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Background:
Grebe dysplasia, Hunter-Thompson dysplasia, and du Pan dysplasia constitute a spectrum of skeletal dysplasias inherited as an autosomal recessive trait characterized by short stature, severe acromesomelic shortening of the limbs, and normal axial skeleton. The majority of patients with these disorders have biallelic loss-of-function mutations of GDF5. In single instances, Grebe dysplasia and a Grebe dysplasia-like phenotype with genital anomalies have been shown to be caused by mutations in BMPR1B, encoding a GDF5 receptor.
Methods:
We clinically and radiologically characterised an acromesomelic chondrodysplasia in an adult woman born to consanguineous parents. We sequenced GDF5 and BMPR1B on DNA of the proposita. We performed 3D structural analysis and luciferase reporter assays to functionally investigate the identified BMPR1B mutation.
Results:
We extend the genotype-phenotype correlation in the acromesomelic chondrodysplasias by showing that the milder du Pan dysplasia can be caused by a hypomorphic BMPR1B mutation. We show that the homozygous c.91C>T, p.(Arg31Cys) mutation causing du Pan dysplasia leads to a significant loss of BMPR1B function, but to a lesser extent than the previously reported p.Cys53Arg mutation that results in the more severe Grebe dysplasia.
Conclusions:
The phenotypic severity gradient of the clinically and radiologically related acromesomelic chondrodysplasia spectrum of skeletal disorders may be due to the extent of functional impairment of the ligand-receptor pair GDF5-BMPR1B.
The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for \(H^+\) efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit \(H^+\) efflux (\(H^+\)-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (\(H^+\) efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to \(K^+\) efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the \(H^+\)-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of (\(H^+\)-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of (\(H^+\)-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.
Plants extract mineral nutrients from the soil, or from interactions with mutualistic soil microbes via their root systems. Adapting root architecture to nutrient availability enables efficient resource utilization, particularly in patchy and dynamic environments. Root growth responses to soil nitrogen levels are shoot-mediated, but the identity of shoot-derived mobile signals regulating root growth responses has remained enigmatic. Here we show that a shoot-derived micro RNA, miR2111, systemically steers lateral root initiation and nitrogen responsiveness through its root target TML (TOO MUCH LOVE) in the legume Lotus japonicus, where miR2111 and TML were previously shown to regulate symbiotic infections with nitrogen fixing bacteria. Intriguingly, systemic control of lateral root initiation by miR2111 and TML/HOLT (HOMOLOGUE OF LEGUME TML) was conserved in the nonsymbiotic ruderal Arabidopsis thaliana, which follows a distinct ecological strategy. Thus, the miR2111-TML/HOLT regulon emerges as an essential, conserved factor in adaptive shoot control of root architecture in dicots.
Animal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in the UC-affected population nor can they be used to test the efficacy of inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGFβ1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFNγ might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help to increase the translation of animal and clinical studies.
A novel cost effective and high-throughput isolation and identification method for marine microalgae
(2014)
BACKROUND:
Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures.
RESULTS:
In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms.
CONCLUSIONS:
We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures."
Olfactory receptors (ORs) are G protein-coupled receptors which serve important sensory functions beyond their role as odorant detectors in the olfactory epithelium. Here we describe a novel role for one of these ORs, Olfr1393, as a regulator of renal glucose handling. Olfr1393 is specifically expressed in the kidney proximal tubule, which is the site of renal glucose reabsorption. Olfr1393 knockout mice exhibit urinary glucose wasting and improved glucose tolerance, despite euglycemia and normal insulin levels. Consistent with this phenotype, Olfr1393 knockout mice have a significant decrease in luminal expression of Sglt1, a key renal glucose transporter, uncovering a novel regulatory pathway involving Olfr1393 and Sglt1. In addition, by utilizing a large scale screen of over 1400 chemicals we reveal the ligand profile of Olfr1393 for the first time, offering new insight into potential pathways of physiological regulation for this novel signaling pathway.
Background: Members of the TGF-b superfamily are characterized by a highly promiscuous ligand-receptor interaction as is readily apparent from the numeral discrepancy of only seven type I and five type II receptors available for more than 40 ligands. Structural and functional studies have been used to address the question of how specific signals can be deduced from a limited number of receptor combinations and to unravel the molecular mechanisms underlying the protein-protein recognition that allow such limited specificity. Principal Findings: In this study we have investigated how an antigen binding antibody fragment (Fab) raised against the extracellular domain of the BMP receptor type IA (BMPR-IA) recognizes the receptor’s BMP-2 binding epitope and thereby neutralizes BMP-2 receptor activation. The crystal structure of the complex of the BMPR-IA ectodomain bound to the Fab AbD1556 revealed that the contact surface of BMPR-IA overlaps extensively with the contact surface for BMP-2 interaction. Although the structural epitopes of BMPR-IA to both binding partners coincides, the structures of BMPR-IA in the two complexes differ significantly. In contrast to the structural differences, alanine-scanning mutagenesis of BMPR-IA showed that the functional determinants for binding to the antibody and BMP-2 are almost identical. Conclusions: Comparing the structures of BMPR-IA bound to BMP-2 or bound to the Fab AbD1556 with the structure of unbound BMPR-IA shows that binding of BMPR-IA to its interaction partners follows a selection fit mechanism, possibly indicating that the ligand promiscuity of BMPR-IA is inherently encoded by structural adaptability. The functional and structural analysis of the BMPR-IA binding antibody AbD1556 mimicking the BMP-2 binding epitope may thus pave the way for the design of low-molecular weight synthetic receptor binders/inhibitors.
Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca\(^{2+}\) signalling has been intensively studied, the role of the vacuolar membrane remains elusive.
A newly developed vacuolar voltage clamp technique was used in combination with live-cell imaging, to study the role of the vacuolar membrane in Ca\(^{2+}\) and pH homeostasis of bulging root hair cells of Arabidopsis.
Depolarisation of the vacuolar membrane caused a rapid increase in the Ca\(^{2+}\) concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses.
The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H\(^{+}\)/Ca\(^{2+}\) exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage-dependent vacuolar Ca\(^{2+}\) homeostat could contribute to calcium signalling when coupled to a recently discovered K\(^{+}\) channel-dependent module for electrical excitability of the vacuolar membrane.
The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery.
Mycotoxins in agriculturally used plants can cause intoxication in animals and can lead to severe financial losses for farmers. The endophytic fungus Epichloë festucae var. lolii living symbiotically within the cool season grass species Lolium perenne can produce vertebrate and invertebrate toxic alkaloids. Hence, an exact quantitation of alkaloid concentrations is essential to determine intoxication risk for animals. Many studies use different methods to detect alkaloid concentrations, which complicates the comparability. In this study, we showed that alkaloid concentrations of individual plants exceeded toxicity thresholds on real world grasslands in Germany, but not on the population level. Alkaloid concentrations on five German grasslands with high alkaloid levels peaked in summer but were also below toxicity thresholds on population level. Furthermore, we showed that alkaloid concentrations follow the same seasonal trend, regardless of whether plant fresh or dry weight was used, in the field and in a common garden study. However, alkaloid concentrations were around three times higher when detected with dry weight. Finally, we showed that alkaloid concentrations can additionally be biased to different alkaloid detection methods. We highlight that toxicity risks should be analyzed using plant dry weight, but concentration trends of fresh weight are reliable.
The origins of multicellular physiology are tied to evolution of gene expression. Genes can shift expression as organisms evolve, but how ancestral expression influences altered descendant expression is not well understood. To examine this, we amalgamate 1,903 RNA-seq datasets from 182 research projects, including 6 organs in 21 vertebrate species. Quality control eliminates project-specific biases, and expression shifts are reconstructed using gene-family-wise phylogenetic Ornstein-Uhlenbeck models. Expression shifts following gene duplication result in more drastic changes in expression properties than shifts without gene duplication. The expression properties are tightly coupled with protein evolutionary rate, depending on whether and how gene duplication occurred. Fluxes in expression patterns among organs are nonrandom, forming modular connections that are reshaped by gene duplication. Thus, if expression shifts, ancestral expression in some organs induces a strong propensity for expression in particular organs in descendants. Regardless of whether the shifts are adaptive or not, this supports a major role for what might be termed preadaptive pathways of gene expression evolution.
Background
Microbial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized.
Results
Chlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm2 of 380 nm (~ 73 J/m2) or inhibited by ~ 40 photons/nm\(^2\) of 473 nm (~ 18 J/m\(^2\)).
Conclusions
This engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.
Background
The plant endophytic fungus Serendipita indica colonizes roots of a wide range of plant species and can enhance growth and stress resistance of these plants. Due to its ease of axenic cultivation and its broad host plant range including the model plant Arabidopsis thaliana and numerous crop plants, it is widely used as a model fungus to study beneficial fungus-root interactions. In addition, it was suggested to be utilized for commercial applications, e.g. to enhance yield in barley and other species. To produce inoculum, S. indica is mostly cultivated in a complex Hill-Kafer medium (CM medium), however, growth in this medium is slow, and yield of chlamydospores, which are often used for plant root inoculation, is relatively low.
Results
We tested and optimized a simple vegetable juice-based medium for an enhanced yield of fungal inoculum. The described vegetable juice (VJ) medium is based on commercially available vegetable juice and is easy to prepare. VJ medium was superior to the currently used CM medium with respect to biomass production in liquid medium and hyphal growth on agar plates. Using solid VJ medium supplemented with sucrose (VJS), a high amount of chlamydospores developed already after 8 days of cultivation, producing significantly more spores than on CM medium. Use of VJ medium is not restricted to S. indica, as it also supported growth of two pathogenic fungi often used in plant pathology experiments: the ascomycete Fusarium graminearum, the causal agent of Fusarium head blight disease on wheat and barley, and Verticillium longisporum, the causal agent of verticillium wilt.
Conclusions
The described VJ medium is recommended for streamlined and efficient production of inoculum for the plant endophytic fungus Serendipita indica and might prove superior for the propagation of other fungi for research purposes.
Whereas the role of calcium ions (Ca\(^{2+}\)) in plant signaling is well studied, the physiological significance of pH‐changes remains largely undefined.
Here we developed CapHensor, an optimized dual‐reporter for simultaneous Ca\(^{2+}\) and pH ratio‐imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio‐temporal relationships between membrane voltage, Ca\(^{2+}\)‐ and pH‐dynamics revealed interconnections previously not described.
In tobacco PTs, we demonstrated Ca\(^{2+}\)‐dynamics lag behind pH‐dynamics during oscillatory growth, and pH correlates more with growth than Ca\(^{2+}\). In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA‐responses and even opened stomata in the presence of ABA, disclosing an important pH‐dependent GC signaling node. In MCs, a flg22‐induced membrane depolarization preceded Ca2+‐increases and cytosolic acidification by c. 2 min, suggesting a Ca\(^{2+}\)/pH‐independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage‐, Ca\(^{2+}\)‐ and pH‐responses.
We propose close interrelation in Ca\(^{2+}\)‐ and pH‐signaling that is cell type‐ and stimulus‐specific and the pH having crucial roles in regulating PT growth and stomata movement.
In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.
The green synthesis of silver nanoparticles (SNPs) using plant extracts is an eco-friendly method. It is a single step and offers several advantages such as time reducing, cost-effective and environmental non-toxic. Silver nanoparticles are a type of Noble metal nanoparticles and it has tremendous applications in the field of diagnostics, therapeutics, antimicrobial activity, anticancer and neurodegenerative diseases. In the present work, the aqueous extracts of aerial parts of Lampranthus coccineus and Malephora lutea F. Aizoaceae were successfully used for the synthesis of silver nanoparticles. The formation of silver nanoparticles was early detected by a color change from pale yellow to reddish-brown color and was further confirmed by transmission electron microscope (TEM), UV–visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), and energy-dispersive X-ray diffraction (EDX). The TEM analysis of showed spherical nanoparticles with a mean size between 12.86 nm and 28.19 nm and the UV- visible spectroscopy showed λ\(_{max}\) of 417 nm, which confirms the presence of nanoparticles. The neuroprotective potential of SNPs was evaluated by assessing the antioxidant and cholinesterase inhibitory activity. Metabolomic profiling was performed on methanolic extracts of L. coccineus and M. lutea and resulted in the identification of 12 compounds, then docking was performed to investigate the possible interaction between the identified compounds and human acetylcholinesterase, butyrylcholinesterase, and glutathione transferase receptor, which are associated with the progress of Alzheimer’s disease. Overall our SNPs highlighted its promising potential in terms of anticholinesterase and antioxidant activity as plant-based anti-Alzheimer drug and against oxidative stress.
Actinomycetes are prolific producers of pharmacologically important compounds accounting for about 70% of the naturally derived antibiotics that are currently in clinical use. In this study, we report on the isolation of Streptomyces sp. strains from Mediterranean sponges, on their secondary metabolite production and on their screening for anti-infective activities. Bioassay-guided isolation and purification yielded three previously known compounds namely, cyclic depsipeptide valinomycin, indolocarbazole alkaloid staurosporine and butenolide. This is the first report of the isolation of valinomycin from a marine source. These compounds exhibited novel anti-parasitic activities specifically against Leishmania major (valinomycin IC50 < 0.11 μM; staurosporine IC50 5.30 μM) and Trypanosoma brucei brucei (valinomycin IC50 0.0032 μM; staurosporine IC50 0.022 μM; butenolide IC50 31.77 μM). These results underscore the potential of marine actinomycetes to produce bioactive compounds as well as the re-evaluation of previously known compounds for novel anti-infective activities.
Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with \([^{125} I]\) Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.
Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70–90 μM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 μM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.
The number of plants pollinated by ants is surprisingly low given the abundance of ants and the fact that they are common visitors of angiosperms. Generally ants are considered as nectar robbers that do not provide pollination service. We studied the pollination system of the endangered dry grassland forb Euphorbia seguieriana and found two ant species to be the most frequent visitors of its flowers. Workers of Formica cunicularia carried five times more pollen than smaller Tapinoma erraticum individuals, but significantly more viable pollen was recovered from the latter. Overall, the viability of pollen on ant cuticles was significantly lower (p < 0.001)-presumably an antibiotic effect of the metapleural gland secretion. A marking experiment suggested that ants were unlikely to facilitate outcrossing as workers repeatedly returned to the same individual plant. In open pollinated plants and when access was given exclusively to flying insects, fruit set was nearly 100%. In plants visited by ants only, roughly one third of flowers set fruit, and almost none set fruit when all insects were excluded. The germination rate of seeds from flowers pollinated by flying insects was 31 +/- 7% in contrast to 1 +/- 1% resulting from ant pollination. We conclude that inbreeding depression may be responsible for the very low germination rate in ant pollinated flowers and that ants, although the most frequent visitors, play a negligible or even deleterious role in the reproduction of E. seguieriana. Our study reiterates the need to investigate plant fitness effects beyond seed set in order to confirm ant-plant mutualisms.
Bulb, leaf, scape and flower samples of British bluebells (Hyacinthoides non-scripta) were collected regularly for one growth period. Methanolic extracts of freeze-dried and ground samples showed antitrypanosomal activity, giving more than 50% inhibition, for 20 out of 41 samples. High-resolution mass spectrometry was used in the dereplication of the methanolic extracts of the different plant parts. The results revealed differences in the chemical profile with bulb samples being distinctly different from all aerial parts. High molecular weight metabolites were more abundant in the flowers, shoots and leaves compared to smaller molecular weight ones in the bulbs. The anti-trypanosomal activity of the extracts was linked to the accumulation of high molecular weight compounds, which were matched with saponin glycosides, while triterpenoids and steroids occurred in the inactive extracts. Dereplication studies were employed to identify the significant metabolites via chemotaxonomic filtration and considering their previously reported bioactivities. Molecular networking was implemented to look for similarities in fragmentation patterns between the isolated saponin glycoside at m/z 1445.64 [M + formic-H](-) equivalent to C64H104O33 and the putatively found active metabolite at m/z 1283.58 [M + formic-H](-) corresponding to scillanoside L-1. A combination of metabolomics and bioactivity-guided approaches resulted in the isolation of a norlanostane-type saponin glycoside with antitrypanosoma I activity of 98.9% inhibition at 20 mu M.
Polygonum cuspidatum (Japanese knotweed, also known as Huzhang in Chinese), a plant that produces bioactive components such as stilbenes and quinones, has long been recognized as important in traditional Chinese herbal medicine. To better understand the biological features of this plant and to gain genetic insight into the biosynthesis of its natural products, we assembled a draft genome of P. cuspidatum using Illumina sequencing technology. The draft genome is ca. 2.56 Gb long, with 71.54% of the genome annotated as transposable elements. Integrated gene prediction suggested that the P. cuspidatum genome encodes 55,075 functional genes, including 6,776 gene families that are conserved in the five eudicot species examined and 2,386 that are unique to P. cuspidatum. Among the functional genes identified, 4,753 are predicted to encode transcription factors. We traced the gene duplication history of P. cuspidatum and determined that it has undergone two whole-genome duplication events about 65 and 6.6 million years ago. Roots are considered the primary medicinal tissue, and transcriptome analysis identified 2,173 genes that were expressed at higher levels in roots compared to aboveground tissues. Detailed phylogenetic analysis demonstrated expansion of the gene family encoding stilbene synthase and chalcone synthase enzymes in the phenylpropanoid metabolic pathway, which is associated with the biosynthesis of resveratrol, a pharmacologically important stilbene. Analysis of the draft genome identified 7 abscisic acid and water deficit stress-induced protein-coding genes and 14 cysteine-rich transmembrane module genes predicted to be involved in stress responses. The draft de novo genome assembly produced in this study represents a valuable resource for the molecular characterization of medicinal compounds in P. cuspidatum, the improvement of this important medicinal plant, and the exploration of its abiotic stress resistance.
Technical features of a novel multi-color pulse amplitude modulation (PAM) chlorophyll fluorometer as well as the applied methodology and some typical examples of its practical application with suspensions of Chlorella vulgaris and Synechocystis PCC 6803 are presented. The multi-color PAM provides six colors of pulse-modulated measuring light (peak-wavelengths at 400, 440, 480, 540, 590, and 625 nm) and six colors of actinic light (AL), peaking at 440, 480, 540, 590, 625 and 420–640 nm (white). The AL can be used for continuous illumination, maximal intensity single-turnover pulses, high intensity multiple-turnover pulses, and saturation pulses. In addition, far-red light (peaking at 725 nm) is provided for preferential excitation of PS I. Analysis of the fast fluorescence rise kinetics in saturating light allows determination of the wavelength- and sample-specific functional absorption cross section of PS II, Sigma(II)λ, with which the PS II turnover rate at a given incident photosynthetically active radiation (PAR) can be calculated. Sigma(II)λ is defined for a quasi-dark reference state, thus differing from σPSII used in limnology and oceanography. Vastly different light response curves for Chlorella are obtained with light of different colors, when the usual PAR-scale is used. Based on Sigma(II)λ the PAR, in units of μmol quanta/(m2 s), can be converted into PAR(II) (in units of PS II effective quanta/s) and a fluorescence-based electron transport rate ETR(II) = PAR(II) · Y(II)/Y(II)max can be defined. ETR(II) in contrast to rel.ETR qualifies for quantifying the absolute rate of electron transport in optically thin suspensions of unicellular algae and cyanobacteria. Plots of ETR(II) versus PAR(II) for Chlorella are almost identical using either 440 or 625 nm light. Photoinhibition data are presented suggesting that a lower value of ETR(II)max with 440 nm possibly reflects photodamage via absorption by the Mn-cluster of the oxygen-evolving complex.
Marine sponges are known as a rich source for novel bioactive compounds with valuable pharmacological potential. One of the most predominant sponge genera is Hyrtios, reported to have various species such as Hyrtios erectus, Hyrtios reticulatus, Hyrtios gumminae, Hyrtios communis, and Hyrtios tubulatus and a number of undescribed species. Members of the genus Hyrtios are a rich source of natural products with diverse and valuable biological activities, represented by different chemical classes including alkaloids, sesterterpenes and sesquiterpenes. This review covers the literature until June 2016, providing a complete survey of all compounds isolated from the genus Hyrtios with their corresponding biological activities whenever applicable.
Marine sponge–associated actinomycetes are considered as promising sources for the discovery of novel biologically active compounds. In the present study, a total of 64 actinomycetes were isolated from 12 different marine sponge species that had been collected offshore the islands of Milos and Crete, Greece, eastern Mediterranean. The isolates were affiliated to 23 genera representing 8 different suborders based on nearly full length 16S rRNA gene sequencing. Four putatively novel species belonging to genera Geodermatophilus, Microlunatus, Rhodococcus and Actinomycetospora were identified based on a 16S rRNA gene sequence similarity of < 98.5% to currently described strains. Eight actinomycete isolates showed bioactivities against Trypanosma brucei brucei TC221 with half maximal inhibitory concentration (IC50) values <20 μg/mL. Thirty four isolates from the Milos collection and 12 isolates from the Crete collection were subjected to metabolomic analysis using high resolution LC-MS and NMR for dereplication purposes. Two isolates belonging to the genera Streptomyces (SBT348) and Micromonospora (SBT687) were prioritized based on their distinct chemistry profiles as well as their anti-trypanosomal activities. These findings demonstrated the feasibility and efficacy of utilizing metabolomics tools to prioritize chemically unique strains from microorganism collections and further highlight sponges as rich source for novel and bioactive actinomycetes.
Background: In higher plants, a diverse array of developmental and growth-related processes is regulated by the plant hormone auxin. Recent publications have proposed that besides the well-characterized Auxin Response Factors (ARFs) that bind Auxin Response Elements (AuxREs), also members of the bZIP- and MYB-transcription factor (TF) families participate in transcriptional control of auxin-regulated genes via bZIP Response Elements (ZREs) or Myb Response Elements (MREs), respectively. Results: Applying a novel bioinformatic algorithm, we demonstrate on a genome-wide scale that singular motifs or composite modules of AuxREs, ZREs, MREs but also of MYC2 related elements are significantly enriched in promoters of auxin-inducible genes. Despite considerable, species-specific differences in the genome structure in terms of the GC content, this enrichment is generally conserved in dicot (Arabidopsis thaliana) and monocot (Oryza sativa) model plants. Moreover, an enrichment of defined composite modules has been observed in selected auxin-related gene families. Consistently, a bipartite module, which encompasses a bZIP-associated G-box Related Element (GRE) and an AuxRE motif, has been found to be highly enriched. Making use of transient reporter studies in protoplasts, these findings were experimentally confirmed, demonstrating that GREs functionally interact with AuxREs in regulating auxin-mediated transcription. Conclusions: Using genome-wide bioinformatic analyses, evolutionary conserved motifs have been defined which potentially function as AuxRE-dependent coupling elements to establish auxin-specific expression patterns. Based on these findings, experimental approaches can be designed to broaden our understanding of combinatorial, auxin-controlled gene regulation.
Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C\(_1\)) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus.
In Brassicaceae, tissue damage triggers the mustard oil bomb i.e., activates the degradation of glucosinolates by myrosinases leading to a rapid accumulation of isothiocyanates at the site of damage. Isothiocyanates are reactive electrophilic species (RES) known to covalently bind to thiols in proteins and glutathione, a process that is not only toxic to herbivores and microbes but can also cause cell death of healthy plant tissues. Previously, it has been shown that subtoxic isothiocyanate concentrations can induce transcriptional reprogramming in intact plant cells. Glutathione depletion by RES leading to breakdown of the redox potential has been proposed as a central and common RES signal transduction mechanism. Using transcriptome analyses, we show that after exposure of Arabidopsis seedlings (grown in liquid culture) to subtoxic concentrations of sulforaphane hundreds of genes were regulated without depletion of the cellular glutathione pool. Heat shock genes were among the most highly up-regulated genes and this response was found to be dependent on the canonical heat shock factors A1 (HSFA1). HSFA1-deficient plants were more sensitive to isothiocyanates than wild type plants. Moreover, pretreatment of Arabidopsis seedlings with subtoxic concentrations of isothiocyanates increased resistance against exposure to toxic levels of isothiocyanates and, hence, may reduce the autotoxicity of the mustard oil bomb by inducing cell protection mechanisms.
Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and \(CO_2\) uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal \(CO_2\) concentrations, except for very low \(CO_2\), where the rate of charge flux distinctly exceeded the rate of CO2 uptake. Parallel oscillations in \(CO_2\) uptake and charge flux were induced by high \(CO_2\) and \(O_2\). The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.
Thylakoid and cytoplasmic membranes of the cyanobacterium Syncchocystis sp. PCC 6803 were purified by sucrose gradient centrifugation. Both membranes oxidize NADH in a rotenone-sensitive reaction. Antibodies prepared against psbG/ndhKand ndhJ fusion proteins detect the corresponding polypeptides in both membrane preparations. This demonstrates that a NADH-dehydrogenase, homologous to the mitochondrial NADHubiquinone-oxidoreductase (complex I of the respiratory chain) is present in cyanobacteria, The NADH-dehydrogenase can be solubilized with the detergent /-D-dodecylmaltoside. Sedimentation analysis of the solubilized enzyme on a sucrose gradient indicates that it is a multisubunit protein complex.
A newly developed compact measuring system for assessment of transmittance changes in the near-infrared spectral region is described; it allows deconvolution of redox changes due to ferredoxin (Fd), P700, and plastocyanin (PC) in intact leaves. In addition, it can also simultaneously measure chlorophyll fluorescence. The major opto-electronic components as well as the principles of data acquisition and signal deconvolution are outlined. Four original pulse-modulated dual-wavelength difference signals are measured (785-840 nm, 810-870 nm, 870-970 nm, and 795-970 nm). Deconvolution is based on specific spectral information presented graphically in the form of 'Differential Model Plots' (DMP) of Fd, P700, and PC that are derived empirically from selective changes of these three components under appropriately chosen physiological conditions. Whereas information on maximal changes of Fd is obtained upon illumination after dark-acclimation, maximal changes of P700 and PC can be readily induced by saturating light pulses in the presence of far-red light. Using the information of DMP and maximal changes, the new measuring system enables on-line deconvolution of Fd, P700, and PC. The performance of the new device is demonstrated by some examples of practical applications, including fast measurements of flash relaxation kinetics and of the Fd, P700, and PC changes paralleling the polyphasic fluorescence rise upon application of a 300-ms pulse of saturating light.
High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC₅₀ value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.
Design and validation of a disease network of inflammatory processes in the NSG-UC mouse model
(2017)
Background: Ulcerative colitis (UC) is a highly progressive inflammatory disease that requires the interaction of epithelial, immune, endothelial and muscle cells and fibroblasts. Previous studies suggested two inflammatory conditions in UC-patients: ‘acute’ and ‘remodeling’ and that the design of a disease network might improve the understanding of the inflammatory processes. The objective of the study was to design and validate a disease network in the NOD-SCID IL2rγ\(^{null}\) (NSG)-UC mouse model to get a better understanding of the inflammatory processes.
Methods: Leukocytes were isolated from the spleen of NSG-UC mice and subjected to flow cytometric analysis. RT-PCR and RNAseq analysis were performed from distal parts of the colon. Based on these analyses and the effects of interleukins, chemokines and growth factors described in the literature, a disease network was designed. To validate the disease network the effect of infliximab and pitrakinra was tested in the NSG-UC model. A clinical- and histological score, frequencies of human leukocytes isolated from spleen and mRNA expression levels from distal parts of the colon were determined.
Results: Analysis of leukocytes isolated from the spleen of challenged NSG-UC mice corroborated CD64, CD163 and CD1a expressing CD14+ monocytes, CD1a expressing CD11b+ macrophages and HGF, TARC, IFNγ and TGFß1 mRNA as inflammatory markers. The disease network suggested that a proinflammatory condition elicited by IL-17c and lipids and relayed by cytotoxic T-cells, Th17 cells and CD1a expressing macrophages and monocytes. Conversely, the remodeling condition was evoked by IL-34 and TARC and promoted by Th2 cells and M2 monocytes. Mice benefitted from treatment with infliximab as indicated by the histological- and clinical score. As predicted by the disease network infliximab reduced the proinflammatory response by suppressing M1 monocytes and CD1a expressing monocytes and macrophages and decreased levels of IFNγ, TARC and HGF mRNA. As predicted by the disease network inflammation aggravated in the presence of pitrakinra as indicated by the clinical and histological score, elevated frequencies of CD1a expressing macrophages and TNFα and IFNγ mRNA levels.
Conclusions: The combination of the disease network and the NSG-UC animal model might be developed into a powerful tool to predict efficacy or in-efficacy and potential mechanistic side effects.
Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.
The phyllosphere of plants is inhabited by diverse microorganisms, however, the factors shaping their community composition are not fully elucidated. The plant cuticle represents the initial contact surface between microorganisms and the plant. We thus aimed to investigate whether mutations in the cuticular wax biosynthesis would affect the diversity of the phyllosphere microbiota. A set of four Arabidopsis thaliana eceriferum mutants (cer1, cer6, cer9, cer16) and their respective wild type (Landsberg erecta) were subjected to an outdoor growth period and analysed towards this purpose. The chemical distinctness of the mutant wax phenotypes was confirmed by gas chromatographic measurements. Next generation amplicon pyrosequencing of the bacterial communities showed distinct community patterns. This observation was supported by denaturing gradient gel electrophoresis experiments. Microbial community analyses revealed bacterial phylotypes that were ubiquitously present on all plant lines (termed “core” community) while others were positively or negatively affected by the wax mutant phenotype (termed “plant line-specific“ community). We conclude from this study that plant cuticular wax composition can affect the community composition of phyllosphere bacteria.
Diversity of Nonribosomal Peptide Synthetase Genes in the Microbial Metagenomes of Marine Sponges
(2012)
Genomic mining revealed one major nonribosomal peptide synthetase (NRPS) phylogenetic cluster in 12 marine sponge species, one ascidian, an actinobacterial isolate and seawater. Phylogenetic analysis predicts its taxonomic affiliation to the actinomycetes and hydroxy-phenyl-glycine as a likely substrate. Additionally, a phylogenetically distinct NRPS gene cluster was discovered in the microbial metagenome of the sponge Aplysina aerophoba, which shows highest similarities to NRPS genes that were previously assigned, by ways of single cell genomics, to a Chloroflexi sponge symbiont. Genomic mining studies such as the one presented here for NRPS genes, contribute to on-going efforts to characterize the genomic potential of sponge-associated microbiota for secondary metabolite biosynthesis.
DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding
(2014)
The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.
DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors
(2013)
Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene expression, physiological processes, and the development of crown gall tumors.
Draft genome of the \(Arabidopsis\) \(thaliana\) phyllosphere bacterium, \(Williamsia\) sp. ARP1
(2016)
The Gram-positive actinomycete \(Williamsia\) sp. ARP1 was originally isolated from the \(Arabidopsis\) \(thaliana\) phyllosphere. Here we describe the general physiological features of this microorganism together with the draft genome sequence and annotation. The 4,745,080 bp long genome contains 4434 protein-coding genes and 70 RNA genes. To our knowledge, this is only the second reported genome from the genus \(Williamsia\) and the first sequenced strain from the phyllosphere. The presented genomic information is interpreted in the context of an adaptation to the phyllosphere habitat.
The marine sponge-associated bacterium Actinokineospora sp. strain EG49 produces the antitrypanosomal angucycline-like compound actinosporin A. The draft genome of Actinokineospora sp. EG49 has a size of 7.5 megabases and a GC content of 72.8% and contains 6,629 protein-coding sequences (CDS). antiSMASH predicted 996 genes residing in 36 secondary metabolite gene clusters.
Maintaining the integrity of the cuticular transpiration barrier even at elevated temperatures is of vital importance especially for hot-desert plants. Currently, the temperature dependence of the leaf cuticular water permeability and its relationship with the chemistry of the cuticles are not known for a single desert plant. This study investigates whether (i) the cuticular permeability of a desert plant is lower than that of species from non-desert habitats, (ii) the temperature-dependent increase of permeability is less pronounced than in those species and (iii) whether the susceptibility of the cuticular permeability barrier to high temperatures is related to the amounts or properties of the cutin or the cuticular waxes. We test these questions with Rhazya stricta using the minimum leaf water vapour conductance (gmin) as a proxy for cuticular water permeability. gmin of R. stricta (5.41 × 10\(^{-5}\) m s\(^{-1}\) at 25 °C) is in the upper range of all existing data for woody species from various non-desert habitats. At the same time, in R. stricta, the effect of temperature (15-50 °C) on gmin (2.4-fold) is lower than in all other species (up to 12-fold). Rhazya stricta is also special since the temperature dependence of gmin does not become steeper above a certain transition temperature. For identifying the chemical and physical foundation of this phenomenon, the amounts and the compositions of cuticular waxes and cutin were determined. The leaf cuticular wax (251.4 μg cm\(^{-2}\)) is mainly composed of pentacyclic triterpenoids (85.2% of total wax) while long-chain aliphatics contribute only 3.4%. In comparison with many other species, the triterpenoid-to-cutin ratio of R. stricta (0.63) is high. We propose that the triterpenoids deposited within the cutin matrix restrict the thermal expansion of the polymer and, thus, prevent thermal damage to the highly ordered aliphatic wax barrier even at high temperatures.
While much research has addressed the aboveground response of trees to climate warming and related water shortage, not much is known about the drought sensitivity of the fine root system, in particular of mature trees. This study investigates the response of topsoil (0–10 cm) fine root biomass (FRB), necromass (FRN), and fine root morphology of five temperate broadleaf tree species (Acer platanoides L., Carpinus betulus L., Fraxinus excelsior L., Quercus petraea (Matt.) Liebl., Tilia cordata Mill.) to a reduction in water availability, combining a precipitation gradient study (nine study sites; mean annual precipitation (MAP): 920–530 mm year\(^{−1}\)) with the comparison of a moist period (average spring conditions) and an exceptionally dry period in the summer of the subsequent year. The extent of the root necromass/biomass (N/B) ratio increase was used as a measure of the species’ belowground sensitivity to water deficits. We hypothesized that the N/B ratio increases with long-term (precipitation gradient) and short-term reductions (moist vs. dry period) of water availability, while FRB changes only a little. In four of the five species (exception: A. platanoides), FRB did not change with a reduction in MAP, whereas FRN and N/B ratio increased toward the dry sites under ample water supply (exception: Q. petraea). Q. petraea was also the only species not to reduce root tip frequency after summer drought. Different slopes of the N/B ratio-MAP relation similarly point at a lower belowground drought sensitivity of Q. petraea than of the other species. After summer drought, all species lost the MAP dependence of the N/B ratio. Thus, fine root mortality increased more at the moister than the drier sites, suggesting a generally lower belowground drought sensitivity of the drier stands. We conclude that the five species differ in their belowground drought response. Q. petraea follows the most conservative soil exploration strategy with a generally smaller FRB and more drought-tolerant fine roots, as it maintains relatively constant FRB, FRN, and morphology across spatial and temporal dimensions of soil water deficits.
Fungal endophytes of the genus Epichloë live symbiotically in cool season grass species and can produce alkaloids toxic to insects and vertebrates, yet reports of intoxication of grazing animals have been rare in Europe in contrast to overseas. However, due to the beneficial resistance traits observed in Epichloë infected grasses, the inclusion of Epichloë in seed mixtures might become increasingly advantageous. Despite the toxicity of fungal alkaloids, European seed mixtures are rarely tested for Epichloë infection and their infection status is unknown for consumers. In this study, we tested 24 commercially available seed mixtures for their infection rates with Epichloë endophytes and measured the concentrations of the alkaloids ergovaline, lolitrem B, paxilline, and peramine. We detected Epichloë infections in six seed mixtures, and four contained vertebrate and insect toxic alkaloids typical for Epichloë festucae var. lolii infecting Lolium perenne. As Epichloë infected seed mixtures can harm livestock, when infected grasses become dominant in the seeded grasslands, we recommend seed producers to test and communicate Epichloë infection status or avoiding Epichloë infected seed mixtures.
Plants do not have neurons but operate transmembrane ion channels and can get electrical excited by physical and chemical clues. Among them the Venus flytrap is characterized by its peculiar hapto-electric signaling. When insects collide with trigger hairs emerging the trap inner surface, the mechanical stimulus within the mechanosensory organ is translated into a calcium signal and an action potential (AP). Here we asked how the Ca\(^{2+}\) wave and AP is initiated in the trigger hair and how it is feed into systemic trap calcium-electrical networks. When Dionaea muscipula trigger hairs matures and develop hapto-electric excitability the mechanosensitive anion channel DmMSL10/FLYC1 and voltage dependent SKOR type Shaker K\(^{+}\) channel are expressed in the sheering stress sensitive podium. The podium of the trigger hair is interface to the flytrap’s prey capture and processing networks. In the excitable state touch stimulation of the trigger hair evokes a rise in the podium Ca2+ first and before the calcium signal together with an action potential travel all over the trap surface. In search for podium ion channels and pumps mediating touch induced Ca\(^{2+}\) transients, we, in mature trigger hairs firing fast Ca\(^{2+}\) signals and APs, found OSCA1.7 and GLR3.6 type Ca\(^{2+}\) channels and ACA2/10 Ca\(^{2+}\) pumps specifically expressed in the podium. Like trigger hair stimulation, glutamate application to the trap directly evoked a propagating Ca\(^{2+}\) and electrical event. Given that anesthetics affect K\(^+\) channels and glutamate receptors in the animal system we exposed flytraps to an ether atmosphere. As result propagation of touch and glutamate induced Ca\(^{2+}\) and AP long-distance signaling got suppressed, while the trap completely recovered excitability when ether was replaced by fresh air. In line with ether targeting a calcium channel addressing a Ca\(^{2+}\) activated anion channel the AP amplitude declined before the electrical signal ceased completely. Ether in the mechanosensory organ did neither prevent the touch induction of a calcium signal nor this post stimulus decay. This finding indicates that ether prevents the touch activated, glr3.6 expressing base of the trigger hair to excite the capture organ.
Donor CD4\(^+\)Foxp3\(^+\) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2-and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.
Background:
Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells.
Methods:
FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function.
Results:
SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3.
Conclusions:
SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.
Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.
We did not expect that research on the molecular mechanism of algal phototaxis or archaeal light‐driven ion transport might interest readers of a medical journal when we conceived and performed our experiments a decade ago. On the other hand, it did not escape our attention that channelrhodopsin is helping an ever‐increasing number of researchers to address their specific questions. For example, the channelrhodopsin approach is used to study the molecular events during the induction of synaptic plasticity or to map long‐range connections from one side of the brain to the other, and to map the spatial location of inputs on the dendritic tree of individual neurons. The current applications have been summarized in a number of recent reviews (Fenno et al, 2011; Yizhar et al, 2011; Zhang et al, 2011). Here, we give personal insight into the history of the discovery of channelrhodopsin and a biophysical perspective on this remarkable class of proteins that has been the main topic of our research since the 1990s.
Sodium-glucose transporters (SGLT) belong to the solute carrier 5 family, which is characterized by sodium dependent transport of sugars and other solutes. In contrast, the human SGLT3 (hSGLT3) isoform, encoded by SLC5A4, acts as a glucose sensor that does not transport sugar but induces membrane depolarization by Na\(^{+}\) currents upon ligand binding. Whole-exome sequencing (WES) of several extended pedigrees with high density of attention-deficit/hyperactivity disorder (ADHD) identified a triplet ATG deletion in SLC5A4 leading to a single amino acid loss (ΔM500) in the hSGLT3 protein imperfectly co-segregating with the clinical phenotype of ADHD. Since mutations in homologous domains of hSGLT1 and hSGLT2 were found to affect intestinal and renal function, respectively, we analyzed the functional properties of hSGLT3[wt] and [ΔM500] by voltage clamp and current clamp recordings from cRNA-injected Xenopus laevis oocytes.
The cation conductance of hSGLT3[wt] was activated by application of glucose or the specific agonist 1-desoxynojirimycin (DNJ) as revealed by inward currents in the voltage clamp configuration and cell depolarization in the current clamp mode. Almost no currents and changes in membrane potential were observed when glucose or DNJ were applied to hSGLT3[ΔM500]-injected oocytes, demonstrating a loss of function by this amino acid deletion in hSGLT3. To monitor membrane targeting of wt and mutant hSGLT3, fusion constructs with YFP were generated, heterologously expressed in Xenopus laevis oocytes and analyzed for membrane fluorescence by confocal microscopy. In comparison to hSGLT3[wt] the fluorescent signal of mutant [ΔM500] was reduced by 43% indicating that the mutant phenotype might mainly result from inaccurate membrane targeting. As revealed by homology modeling, residue M500 is located in TM11 suggesting that in addition to the core structure (TM1-TM10) of the transporter, the surrounding TMs are equally crucial for transport/sensor function.
In conclusion, our findings indicate that the deletion [ΔM500] in hSGLT3 inhibits membrane targeting and thus largely disrupts glucose-induced sodium conductance, which may, in interaction with other ADHD risk-related gene variants, influence the risk for ADHD in deletion carriers.