EGCG directly targets intracellular amyloid-β(1-42) aggregates and promotes their lysosomal degradation

Introduction: Several lines of evidence indicate that aggregation and accumulation of amyloid-β(1-42) (Aβ42) causes cellular dysfunction and toxicity. Besides extracellular Aβ42 plaque formation and the intraneuronal deposition of hyperphosphorylated tau protein in Alzheimer’s disease (AD) patient brains, intracellular Aβ42 aggregation plays an important role in AD pathogenesis and even precedes extracellular Aβ42 plaque formation. The fact that neuronal cells internalize secreted Aβ42 whereby intracellular Aβ42 aggregation is induced by a prion-like seeding process, has further highlighted the importance of intraneuronal Aβ42 aggregation in disease progression. A potential causal therapy of AD involves slowing disease progression by targeting seeding-competent intracellular Aβ42 aggregates and enhancing their degradation in neuronal cells. Method: Therefore, a cell-based screening assay was established that allows the identification of small molecules effectively promoting the degradation of endocytotically internalized Aβ42 aggregates. This cell-based assay was intensively characterized and used in a proof-of-principle approach to screen a focused library of polyphenolic compounds preselected based on their anti-amyloidogenic properties in vitro. Further, atomic force, electron and confocal microscopy, biochemical analysis and enzyme activity assays were used and structure-activity relationship studies were performed to elucidate the mechanisms underlying the promotion of cellular aggregate degradation by direct amyloid-targeting compounds. Results: The polyphenol epigallocatechin gallate (EGCG) most potently promoted the clearance of intracellular Aβ42 aggregates in the neuroblastoma cell model. As a consequence of EGCG treatment, intracellular Aβ42 aggregate load is reduced, Aβ42 seeding-activity of cellular extracts is mitigated and Aβ42-induced mitochondrial metabolic impairment is rescued in neuroblastoma cells as well in primary neurons. Mechanistic studies revealed that EGCG directly targets intracellular Aβ42 aggregates and that the EGCG-mediated increase in Aβ42 degradation is primarily dependent on lysosomal enzyme activity. Furthermore, EGCG-induced structural remodeling of fibrillar Aβ42 aggregates into amorphous subspecies showed to enhance lysosomal enzymatic cleavage by the cysteine-protease cathepsin B (CatB), presenting a potential mechanism for the strong increase of cellular Aβ42 aggregate degradation. Take home points: Thus, this study shows that the direct targeting of intracellular Aβ42 aggregates with amyloid-remodeling compounds can be a promising approach to promote the degradation of proteotoxic peptide species. This can be an effective strategy to support the endogenous autophago-lysosomal degradation of intracellularly accumulating proteins in neurodegenerative proteinopathies such as amyotrophic lateral sclerosis, Huntington’s, Parkinson’s and Alzheimer’s disease.Einleitung: Zahlreiche Studien haben gezeigt, dass die Aggregation und Akkumulation von Amyloid-β(1-42) (Aβ42) eine zelluläre Dysfunktion und Zelltoxizität zur Folge haben. Neben der Entstehung von extrazellulären Aβ42 Plaques und der intrazellulären Ablagerung von hyperphosphorylierten Tau Proteinen im neuronalen Gewebe von Alzheimer Patienten, spielt die intrazelluläre Ablagerung von Aβ42 Aggregaten eine zentrale Rolle in der Pathogenese der Alzheimer-Demenz (AD) und geht der extrazellulären Plaque-Bildung sogar voraus. Die Erkenntnis, dass neuronale Zellen sekretiertes Aβ42 aufnehmen und dadurch eine intrazelluläre Fehlfaltung in einem Prionen-ähnlichen Seeding-Prozess induziert werden kann, lässt der intraneuronalen Aβ42 Ablagerung in der molekularen Pathogenese der Krankheitsprogression eine besondere Wichtigkeit zukommen. Methodik: Aufgrund dessen haben wir ein zellbasiertes Testverfahren entwickelt, mit dem niedermolekulare Substanzen identifiziert werden können, welche den Abbau von endozytotisch internalisierten, seeding-kompetenten Aβ42 Aggregaten fördern. Mittels diesem zellbasierten Screeningverfahren haben wir eine fokussierte Bibliothek aus Polyphenol-Verbindungen getestet, die aufgrund ihrer bekannten, anti-amyloidogenen Wirkung vorselektiert wurden. Darüber hinaus wurden Rasterkraft-, Elektronen- und Konfokalmikroskopie sowie biochemische Proteinanalyse- und Enzymaktivitätsverfahren verwendet, um den zugrundeliegenden Mechanismus der Förderung des Abbaus von intrazellulären Proteinaggregatspezies durch anti-amyloidogene, niedermolekulare Substanzen aufzuklären. Ergebnisse: Das Polyphenol Epigallocatechingallat (EGCG) zeigte die potenteste Wirkung auf die Reduktion von intrazellulären Aβ42 Aggregaten in unserem Modellsystem. Durch die EGCG-Behandlung wird die Menge an intrazellulären Aβ42 Aggregaten stark reduziert, die Seeding-Kapazität zellulärer Extrakte abgeschwächt und der durch Aβ42 induzierten verminderten mitochondrialen Stoffwechselaktivität sowohl in Neuroblastoma-Zellen als auch in primären Neuronen entgegengewirkt. Mechanistische Untersuchungen offenbarten zudem, dass EGCG direkt an intrazellulären Aβ42 Aggregaten ansetzt, dass die Abbau-fördernde Wirkung abhängig von der lysosomalen Enzymaktivität ist und, dass die durch EGCG bewirkte, strukturelle Umlagerung von fibrillären Aggregaten in amorphe Subspezies den Aβ42 Abbau durch das lysosomale Enzym Cathepsin B erleichtert. Fazit: Diese experimentellen Arbeiten zeigen somit, dass Amyloidstruktur-verändernde, niedermolekulare Substanzen, die direkt an intrazelluläre Aβ42 Aggregate binden, einen vielversprechenden Ansatz zur Reduktion der intrazellulären Aβ42 Last darstellen. Dieser Ansatz könnte genutzt werden, um bei proteinopathischen neurodegenerativen Erkrankungen wie der amyotrophen Lateralsklerose, Chorea Huntington, Parkinson und der Alzheimer Erkrankung die zelleigene Verstoffwechslung von fehlgefalteten, intrazellulär-akkumulierenden Proteinen im lysosomalen Kompartiment zu stärken

Novel multi-objective affinity approach allows to identify pH-specific μ-opioid receptor agonists

Opioids are essential pharmaceuticals due to their analgesic properties, however, lethal side effects, addiction, and opioid tolerance are extremely challenging. The development of novel molecules targeting the -opioid receptor (MOR) in inflamed, but not in healthy tissue, could significantly reduce these unwanted effects. Finding such novel molecules can be achieved by maximizing the binding affinity to the MOR at acidic pH while minimizing it at neutral pH, thus combining two conflicting objectives. Here, this multi-objective optimal affinity approach is presented, together with a virtual drug discovery pipeline for its practical implementation. When applied to finding pH-specific drug candidates, it combines protonation state-dependent structure and ligand preparation with high-throughput virtual screening. We employ this pipeline to characterize a set of MOR agonists identifying a morphine-like opioid derivative with higher predicted binding affinities to the MOR at low pH compared to neutral pH. Our results also confirm existing experimental evidence that NFEPP, a previously described fentanyl derivative with reduced side effects, and recently reported -fluorofentanyls and -morphines show an increased specificity for the MOR at acidic pH when compared to fentanyl and morphine. We further applied our approach to screen a >50K ligand library identifying novel molecules with pH-specific predicted binding affinities to the MOR. The presented differential docking pipeline can be applied to perform multi-objective affinity optimization to identify safer and more specific drug candidates at large scale

Methods of a national colorectal cancer cohort study: the PIPER Project

A national study looking at bowel cancer in New Zealand has previously been completed (the PIPER Project). The study included 5,610 patients and collected medical information about how each person was found to have bowel cancer and the treatment they received. This paper reports how the study was carried out. The information collected in the study will be used to look at the quality of care being provided to New Zealand patients with bowel cancer, and to find out if differences in care occur based on where people live, their ethnicity and their socioeconomic status

GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema

Information literacy and RSS feeds at LSE

This chapter describes how RSS has been used at the London School of Economic and Political Science (LSE) to enhance access to information on training courses, including information literacy classes, for staff and students. RSS underpins much of what we recognise as Web 2.0 and social software. In his recent book, Bradley (2007) argues that in order to fully exploit social software in libraries, it is essential to understand RSS technology. Even a basic understanding can allow all librarians, not just those running a schedule of teaching or training, to make their information more accessible. However, our experiences show that RSS has information literacy implications: users must re-think how they access information on the web. Rather than visiting a website to see what is new, users are afforded a mechanism for picking up new information automatically. The tools required, while simple to use, arguably do require a greater level of information literacy on the part of users. Therefore, paradoxically, while RSS has provided LSE with an opportunity to make training information more widely available, staff and students need greater information literacy skills to fully exploit the technolog

Novel multi-objective affinity approach allows to identify pH-specific μ-opioid receptor agonists

Abstract Opioids are essential pharmaceuticals due to their analgesic properties, however, lethal side effects, addiction, and opioid tolerance are extremely challenging. The development of novel molecules targeting the $$\mu$$ μ -opioid receptor (MOR) in inflamed, but not in healthy tissue, could significantly reduce these unwanted effects. Finding such novel molecules can be achieved by maximizing the binding affinity to the MOR at acidic pH while minimizing it at neutral pH, thus combining two conflicting objectives. Here, this multi-objective optimal affinity approach is presented, together with a virtual drug discovery pipeline for its practical implementation. When applied to finding pH-specific drug candidates, it combines protonation state-dependent structure and ligand preparation with high-throughput virtual screening. We employ this pipeline to characterize a set of MOR agonists identifying a morphine-like opioid derivative with higher predicted binding affinities to the MOR at low pH compared to neutral pH. Our results also confirm existing experimental evidence that NFEPP, a previously described fentanyl derivative with reduced side effects, and recently reported $$\beta$$ β -fluorofentanyls and -morphines show an increased specificity for the MOR at acidic pH when compared to fentanyl and morphine. We further applied our approach to screen a >50K ligand library identifying novel molecules with pH-specific predicted binding affinities to the MOR. The presented differential docking pipeline can be applied to perform multi-objective affinity optimization to identify safer and more specific drug candidates at large scale

A proof‐of‐concept study of the removal of early and late phase biofilm from skin wound models using a liquid acoustic stream

Chronic wounds fail to progress through the normal stages of healing, with the largest remediable cause of chronicity being presence of a multi-species biofilm. Removal of biofilm from the wound environment is central to wound care. A device for mechanically removing biofilms from wounds has been devised. The removal is caused by small-scale liquid currents and shear, generated by acoustically activated microscopic air bubbles. These bubbles and acoustic waves are delivered onto the wound by a gentle liquid stream, allowing cleaning in situ and removal of debris in the run-off liquid. We have investigated if this liquid acoustic wound stream (LAWS) can remove bacterial biofilm from soft biological wound models and studied the effect of LAWS on the cellular tissues of the substrate. LAWS will efficiently remove early Pseudomonas aeruginosa biofilm from an artificial wound in a pig's trotter, 24 hours-mature biofilm of P. aeruginosa from a pre-wounded human full thickness skin model (EpiDerm FT), and 3-day mature biofilm of P. aeruginosa or Staphylococcus aureus from a porcine skin explant. Histological examinations of uninfected EpiDerm models that had been treated by LAWS and then stained with Haematoxylin and Eosin, demonstrated no damage to the human tissue, and wound diameter was smaller in the treated skin models compared with untreated samples. Immunofluorescence staining for cytokeratin 14 showed that keratinocytes had migrated further across the wound in the uninfected samples treated by LAWS. We discuss the implications for wound healing and propose further laboratory and clinical studies to demonstrate the removal of biofilm from patients with chronic leg ulcers and the impact on healing.</p

Dataset for: &quot;A proof&#x2010;of&#x2010;concept study of the removal of early and late phase biofilm from skin wound models using a liquid acoustic stream&quot;

This dataset contains: The raw data of the image analysis of the percentage coverage measurements in figures 3 and 4, and the measurements of re-epithelialisation in figures 6 and 7</span

Diagnosis, Treatment, and Outcome of Arterioureteral Fistula: The Urologist's Perspective

Introduction: Arterioureteral fistula (AUF) is a rare but potentially life-threatening disease that primarily arises as a long-term complication in oncological patients who have permanent ureteral stenting. The incidence is rising. The objective of this study was to outline the risk factors for management and outcome of AUF in a large individual case series. Patients and Methods: Twenty-six AUF cases in 24 patients from six German tertiary referral centers occurring between 2008 and 2016 were identified retrospectively and entered into a dedicated database by using patient notes and out-patient visits. Results: Of 24 patients, 23 had a history of abdominopelvic surgery for oncological disease, 21/24 had undergone radiotherapy, and 23/24 had long-term ureteral stenting. All cases presented with visible hematuria, 11/26 at the time of a stent exchange. Blood transfusions were required in 92.3%, and intravenous inotropes were needed in 46.2%. Of 26 patients, 11 had flank pain. CT angiogram was positive in 35.7%. Angiography and endovascular fistula repair was performed in 88.5%, and the rest received open surgical repair. Mortality was 7.7%. Endovascular treatment was technically successful in 91.3%, and open surgery was successful in 3/4 cases. Recurrent AUF developed in 3/24 patients. Stent-related complications occurred in 15%. Vascular complications were common. Long-term survival was limited due to progression of the underlying malignant disease. Conclusion: AUF results in major hemorrhage and warrants time-efficient diagnosis and treatment. Awareness is key. When AUF is considered, interventional angiography should promptly be performed. Fistula detection can be improved by guidewire manipulation. Pre-interventional CT angiogram may be omitted due to low sensitivity. Endovascular repair with stenting and/or coiling is effective and safe