The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins.

TIR-domain-containing adapter-inducing interferon-ß (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.

Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins.

The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs. The core tetrad sequence of the ICP6 RHIM is important for both amyloid formation and interaction with host RHIM-containing proteins. Notably, we show that the amyloid forming capacity of the ICP6 RHIM is affected by the redox environment. We propose that the formation of an intramolecular disulfide bond within ICP6 triggers the formation of amyloid assemblies that are distinct from previously characterised viral amyloids M45 and ORF20. Formation of viral-host heteromeric amyloid assemblies may underlie a general mechanism of viral adaptation against host immune machineries.

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death.

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies.

RHIM-based protein:protein interactions in microbial defence against programmed cell death by necroptosis.

The Receptor-interacting protein kinase Homotypic Interaction Motif (RHIM) is an amino acid sequence that mediates multiple protein:protein interactions in the mammalian programmed cell death pathway known as necroptosis. At least one key RHIM-based complex has been shown to have a functional amyloid fibril structure, which provides a stable hetero-oligomeric platform for downstream signaling. RHIMs and related motifs are present in immunity-related proteins across nature, from viruses to fungi to metazoans. Necroptosis is a hallmark feature of cellular clearance of infection. For this reason, numerous pathogens, including viruses and bacteria, have developed varied methods to modulate necroptosis, focusing on inhibiting RHIM:RHIM interactions, and thus their downstream cell death effects. This review will discuss current understanding of RHIM:RHIM interactions in normal cellular activation of necroptosis, from a structural and cell biology perspective. It will compare the mechanisms by which pathogens subvert these interactions in order to maintain their replicative and infective cycles and consider the similarities between RHIMs and other functional amyloid-forming proteins associated with cell death and innate immunity. It will discuss the implications of the heteromeric nature and structure of RHIM-based amyloid complexes in the context of other functional amyloids.

Microbial functional amyloids serve diverse purposes for structure, adhesion and defence.

The functional amyloid state of proteins has in recent years garnered much attention for its role in serving crucial and diverse biological roles. Amyloid is a protein fold characterised by fibrillar morphology, binding of the amyloid-specific dyes Thioflavin T and Congo Red, insolubility and underlying cross-ß structure. Amyloids were initially characterised as an aberrant protein fold associated with mammalian disease. However, in the last two decades, functional amyloids have been described in almost all biological systems, from viruses, to bacteria and archaea, to humans. Understanding the structure and role of these amyloids elucidates novel and potentially ancient mechanisms of protein function throughout nature. Many of these microbial functional amyloids are utilised by pathogens for invasion and maintenance of infection. As such, they offer novel avenues for therapies. This review examines the structure and mechanism of known microbial functional amyloids, with a particular focus on the pathogenicity conferred by the production of these structures and the strategies utilised by microbes to interfere with host amyloid structures. The biological importance of microbial amyloid assemblies is highlighted by their ubiquity and diverse functionality.

Auricular vagus nerve stimulation enhances central serotonergic function and inhibits diabetic neuropathy development in Zucker fatty rats.

Painful neuropathy is a frequent comorbidity in diabetes. Zucker diabetic fatty (fa/fa) rats develop type 2 diabetes spontaneously with aging and show nociceptive hypersensitivity at the age of 13 weeks. In preclinical and clinical studies, the treatment of diabetic neuropathy is challenging, but complementary medicine such as transcutaneous auricular vagus nerve stimulation (taVNS) appears beneficial to the relief of neuropathic pain. However, the mechanism behind the effectiveness of taVNS remains unclear. In this study, we show that daily 30-min taVNS (2/15 Hz, 2 mA) for consecutive 27 days effectively inhibited the development of nociceptive hypersensitivity in Zucker diabetic fatty rats as detected by thermal hyperalgesia and mechanical allodynia in hindpaw. We also demonstrated that this beneficial effect in nociceptive behavior is related to an elevated serotonin (5-HT) plasma concentration and an upregulated expression of 5-HT receptor type 1A (5-HT1AR) in hypothalamus. We conclude that daily 30-min taVNS sessions lessen diabetic neuropathy development by enhancing serotonergic function in genetically diabetes prone individuals. Perspective This article presents taVNS as a new approach to inhibit the development of diabetic neuropathy in genetically prone individuals. This approach could potentially help clinicians who seek to avoid the complication of neuropathic pain in diabetic patient or to relieve the pain if there was one.

Seizure prevention by the naturally occurring phenols, carvacrol and thymol in a partial seizure-psychomotor model.

The natural compounds carvacrol and thymol completely prevented seizures in the 6 Hz, 32 mA partial seizure model. Carvacrol and thymol, both exhibited an ED50 = 35.8 mg/kg, ip and yielded protective indices of 5.3 and 3.4, respectively. At 44 mA current intensity, carvacrol and thymol exhibited ED50s of 88.82 mg/kg (PI = 2.15) and 73.0 mg/kg (PI = 1.65), respectively. Thymol, but not carvacrol showed partial inhibitory activity in the maximal electroshock (MES), sc Metrazol (scMET) and Corneal-kindled models. These results suggest that carvacrol and thymol are more efficacious anticonvulsants than suggested by their lower efficacies in the conventional MES and scMET tests.

ortho Substituent effects on the anticonvulsant properties of 4-hydroxy-trifluoroethyl phenols.

2,6-Dialkylphenols with isopropyl and sec-butyl substituent are well known anesthetic compounds. The 4-substitution with 1-hydroxy-2,2,2-trifluoroethyl (4-HTFE) group in these compounds led to the discovery of compounds with anticonvulsant activity in the 6 Hz (32 mA) model of partial epilepsy. In the present study a series of 2-alkyl-4-HTFE phenols with the 6-position being replaced with either hydrogen and bromine were designed, synthesized and tested to evaluate the effect of ortho-substitution on the anticonvulsant property. The studies show that 2-substituted branched alkyl chain (iso-propyl and sec-butyl) is necessary for the anti-seizure effect. Phenols with 2-substituted linear alkyl groups (methyl, ethyl and n-propyl) having no substitution at 6-position were found to be devoid of antiseizure effects. The 6-substitution with bromine moderately reduces the anticonvulsant effect in the compounds with branched alkyl chains, but led to enhanced anticonvulsant effect in the compound with a linear alkyl chain. This study shows that 4-HTFE phenols having isopropyl or sec-butyl ortho groups produce good antiseizure protection in the 6 Hz therapy-resistant mouse model.

The anticonvulsant effects of propofol and a propofol analog, 2,6-diisopropyl-4-(1-hydroxy-2,2,2-trifluoroethyl)phenol, in a 6 Hz partial seizure model.

BACKGROUND: Propofol is a general anesthetic having good anticonvulsant properties, but is limited in antiseizure use because of its potent anesthetic/sedative properties. It is proposed that substitution of the propofol molecule in the para position may yield compounds having less toxicity, yet possessing anticonvulsant properties because of retention of the 2,6-diisopropylphenol configuration. Reported herein is the synthesis of a para-substituted analog of propofol, 2,6-diisopropyl-4-(1-hydroxy-2,2,2-trifluoroethyl)phenol (MB003), and a similar analog of 2,6-di-sec-butylphenol (MB050), and their comparative anticonvulsant effects in National Institute of Neurological Disorders and Stroke screening models. METHODS: MB003 and MB050 were synthesized by the reaction of propofol or 2,6-di-sec-butylphenol, respectively, with trifluoroacetaldehyde ethyl hemiacetal in the presence of catalytic amounts of K(2)CO(3). Compounds were purified to >98% purity. Propofol, MB003, 2,6-di-sec-butylphenol, and MB050 were screened for protective effects by the National Institute of Neurological Disorders and Stroke Anticonvulsant Screening Program in the mouse maximal electroshock, subcutaneous metrazol, and 6 Hz (32 mA) partial seizure models. All compounds were administered by i.p. injection. The toxicity of each compound was assessed by the ability of the animals to stay on a Rotorod after dosing. RESULTS: Propofol, MB003, and MB050 were found to be most protective in the 6 Hz model with lesser protective effects in the mouse maximal electroshock and subcutaneous metrazol models. In the 6 Hz model, propofol yielded a 50% effective dose of 32.8 mg/kg; MB003, 38.4 mg/kg; and MB050, 74.0 mg/kg. Propofol, and to a greater degree, 2,6-di-sec-butylphenol, exhibited high toxicity. The corresponding 2,6-dimethylphenol analog to MB003 and MB050, 2,6-dimethyl-4-(1-hydroxy-2,2,2-trifluoroethyl)phenol, was not protective in the 6 Hz model and exhibited no toxicity at any dose tested. CONCLUSION: These results show that the anesthetics propofol and 2,6-di-sec-butylphenol may be substituted in the para position with a 1-hydroxy-2,2,2-trifluoroethyl moiety and the resulting molecules have anticonvulsant activity in the 6 Hz model while exhibiting less toxicity (ataxia) than the parent 2,6-dialkylphenols. The effectiveness of propofol, MB003, 2,6-di-sec-butylphenol, and MB050 and the ineffectiveness of 2,6-dimethyl-4-(1-hydroxy-2,2,2-trifluoroethyl)phenol, in the 6 Hz model shows that the 2,6-diisopropyl or 2,6-di-sec-butyl phenolic configuration is more important to anticonvulsant activity than having the phenolic para position free of substituents. These results suggest that 1-hydroxy-2,2,2-trifluoroethyl substituted 2,6-di-alkylphenols may have useful anticonvulsant activities.

Pharmacokinetic characteristics of bolus-administered mannitol in patients undergoing elective craniotomy.

To better understand mannitol pharmacokinetics, the authors constructed and compared population models for high-versus low-dose bolus infusions in humans. Patients (aged 18-75, American Society of Anesthesiologists physical status 1-3) scheduled for elective craniotomy with an anticipated need for intraoperative mannitol were randomly assigned to receive either 0.5 (n = 10) or 1.0 (n = 12) g/kg of 20% mannitol over 15 minutes. Serial blood samples were collected at the predetermined intervals over 12 hours. Plasma mannitol concentrations were measured by gas chromatography and subjected to pharmacokinetic analysis; a 3-compartment model best described mannitol disposition characteristics. Weight and dose were the important covariates for rapid peripheral volume of distribution (V2) and central clearance (CL1), respectively. Estimated population means were 2.80, 8.86, and 12.0 L for central (V1), rapid (V2), and slow (V3) volumes of distribution, respectively. Clearances of the central compartments (CL1) were 0.07 versus 0.04 L/min in the high-versus low-dose group, respectively. Thus, mannitol kinetics can be considered as nonlinear. Clearances of the rapid peripheral (CL2) and slow peripheral compartments (CL3) were identical (2.07 and 0.16 L/min) in both. The current weight-based dosing guidelines yielded greater than expected plasma drug concentrations in obese patients.

Ketoprofen produces modality-specific inhibition of pain behaviors in rats after plantar incision.

BACKGROUND: Postoperative pain remains a significant problem despite optimal treatment with current drugs. Nonsteroidal antiinflammatory drugs reduce inflammation and provide analgesia but are associated with adverse side effects. METHODS: We tested low doses (0.5-5 mg/kg) of parenteral ketoprofen against pain-related behaviors after plantar incision in rats. To further evaluate the potential sites of action of ketoprofen in our model, a novel, sustained-release microparticle formulation of ketoprofen was placed into the wound, and tested for its effects on pain behaviors. Intrathecal ketoprofen (150 microg) was also studied. Plasma samples were assayed for drug concentrations. RESULTS: We found that low doses of parenterally administered ketoprofen produced a modality-specific effect on pain behaviors; guarding after incision was decreased, whereas no inhibition of exaggerated responses to heat or mechanical stimuli was evident. Very low doses, 0.5 mg/kg, could produce inhibition of guarding. The locally applied sustained-release ketoprofen-eluting microparticles and intrathecally administered ketoprofen also produced a modality-specific effect on pain behaviors after incision, inhibiting only guarding. Plasma levels of ketoprofen after parenteral or local administration were in the range of therapeutic blood levels in postoperative patients. CONCLUSIONS: This study demonstrates that ketoprofen is an effective analgesic for nonevoked guarding in rats after plantar incision. There was no effect on mechanical or heat responses, which highlights the importance of multiple-modality testing of pain behaviors for drug evaluation. We found efficacy at doses used clinically in postoperative patients.

Sevoflurane: are there differences in products?

Sevoflurane is currently available in the United States from two manufacturers: Ultane (Abbott Laboratories, Inc.) and a generic product, Sevoflurane Inhalation Anesthetic (Baxter Healthcare Corp.). These products are rated therapeutically equivalent by the Food and Drug Administration, but there are some differences. Ultane is made in a single-step synthetic process and generic sevoflurane is manufactured using a three-step process. Ultane contains >300 ppm water and generic sevoflurane contains < or =130 ppm water. Ultane is supplied in a plastic polyethylene naphthalate polymer bottle, while generic sevoflurane is supplied in lacquer-lined aluminum bottles. The manufacturing processes and impurities, sevoflurane degradation resulting from Lewis acid reactions, and suitability of nonglass containers for sevoflurane are discussed.

Propofol: the challenges of formulation.

Propofol is a potent lipophilic anesthetic that was initially formulated in Cremophor El for human use. Because of the occurrence of Cremophor EL anaphylaxis and improvements in the quality of lipid emulsions, it was ultimately brought to market as 1% propofol formulated in 10% soybean oil emulsion. Emulsions represent complex formulation compositions whose suitability for intravenous administration is dependent on a number of factors. Despite the success of propofol emulsions, drawbacks to such formulations include inherent emulsion instability, injection pain, a need for antimicrobial agents to prevent sepsis, and a concern of hyperlipidemia-related side effects. Efforts to overcome such drawbacks have involved the development of propofol emulsions with altered propofol and lipid contents, the addition of different excipients to emulsions for antimicrobial activity, and study of nonemulsion formulations including propofol-cyclodextrin and propofol-polymeric micelle formulations. In addition, a number of propofol prodrugs have been made and evaluated.

Role of lipid in sulfite-dependent propofol dimerization.

BACKGROUND: During long-term intravenous infusions, sulfite in sulfite-containing propofol emulsions can cause the peroxidation of lipid and dimerization of propofol. This study evaluated the role of lipid in sulfite-dependent propofol dimerization by determining the effects of individual fatty acids in soybean oil emulsion and peroxidized lipids in a model system. METHODS: Individual fatty acids, stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), and arachidonic (20:4), were added to sulfite-containing propofol emulsion and incubated for 90 min at 37 degrees C. Model systems containing soybean oil (100 microl), water (900 microl), propofol (10 mg/ml), and sulfite (0.25 mg/ml) composed of oils with different peroxide values were allowed to react for 60 min at room temperature. After the reactions, propofol dimer and propofol dimer quinone were analyzed by reversed-phase high-pressure liquid chromatography. RESULTS: Propofol did not dimerize when added to aqueous sulfite unless soybean oil was also included. The addition of the polyunsaturated fatty acids (linoleic, linolenic, arachidonic) to sulfite-containing propofol emulsion resulted in large increases of propofol dimerization compared with stearic or oleic acid. Using biphasic mixtures of soybean oil and aqueous sulfite, propofol dimerization increased with increasing peroxide content of the oil. In propofol emulsion, lipoxidase and ferrous iron in the absence of sulfite also caused the dimerization of propofol. CONCLUSIONS: These results show that lipid can play a significant role in sulfite-dependent propofol dimerization. The relation of dimerization to polyunsaturated fatty acid and soybean oil peroxide content suggests that sulfite reacts with unsaturated lipid or peroxide-modified lipid to facilitate propofol dimerization.

The hypnotic and analgesic effects of 2-bromomelatonin.

2-bromomelatonin is an analog of melatonin with a higher melatonin receptor affinity. We tested the hypnotic and analgesic properties of 2-bromomelatonin and compared them with those of propofol. Sprague-Dawley rats were assigned to receive 2-bromomelatonin or propofol IV, or morphine intraperitoneally. Righting reflex and response to tail clamping were assessed. Both 2-bromomelatonin and propofol caused a dose-dependent increase in the percent of rats displaying loss of both the righting reflex and the response to tail clamping. 2-Bromomelatonin was comparable to propofol in terms of its rapid onset and short duration of hypnosis. The 50% effective dose (95% confidence interval) for loss of righting reflex for propofol and 2-bromomelatonin were 3.7 (3.4-4.0) and 38 (35-41) mg/kg, respectively. Corresponding values for loss of response to tail clamp were 2.9 (3.5-4.0) and 21 (15-30) mg/kg, respectively. 2-bromomelatonin is approximately 6-10 times less potent than propofol depending on the end-point used. Intraperitoneal 30 mg/kg morphine did not affect the righting reflex, but resulted in loss of response to tail clamping in all animals. 2-bromomelatonin can exert hypnotic and antinocifensive effects similar to that observed with propofol. Unlike propofol, the reduced nocifensive behavior persisted after the animals had regained their righting reflex. This study provides evidence that 2-bromomelatonin has properties that are desirable in anesthetics or anesthetic adjuvants.

The electroencephalographic effects of IV anesthetic doses of melatonin: comparative studies with thiopental and propofol.

UNLABELLED: We have demonstrated that large-dose IV melatonin can exert hypnotic effects similar to those caused by thiopental and propofol. In this study, we compared the electroencephalographic (EEG) effects of melatonin with those of thiopental and propofol. Sprague-Dawley rats were assigned to receive equipotent bolus doses of thiopental (23.8 mg/kg), propofol (14.9 mg/kg), or melatonin (312 mg/kg). EEG effects were recorded at periodic intervals over 10 minutes. Of eight processed EEG variables analyzed, only relative total power (rTP), relative spectral edge 95% (rSE95), and relative approximate entropy (rAE) were altered by all drugs compared with their control vehicles. Drug administration decreased the values relative to baseline, with subsequent return toward baseline during the 10-min time course. Thiopental significantly increased rTP, whereas propofol and melatonin did not. All drugs significantly decreased rSE95. However, the time course of peak effect and duration differed for each, with melatonin exhibiting a slower onset and a more sustained EEG effect. All drugs significantly decreased rAE, with similar time courses for thiopental and propofol and a slower onset/longer duration for melatonin. Melatonin produced effects on processed EEG variables similar to those of thiopental and propofol, specifically a decrease in the rSE95 and a decrease in the rAE. IMPLICATIONS: Anesthetic doses of melatonin produced effects on processed electroencephalographic variables similar to those of thiopental and propofol.

Free radical and drug oxidation products in an intensive care unit sedative: propofol with sulfite.

OBJECTIVES: Some propofol emulsion formulations contain EDTA or sodium metabisulfite to inhibit microbe growth on extrinsic contamination. EDTA is not known to react with propofol formulation components; however, sulfite has been shown to support some oxidation processes and may react with propofol. This study compared the oxidation of propofol and the formation of free radicals by electron paramagnetic resonance analysis in EDTA and sulfite propofol emulsions during a simulated intensive care unit 12-hr intravenous infusion. DESIGN: Controlled laboratory study. SETTING: University laboratory. MEASUREMENTS AND MAIN RESULTS: Propofol emulsions (3.5 mL) were dripped from spiked 50-mL vials at each hour for 12 hrs. Two propofol oxidation products, identified as propofol dimer and propofol dimer quinone, were detected in sulfite and EDTA propofol emulsions; however, sulfite propofol emulsion contained higher quantities of both compounds. After initiation of the simulated infusion, the quantities of propofol dimer and propofol dimer quinone increased in the sulfite propofol emulsion, but the lower levels in the EDTA propofol emulsion remained constant. Sulfite propofol emulsion began to visibly yellow at about 6-7 hrs. The EDTA propofol emulsion remained white at all times. The absorbance spectra of the propofol dimer and propofol dimer quinone extracted from sulfite propofol emulsion showed that propofol dimer did not absorb in the visible spectrum, but the propofol dimer quinone had an absorbance peak at 421 nm, causing it to appear yellow. Electron paramagnetic resonance analysis of the propofol emulsion containing metabisulfite revealed that the sulfite propofol emulsion yielded a strong free radical signal consistent with the formation of the sulfite anion radical (SO3*-). The EDTA propofol emulsion yielded no free radical signal above background. CONCLUSION: Sulfite from the metabisulfite additive in propofol emulsion creates an oxidative environment when these emulsions are exposed to air during a simulated intravenous infusion. This oxidation results in propofol dimerization and emulsion yellowing, the latter of which is caused by the formation of propofol dimer quinone. These processes can be attributed to the rapid formation of the reactive sulfite free radical.