Charakterisierung eines minimierten eukaryoten Cytoplasmas - das periplastidäre Kompartiment der Diatomee Phaeodactylum tricornutum

Diatomeen besitzen zwei divergente eukaryote Cytoplasmen: (i) das Cytosol der Wirtszelle, und (ii) das periplastidäre Kompartiment (PPC), welches das reduzierte Cytoplasma eines rhodophytischen Endosymbionten (komplexe Plastide) darstellt. Letzteres findet sich bei einer Vielzahl ökologisch höchst relevanter Mikroalgen und pathogener Protisten (Apicomplexa), welche komplexe Plastiden mit vier Hüllmembranen besitzen. Über die Proteinzusammensetzung und Funktion des PPC war bisher nur wenig bekannt. Im Rahmen dieser Arbeit wurde die Proteinkomposition des PPC der Diatomee Phaeodactylum tricornutum über einen kombinierten in silico/in vivo Ansatz untersucht. PPC-Kandidaten wurden dabei anhand der Präsenz spezifischer N-terminaler Zielsteuerungssequenzen genomisch identifiziert und über die Expression von eGFP-Fusionsproteinen auf ihre subzelluläre Lokalisation untersucht. Insgesamt konnten dadurch in dieser Arbeit 29 neue PPC-Proteine detektiert werden, welche sowohl bereits bekannte Funktionen im PPC, wie Proteintransport (SELMA), Proteinfaltung und Metabolismus, ergänzen, als auch auf bisher unbekannte putative Prozesse im PPC hindeuten. Insbesondere sind dabei Proteolyse und Degradation, Plastidenteilung, Lipidtransfer und eine potentielle strukturelle Organisation zu nennen. In gleichem Maße relevant sind Prozesse, die im Zuge bioinformatischer Analysen nicht im PPC der Diatomee detektiert werden konnten, was für klassische eukaryote Cytoskelett- und Vesikelkomponenten, sowie typische Signaltransduktionsfaktoren zutrifft. Die Ergebnisse dieser Arbeit legen nahe, dass es sich beim PPC von P. tricornutum um ein auf natürliche Weise minimiertes eukaryotes Cytoplasma mit stark begrenzten metabolischen und zellulären Kapazitäten handelt, welches mit einem autonomen eukaryoten Cytoplasma nur wenige Gemeinsamkeiten aufweist und dessen Funktion höchstwahrscheinlich vor allem der Selbsterhaltung und Homöostase der ehemals primären Plastide dient. Ein weiteres Thema dieser Arbeit beschäftigte sich mit der Identifizierung und Lokalisation von Metabolittranslokatoren in P. tricornutum, die eine Verbindung zwischen den Stoffwechselprozessen von Wirt und Symbiont ermöglichen. Dabei gelang es mittels in silico Analysen und anschließenden in vivo GFP-Lokalisationsstudien mehrere putative Triosephosphat/Phosphat Translokatoren in den Membranen der komplexen Plastide von P. tricornutum nachzuweisen. Die Ergebnisse deuten darauf hin, dass diese, möglicherweise durch den Transport von Triosephosphaten, eine metabolische Kommunikation zwischen Wirt und Symbiont gewährleisten könnten

ERAD Components in Organisms with Complex Red Plastids Suggest Recruitment of a Preexisting Protein Transport Pathway for the Periplastid Membrane

The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis

Optimum Design and Control of Heat Pumps for Integration into Thermohydraulic Networks

Germany has become one of the leading players in the transformation of the electricity sector, now having up to 42% of electricity coming from renewable sources. However, the transformation of the heating sector is still in its infancy, and especially the provision of industrial process heating is highly dependent on unsustainable fuels. One of the most promising heating technologies for renewable energies is power-to-heat, especially heat pump technology, as it can use renewable electricity to generate heat efficiently. This research explores the economic and technical boundary conditions regarding the integration of heat pumps into existing industrial thermohydraulic heating and cooling networks. To calculate the optimum design and control of heat pumps, a mixed-integer linear programming model (MILP) is developed. The model seeks the most cost-efficient configuration of heat pumps and stratified thermal storage tanks. Additionally, it optimizes the operation of all energy converters and stratified thermal storage tanks to meet a specified heating and cooling demand over one year. The objective function is modeled after the net present value (NPV) method and considers capital expenditures (costs for heat pumps and stratified thermal storage tanks) and operational expenditures (electricity costs and costs for conventional heating and cooling). The comparison of the results via a simulation model reveals an accuracy of more than 90%

Probing vulnerability of the gp41 C-terminal heptad repeat as target for miniprotein HIV inhibitors

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.Spanish Ministry of Economy and Competitiveness (grant: BIO2016-76640-R), ANRS and the Vaccine Research Institute for the Investissements d'Avenir program to C.M. and by the European Fund for Research and Development from the European Union.Departamento de Química Física, Facultad de Ciencias, Universidad de Granada. Grupo FQM-171 "Biofísica y Biotecnología Molecular

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.This work was supported by grants CV20.26565 from the Consejería de Economía y Conocimiento, Junta de Andalucía (Spain), PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033), and co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future”. The work performed in C.M.'s laboratory was supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA et les hépatites virales), the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (No. 681032, Horizon 2020). Work in S.B.'s laboratory was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR-11-LABX-0070_TRANSPLANTEX), the INSERM (UMR_S 1109), the Institut Universitaire de France (IUF), all the University of Strasbourg (IDEX UNISTRA), the European Regional Development Fund (European Union) INTERREG V program (project no. 3.2 TRIDIAG) and MSD-Avenir grant AUTOGEN. We are grateful to the Spanish Radiation Synchrotron Source (ALBA), Barcelona, Spain and the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and staff assistance at XALOC (ALBA) and ID30B and ID23-2 (ESRF) beamlines during diffraction data collection. We thank María Carmen Salinas-García for her assistance in carrying out the crystallization screenings. We also thank Pilar González-García for helping us with the statistical analysis

Simulation Modeling for Energy-Flexible Manufacturing: Pitfalls and How to Avoid Them

Due to the high share of industry in total electricity consumption, industrial demand-side management can make a relevant contribution to the stability of power systems. At the same time, companies get the opportunity to reduce their electricity procurement costs by taking advantage of increasingly fluctuating prices on short-term electricity markets, the provision of system services on balancing power markets, or by increasing the share of their own consumption from on-site generated renewable energy. Demand-side management requires the ability to react flexibly to the power supply situation without negatively affecting production targets. It also means that the management and operation of production must consider not only production-related parameters but also parameters of energy availability, which further increase the complexity of decision-making. Although simulation studies are a recognized tool for supporting decision-making processes in production and logistics, the simultaneous simulation of material and energy flows has so far been limited mainly to issues of energy efficiency as opposed to energy flexibility, where application-oriented experience is still limited. We assume that the consideration of energy flexibility in the simulation of manufacturing systems will amplify already known pitfalls in conducting simulation studies. Based on five representative industrial use cases, this article provides practitioners with application-oriented experiences of the coupling of energy and material flows in simulation modeling of energy-flexible manufacturing, identifies challenges in the simulation of energy-flexible production systems, and proposes approaches to face these challenges. Seven pitfalls that pose a particular challenge in simulating energy-flexible manufacturing have been identified, and possible solutions and measures for avoiding them are shown. It has been found that, among other things, consistent management of all parties involved, early clarification of energy-related, logistical, and resulting technical requirements for models and software, as well as the application of suitable methods for validation and verification are central to avoiding these pitfalls. The identification and characterization of challenges and the derivation of recommendations for coping with them can raise awareness of typical pitfalls. This paper thus helps to ensure that simulation studies of energy-flexible production systems can be carried out more efficiently in the future

In Silico and In Vivo Investigations of Proteins of a Minimized Eukaryotic Cytoplasm

Algae with secondary plastids such as diatoms maintain two different eukaryotic cytoplasms. One of them, the so-called periplastidal compartment (PPC), is the naturally minimized cytoplasm of a eukaryotic endosymbiont. In order to investigate the protein composition of the PPC of diatoms, we applied knowledge of the targeting signals of PPC-directed proteins in searches of the genome data for proteins acting in the PPC and proved their in vivo localization via expressing green fluorescent protein (GFP) fusions. Our investigation increased the knowledge of the protein content of the PPC approximately 3-fold and thereby indicated that this narrow compartment was functionally reduced to some important cellular functions with nearly no housekeeping biochemical pathways

Genome sequencing reveals metabolic and cellular interdependence in an amoeba-kinetoplastid symbiosis

Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba, amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans-splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive ‘cross-talk’ between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba

Longitudinal river zonation in the tropics: examples of fish and caddisflies from endorheic Awash river, Ethiopia

Primary Research PaperSpecific concepts of fluvial ecology are well studied in riverine ecosystems of the temperate zone but poorly investigated in the Afrotropical region. Hence, we examined the longitudinal zonation of fish and adult caddisfly (Trichoptera) assemblages in the endorheic Awash River (1,250 km in length), Ethiopia. We expected that species assemblages are structured along environmental gradients, reflecting the pattern of large-scale freshwater ecoregions. We applied multivariate statistical methods to test for differences in spatial species assemblage structure and identified characteristic taxa of the observed biocoenoses by indicator species analyses. Fish and caddisfly assemblages were clustered into highland and lowland communities, following the freshwater ecoregions, but separated by an ecotone with highest biodiversity. Moreover, the caddisfly results suggest separating the heterogeneous highlands into a forested and a deforested zone. Surprisingly, the Awash drainage is rather species-poor: only 11 fish (1 endemic, 2 introduced) and 28 caddisfly species (8 new records for Ethiopia) were recorded from the mainstem and its major tributaries. Nevertheless, specialized species characterize the highland forests, whereas the lowlands primarily host geographically widely distributed species. This study showed that a combined approach of fish and caddisflies is a suitable method for assessing regional characteristics of fluvial ecosystems in the tropicsinfo:eu-repo/semantics/publishedVersio