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Aims Critical Limb Ischaemia (CLI) patients often present with ischaemic rest pain and uncontrolled pain can impact on quality of life. To improve pain control, a set a of trust-wide guidelines were developed to standardise pain management for CLI patients. Methods This was a prospective cross-sectional audit, which initially involved 30 patients admitted under vascular surgery at a tertiary centre with CLI between May and June 2020. The improvements implemented included an analgesia education session for junior doctors, inclusion of an analgesia dosing table in the vascular clerking proforma and a simplified analgesia table as a poster. The re-audit included 22 patients with CLI between September and October 2020. Data was collected from drug charts and Fisher’s exact test was used for the statistical analysis. Results Baseline data suggested poor compliance with guidelines with only 3% of patients prescribed the guideline recommended PRN opiate doses. This improved post-intervention to 52% (p Conclusion This audit has demonstrated the successful implementation of targeted changes to improve compliance with trust pain management guidelines for patients with CLI. The challenge remains of overcoming the unknown eGFR at time of prescription and unknown background analgesia that can lead to over and under prescribing.

Studies on the amyloidogenic N-terminal domain of the E. coli HypF protein (HypF-N) have contributed significantly to a detailed understanding of the pathogenic mechanisms in neurodegenerative diseases characterised by the formation of misfolded oligomers, by proteins such as amyloid-β, α-synuclein and tau. Given that both cell membranes and mitochondria are increasingly recognised as key targets of oligomer toxicity, we investigated the damaging effects of aggregates of HypF-N on mitochondrial membranes. Essentially, we found that HypF-N oligomers characterised by high surface hydrophobicity (type A) were able to trigger a robust permeabilisation of mito-mimetic liposomes possessing cardiolipin-rich membranes and dysfunction of isolated mitochondria, as demonstrated by a combination of mitochondrial shrinking, lowering of mitochondrial membrane potential and cytochrome c release. Furthermore, using single-channel electrophysiology recordings we obtained evidence that the type A aggregates induced currents reflecting formation of ion-conducting pores in mito-mimetic planar phospholipid bilayers, with multi-level conductances ranging in the hundreds of pS at negative membrane voltages. Conversely, HypF-N oligomers with low surface hydrophobicity (type B) could not permeabilise or porate mitochondrial membranes. These results suggest an inherent toxicity of membrane-active aggregates of amyloid-forming proteins to mitochondria, and that targeting of oligomer-mitochondrial membrane interactions might therefore afford protection against such damage., peer-reviewed

A signature feature of age-related neurodegenerative proteinopathies is the misfolding and aggregation of proteins, typically amyloid-β (Aβ) in Alzheimer's disease (AD) and α-synuclein (α-syn) in Parkinson's disease (PD), into soluble oligomeric structures that are highly neurotoxic. Cellular and animal models that faithfully replicate the hallmark features of these disorders are being increasing exploited to identify disease-modifying compounds. Natural compounds have been identified as a useful source of bioactive molecules with promising neuroprotective capabilities. In the present report, we investigated whether extracts derived from two ubiquitous Mediterranean plants namely, the prickly pear Opuntia ficus-indica (EOFI) and the brown alga Padina pavonica (EPP) alleviate neurodegenerative phenotypes in yeast (Saccharomyces cerevisiae) and fly (Drosophila melanogaster) models of AD and PD. Pre-treatment with EPP or EOFI in the culture medium significantly improved the viability of yeast expressing the Arctic Aβ42 (E22G) mutant. Supplementing food with EOFI or EPP dramatically ameliorated lifespan and behavioural signs of flies with brain-specific expression of wild-type Aβ42 (model of late-onset AD) or the Arctic Aβ42 variant (model of early-onset AD). Additionally, we show that either extract prolonged the survival of a PD fly model based on transgenic expression of the human α-syn A53T mutant. Taken together, our findings suggest that the plant-derived extracts interfere with shared mechanisms of neurodegeneration in AD and PD. This notion is strengthened by evidence demonstrating that EOFI and to a greater extent EPP, while strongly inhibiting the fibrillogenesis of both Aβ42 and α-syn, accumulate remodelled oligomeric aggregates that are less effective at disrupting lipid membrane integrity. Our work therefore opens new avenues for developing therapeutic applications of these natural plant extracts in the treatment of amyloidogenic neurodegenerative disorders.

Misfolding and aggregate formation by the tau protein has been closely related with neurotoxicity in a large group of human neurodegenerative disorders, which includes Alzheimer's disease. Here, we investigate the membrane-active properties of tau oligomers on mitochondrial membranes, using minimalist in vitro model systems. Thus, exposure of isolated mitochondria to oligomeric tau evoked a disruption of mitochondrial membrane integrity, as evidenced by a combination of organelle swelling, efflux of cytochrome c and loss of the mitochondrial membrane potential. Tau-induced mitochondrial dysfunction occurred independently of the mitochondrial permeability transition (mPT) pore complex. Notably, mitochondria were rescued by pre-incubation with 10-N-nonyl acridine orange (NAO), a molecule that specifically binds cardiolipin (CL), the signature phospholipid of mitochondrial membranes. Additionally, NAO prevented direct binding of tau oligomers to isolated mitochondria. At the same time, tau proteins exhibited high affinity to CL-enriched membranes, whilst permeabilisation of lipid vesicles also strongly correlated with CL content. Intriguingly, using single-channel electrophysiology, we could demonstrate the formation of non-selective ion-conducting tau nanopores exhibiting multilevel conductances in mito-mimetic bilayers. Taken together, the data presented here advances a scenario in which toxic cytosolic entities of tau protein would target mitochondrial organelles by associating with their CL-rich membrane domains, leading to membrane poration and compromised mitochondrial structural integrity.

The identification of compounds which protect the double-membrane of mitochondrial organelles from disruption by toxic confomers of amyloid proteins may offer a therapeutic strategy to combat human neurodegenerative diseases. Here, we exploited an extract from the marine brown seaweed Padina pavonica (PPE) as a vital source of natural bioactive compounds to protect mitochondrial membranes against insult by oligomeric aggregates of the amyloidogenic proteins amyloid-β (Aβ), α-synuclein (α-syn) and tau, which are currently considered to be major targets for drug discovery in Alzheimer's disease (AD) and Parkinson's disease (PD). We show that PPE manifested a significant inhibitory effect against swelling of isolated mitochondria exposed to the amyloid oligomers, and attenuated the release of cytochrome c from the mitochondria. Using cardiolipin-enriched synthetic lipid membranes, we also show that dye leakage from fluorophore-loaded vesicles and formation of channel-like pores in planar bilayer membranes are largely prevented by incubating the oligomeric aggregates with PPE. Lastly, we demonstrate that PPE curtails the ability of Aβ42 and α-syn monomers to self-assemble into larger β-aggregate structures, as well as potently disrupts their respective amyloid fibrils. In conclusion, the mito-protective and anti-aggregator biological activities of Padina pavonica extract may be of therapeutic value in neurodegenerative proteinopathies, such as AD and PD., peer-reviewed

Alzheimer’s disease is a devastating neurodegenerative disease eventually leading to dementia. An effective treatment does not yet exist. Here we show that oral application of the compound anle138b restores hippocampal synaptic and transcriptional plasticity as well as spatial memory in a mouse model for Alzheimer’s disease, when given orally before or after the onset of pathology. At the mechanistic level, we provide evidence that anle138b blocks the activity of conducting Ab pores without changing the membrane embedded Ab-oligomer structure. In conclusion, our data suggest that anle138b is a novel and promising compound to treat AD-related pathology that should be investigated further., peer-reviewed

Parkinson's disease (PD) is a common motor neurodegenerative disorder with multifactorial etiology that is an increasing burden on our aging society. PD is characterized by nigrostriatal degeneration which might involve oxidative stress, α-synuclein (αS) aggregation, dysregulation of redox metal homeostasis and neurotoxicity. Although the exact cause remains unknown, both genetic and environmental factors have been implicated. Among the various environmental factors tea consumption has attracted increasing interest, as besides being one of the most consumed beverages in the world, tea contains specific polyphenols which can play an important role in delaying the onset or halting the progression of PD. Green and black teas are rich sources of polyphenols, the most abundant being epigallocatechin-3-gallate (EGCG) and theaflavins. There is now consistent mechanistic data on the neuroprotective and neuroregenerative effects of tea polyphenols, indicating that they do not just possess anti-oxidant or anti-chelating properties but may directly interfere with aggregation of the αS protein and modulate intracellular signalling pathways, both in vitro and in animal models. EGCG in green tea has been by far the most studied compound and therefore future investigations should address more the effects of other polyphenols, especially theaflavins in black tea. Nevertheless, despite significant data on their potential neuroprotective effects, clinical studies are still very limited and to date only EGCG has reached phase II trials. This review collates the current knowledge of tea polyphenols and puts into perspective their potential to be considered as nutraceuticals that target various pathologies in PD.

Parkinson's disease (PD) is a common motor neurodegenerative disorder with multifactorial etiology that is an increasing burden on our aging society. PD is characterized by nigrostriatal degeneration which might involve oxidative stress, α-synuclein (αS) aggregation, dysregulation of redox metal homeostasis and neurotoxicity. Although the exact cause remains unknown, both genetic and environmental factors have been implicated. Among the various environmental factors tea consumption has attracted increasing interest, as besides being one of the most consumed beverages in the world, tea contains specific polyphenols which can play an important role in delaying the onset or halting the progression of PD. Green and black teas are rich sources of polyphenols, the most abundant being epigallocatechin-3-gallate (EGCG) and theaflavins. There is now consistent mechanistic data on the neuroprotective and neuroregenerative effects of tea polyphenols, indicating that they do not just possess anti-oxidant or anti-chelating properties but may directly interfere with aggregation of the αS protein and modulate intracellular signalling pathways, both in vitro and in animal models. EGCG in green tea has been by far the most studied compound and therefore future investigations should address more the effects of other polyphenols, especially theaflavins in black tea. Nevertheless, despite significant data on their potential neuroprotective effects, clinical studies are still very limited and to date only EGCG has reached phase II trials. This review collates the current knowledge of tea polyphenols and puts into perspective their potential to be considered as nutraceuticals that target various pathologies in PD.

Cumulative evidence now suggests that the abnormal aggregation of the protein α-synuclein (αS) is a critical factor in triggering neurodegeneration in Parkinson's disease (PD). In particular, a fundamental pathogenetic mechanism appears to involve targeting of neuronal membranes by soluble oligomeric intermediates of αS, leading to their disruption or permeabilisation. Therefore, a model assay was developed in which fluorophore-loaded unilamellar vesicles were permeabilised by soluble oligomers, the latter formed by aggregation of human recombinant αS protein. The αS oligomers induced an impairment of membrane integrity similar to that of the pore-forming bacterial peptide gramicidin. The lipid vesicle permeabilisation assay was then utilised to screen 11 natural polyphenolic compounds, 8 synthetic N′-benzylidene-benzohydrazide compounds and black tea extract for protection against membrane damage by wild-type and mutant (A30P, A53T) synuclein aggregates. A select group of potent inhibitory compounds included apigenin, baicalein, morin, nordihydroguaiaretic acid, and black tea extract. Structure–activity analysis further suggests that a 5,7-dihydroxy-chromen-4-one moiety appears to be favourable for the inhibition reaction. In conclusion, we have identified a group of polyphenols that can effectively hinder membrane damage by αS aggregates. These may serve as a viable source of lead compounds for the development and design of novel therapeutic agents in PD.

Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe 3+) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease., peer-reviewed

Amyloid-β (Aβ) aggregation is a recognized key process in the pathogenesis of Alzheimer's disease (AD). Misfolded Aβ peptides self-assemble into higher-order oligomers that compromise membrane integrity, leading to synaptic degeneration and neuronal cell death. The main aim of this study was to explore whether small-molecule compounds and black tea extract can protect phospholipid membranes from disruption by Aβ aggregates. We first established a robust protocol for aggregating Aβ₄₂ peptides into a range of oligomers that efficiently permeabilized small unilamellar liposomes. Next, 15 natural plant polyphenolic compounds, 8 N'-benzylidene-benzohydrazide (NBB) compounds and black tea extract were assessed for their ability to antagonize liposome permeabilization by the Aβ₄₂ oligomers. Our data indicates that black tea extract, the flavones apigenin and baicalein, and the stilbene nordihydroguaiaretic acid (NDGA) are indeed potent inhibitors. Taking into consideration the results of all the small-molecule polyphenols and NBB compounds, it can be proposed that a dihydroxyphenyl ring structure, alone or as part of a flavone scaffold, is particularly effective for protection against membrane damage by the Aβ₄₂ oligomers. Given the critical role of membrane perforation in the neurodegenerative cascade, these conclusions may guide the design and development of novel therapeutic drugs in AD.

Amyloid-β (Aβ) aggregation is a recognized key process in the pathogenesis of Alzheimer's disease (AD). Misfolded Aβ peptides self-assemble into higher-order oligomers that compromise membrane integrity, leading to synaptic degeneration and neuronal cell death. The main aim of this study was to explore whether small-molecule compounds and black tea extract can protect phospholipid membranes from disruption by Aβ aggregates. We first established a robust protocol for aggregating Aβ₄₂ peptides into a range of oligomers that efficiently permeabilized small unilamellar liposomes. Next, 15 natural plant polyphenolic compounds, 8 N'-benzylidene-benzohydrazide (NBB) compounds and black tea extract were assessed for their ability to antagonize liposome permeabilization by the Aβ₄₂ oligomers. Our data indicates that black tea extract, the flavones apigenin and baicalein, and the stilbene nordihydroguaiaretic acid (NDGA) are indeed potent inhibitors. Taking into consideration the results of all the small-molecule polyphenols and NBB compounds, it can be proposed that a dihydroxyphenyl ring structure, alone or as part of a flavone scaffold, is particularly effective for protection against membrane damage by the Aβ₄₂ oligomers. Given the critical role of membrane perforation in the neurodegenerative cascade, these conclusions may guide the design and development of novel therapeutic drugs in AD., peer-reviewed

A method is described for isolating human hepatocytes from tissue fragments after warm and cold ischemia as experienced during hepatic resections. Cells with a high trypan blue dye exclusion and good culture characteristics were isolated by employing an initial tissue perfusion with UW solution. The method could facilitate transfer of liver tissues between distant centers for cell isolation studies.

Alzheimer's disease and Parkinson's disease are neurodegenerative disorders characterised by the misfolding of proteins into soluble prefibrillar aggregates. These aggregate complexes disrupt mitochondrial function, initiating a pathophysiological cascade leading to synaptic and neuronal degeneration. In order to explore the interaction of amyloid aggregates with mitochondrial membranes, we made use of two in vitro model systems, namely: (i) lipid vesicles with defined membrane compositions that mimic those of mitochondrial membranes, and (ii) respiring mitochondria isolated from neuronal SH-SY5Y cells. External application of soluble prefibrillar forms, but not monomers, of amyloid-beta (Aβ42 peptide), wild-type α-synuclein (α-syn), mutant α-syn (A30P and A53T) and tau-441 proteins induced a robust permeabilisation of mitochondrial-like vesicles, and triggered cytochrome c release (CCR) from isolated mitochondrial organelles. Importantly, the effect on mitochondria was shown to be dependent upon cardiolipin, an anionic phospholipid unique to mitochondria and a well-known key player in mitochondrial apoptosis. Pharmacological modulators of mitochondrial ion channels failed to inhibit CCR. Thus, we propose a generic mechanism of thrilling mitochondria in which soluble amyloid aggregates have the intrinsic capacity to permeabilise mitochondrial membranes, without the need of any other protein. Finally, six small-molecule compounds and black tea extract were tested for their ability to inhibit permeation of mitochondrial membranes by Aβ42, α-syn and tau aggregate complexes. We found that black tea extract and rosmarinic acid were the most potent mito-protectants, and may thus represent important drug leads to alleviate mitochondrial dysfunction in neurodegenerative diseases.

Aggregation of alpha-synuclein (αS) into oligomers is critically involved in the pathogenesis of Parkinson’s disease (PD). Using confocal single-molecule fluorescence spectroscopy, we have studied the effects of 14 naturally-occurring polyphenolic compounds and black tea extract on αS oligomer formation. We found that a selected group of polyphenols exhibited potent dose-dependent inhibitory activity on αS aggregation. Moreover, they were also capable of robustly disaggregating pre-formed αS oligomers. Based upon structure–activity analysis, we propose that the key molecular scaffold most effective in inhibiting and destabilizing self-assembly by αS requires: (i) aromatic elements for binding to the αS monomer/oligomer and (ii) vicinal hydroxyl groups present on a single phenyl ring. These findings may guide the design of novel therapeutic drugs in PD.Structured summary of protein interactionsAlpha-synuclein binds to Alpha-synuclein by biophysical (View Interaction 1, 2)