Transcriptomic response to prolonged ethanol production in the cyanobacterium Synechocystis sp. PCC6803

BACKGROUND: The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. RESULTS: The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d(-1) and 0.0303% (v/v) ethanol d(-1) were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. CONCLUSIONS: Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected

More than just an eagle killer: The freshwater cyanobacterium Aetokthonos hydrillicola produces highly toxic dolastatin derivatives

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the “eagle killer” neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans. Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product

Analisa Pondasi Pile Raft Pada Tanah Lunak Dengan Plaxis 2d

Permasalahan penurunan menjadi salah satu masalah yang sering dihadapi para perencana pondasi bangunan dikarenakan oleh kondisi tanah yang lunak. Untuk mengatasi permasalahan yang ada, banyak perencana menggunakan pondasi raft atau pondasi rakit, karena dianggap mampu memberikan faktor keamanan yang memadai dalam menghadapi kegagalan daya dukung ultimate. Namun diperkirakan pondasi raft ini akan mengalami penurunan yang besar. Permasalahan tersebut mungkin dapat berkurang jika adanya penambahan pile pada pondasi raft sehingga menjadi pondasi pile raft. Dengan penambahan pile pada pondasi raft diharapkan perencanaannya mempertimbangkan segi ekonomis. Dengan menggunakan beban merata 6 t/m2, dilakukan penelitian pada pondasi pile raft dengan memvariasikan tebal raft yakni 80 cm, 100 cm, 120 cm dan 140 cm. Untuk panjang pile divariasikan dari panjang 5 m, 7 m, 9 m, 13 m dan 15 m. Analisis penurunan dilakukan dengan menggunakan software Plaxis 2D dan Metode Poulos. Hasil dari penelitian ini menunjukkan bahwa Penambahan jumlah pile pada pondasi raft menghasilkan profil penurunan yang berkurang namun pada suatu keadaan tertentu penambahan pile tidak memberikan kontribusi yang lebih signifikan. Begitupun dengan perhitungan Poulos, pada konfigurasi pile tertentu tidak memberi kontribusi lagi. Sehingga desain yang ekonomis pada penelitian ini adalah dengan menggunakan tebal raft 80 cm dengan panjang pile 13 m dan konfigurasi pile 7x7

Research Data Management Organiser: A tool to support the planning, implementation and organisation of research data management

The project Research Data Management Organiser (RDMO)1 develops a tool to support the planning, implementation and organisation of research data management. The multilingual open source tool can be installed locally and adapted to institutional or discipline-specific needs with regards to contents. It provides interfaces to institutional authentication procedures. Key features of the first version, released in April 2017, include: the ability to continuously update and augment the information in the course of a project; access for different stakeholders, such as researchers, project coordinators, the IT department, data managers etc., with customized views; export formats for different purposes including data management plans (DMPs) according to funder requirements. Planned future developments include, among other things, features to support actual data management, e.g. tasks (with deadlines and a reminder functionality) that can be linked with dedicated stakeholder responsibilities

Success Criteria for the Development and Sustainable Operation of Virtual Research Environments

In many areas of research, virtual research environments (VREs) have become an essential part of modern research processes. The providers of VREs need to respond to this growing importance with functioning and efficient processes for the development, operation and quality assurance of VREs. We have developed a life-cycle model for VREs, which focuses in particular on the success-critical points for the transition to a VRE's sustainable operation. Furthermore, we discuss a set of success criteria that enables all involved in the VRE (operators, funding bodies, users) to identify which aspects will be relevant to their specific needs prior to the creation of a new VRE. In light of the heterogeneity of VREs, this set of criteria is supplemented in individual cases by discipline-specific criteria

Hapalindoles from the Cyanobacterium Hapalosiphon sp. Inhibit T Cell Proliferation

Novel immunomodulating agents are currently sought after for the treatment of autoimmune diseases and cancers. In this context, a screening campaign of a collection of 575 cyanobacteria extracts for immunomodulatory effects has been conducted. The screening resulted in several active extracts. Here we report the results of subsequent studies on an extract from the cyanobacterium; Hapalosiphon; sp. CBT1235. We identified 5 hapalindoles as the compounds responsible for the observed immunomodulatory effect. These indole alkaloids are produced by several strains of the cyanobacterial family Hapalosiphonaceae. They are known for their anti-infective, cytotoxic, and other bioactivities. Modulation of the activity of human immune cells has not yet been described. The immunomodulatory activity of the hapalindoles was characterized; in vitro; using flow cytometry-based measurements of T cell proliferation after carboxyfluorescein diacetate succinimidyl ester staining, and apoptosis and necrosis induction after annexin V/propidium iodide staining. The most potent compound, hapalindole A, reduced T cell proliferation with an IC; 50; of 1.56 µM, while relevant levels of apoptosis were measurable only at 10-fold higher concentrations. Hapalindole A-formamide and hapalindole J-formamide, isolated for the first time from a natural source, had much lower activity than the nonformylated derivatives while, at the same time, being less selective for antiproliferative over apoptotic effects

Placental immune response to apple allergen in allergic mothers

The immunological milieu in the placenta may be crucial for priming the developing foetal immune system. Early imbalances may promote the establishment of immune-mediated diseases in later life, including allergies. The initial exposure to allergens seems to occur in utero, but little is known about allergen-induced placental cytokine and chemokine release. The release of several cytokines and chemokines from placenta tissue after exposure to mast cell degranulator compound 48/80 or apple allergen in placentas from allergic and healthy mothers was to be analysed. Four placentas from women with apple allergy and three controls were applied in a placental perfusion model with two separate cotyledons simultaneously perfused with and without apple allergen (Mal d 1). Two control placentas were perfused with compound 48/80. In outflow, histamine was quantified spectrophotofluorometrically, IL-2, IL-4, IL-6, IL-10, TNF and IFN-gamma by a cytometric multiplex bead array and IL-13 and CXCL10, CXCL11, CCL17 and CCL22 with an in-house multiplex Luminex assay. Compound 48/80 induced a rapid release of histamine, CXCL10, CXCL11, CCL17 and CCL22, but not of the other factors. Apple allergen induced a time-dependent release of IL-6 and TNF, but not of histamine, in placentas of women with apple allergy compared with the unstimulated cotyledon. CCL17 levels were slightly increased after allergen stimulation in control placentas. Allergens can induce placental cytokines and chemokines distinctly in allergic and healthy mothers. These mediators may affect the prenatal development of the immune system and modify the risk of diseases related to immune disorders in childhood such as allergies.Funding Agencies|Institut Danone (Haar, Germany); European Network of Excellence within the 6th Framework Programme of the European Union [512040]; Swedish Research Council [K2011-56X-21854-01-06]; Cancer and Allergy Association; Olle Engkvist Foundation</p