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Summary: Background: A better understanding of the molecular events during liver normothermic machine perfusion (NMP) is warranted to develop a data-based approach for the identification of biomarkers representative of graft quality and posttransplant outcome. We analysed the dynamic transcriptional changes during NMP and linked them to clinical and biochemical parameters. Methods: 50 livers subjected to NMP for up to 24 h were enrolled. Bulk RNA sequencing was performed in serial biopsies collected pre and during NMP, and after reperfusion. Perfusate was sampled to monitor liver function. qPCR and immunohistochemistry were performed to validate findings. Molecular profiles were compared between transplanted and non-transplanted livers, and livers with and without early allograft dysfunction. Findings: Pathways related to immune and cell stress responses, cell trafficking and cell regulation were activated during NMP, while cellular metabolism was downregulated over time. Anti-inflammatory responses and genes involved in tissue remodelling were induced at later time-points, suggesting a counter-response to the immediate damage. NMP strongly induced a gene signature associated with ischemia-reperfusion injury. A 7-gene signature corresponds with the benchmarking criteria for transplantation or discard at 6 h NMP (area under curve 0.99). CD274 gene expression (encoding programmed cell-death ligand-1) showed the highest predictive value. LEAP2 gene expression at 6 h NMP correlated with impaired graft function. Interpretation: Assessment of gene expression markers could serve as a reliable tool to evaluate liver quality during NMP and predicts early graft function after transplantation. Funding: The research was supported by “In Memoriam Dr. Gabriel Salzner Stiftung”, Tiroler Wissenschaftsfond, Jubiläumsfonds-Österreichische Nationalbank and MUI Start grant.

The current dropout rate in physics studies in Germany is about 60%, with the majority of dropouts occurring in the first year. Consequently, the physics study entry phase poses a significant challenge for many students. Students’ stress perceptions can provide more profound insights into the processes and challenges during that period. In a panel study featuring 67 measuring points involving up to 128 participants at each point, we investigated students’ stress perceptions with the perceived stress questionnaire (PSQ), identified underlying sources of stress, and assessed self-estimated workloads across two different cohorts. This examination occurred almost every week during the first semester, and for one cohort also in the second semester, yielding a total of 3241 PSQ data points and 5823 stressors. The PSQ data indicate a consistent stress trajectory across all three groups studied that is characterized by significant dynamics between measuring points, spanning from M=20.1,SD=15.9 to M=63.6,SD=13.4 on a scale from 0 to 100. Stress levels rise in the first weeks of the lecture, followed by stable, elevated stress levels until the exams and a relaxation phase afterward during the lecture-free time and Christmas vacation. In the first half of the lecture period, students primarily indicated the weekly exercise sheets, the physics lab course, and math courses as stressors; later on, preparation for exams and the exams themselves emerged as the most important stressors. Together with the students’ self-estimated workloads that correlate with the PSQ scores, we can create a coherent picture of stress perceptions among first-year physics students, which builds the basis for supportive measures and interventions.

Organ quality can be assessed prior to transplantation, during normothermic machine perfusion (NMP) of the liver. Evaluation of mitochondrial function by high-resolution respirometry (HRR) may serve as a viability assessment concept in this setting. Freshly collected tissue is considered as optimal sample for HRR, but due to technical and personnel requirements, more flexible and schedulable measurements are needed. However, the impact of cold storage following NMP before processing biopsy samples for mitochondrial analysis remains unknown. We aimed at establishing an appropriate storage protocol of liver biopsies for HRR. Wedge biopsies of 5 human livers during NMP were obtained and assessed by HRR. Analysis was performed after 0, 4, 8, and 12 h of hypothermic storage (HTS) in HTK organ preservation solution at 4°C. With HTS up to 4 h, mitochondrial performance did not decrease in HTS samples compared with 0 h (OXPHOS, 44.62 [34.75–60.15] pmol·s−1·mg wet mass−1 vs. 43.73 [40.69–57.71], median [IQR], p > 0.999). However, at HTS beyond 4 h, mitochondrial respiration decreased. We conclude that HTS can be safely applied for extending the biopsy measurement window for up to 4 h to determine organ quality, but also that human liver respiration degrades beyond 4 h HTS following NMP.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (−0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

Mitochondrial quality control mechanisms prevent damage accumulation, including in mitochondrial DNA (mtDNA). Here, Sen et al. show that altered mtDNA elicits local rearrangements in mitochondrial membrane potential and cristae structure, with mtDNA eliminated through VPS35 endosomes.

Summary: Background: Reliable biomarkers for organ quality assessment during normothermic machine perfusion (NMP) are desired. ATP (adenosine triphosphate) production by oxidative phosphorylation plays a crucial role in the bioenergetic homeostasis of the liver. Thus, detailed analysis of the aerobic mitochondrial performance may serve as predictive tool towards the outcome after liver transplantation. Methods: In a prospective clinical trial, 50 livers were subjected to NMP (OrganOx Metra) for up to 24.ßh. Biopsy and perfusate samples were collected at the end of cold storage, at 1.ßh, 6.ßh, end of NMP, and 1.ßh after reperfusion. Mitochondrial function and integrity were characterized by high-resolution respirometry (HRR), AMP, ADP, ATP and glutamate dehydrogenase analysis and correlated with the clinical outcome (L-GrAFT score). Real-time confocal microscopy was performed to assess tissue viability. Structural damage was investigated by histology, immunohistochemistry and transmission electron microscopy. Findings: A considerable variability in tissue viability and mitochondrial respiration between individual livers at the end of cold storage was observed. During NMP, mitochondrial respiration with succinate and tissue viability remained stable. In the multivariate analysis of the 35 transplanted livers (15 were discarded), area under the curve (AUC) of LEAK respiration, cytochrome c control efficiency (mitochondrial outer membrane damage), and efficacy of the mitochondrial ATP production during the first 6.ßh of NMP correlated with L-GrAFT. Interpretations: Bioenergetic competence during NMP plays a pivotal role in addition to tissue injury markers. The AUC for markers of outer mitochondrial membrane damage, ATP synthesis efficiency and dissipative respiration (LEAK) predict the clinical outcome upon liver transplantation. Funding: This study was funded by a Grant from the In Memoriam Dr. Gabriel Salzner Stiftung awarded to SS and the Tiroler Wissenschaftsfond granted to TH.

The majority of organs used for liver transplantation come from brain-dead donors (DBD). In order to overcome the organ shortage, increasingly donation after circulatory death (DCD) organs are also considered. Since normothermic machine perfusion (NMP) restores metabolic activity and allows for in-depth assessment of organ quality and function prior to transplantation, such organs may benefit from NMP. We herein compare the bioenergetic performance through a comprehensive evaluation of mitochondria by high-resolution respirometry in tissue biopsies and the inflammatory response in DBD and DCD livers during NMP. While livers were indistinguishable by perfusate biomarker assessment and histology, our findings revealed a greater impairment of mitochondrial function in DCD livers after static cold storage compared to DBD livers. During subsequent NMPs, DCD organs recovered and eventually showed a similar performance as DBD livers. Cytokine expression analysis showed no differences in the early phase of NMP, while towards the end of NMP, significantly elevated levels of IL-1β, IL-5 and IL-6 were found in the perfusate of DCD livers. Based on our results, we find it worthwhile to reconsider more DCD organs for transplantation to further extend the donor pool. Therefore, donor organ quality criteria must be developed, which may include an assessment of bioenergetic function and cytokine quantification.

Abstract Background Muscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration. Methods A clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration. Results 2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI. Conclusion WIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

The implementation of ex vivo organ machine perfusion (MP) into clinical routine undoubtedly helped to increase the donor pool. It enables not just organ assessment, but potentially regeneration and treatment of marginal organs in the future. During organ procurement, redox-stress triggered ischemia-reperfusion injury (IRI) is inevitable, which in addition to pre-existing damage negatively affects such organs. Ex vivo MP enables to study IRI-associated tissue damage and its underlying mechanisms in a near to physiological setting. However, research using whole organs is limited and associated with high costs. Here, in vitro models well suited for early stage research or for studying particular disease mechanisms come into play. While cell lines convince with simplicity, they do not exert all organ-specific functions. Tissue slice cultures retain the three-dimensional anatomical architecture and cells remain within their naïve tissue-matrix configuration. Organoids may provide an even closer modelling of physiologic organ function and spatial orientation. In this review, we discuss the role of oxidative stress during ex vivo MP and the suitability of currently available in vitro models to further study the underlying mechanisms and to pretest potential treatment strategies.

In diesem Beitrag wird die strategische Partnerschaft zwischen dem Institut für Erziehungswissenschaft der Universität Kassel und Versuchsschulen des Landes Hessen präsentiert. Dazu werden die Akteure der Zusammenarbeit vorgestellt. Es wird die Form der Zusammenarbeit erläutert und an zwei Forschungsprojekten illustriert. Erkennbar wird, dass durch die unterschiedlichen Formate der Zusammenarbeit Impulse für die Entwicklung von Schulen und für die Gewinnung erziehungswissenschaftlicher Erkenntnisse entstehen. The article presents the strategic partnership between the Institute of Educational Science of the University of Kassel and experimental schools of the state of Hesse. For this purpose, the actors of the cooperation are presented. The form of cooperation is explained and illustrated by two research projects. It becomes apparent that the different formats of cooperation generate impulses for school development and for the acquisition of educational science knowledge.

Bio-polymer gelatine can be found in a broad variety of applications, mostly in the food industry. Moreover, it is used in the encapsulation of active pharmaceutical ingredients. In electrospinning, it is used for drug release, but can also strongly influence the morphologies of nanofiber mats when blended with other polymers. In a recent project, we studied the influence of adding citric acid and coconut oil to gelatine electrospinning solutions. While the former can be used to modify gelatine nanofiber diameters and create diverse morphologies between the fibres and sprayed droplets of different shapes, the latter results in an electrospraying process and additionally increases water resistance, suggesting a possible use of the combination for tailored drug release.

The liver, in combination with a functional biliary system, is responsible for maintaining a great number of vital body functions. However, acute and chronic liver diseases may lead to irreversible liver damage and, ultimately, liver failure. At the moment, the best curative option for patients suffering from end-stage liver disease is liver transplantation. However, the number of donor livers required by far surpasses the supply, leading to a significant organ shortage. Cellular therapies play an increasing role in the restoration of organ function and can be integrated into organ transplantation protocols. Different types and sources of stem cells are considered for this purpose, but highly specific immune cells are also the focus of attention when developing individualized therapies. In-depth knowledge of the underlying mechanisms governing cell differentiation and engraftment is crucial for clinical implementation. Additionally, novel technologies such as ex vivo machine perfusion and recent developments in tissue engineering may hold promising potential for the implementation of cell-based therapies to restore proper organ function.

Transplantation represents the treatment of choice for many end-stage diseases but is limited by the shortage of healthy donor organs. Ex situ normothermic machine perfusion (NMP) has the potential to extend the donor pool by facilitating the use of marginal quality organs such as those from donors after cardiac death (DCD) and extended criteria donors (ECD). NMP provides a platform for organ quality assessment but also offers the opportunity to treat and eventually regenerate organs during the perfusion process prior to transplantation. Due to their anti-inflammatory, immunomodulatory and regenerative capacity, mesenchymal stem cells (MSCs) are considered as an interesting tool in this model system. Only a limited number of studies have reported on the use of MSCs during ex situ machine perfusion so far with a focus on feasibility and safety aspects. At this point, no clinical benefits have been conclusively demonstrated, and studies with controlled transplantation set-ups are urgently warranted to elucidate favorable effects of MSCs in order to improve organs during ex situ machine perfusion.

Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders in mammals that are caused by unconventional agents predominantly composed of aggregated misfolded prion protein (PrP). Prions self-propagate by recruitment of host-encoded PrP into highly ordered b-sheet rich aggregates. Prion strains differ in their clinical, pathological and biochemical characteristics and are likely to be the consequence of distinct abnormal prion protein conformers that stably replicate their alternate states in the host cell. Understanding prion cell biology is fundamental for identifying potential drug targets for disease intervention. The development of permissive cell culture models has greatly enhanced our knowledge on entry, propagation and dissemination of TSE agents. However, despite extensive research, the precise mechanism of prion infection and potential strain effects remain enigmatic. This review summarizes our current knowledge of the cell biology and propagation of prions derived from cell culture experiments. We discuss recent findings on the trafficking of cellular and pathologic PrP, the potential sites of abnormal prion protein synthesis and potential co-factors involved in prion entry and propagation.

Infective second-stage juveniles of the obligate plant-parasitic root-knot and cyst nematodes invade plant roots to induce specialized feeding structures. Here, we present data on the distribution of plasmodesmata in cell walls of syncytia and giant cells induced by cyst and root-knot nematodes. An Arabidopsis and a tobacco line were used, containing viral movement proteins fused to green fluorescent protein as a localization marker for plasmodesmata. Plasmodesmata were detected in walls between giant cells but also in walls toward neighboring cells. In syncytia, plasmodesmata were mainly detected at later stages. In young syncytia, few plasmodesmata were observed and a specific temporal callose deposition along plasmodesmata indicated impaired symplasmic exchange. In order to study the relevance of callose deposition for successful cyst nematode development in Arabidopsis, two mutant lines inhibited in callose synthesis and degradation, respectively, were used in nematode infection assays. Histological analyses showed that syncytia were smaller when callose degradation was reduced, indicating a significant importance of this process to cyst nematode development.

Abstract Following publication of the original article [1], the authors reported that there was a mistake in the Methods section → Measurements of biomarkers (page 6, line 3): 2000g should read 2500g. So, the correct sentence should be “All samples were obtained by venipuncture into serum monovettes® and centrifuged at 2500g for 10 min at 20 °C.”

ABSTRACT Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular “nanotraps,” which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. IMPORTANCE Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for building synthetic cellular structures, manipulating protein function, and creating gradual targeted knockdowns. Our findings provide a proof of principle for the universal use of fluorescently tagged proteins as targets for nanotraps to fulfill these tasks.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that affect mammals including humans. The proteinaceous nature of the infectious agent, the prion, and its propagation, challenge established dogmas in biology. It is now widely accepted that prion diseases are caused by unconventional agents principally composed of a misfolded host-encoded protein, PrP. Surprisingly, major break-throughs in prion research came from studies on functionally unrelated proteins in yeast and filamentous fungi. Aggregates composed of these proteins act as epigenetic elements of inheritance that can propagate their alternative states by a conformational switch into an ordered ß-sheet rich polymer just like mammalian prions. Since their discovery prions of lower eukaryotes have provided invaluable insights into all aspects of prion biogenesis. Importantly, yeast prions provide proof-of-principle that distinct protein conformers can be infectious and can serve as genetic elements that have the capacity to encipher strain specific information. As a powerful and tractable model system, yeast prions will continue to increase our understanding of prion-host cell interaction and potential mechanisms of protein-based epigenetic inheritance.

Nerve growth factor (NGF) regulates sensory neuron phenotype by elevated expression of ion channels and receptors contributing to pain. Peripheral opioid antinociception is dependent on sensory neuron μ opioid receptor (MOR) expression, coupling and efficacy. This study investigates the role of NGF in the upregulation of the number and efficacy of sensory MORs rendering sites of painful inflammation more susceptible to opioids. We identified co-localization of MOR with calcitonin gene-related peptides (CGRP) and with the NGF receptors tyrosine receptor kinase (TrkA) and p75NTR within rat dorsal root ganglia (DRG). We showed that unilateral hind paw inflammation induced with Freund's complete adjuvant (FCA) or intraplantar (i.pl.) NGF increased NGF's retrograde transport and MOR expression in TrkA positive DRG which was prevented by the disruption of this NGF transport. MOR upregulation in DRG was followed by enhanced axonal MOR transport towards peripheral nerve terminals and subsequent increase of MOR-ir nerve fibres within skin. Furthermore, peripheral antinociception elicited by i.pl. fentanyl was naloxone reversible and potentiated exclusively in inflamed and NGF-treated paws. Both FCA- and NGF-induced effects occurring through DRG to peripheral nerve fibres and the potentiation of antinociception were abrogated by NGF neutralization. Therefore, our results suggest that NGF not only contributes to inflammatory pain but also governs the upregulation in the number and efficacy of sensory neuron MOR, resulting in enhanced opioid susceptibility towards better pain control. This suggests the potential to overcome the unresponsiveness to opioids of certain neuropathic pain states. [ABSTRACT FROM AUTHOR]

Prokaryotic protein–protein interactions are underrepresented in currently available databases. Here, we describe a ‘gold standard’ dataset (MPI-LIT) focusing on microbial binary protein–protein interactions and associated experimental evidence that we have manually curated from 813 abstracts and full texts that were selected from an initial set of 36 852 abstracts. The MPI-LIT dataset comprises 1237 experimental descriptions that describe a non-redundant set of 746 interactions of which 659 (88%) are not reported in public databases. To estimate the curation quality, we compared our dataset with a union of microbial interaction data from IntAct, DIP, BIND and MINT. Among common abstracts, we achieve a sensitivity of up to 66% for interactions and 75% for experimental methods. Compared with these other datasets, MPI-LIT has the lowest fraction of interaction experiments per abstract (0.9) and the highest coverage of strains (92) and scientific articles (813). We compared methods that evaluate functional interactions among proteins (such as genomic context or co-expression) which are implemented in the STRING database. Most of these methods discriminate well between functionally relevant protein interactions (MPI-LIT) and high-throughput data. Availability: http://www.jcvi.org/mpidb/interaction.php?dbsource=MPI-LIT. Contact: raja@jcvi.org Supplementary information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]