From cell penetrating peptides to peptoids and polyamines as novel artificial molecular transporters

In recent years, RNA interference has gained a lot of importance as a tool for posttranscriptional silencing of genes due to its high specificity, efficiency and ease of application. In mammalian cells RNAi is triggered by the application of 21 bp short dsRNAs, so-called short interfering RNAs (siRNAs). Numerous studies indicate the great potential of RNAi in the therapy of viral infections and inherited diseases. Cell-penetrating peptides can be used to apply siRNAs to primary cells, non-dividing cells or fully grown organisms, that are diffcult to transfect. These short positively charged peptides are internalized by cells. They can be detected in the endosomes, lysosomes but also in the cytosol. If attached to CPPs, large cargo molecules can be taken up with a high efficiency that surpasses that of most conventional transfection methods. In the work presented here, peptide-coupled siRNAs (pepsiRNAs) have been developed as a novel tool for transient RNAi in mammalian cells. The peptides were attached to the siRNA by a disulfide bond, that is cleaved under the reducing conditions of the cytosol and thus releases the siRNA cargo. PepsiRNAs are readily taken up by many cell types that are difficult to address by conventional transfection methods. An siRNA-induced downregulation of the targeted genes was observed at concentrations between 10 and 100 nM. SiRNAs with a 5´-thiol modification upon their sense-strand were generated by in vitro transcription, for which a thiol-modified nucleotide was synthesized via an optimized route. The cell-penetrating peptides Penetratin and Tat were recombinantly expressed as fusion-proteins with glutathione-S-transferase (GST) and purified with a modified procedure to overcome the strong membrane interaction of the GST-tagged CPPs. Recombinant TEV protease was expressed to cleave the CPPs from the fusion tag, and the cleavage activity was assessed by comparison with commercially obtained TEV protease. Thus, alternative routes to the building blocks for pepsiRNAs have been provided to scale up the amount of pepsiRNAs. Finally, small molecules with cell-penetrating properties have been developed as a future replacement of the peptide moiety. Fluorescently labeled peptoids of differing length with amine-functionalized side chains have been shown to enter different mammalian cells lines at concentrations in the lower micromolar range by an endocytosis dependent mechanism. Cationic molecules as small as spermine attached to fluorescein are taken up by an endocytosis-related mechanism. By the same approach porphyrins were delivered into the interior of the cells, where they exhibited a cytotoxic effect upon illumination, so that spermine-coupled porphyrins may be developed into a novel drug for photodynamic therapy.Entwicklung neuer Delivery-Strategien für siRNAs - Von zellpenetrierenden Peptiden zu Peptoiden und Polyaminen als neuartige molekulare Transporter In den vergangenen Jahren, hat die RNA Interferenz (RNAi) aufgrund ihrer Spezifität, Effizienz und einfachen Anwendbarkeit eine große Bedeutung als Technik für das posttranskriptionale Gene-Silencing gewonnen. In Säugerzellen wird RNAi durch die Gabe von 21 bp kurzen doppelsträngigen RNAs, sogenannten short interfering RNAs (siRNAs), ausgelöst. Zahlreiche Studien belegen das große Potential dieser Methode für die Therapie viraler Infektionen und Erbkrankheiten. Um siRNAs auch in schlecht transfizierbare Systeme wie primäre und nicht-teilende Zellen bzw. ausgewachsene Organismen einzubringen, können zellpenetrierende Peptide (CPPs) verwendet werden. Diese kurzen positiv geladenen Peptide werden von Zellen aufgenommen, wo sie in Endosomen, Phagosomen, aber auch im Zytosol detektierbar sind. Verknüpft mit CPPs werden große Moleküle mit hoher Effizienz von Zellen aufgenommen, die die der meisten konventionellen Transfektionsmethoden übertrifft. In dieser Arbeit wurden peptidgekuppelte siRNAs (pepsiRNAs) als neue Methode zur transienten RNAi in Säugerzellen entwickelt. Hierzu wurden die Peptide über eine Disulfidbrücke mit den siRNAs verknüpft, unter den reduzierenden Bedingungen des Zytosols gespalten wird und , so dass die siRNA im Innern der Zelle freigesetzt wird. PepsiRNAs werden von einer Reihe von Zelltypen aufgenommen, die mit konventionellen Techniken nur schlecht behandelbar sind. Eine deutliche Herunterregulation der Ziel-Genprodukte wurde in einem Konzentrationsbereich von 10 -100 nM beobachtet. SiRNAs mit einer 5´-Thiolmodifikation am sense-Strang wurden durch in vitro Transkription gewonnen, für die ein thiolmodifizierten Nucleotid synthetisiert und dessen Synthese optimiert wurde. Die zellpenetrierenden Peptide Penetratin und Tat wurden rekombinant als Fusionproteine mit Gluatathion-S-Transferase (GST) exprimiert und mit modifizierten Verfahren gereinigt, um die starken Wechselwirkungen der GST-CPPs mit den Zellmembranen zu überwinden. Um die CPPs vom GST-Fusionstag zu trennen, wurde rekombinante TEV-Protease exprimiert und die Aktivität mit der von kommerziell erhältlicher TEV-Protease verglichen. Somit stehen nun Alternativen zur Festphasensynthese zur Verfügung, um die Bestandteile der pepsiRNAs in größeren Mengen herzustellen. Schließlich wurden kleine Moleküle mit zellpenetrierenden Eigenschaften als Ersatz für die Peptideinheit entwickelt. Es konnte gezeigt werden, dass fluoreszenzmarkierte Peptoide verschiedener Länge mit aminofunktionalisierten Seitenketten in Konzentrationen von einigen 10 µM von verschiedenen Säugerzelllinien durch einen endozytoseabhängigen Mechanismus aufgenommen werden. Selbst kleine positiv geladene Moleküle wie fluoresceinmarkiertes Spermin werden über einen endozytoseartigen Mechanismus aufgenommen. Auf diese Weise konnten auch Porphyrine ins Innere der Zellen gebracht werden, wo sie bei Belichtung ihre zytotoxische Wirkung entfalten, so dass sie als neuartiger Wirkstoff für die Photodynamische Therapie weiterentwickelt werden können

Lesiones no intencionadas infantiles en asentamientos precarios: campo de refugiados

Las lesiones no intencionadas son una causa de morbimortalidad emergente en niños y adultos jóvenes. El riesgo de lesiones aumenta en entornos precarios como lo son los campos de refugiados en los que, además, más de la mitad de la población desplazada en el mundo son niños. Los pocos datos publicados sobre lesiones no intencionadas en campos de refugiados sugieren una incidencia entre un 2,2 y un 39% de las consultas, siendo la causa más frecuente las caídas. El objetivo de este trabajo es determinar la incidencia de lesiones no intencionadas, sus condicionantes y las medidas preventivas en el asentamiento de Lóvua (Angola). Se realizó un análisis descriptivo retrospectivo de datos epidemiológicos de la actividad de 5 meses de las clínicas del campo y un análisis descriptivo cuantitativo y cualitativo de los factores de riesgo y medidas preventivas en el asentamiento. En Lóvua las lesiones no intencionadas representan el 1,02 % de las primeras consultas médicas, pero producen un elevado número de visitas en enfermería suponiendo un 88% de todas las curas. El 72% de las consultas de menores de 18 años por lesiones en enfermería son por traumáticas no intencionadas y el 28% por quemaduras. Los menores varones consultaron 1,56 veces más por lesiones traumáticas no intencionadas que las niñas (3,5 veces más en adultos). En el caso de las quemaduras las niñas consultaron 1,3 veces más que los niños (1,7 veces más en adultos). Se detectó un número relativamente alto de lesiones por animales incluyendo 2 fallecimientos por serpiente. En el asentamiento destacan factores de riesgo como la baja supervisión de los niños, cocinas a fuego abierto, fosas familiares no valladas, ausencia de zonas de juego seguras y presencia de animales venenosos. Factores protectores destacables son el adecuado espaciamiento y el empleo de energía solar. Para un análisis más preciso de las lesiones no intencionadas en el asentamiento de Lóvua haría falta una recogida de datos más detallada. Parece útil incluir el análisis de riesgos de lesiones no intencionadas en la planificación de campos de refugiados para poder aplicar medidas de prevención y mitigación adicionales a las recogidas en las guías según cada contexto específico.Unintentional injuries are an emerging cause of morbidity and mortality in children and young adults. The risk of injuries increases in precarious environments such as refugee camps. Moreover, more than half of the displaced population in the world are children. The few published data on unintentional injuries in refugee camps suggest an incidence between 2.2% and 39% of consultations and that falls are the leading cause. The goal of this work is to explore the incidence of unintentional injuries, their conditioners and preventive measures in the settlement of Lóvua (Angola). A retrospective descriptive analysis of epidemiological data from 5 months clinical activity and a quantitative and qualitative descriptive analysis of risk factors and preventive measures in the settlement were performed. In Lóvua unintentional injuries represent 1.02% of the first medical consultations, but they produce a high number of visits in nursing assuming 88% of all dressings. 72% of consultations of children (under 18 years of age) due to unintentional injuries in nursing are traumatic events and 28% are burns. Male minors consulted 1,56 times more for unintentional traumatic injuries than women (3,5 times in adults). In the case of burns feminine minors consulted 1,3 times more than males (1,7 times in adults). A relatively high number of animal-caused injuries was detected, including 2 deaths per snake. In the settlement of Lóvua the outstanding risk factors found were low supervision of children, open fire cooking, unfenced family pits, absence of safe play areas and the presence of poisonous animals. Outstanding protection factors were the adequate spacing and the use of solar energy. For a more accurate analysis of unintentional injuries in the settlement of Lóvua, further studies with detailed data collection would be necessary. It seems useful to include injury risk assessment in refugee camp planning, in order to apply additional prevention and mitigation measures to those already included in the humanitarian guides, according to each specific context.Máster Universitario en Acción Humanitaria Sanitaria (M161

Making sure that the emigration of healthcare personnel from Albania and BiH works for all: what Germany can do

The migration of healthcare professionals has detrimental impacts on socio-economic development in Albania and Bosnia-Herzegovina (BiH). Germany’s active recruitment of healthcare professionals contributes to this trend and thus adversely affects the goals of bilateral development cooperation. The Ministry of Economic Cooperation and Development should engage with the Ministries of Health and of Labour and Social Affairs to bolster the sustainability of the government’s inter-agency strategy on the recruitment of qualified workers (2019). A detailed whole-of-government approach needs to be put in place that further assesses and prevents adverse long-term demographic and socio-economic effects of emigration of care personnel in origin countries. The German Ministry of Health must ensure that legal and ethical standards for the recruitment of international personnel are observed. It should support the governments of Albania and BiH in monitoring the private mediation companies’ training and recruitment practices of nurses and medical technicians to avoid exploitative practices and adhesion contracts. The German Ministry for Economic Cooperation and Development should support programmes that provide equipment and know-how. Options for career advancement and training, safety measures and legal protection are the most urgent issues to prevent a care drain and need for medical tourism. The German Ministry for Economic Cooperation and Development should support such reforms through bilateral cooperation (technical advice) with Albania and BiH to counteract distortions of the labour market due to out-migration and to aid structural economic recovery. The attractiveness of alternative apprenticeships and job profiles needs to be increased, not least because the long-term attachment and increase of skilled workforce will positively affect the investment climate

Comparison of Calculated with Measured Oxygen Consumption in Children Undergoing Cardiac Catheterization

Our objective was to compare calculated (LaFarge) with measured oxygen consumption (VO2) using the AS/3 TM Compact Airway Module M-CAiOVX (Datex-Ohmeda, Helsinki, Finland; AS/3 TM) in children without cardiac shunts in a prospective, observational study. VO2 was determined at the end of the routine diagnostic and/or interventional catheterization. VO2was calculated according to the formula of LaFarge and Miettinen for each child and compared with the measured VO2. Data were compared using simple regression and Bland Altman analysis. Fifty-two children aged from 0.5 to 16years (median, 6.9years) and weighing 3.4 to 59.4kg (median, 22.9kg) were investigated. Calculated VO2values ranged from 59.0 to 230.8ml/min, and measured VO2 values from 62.7 to 282.2ml/min. Comparison of calculated versus measured VO2 values revealed a significant correlation (r=0.90, p<0.0001). Bias and precision were 8.9 and 48.3ml/min, respectively (95% limits of agreement: −39.4 to 57.2ml/min). Comparison of calculated VO2 in children older than 3 years (n=41), as restricted to the formula, with measured VO2, revealed a slightly reduced correlation (r=0.86, p<0.0001). Bias and precision were 10.0 and 52.5ml/min, respectively (95% limits of agreement: -42.4 to 62.5ml/min). We conclude that calculation of VO2 by the LaFarge formula does not provide reliable values compared to measured values. In clinical routine, measured rather than calculated VO2 values should be used for the estimation of cardiac output and related variable

Reduced vagal activity in borderline personality disorder is unaffected by intranasal oxytocin administration, but predicted by the interaction between childhood trauma and attachment insecurity

Individuals with borderline personality disorder (BPD) show self-regulatory deficits, associated with reduced heart-rate variability (HRV). However, results on reduced HRV in BPD remain heterogeneous, thus encouraging the search for developmental constructs explaining this heterogeneity. The present study first examined predictors of reduced resting-state HRV in BPD, namely the interaction between self-reported adult attachment insecurity and childhood trauma. Second, we investigated if alterations in resting-state HRV are modified by intranasal oxytocin administration, as oxytocin may enhance HRV and is implicated in the interaction between childhood trauma and disturbed attachment for the pathogenesis of BPD. In a randomized, placebo-controlled trial, 53 unmedicated women with BPD and 60 healthy controls (HC) self-administered either 24 I.U. of oxytocin or placebo and underwent a 4-min electrocardiogram. Our results replicate significantly reduced HRV in women with BPD, explained up to 16% by variations in childhood trauma and attachment insecurity. At high levels of acute attachment insecurity, higher levels of childhood trauma significantly predicted reduced HRV in BPD. However, our results do not support a significant effect of oxytocin on mean HRV, and no interaction effect emerged including childhood trauma and attachment insecurity. Our findings highlight a complex interaction between reduced vagal activity and developmental factors in BPD

An Additively Manufactured Sample Holder to Measure the Controlled Release of Vancomycin from Collagen Laminates

The controlled release of antibiotics prevents the spread of pathogens and thereby improves healing processes in regenerative medicine. However, high concentrations may interfere with healing processes. It is therefore advantageous to use biodegradable materials for a controlled release. In particular, multilayer materials enable differential release at different surfaces. For this purpose, collagen sheets of different properties can be bonded by photochemical crosslinking. Here, we present the development and application of an easily accessible, additively manufactured sample holder to study the controlled release of vancomycin from modularly assembled collagen laminates in two directions. As proof-of-concept, we show that laminates of collagen sheets covalently linked by rose bengal and green light crosslinking (RGX) can be tightly inserted into the device without leakage from the upper to lower cavity. We used this sample holder to detect the release of vancomycin from symmetrically and asymmetrically loaded two-layer and three-layer collagen laminates into the upper and lower cavity of the sample holder. We show that these collagen laminates are characterized by a collagen type-dependent vancomycin release, enabling the control of antibiotic release profiles as well as the direction of antibiotic release

Oxygen carriers affect kidney immunogenicity during ex-vivo machine perfusion

Normothermic ex-vivo machine perfusion provides a powerful tool to improve donor kidney preservation and a route for the delivery of pharmacological or gene therapeutic interventions prior to transplantation. However, perfusion at normothermic temperatures requires adequate tissue oxygenation to meet the physiological metabolic demand. For this purpose, the addition of appropriate oxygen carriers (OCs) to the perfusion solution is essential to ensure a sufficient oxygen supply and reduce the risk for tissue injury due to hypoxia. It is crucial that the selected OCs preserve the integrity and low immunogenicity of the graft. In this study, the effect of two OCs on the organ's integrity and immunogenicity was evaluated. Porcine kidneys were perfused ex-vivo for four hours using perfusion solutions supplemented with red blood cells (RBCs) as conventional OC, perfluorocarbon (PFC)-based OC, or Hemarina-M101 (M101), a lugworm hemoglobin-based OC named HEMO2life®, recently approved in Europe (i.e., CE obtained in October 2022). Perfusions with all OCs led to decreased lactate levels. Additionally, none of the OCs negatively affected renal morphology as determined by histological analyses. Remarkably, all OCs improved the perfusion solution by reducing the expression of pro-inflammatory mediators (IL-6, IL-8, TNFα) and adhesion molecules (ICAM-1) on both transcript and protein level, suggesting a beneficial effect of the OCs in maintaining the low immunogenicity of the graft. Thus, PFC-based OCs and M101 may constitute a promising alternative to RBCs during normothermic ex-vivo kidney perfusion

No Evidence of Persisting Unrepaired Nuclear DNA Single Strand Breaks in Distinct Types of Cells in the Brain, Kidney, and Liver of Adult Mice after Continuous Eight-Week 50 Hz Magnetic Field Exposure with Flux Density of 0.1 mT or 1.0 mT

BACKGROUND: It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields (50 or 60 Hz) may lead to human health effects such as childhood leukemia or brain tumors. In a previous study investigating multiple types of cells from brain and kidney of the mouse (Acta Neuropathologica 2004; 107: 257-264), we found increased unrepaired nuclear DNA single strand breaks (nDNA SSB) only in epithelial cells of the choroid plexus in the brain using autoradiographic methods after a continuous eight-week 50 Hz magnetic field (MF) exposure of adult mice with flux density of 1.5 mT. METHODS: In the present study we tested the hypothesis that MF exposure with lower flux densities (0.1 mT, i.e., the actual exposure limit for the population in most European countries, and 1.0 mT) shows similar results to those in the previous study. Experiments and data analysis were carried out in a similar way as in our previous study. RESULTS: Continuous eight-week 50 Hz MF exposure with 0.1 mT or 1.0 mT did not result in increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice. MF exposure with 1.0 mT led to reduced unscheduled DNA synthesis (UDS) in epithelial cells in the choroid plexus of the fourth ventricle in the brain (EC-CP) and epithelial cells of the cortical collecting duct in the kidney, as well as to reduced mtDNA synthesis in neurons of the caudate nucleus in the brain and in EC-CP. CONCLUSION: No evidence was found for increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT