (antioxidants) and Aloe vera and d-panthenol (moisturizers), and hydroglycolic solutions of disodium cromoglycate. Our results show that clear inhibition of ACD took place. The target of this formulation was PAR-2, TRPV4, and other mediators of the inflammatory and pain responses. However, this formulation must be evaluated in other models besides the mouse to confirm its effectiveness. Conclusion The formulation presented here may provide new ACD therapies that do not involve the use of corticosteroids. Electronic supplementary material The online version of this article (10.1007/s13555-018-0223-8) contains supplementary material, which is available to authorized users.

Docosahexaenoic acid (DHA, 22:6 n-3) is abundant in the retina and is enzymatically converted into pro-homeostatic docosanoids. The DHA- or eicosapentaenoic acid (EPA)-derived 26 carbon fatty acid is a substrate of elongase ELOVL4, which is expressed in photoreceptor cells and generates very long chain (≥C28) polyunsaturated fatty acids including n-3 (VLC-PUFAs,n-3). While ELOVL4 mutations are linked to vision loss and neuronal dysfunctions, the roles of VLC-PUFAs remain unknown. Here we report a novel class of lipid mediators biosynthesized in human retinal pigment epithelial (RPE) cells that are oxygenated derivatives of VLC-PUFAs,n-3; we termed these mediators elovanoids (ELV). ELVs have structures reminiscent of docosanoids but with different physicochemical properties and alternatively-regulated biosynthetic pathways. The structures, stereochemistry, and bioactivity of ELVs were determined using synthetic materials produced by stereo-controlled chemical synthesis. ELVs enhance expression of pro-survival proteins in cells undergoing uncompensated oxidative stress. Our findings unveil a novel autocrine/paracrine pro-homeostatic RPE cell signaling that aims to sustain photoreceptor cell integrity and reveal potential therapeutic targets for retinal degenerations.

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium–derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea–TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.

Efficient biomarkers for early prediction and diagnosis of an acutely symptomatic carotid plaque rupture event are currently lacking, impairing the ability to diagnose and treat patients with an acute plaque rupture events in a timely fashion. Resolvins are endogenous inflammation-resolving lipid mediators that are induced by inflammatory insults. We hypothesized that resolvin and other lipid profiles in sera likely mark the process towards plaque rupture.Circulating lipids associated with plaque rupture events were quantitatively profiled via targeted mediator-lipidomics using ultraperformance liquid chromatography tandem mass spectrometry in patients with acutely symptomatic and asymptomatic carotid disease.Resolvin D1 (RvD1, 82 ± 11pM vs. 152 ± 17pM, p = 0.001) and docosahexaenoic acid (DHA) (0.052 ± 0.007µM versus 0.076 ± 0.008µM, p = 0.025) levels are decreased in the sera of patients presenting with an acutely symptomatic carotid plaque rupture event (n = 21) compared to patients with asymptomatic (n = 24) high-grade carotid stenosis. Circulating arachidonic acid (AA) levels, however, were higher (0.429 ± 0.046µM versus 0.257 ± 0.035µM, p0.01) in acutely symptomatic compared to asymptomatic carotid patients. ROC curve analysis demonstrates that the serum ratio AA:RvD1 (AUC 0.84, sensitivity 0.71, specificity 0.92) and AA:DHA (AUC 0.86, sensitivity 0.90, specificity 0.71) are biomarkers for the risk of atherosclerotic plaque rupture.A circulating pro-inflammatory lipid profile, characterized by high AA:RvD1 and AA:DHA, is associated with acutely symptomatic carotid disease and stroke.

We report the discovery of a novel family of prohomeostatic and neuroprotective lipid mediators called elovanoids., We report the characterization of a novel class of lipid mediators termed elovanoids (ELVs) (ELV-N32 and ELV-N34), which are dihydroxylated derivatives of 32:6n3 and 34:6n3, respectively. The precursors of ELVs are made by elongation of a 22:6n3 fatty acid and catalyzed by ELOVL4 (elongation of very-long-chain fatty acids–4). The structure and stereochemistry of ELVs were established using synthetic compounds produced by stereocontrolled total synthesis. We report that ELV-mediated protection is induced in neuronal cultures undergoing either oxygen/glucose deprivation or N-methyl-d-aspartate receptor–mediated excitotoxicity, as well as in experimental ischemic stroke. The methyl ester or sodium salt of ELV-N32 and ELV-N34 resulted in reduced infarct volumes, promoted cell survival, and diminished neurovascular unit disruption when administered 1 hour following 2 hours of ischemia by middle cerebral artery occlusion. Together, our data reveal a novel prohomeostatic and neuroprotective lipid-signaling mechanism aiming to sustain neural cell integrity.

Acute ischemic stroke triggers complex neurovascular, neuroinflammatory and synaptic alterations. Aspirin and docosahexaenoic acid (DHA), an omega-3 essential fatty acid family member, have beneficial effects on cerebrovascular diseases. DHA is the precursor of neuroprotectin D1 (NPD1), which downregulates apoptosis and, in turn, promotes cell survival. Here we have tested the effect of aspirin plus DHA administration and discovered the synthesis of aspirin-triggered NPD1 (AT-NPD1) in the brain. Then we performed the total chemical synthesis of this molecule and tested in the setting of 2h middle cerebral artery occlusion (MCAo) in Sprague-Dawley rats. Neurological status was evaluated at 24h, 48 h, 72 h, and 7 days. At 3h post-stroke onset, an intravenous administration of 333 μg/kg of AT-NPD1 sodium salt (AT-NPD1-SS) or methyl-ester (AT-NPD1-ME) or vehicle (saline) as treatment was given. On day 7, ex vivo magnetic resonance imaging (MRI) of the brains was conducted on 11.7 T MRI. T2WI, 3D volumes, and apparent diffusion coefficient (ADC) maps were generated. In addition, infarct volumes and number of GFAP (reactive astrocytes), ED-1 (activated microglia/macrophages) and SMI-71-positive vessels were counted in the cortex and striatum at the level of the central lesion. All animals showed similar values for rectal and cranial temperatures, arterial blood gases, and plasma glucose during and after MCAo. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly improved neurological scores compared to saline treatment at 24h, 48 h, 72 h and 7 days. Total lesion volumes computed from T2WI images were significantly reduced by both AT-NPD1-SS and AT-NPD1-ME treatment in the cortex (by 44% and 81%), striatum (by 61% and 77%) and total infarct (by 48% and 78%, respectively). Brain edema, computed from T2WI in the cortex (penumbra) and striatum (core), was elevated in the saline group. In contrast, both AT-NPD1 decreased water content in the striatum on day 7. 3D volumes, computed from T2WI, were dramatically reduced with both AT-NPD1 and the lesion was mostly localized in the subcortical areas. Treatment with both AT-NPD1-SS and AT-NPD1-ME significantly reduced cortical (by 76% and 96%), subcortical (by 61% and 70%) and total (69% and 84%, respectively) infarct volumes as defined by histopathology. In conclusion, a novel biosynthetic pathway that leads to the formation of AT-NPD1 mediator in the brain was discovered. In addition, administration of synthetic AT-NPD1, in either its sodium salt or as the methyl ester, was able to attenuate cerebral ischemic injury which leads to a novel approach for pharmaceutical intervention and clinical translation.

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells' functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1(-/-) mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1(-/-) mice. RPE-rich eyecup cultures from AdipoR1(-/-) reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity., Docosahexaenoic acid is a major and important retinal fatty acid that is recruited and retained in the photoreceptor membrane via an unknown mechanism. Here, Rice et al. show that adiponectin receptor 1 is a key molecular switch for docosahexaenoic acid membrane homeostasis and photoreceptor cell function.

A rapid and reliable method was presented for studying hydrogel dynamics/kinetics. Two temperature-sensitive hydrogels, poly- N -isopropylacrylamide (poly(NIPAAm)) and the copolymer of N , N -diethylacrylamide and sodium methacrylate (molar ratio=97:3, poly(NDEAAm- co -MAA)) were synthesized. The thermal-behaviors of the gels were studied through the absorbance intensities of both swollen water and gel frame components, and the peak positions of amide band along heating/cooling pathways under dynamic Fourier transform infrared (FTIR) probing. The results showed that the lower critical solution temperature (LCST) of poly(NIPAAm) is about 33–35 °C, which is consistent with reported value of ∼34 °C. Compared to poly(NIPAAm), poly(NDEAAm- co -MAA) has relatively continuous volume phase transition, starting at ∼35 °C and a better thermal-reversibility with similar swelling and deswelling profiles over a larger temperature range (10–80 °C for poly(NDEAAm- co -MAA) vs. 10–33 °C for poly(NIPAAm)). The H-bonding water along phase transition was also studied, showing a less reversibility of poly(NIPAAm) compared to poly(NDEAAm- co -MAA). In addition, FTIR spectrometer was also used to study the volume changes of poly(NDEAAm- co -MAA) under variations in environmental salinity.

The assembly of monomeric protein subunits into a viral capsid is a finely tuned molecular process. In response to subtle changes in environmental conditions, this supramolecular complex can dramatically reorganize. Defining the forces that control this structure and the cooperative action of subunits has implications for biology and nanotechnology. The small icosahedral RNA tetravirus family members Nudaurelia omega capensis (NomegaV) and Helicoverpa armigera stunt virus (HaSV) can be purified as provirions, and maturation to capsids can be induced by a drop in pH. In this study, a comparison of capsid secondary structure using FT-IR revealed that the procapsid has more alpha-helical content than the capsid, supporting the proposal that helix to coil transition may be important for maturation. The dynamic properties of the two states were probed using limited proteolysis and peptide mass mapping to identify regions of significant flexibility. Interestingly, the initial sites of protease cleavage were the N and C terminal domains that are internal in high-resolution models, and to inter-subunit surfaces. Further comparison of the two particle forms using FT-IR revealed that in response to thermal stress, the provirion disassembles and unfolds in a cooperative manner over a narrow temperature range (approximately 5 degrees C). Paradoxically, the capsid form, which is stable in a wide range of pH and ionic conditions and is more resistant to proteolysis, responds to thermal stress at a lower temperature than the procapsid form. This suggests that a metastable state is the end product of assembly.

The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amyloid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thioflavin T showed that p53tet-wt and p53tet-R337H adopt an alternative beta-sheet conformation (p53tet-wt-beta and p53tet-R337H-beta, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elevated temperatures. Electron micrographs showed that the alternative conformations for p53tet-wt (p53tet-wt-beta) and p53tet-R337H (p53tet-R337H-beta) were supramolecular structures best described as "molecular ribbons". FT-IR analysis demonstrated that the mechanism of amyloid-like fibril formation involved unfolding of the p53tet-wt beta-strands, followed by unfolding of the alpha-helices, followed finally by formation of beta-strand-containing structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 degrees C) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 degrees C over 20 hours. Interestingly, unlike other amyloid-forming proteins, p53tet-wt-beta and p53tet-R337H-beta disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassembly at pH 8.5 was independent of both. Finally, we propose that the significantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.