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MAIN CONCLUSION: The compatible solute sucrose reduces the efficiency of the enzymatic de-epoxidation of violaxanthin, probably by a direct effect on the protein parts of violaxanthin de-epoxidase which protrude from the lipid phase of the thylakoid membrane. The present study investigates the influence of the compatible solute sucrose on the violaxanthin cycle of higher plants in intact thylakoids and in in vitro enzyme assays with the isolated enzyme violaxanthin de-epoxidase at temperatures of 30 and 10 °C, respectively. In addition, the influence of sucrose on the lipid organization of thylakoid membranes and the MGDG phase in the in vitro assays is determined. The results show that sucrose leads to a pronounced inhibition of violaxanthin de-epoxidation both in intact thylakoid membranes and the enzyme assays. In general, the inhibition is similar at 30 and 10 °C. With respect to the lipid organization only minor changes can be seen in thylakoid membranes at 30 °C in the presence of sucrose. However, sucrose seems to stabilize the thylakoid membranes at lower temperatures and at 10 °C a comparable membrane organization to that at 30 °C can be observed, whereas control thylakoids show a significantly different membrane organization at the lower temperature. The MGDG phase in the in vitro assays is not substantially affected by the presence of sucrose or by changes of the temperature. We conclude that the presence of sucrose and the increased viscosity of the reaction buffers stabilize the protein part of the enzyme violaxanthin de-epoxidase, thereby decreasing the dynamic interactions between the catalytic site and the substrate violaxanthin. This indicates that sucrose interacts with those parts of the enzyme which are accessible at the membrane surface of the lipid phase of the thylakoid membrane or the MGDG phase of the in vitro enzyme assays.
Assuntos
Galactolipídeos , Tilacoides , Sacarose , XantofilasRESUMO
Efficient protein secretion is often a valuable alternative to classic cellular expression to obtain homogenous protein samples. Early on, bacterial type I secretion systems (T1SS) were employed to allow heterologous secretion of fusion proteins. However, this approach was not fully exploited, as many proteins could not be secreted at all or only at low levels. Here, we present an engineered microbial secretion system which allows the effective production of proteins up to a molecular mass of 88 kDa. This system is based on the hemolysin A (HlyA) T1SS of the Gram-negative bacterium Escherichia coli, which exports polypeptides when fused to a hemolysin secretion signal. We identified an A/U-rich enhancer region upstream of hlyA required for effective expression and secretion of selected heterologous proteins irrespective of their prokaryotic, viral, or eukaryotic origin. We further demonstrate that the ribosomal protein S1 binds to the hlyA A/U-rich enhancer region and that this region is involved in the high yields of secretion of functional proteins, like maltose-binding protein or human interferon alpha-2.IMPORTANCE A 5' untranslated region of the mRNA of substrates of type I secretion systems (T1SS) drastically enhanced the secretion efficiency of the endogenously secreted protein. The identification of ribosomal protein S1 as the interaction partner of this 5' untranslated region provides a rationale for the enhancement. This strategy furthermore can be transferred to fusion proteins allowing a broader, and eventually a more general, application of this system for secreting heterologous fusion proteins.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas Hemolisinas/genética , Sistemas de Secreção Tipo I/genética , Interferon-alfa/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Organismos Geneticamente Modificados/genéticaRESUMO
BACKGROUND & AIMS: Besides its critical role during liver regeneration, serotonin (5-HT) has been found to act as a mitogenic factor in several neoplastic entities. Accordingly, we aimed to evaluate whether intra-platelet 5-HT (IP5-HT) was associated with oncological outcome after liver resection and concomitantly evaluate its ability to serve as a therapeutic target to promote liver regeneration. METHODS: A total of 96 patients undergoing liver resection for malignant liver tumors were prospectively included. Optimized plasma and serum preparation were performed and IP5-HT levels were determined. Patients were followed up for postoperative liver dysfunction (LD), morbidity, disease free and overall survival (OS). RESULTS: We found increased preoperative IP5-HT levels in patients with disease recurrence at 6 and 12months (p=0.046, p=0.020, respectively). In clear contrast, patients suffering from postoperative morbidity, severe morbidity or LD had significantly reduced IP5-HT levels (p=0.011, p=0.035, p=0.003, respectively). Patients with high IP5-HT levels (>134ng/ml) suffered from an increased incidence of postoperative disease recurrence at 6 and 12months (p=0.045, p=0.006, respectively) but exhibited a reduction in morbidity, severe morbidity, and LD (p=0.006, p=0.008, p=0.005, respectively). We confirmed these results in our two largest subgroups, demonstrating that they were independent of tumor type. This bivalent effect of IP5-HT was also reflected in patients receiving selective serotonin reuptake inhibitor treatment, who displayed a reduction in disease recurrence accompanied by an increase in postoperative morbidity. Yet, both early disease recurrence and morbidity worsened OS. CONCLUSION: Herein, we present first clinical evidence for IP5-HT being associated with early disease recurrence after liver resection in humans. Thus, pharmacological intervention at the level of platelets and platelet-derived 5-HT to promote liver regeneration should be considered with caution. A careful definition of indications and timing is needed to promote liver regeneration without inducing deleterious effects. LAY SUMMARY: Preoperative intra-platelet serotonin (IP5-HT) levels seem to substantially affect patient outcomes after liver resection for liver tumors. While there is a narrow window of IP5-HT levels where liver regeneration and tumor progression is balanced, excessively high IP5-HT levels (>134ng/ml IP5-HT) lead to an increased incidence of early tumor recurrence and excessively low IP5-HT levels (<73ng/ml IP5-HT) lead to a higher rate of morbidity. Ultimately, overall survival is negatively affected by both postoperative early disease recurrence and morbidity. ClinicalTrials.gov-Identifier: NCT01700231.
Assuntos
Plaquetas/fisiologia , Hepatectomia , Neoplasias Hepáticas/cirurgia , Regeneração Hepática , Recidiva Local de Neoplasia/etiologia , Serotonina/fisiologia , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/fisiopatologia , Masculino , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
Active, widely tunable optical materials have enabled rapid advances in photonics and optoelectronics, especially in the emerging field of meta-devices. Here, we demonstrate that spatially selective defect engineering on the nanometer scale can transform phase-transition materials into optical metasurfaces. Using ion irradiation through nanometer-scale masks, we selectively defect-engineered the insulator-metal transition of vanadium dioxide, a prototypical correlated phase-transition material whose optical properties change dramatically depending on its state. Using this robust technique, we demonstrated several optical metasurfaces, including tunable absorbers with artificially induced phase coexistence and tunable polarizers based on thermally triggered dichroism. Spatially selective nanoscale defect engineering represents a new paradigm for active photonic structures and devices.
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Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.
Assuntos
Carbono/metabolismo , Tilacoides/metabolismo , Chlamydomonas reinhardtii/metabolismo , Microscopia de Fluorescência , Biogênese de Organelas , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/biossíntese , Biossíntese de ProteínasRESUMO
Anaerobic digestion (AD) of sugar beet pressed pulp (SBPP) is a promising treatment concept. It produces biogas as a renewable energy source making sugar production more energy efficient and it turns SBPP from a residue into a valuable resource. In this study one- and two-stage mono fermentation at mesophilic conditions in a continuous stirred tank reactor were compared. Also the optimal incubation temperature for the pre-acidification stage was studied. The fastest pre-acidification, with a hydraulic retention time (HRT) of 4 days, occurred at a temperature of 55 °C. In the methanogenic reactor of the two-stage system stable fermentation at loading rate of 7 kg VS/m³ d was demonstrated. No artificial pH adjustment was necessary to maintain optimum levels in both the pre-acidification and the methanogenic reactor. The total HRT of the two-stage AD was 36 days which is considerably lower compared to the one-stage AD (50 days). The frequently observed problem of foaming at high loading rates was less severe in the two-stage reactor. Moreover the viscosity of digestate in the methanogenic stage of the two-stage fermentation was in average tenfold lower than in the one-stage fermentation. This decreases the energy input for the reactor stirring about 80 %. The observed advantages make the two-stage process economically attractive, despite higher investments for a two reactor system.
Assuntos
Bactérias/metabolismo , Beta vulgaris/metabolismo , Biocombustíveis/análise , Reatores Biológicos/microbiologia , Microbiologia Industrial/instrumentação , Resíduos/análise , Anaerobiose , Beta vulgaris/química , Beta vulgaris/microbiologia , Biodegradação Ambiental , Digestão , Fermentação , ViscosidadeRESUMO
A conspicuous new species of praying mantid, Mythomantis serrata sp. nov., from the Malaysian part of Borneo is described and illustrated. A key to the three known species of the genus Mythomantis and their known geographic distribution is provided. Several morphological characters, most notably those in the male genitals, suggest a close relationship between Mythomantis and the Southeast Asian genera Pseudempusa and Deroplatys. As a consequence, we propose to transfer Mythomantis from Angelinae to Deroplatyinae, and Pseudempusa from Miomantinae Rivetinini to Deroplatyinae, while removing Brancsikia from this subfamily.
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Mantódeos/anatomia & histologia , Mantódeos/classificação , Animais , Bornéu , Demografia , Masculino , Mantódeos/fisiologia , Especificidade da EspécieRESUMO
We present the first checklist of praying mantids (Mantodea) of Borneo, with special reference to the specimens collected during the Scientific Expedition to Lanjak Entimau Wildlife Sanctuary 2008. With 118 confirmed species in 56 genera (including subgenera), Borneo is the island with the highest mantodean diversity known to date. In Lanjak Entimau 38 specimens representing 17 genera and 18 species were collected around the station lights and in surrounding secondary and primary forest. A new synonymy in the genus Deroplatys is established. The observed diversity patterns among Bornean mantids are discussed with reference to the biogeographic history of the Sunda Shelf since the Miocene.
Assuntos
Mantódeos/anatomia & histologia , Mantódeos/classificação , Animais , Bornéu , Demografia , Ecossistema , Malásia , Mantódeos/fisiologia , Especificidade da EspécieRESUMO
Expression of the chloroplast psbD gene encoding the D2 protein of the photosystem II reaction center is regulated by light. In the green alga Chlamydomonas reinhardtii, D2 synthesis requires a high-molecular-weight complex containing the RNA stabilization factor Nac2 and the translational activator RBP40. Based on size exclusion chromatography analyses, we provide evidence that light control of D2 synthesis depends on dynamic formation of the Nac2/RBP40 complex. Furthermore, 2D redox SDS-PAGE assays suggest an intermolecular disulfide bridge between Nac2 and Cys11 of RBP40 as the putative molecular basis for attachment of RBP40 to the complex in light-grown cells. This covalent link is reduced in the dark, most likely via NADPH-dependent thioredoxin reductase C, supporting the idea of a direct relationship between chloroplast gene expression and chloroplast carbon metabolism during dark adaption of algal cells.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Complexo de Proteína do Fotossistema II/metabolismo , Adaptação Fisiológica , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efeitos da radiação , Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Redutases do Citocromo/metabolismo , Escuridão , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Luz , Modelos Moleculares , Complexos Multiproteicos , Oxirredução , Complexo de Proteína do Fotossistema II/genética , Ligação Proteica , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes , Tiorredoxinas/metabolismoRESUMO
We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.
Assuntos
Proteínas de Algas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Chlamydomonas reinhardtii/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Regiões 5' não Traduzidas , Proteínas de Algas/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fotossíntese , Filogenia , Estabilidade de RNA , RNA de Algas/metabolismo , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/genéticaRESUMO
The Como National Park (CNP), located in the north-eastern part of the Ivory Coast, is strongly influenced by the river Como, which is eponymous for the park. Gallery forests along the river, forest islands, open grasslands, bush and tree savannas of various densities offer diverse habitats that harbor both forest and savannah praying mantis species. We present a preliminary checklist of praying mantids (Insecta: Mantodea) of the CNP. Specimens were collected in 2000 and 20172019, resulting in 35 species in 13 families. Two species, Ischnomantis werneri (Giglio-Tos 1916) and Solygia sulcatifrons (Audinet-Serville 1838), were recorded for the first time in Ivory Coast. Additional 47 species, which are expected to occur in the CNP, are listed here.
Assuntos
Mantódeos , Humanos , Animais , Côte d'Ivoire , Parques Recreativos , Insetos , EcossistemaRESUMO
Spartina alterniflora, an invasive plant, was introduced to the Chinese coastal zone in the early 90s. As an eco-engineering species, S. alterniflora not only alters saltmarsh species distributions, previously described as habitat degradation, but it also plays a vital role in coastal protection, especially for the development of recently emerged intertidal shoals. To provide a reference for coastal management under global change, we quantified the impact of the invasion process on provided ecological and coastal protection functions, exemplified at the emerging Jiuduansha Shoals (JDS) in the Yangtze Estuary. Results obtained by high-precision satellite monitoring and numerical modelling showed that the establishment and growth of S. alterniflora can exert considerable changes on local environment. The invasion of S. alterniflora to JDS wetland can be divided into three distinct phases, (1) establishment 1998-2003, (2) expansion 2003-2009, and (3) dominant 2009-2018 stages according to the changes in saltmarsh composition. Spatially, S. alterniflora continuously replaced Scirpus mariqueter, forcing S. mariqueter and Phragmites australis slowly to the lower and higher intertidal habitats, respectively. Notably, S. alterniflora expansion was the main driver that contributed to over 70 % of recent JDS wetland expansion even under sediment deficit conditions. Established S. alterniflora marsh (directly) dampens more waves because of aboveground stems, but it also causes more accretion and indirectly leads to higher "morphological" wave dampening. Thus, it increases coastal defense provided by the saltmarsh in the context of sea-level rise and strengthening storms. In conclusion, the role of S. alterniflora invasion to the local environment under global changes is controversial. For sustainable coastal management, we need context-dependent S. alterniflora management to maximize the benefit of coastal protection and minimize the impact on local ecology, especially in sediment-starving estuaries with expected coastline retreat.
Assuntos
Estuários , Espécies Introduzidas , Poaceae , Áreas Alagadas , China , Mudança ClimáticaRESUMO
Changes in upstream land-use have significantly transformed downstream coastal ecosystems around the globe. Restoration of coastal ecosystems often focuses on local-scale processes, thereby overlooking landscape-scale interactions that can ultimately determine restoration outcomes. Here we use an idealized bio-morphodynamic model, based on estuaries in New Zealand, to investigate the effects of both increased sediment inputs caused by upstream deforestation following European settlement and mangrove removal on estuarine morphology. Our results show that coastal mangrove removal initiatives, guided by knowledge on local-scale bio-morphodynamic feedbacks, cannot mitigate estuarine mud-infilling and restore antecedent sandy ecosystems. Unexpectedly, removal of mangroves enhances estuary-scale sediment trapping due to altered sedimentation patterns. Only reductions in upstream sediment supply can limit estuarine muddification. Our study demonstrates that bio-morphodynamic feedbacks can have contrasting effects at local and estuary scales. Consequently, human interventions like vegetation removal can lead to counterintuitive responses in estuarine landscape behavior that impede restoration efforts, highlighting that more holistic management approaches are needed.
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Ecossistema , Estuários , Humanos , Retroalimentação , Areia , Nova ZelândiaRESUMO
The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.
Assuntos
Proteínas de Algas/metabolismo , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cisteína/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Biossíntese de Proteínas , Proteínas Repressoras/metabolismo , Proteínas de Algas/química , Substituição de Aminoácidos/efeitos da radiação , Animais , Núcleo Celular/efeitos da radiação , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Luz , Mutação/genética , Estresse Oxidativo/efeitos da radiação , Fenótipo , Complexo de Proteína do Fotossistema II/genética , Processos Fototróficos/efeitos da radiação , Ligação Proteica/efeitos da radiação , Biossíntese de Proteínas/efeitos da radiação , Estabilidade Proteica/efeitos da radiação , Estrutura Terciária de Proteína , RNA de Algas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/química , Serina/genética , Relação Estrutura-AtividadeRESUMO
Coastal wetlands fulfil important functions for biodiversity conservation and coastal protection, which are inextricably linked to typical morphological features like tidal channels. Channel network configurations in turn are shaped by bio-geomorphological feedbacks between vegetation, hydrodynamics and sediment transport. This study investigates the impact of two starkly different recruitment strategies between mangroves (fast/homogenous) and salt marshes (slow/patchy) on channel network properties. We first compare channel networks found in salt marshes and mangroves around the world and then demonstrate how observed channel patterns can be explained by vegetation establishment strategies using controlled experimental conditions. We find that salt marshes are dissected by more extensive channel networks and have shorter over-marsh flow paths than mangrove systems, while their branching patterns remain similar. This finding is supported by our laboratory experiments, which reveal that different recruitment strategies of mangroves and salt marshes hamper or facilitate channel development, respectively. Insights of our study are crucial to understand wetland resilience with rising sea-levels especially under climate-driven ecotone shifts.
Assuntos
Mudança Climática , Áreas Alagadas , Ecossistema , Elevação do Nível do MarRESUMO
Mangrove forests are valuable coastal ecosystems that have been shown to persist on muddy intertidal flats through bio-morphodynamic feedbacks. However, the role of coastal conditions on mangrove behavior remains uncertain. This study conducts numerical experiments to systematically explore the effects of tidal range, small wind waves, sediment supply and coastal slope on mangrove development under sea-level rise (SLR). Our results show that mangroves in micro-tidal conditions are more vulnerable because of the gentler coastal equilibrium slope and the limited ability to capture sediment, which leads to substantial mangrove landward displacement even under slow SLR. Macro-tidal conditions with large sediment supply promote accretion along the profile and platform formation, reducing mangrove vulnerability for slow and medium SLR, but still cause rapid mangrove retreat under fast SLR. Small wind waves promote sediment accretion, and exert an extra bed shear stress that confines the mangrove forest to higher elevations with more favorable inundation regimes, offsetting SLR impacts. These processes also have important implications for the development of new landward habitats under SLR. In particular, our experiments show that landward habitat can be created even with limited sediment supply and thus without complete infilling of the available accommodation space. Nevertheless, new accommodation space may be filled over time with sediment originating from erosion of the lower coastal profile. Consistent with field data, model simulations indicate that sediment accretion within the forest can accelerate under SLR, but the timing and magnitude of accretion depend non-linearly on coastal conditions and distance from the mangrove seaward edge.
RESUMO
Staphylococcus aureus-induced infective endocarditis (IE) is a life-threatening disease. Differences in virulence between distinct S. aureus strains, which are partly based on the molecular mechanisms during bacterial adhesion, are not fully understood. Yet, distinct molecular or elemental patterns, occurring during specific steps in the adhesion process, may help to identify novel targets for accelerated diagnosis or improved treatment. Here, we use laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) of post-mortem tissue slices of an established mouse model of IE to obtain fingerprints of element distributions in infected aortic valve tissue. Three S. aureus strains with different virulence due to deficiency in distinct adhesion molecules (fibronectin-binding protein A and staphylococcal protein A) were used to assess strain-specific patterns. Data analysis was performed by t-distributed stochastic neighbor embedding (t-SNE) of mass spectrometry imaging data, using manual reference tissue classification in histological specimens. This procedure allowed for obtaining distinct element patterns in infected tissue for all three bacterial strains and for comparing those to patterns observed in healthy mice or after sterile inflammation of the valve. In tissue from infected mice, increased concentrations of calcium, zinc, and magnesium were observed compared to noninfected mice. Between S. aureus strains, pronounced variations were observed for manganese. The presented approach is sensitive for detection of S. aureus infection. For strain-specific tissue characterization, however, further improvements such as establishing a database with elemental fingerprints may be required.
Assuntos
Endocardite Bacteriana , Endocardite , Infecções Estafilocócicas , Animais , Endocardite Bacteriana/microbiologia , Espectrometria de Massas/métodos , Camundongos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismoRESUMO
The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Escherichia coli/química , Proteínas Hemolisinas/química , Redobramento de Proteína , Cristalização , Cristalografia por Raios X , Multimerização ProteicaRESUMO
In the present study we report on the identification and characterization of three novel chloroplast-targeted DnaJ-like proteins CDJ3-5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In sequence databases we could identify homologues of CDJ3-5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed us to distinguish two clades containing CDJ3/4 and CDJ5 that must have diverged early in the ancestor of the 'green lineage' and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both proteins are weakly expressed and appear to be localized to the stroma and to thylakoid membranes respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after a dark-to-light shift. Accordingly, the Arabidopsis orthologue of CDJ5 was also found to be light-inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli and had redox-active Fe-S clusters. In vitro cross-linking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast ATP-bound HSP70B (heat-shock protein 70B), presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were also in a complex with HSP70B in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of approx. 550-2800 kDa, which appeared to contain RNA. We speculate that the CDJ3-5 proteins might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes.
Assuntos
Chlamydomonas reinhardtii/química , Cloroplastos/química , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Ferro-Enxofre , Proteínas de Protozoários/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Resposta ao Choque Térmico , Luz , Oxirredução , Filogenia , Proteínas de Plantas , Ligação Proteica , Multimerização Proteica , RNA/análise , RNA Mensageiro/análiseRESUMO
Sediment-stabilizing and -destabilizing organisms, i.e. microphytobenthos (biofilms) and macrozoobenthos (bioturbators), affect the erodibility of muddy sediments, potentially altering large-scale estuarine morphology. Using a novel eco-morphodynamic model of an idealized estuary, we investigate eco-engineering effects of microphytobenthos and two macrozoobenthic bioturbators. Local mud erodibility is based on species pattern predicted through hydrodynamics, soil mud content, competition and grazing. Mud resuspension and export is enhanced under bioturbation and prevented under biostabilization through respective exposure and protection of the supra- and intertidal. Bioturbation decreases mud thickness and bed elevations, which increases net mud fluxes. Microphytobenthos reduces erosion, leading to a local mud increase of intertidal sediments. In multi-species scenarios, an effective mud-prone bioturbator strongly alters morphology, exceeding that of a more abundant sand-prone moderate species, showing that morphological change depends on species traits as opposed to abundance. Altering their habitat, the effective mud-prone bioturbator facilitates expansion of the sand-prone moderate bioturbator. Grazing and species competition favor species distributions of dominant bioturbators. Consequently, eco-engineering affects habitat conditions while species interactions determine species dominance. Our results show that eco-engineering species determine the mud content of the estuary, which suggests large effects on the morphology of estuaries with aggravating habitat degradation.