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Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Hepcidin-25 was identified as themain iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II) binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandemmass spectrometry (MS/MS), high-resolutionmass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D)model of hepcidin-25with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or referencematerial comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Say it with double flowers
(2020)
Every year, lovers world-wide rely on mutants to show their feelings on Valentine's Day. This is because many of the most popular ornamental flowering plants have been selected to form extra petals at the expense of reproductive organs to enhance their attractiveness and aesthetic value to humans. This so-called 'double flower' (DF) phenotype, first described more than 2000 years ago (Meyerowitz et al., 1989) is present, for example, in many modern roses, carnations, peonies, and camellias. Gattolin et al. (2020) now identify a unifying explanation for the molecular basis of many of these DF cultivars.
Subsurface microbial communities undertake many terminal electron-accepting processes, often simultaneously. Using a tritium-based assay, we measured the potential hydrogen oxidation catalyzed by hydrogenase enzymes in several subsurface sedimentary environments (Lake Van, Barents Sea, Equatorial Pacific, and Gulf of Mexico) with different predominant electron-acceptors. Hydrogenases constitute a diverse family of enzymes expressed by microorganisms that utilize molecular hydrogen as a metabolic substrate, product, or intermediate. The assay reveals the potential for utilizing molecular hydrogen and allows qualitative detection of microbial activity irrespective of the predominant electron-accepting process. Because the method only requires samples frozen immediately after recovery, the assay can be used for identifying microbial activity in subsurface ecosystems without the need to preserve live material. We measured potential hydrogen oxidation rates in all samples from multiple depths at several sites that collectively span a wide range of environmental conditions and biogeochemical zones. Potential activity normalized to total cell abundance ranges over five orders of magnitude and varies, dependent upon the predominant terminal electron acceptor. Lowest per-cell potential rates characterize the zone of nitrate reduction and highest per-cell potential rates occur in the methanogenic zone. Possible reasons for this relationship to predominant electron acceptor include (i) increasing importance of fermentation in successively deeper biogeochemical zones and (ii) adaptation of H(2)ases to successively higher concentrations of H-2 in successively deeper zones.
Pancreatic steatosis associates with beta-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals.
Functional characterization of ROS-responsive genes, ANAC085 and ATR7, in Arabidopsis thaliana
(2023)
This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.
Etmopteridae (lantern sharks) is the most species-rich family of sharks, comprising more than 50 species.
Many species are described from few individuals, and re-collection of specimens is often hindered by the remoteness of their sampling sites.
For taxonomic studies, comparative morphological analysis of type specimens housed in natural history collections has been the main source of evidence.
In contrast, DNA sequence information has rarely been used.
Most lantern shark collection specimens, including the types, were formalin fixed before long-term storage in ethanol solutions.
The DNA damage caused by both fixation and preservation of specimens has excluded these specimens from DNA sequence-based phylogenetic analyses so far.
However, recent advances in the field of ancient DNA have allowed recovery of wet-collection specimen DNA sequence data.
Here we analyse archival mitochondrial DNA sequences, obtained using ancient DNA approaches, of two wet-collection lantern shark paratype specimens, namely Etmopterus litvinovi and E. pycnolepis, for which the type series represent the only known individuals.
Target capture of mitochondrial markers from single-stranded DNA libraries allows for phylogenetic placement of both species.
Our results suggest synonymy of E. benchleyi with E. litvinovi but support the species status of E. pycnolepis. This revised taxonomy is helpful for future conservation and management efforts, as our results indicate a larger distribution range of E. litvinovi. This study further demonstrates the importance of wet-collection type specimens as genetic resource for taxonomic research.
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.
Development and application of novel genetic transformation technologies in maize (Zea mays L.)
(2007)
Plant genetic engineering approaches are of pivotal importance to both basic and applied research. However, rapid commercialization of genetically engineered crops, especially maize, raises several ecological and environmental concerns largely related to transgene flow via pollination. In most crops, the plastid genome is inherited uniparentally in a maternal manner. Consequently, a trait introduced into the plastid genome would not be transferred to the sexually compatible relatives of the crops via pollination. Thus, beside its several other advantages, plastid transformation provides transgene containment, and therefore, is an environmentally friendly approach for genetic engineering of crop plants. Reliable in vitro regeneration systems allowing repeated rounds of regeneration are of utmost importance to development of plastid transformation technologies in higher plants. While being the world’s major food crops, cereals are among the most difficult-to-handle plants in tissue culture which severely limits genetic engineering approaches. In maize, immature zygotic embryos provide the predominantly used material for establishing regeneration-competent cell or callus cultures for genetic transformation experiments. The procedures involved are demanding, laborious and time consuming and depend on greenhouse facilities. In one part of this work, a novel tissue culture and plant regeneration system was developed that uses maize leaf tissue and thus is independent of zygotic embryos and greenhouse facilities. Also, protocols were established for (i) the efficient induction of regeneration-competent callus from maize leaves in the dark, (ii) inducing highly regenerable callus in the light, and (iii) the use of leaf-derived callus for the generation of stably transformed maize plants. Furthermore, several selection methods were tested for developing a plastid transformation system in maize. However, stable plastid transformed maize plants could not be yet recovered. Possible explanations as well as suggestions for future attempts towards developing plastid transformation in maize are discussed. Nevertheless, these results represent a first essential step towards developing chloroplast transformation technology for maize, a method that requires multiple rounds of plant regeneration and selection to obtain genetically stable transgenic plants. In order to apply the newly developed transformation system towards metabolic engineering of carotenoid biosynthesis, the daffodil phytoene synthase (PSY) gene was integrated into the maize genome. The results illustrate that expression of a recombinant PSY significantly increases carotenoid levels in leaves. The beta-carotene (pro-vitamin A) amounts in leaves of transgenic plants were increased by ~21% in comparison to the wild-type. These results represent evidence for maize to have significant potential to accumulate higher amounts of carotenoids, especially beta-carotene, through transgenic expression of phytoene synthases. Finally, progresses were made towards developing transformation technologies in Peperomia (Piperaceae) by establishing an efficient leaf-based regeneration system. Also, factors determining plastid size and number in Peperomia, whose species display great interspecific variation in chloroplast size and number per cell, were investigated. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. Investigating plastid morphology in Peperomia species with giant chloroplasts, plasmatic connections between chloroplasts (stromules) were observed under the light microscope and in the absence of tissue fixation or GFP overexpression demonstrating the relevance of these structures in vivo. Furthermore, bacteria-like microorganisms were discovered within Peperomia cells, suggesting that this genus provides an interesting model not only for studying plastid biology but also for investigating plant-microbe interactions.
Um das Immunsystem der Pflanze zu manipulieren translozieren gram-negative pathogene Bakterien Typ-III Effektorproteine (T3E) über ein Typ-III Sekretionssystem (T3SS) in die pflanzliche Wirtszelle. Dort lokalisieren T3Es in verschiedenen subzellulären Kompartimenten, wo sie Zielproteine modifizieren und so die Infektion begünstigen. HopZ1a, ein T3E des Pflanzenpathogens Pseudomonas syringae pv. syringae, ist eine Acetyltransferase und lokalisiert über ein Myristolierungsmotiv an der Plasmamembran der Wirtszelle. Obwohl gezeigt wurde, dass HopZ1a die frühe Signalweiterleitung an der Plasmamembran stört, wurde bisher kein mit der Plasmamembran assoziiertes Zielprotein für diesen T3E identifiziert. Um bisher unbekannte HopZ1a-Zieleproteine zu identifizieren wurde im Vorfeld dieser Arbeit eine Hefe-Zwei-Hybrid-Durchmusterung mit einer cDNA-Bibliothek aus Tabak durchgeführt, wobei ein nicht näher charakterisiertes Remorin als Interaktor gefunden wurde.
Bei dem Remorin handelt es sich um einen Vertreter der Gruppe 4 der Remorin-Familie, weshalb es in NbREM4 umbenannt wurde. Durch den Einsatz verschiedener Interaktionsstudien konnte demonstriert werden, dass HopZ1a mit NbREM4 in Hefe, in vitro und in planta wechselwirkt. Es wurde ferner deutlich, dass HopZ1a auf spezifische Weise mit dem konservierten C-Terminus von NbREM4 interagiert, das Remorin jedoch in vitro nicht acetyliert. Analysen mittels BiFC haben zudem ergeben, dass NbREM4 in Homodimeren an der Plasmamembran lokalisiert, wo auch die Interaktion mit HopZ1a stattfindet.
Eine funktionelle Charakterisierung von NbREM4 ergab, dass das Remorin eine spezifische Rolle im Immunsystem der Pflanze einnimmt. Die transiente Expression in N. benthamiana induziert die Expression von Abwehrgenen sowie einen veränderten Blattphänotyp. In A. thaliana wird HopZ1a über das Decoy ZED1 und das R-Protein ZAR1 erkannt, was zur Auslösung einer starken Hypersensitiven Antwort (HR von hypersensitive response) führt. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass ZAR1 in N. benthamiana konserviert ist, NbREM4 jedoch nicht in der ETI als Decoy fungiert. Mit Hilfe einer Hefe-Zwei-Hybrid-Durchmusterung mit NbZAR1 als Köder konnten zwei Proteine, die Catalase CAT1 und der Protonenpumpeninteraktor PPI1, als Interaktoren von NbZAR1 identifiziert werden, welche möglicherweise in der Regulation der HR eine Rolle spielen.
Aus Voruntersuchungen war bekannt, dass NbREM4 mit weiteren, nicht näher charakterisierten Proteinen aus Tabak interagieren könnte. Eine phylogenetische Einordnung hat gezeigt, dass es sich um die bekannte Immun-Kinase PBS1 sowie zwei E3-Ubiquitin-Ligasen, NbSINA1 und NbSINAL3, handelt. PBS1 interagiert mit NbREM4 an der Plasmamembran und phosphoryliert das Remorin innerhalb des intrinsisch ungeordneten N-Terminus. Mittels Massenspektrometrie konnten die Serine an Position 64 und 65 innerhalb der Aminosäuresequenz von NbREM4 als PBS1-abhängige Phosphorylierungsstellen identifiziert wurden.
NbSINA1 und NbSINAL3 besitzen in vitro Ubiquitinierungsaktivität, bilden Homo- und Heterodimere und interagieren ebenfalls mit dem N-terminalen Teil von NbREM4, wobei sie das Remorin in vitro nicht ubiquitinieren.
Aus den in dieser Arbeit gewonnenen Ergebnissen lässt sich ableiten, dass der bakterielle T3E HopZ1a gezielt mit dem Tabak-Remorin NbREM4 an der Plasmamembran interagiert und über einen noch unbekannten Mechanismus mit dem Immunsystem der Pflanze interferiert, wobei NbREM4 möglicherweise eine Rolle als Adapter- oder Ankerprotein zukommt, über welches HopZ1a mit weiteren Immunkomponenten interagiert. NbREM4 ist Teil eines größeren Immunnetzwerkes, zu welchem die bekannte Immun-Kinase PBS1 und zwei E3-Ubiquitin-Ligasen gehören. Mit NbREM4 konnte damit erstmalig ein membranständiges Protein mit einer Funktion im Immunsystem der Pflanze als Zielprotein von HopZ1a identifiziert werden.
The plant pathogen Pseudomonas syringae is a gram-negative bacterium which infects a wide range of plant species including important crops plants. To suppress plant immunity and cause disease P.syringae injects type-III effector proteins (T3Es) into the plant cell cytosol. In this study, we identified a novel target of the well characterized bacterial T3E HopZ1a. HopZ1a is an acetyltransferase that was shown to disrupt vesicle transport during innate immunity by acetylating tubulin. Using a yeast-two-hybrid screen approach, we identified a REMORIN (REM) protein from tobacco as a novel HopZ1a target. HopZ1a interacts with REM at the plasma membrane (PM) as shown by split-YFP experiments. Interestingly, we found that PBS1, a well-known kinase involved in plant immunity also interacts with REM in pull-down assays, and at the PM as shown by BiFC. Furthermore, we confirmed that REM is phosphorylated by PBS1 in vitro. Overexpression of REM provokes the upregulation of defense genes and leads to disease-like phenotypes pointing to a role of REM in plant immune signaling. Further protein-protein interaction studies reveal novel REM binding partners with a possible role in plant immune signaling. Thus, REM might act as an assembly hub for an immune signaling complex targeted by HopZ1a. Taken together, this is the first report describing that a REM protein is targeted by a bacterial effector. How HopZ1a might mechanistically manipulate the plant immune system through interfering with REM function will be discussed.
The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.
The prevalence of contaminant microbial DNA in ancient bone samples represents the principal limiting factor for palaeogenomic studies, as it may comprise more than 99% of DNA molecules obtained. Efforts to exclude or reduce this contaminant fraction have been numerous but also variable in their success. Here, we present a simple but highly effective method to increase the relative proportion of endogenous molecules obtained from ancient bones. Using computed tomography (CT) scanning, we identify the densest region of a bone as optimal for sampling. This approach accurately identifies the densest internal regions of petrous bones, which are known to be a source of high-purity ancient DNA. For ancient long bones, CT scans reveal a high-density outermost layer, which has been routinely removed and discarded prior to DNA extraction. For almost all long bones investigated, we find that targeted sampling of this outermost layer provides an increase in endogenous DNA content over that obtained from softer, trabecular bone. This targeted sampling can produce as much as 50-fold increase in the proportion of endogenous DNA, providing a directly proportional reduction in sequencing costs for shotgun sequencing experiments. The observed increases in endogenous DNA proportion are not associated with any reduction in absolute endogenous molecule recovery. Although sampling the outermost layer can result in higher levels of human contamination, some bones were found to have more contamination associated with the internal bone structures. Our method is highly consistent, reproducible and applicable across a wide range of bone types, ages and species. We predict that this discovery will greatly extend the potential to study ancient populations and species in the genomics era.