Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing

The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker.     

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.     

利用野生青狗尾草的细胞质培育谷子质核互作雄性不育材料

利用野生青狗尾草N10为母本,谷子农家品种大青秸为父本,进行种间杂交,获得了3株杂种。杂种在农艺性状上表现为谷子和青狗尾草的种间类型,花器表现了雄性败育,但雌蕊发育正常,能接受外来花粉结实。细胞形态学观察表明,其雄性败育表现为单核小孢子典败。该谷子雄性不育材料的获得,为利用野生青狗尾草的细胞质培育谷子质核互作雄性不育系,进而为实现谷子三系配套杂种优势利用奠定了基础。     

Evaluation of halophilic microalgae isolated from Rabigh Red Sea coastal area for biodiesel production: Screening and biochemical studies

In the present study, different water samples from Red Sea coastal area at Rabigh city, Saudi Arabia were studied for their dominant algal species. Microalgal isolation was carried out based on dilution method and morphologically examined using F/2 as a growth medium. Dry weight and main biochemical composition (protein, carbohydrates, lipids) of all species were performed at the end of the growth, and biodiesel characteristics were estimated. Nannochloropsis sp., Dunaliella sp., Tetraselmis sp., Prorocentrum sp., Chlorella sp., Nitzschia sp., Coscinodiscus sp., and Navicula sp. were the most dominant species in the collected water samples and were used for further evaluation. Nannochloropsis sp. surpassed all other isolates in concern of biomass production with the maximum recorded dry weight of 0.89 g L^?1, followed by Dunaliella sp. (0.69 g L^?1). The highest crude protein content was observed in Nitzschia sp. (38.21%) and Dunaliella sp. (18.01%), while Nannochloropsis sp. showed 13.38%, with the lowest recorded lipid content in Coscinodiscus sp. (10.09%). Based on the growth, lipid content, and biodiesel characteristics, the present study suggested Dunaliella sp. and Nitzschia sp. as promising candidates for further large-scale biodiesel production.     

From military training area to National Park over 20 years: Indicators for outcome evaluation in a large-scale restoration project in alpine Norway

There is a need for large-scale demonstrations to address the challenges and possibilities for upscaling of ecosystem restoration, and for learning and sharing knowledge across professions and habitats. Large-scale and complex restoration projects need new perspectives on goal formulation, indicators for success, and evaluation to encompass both scientific approaches and the tacit knowledge held by practitioners. The objective of this paper is to use the restoration of a 165 km² former military training area in alpine central Norway into National Park to demonstrate the challenges of upscaling and integration. Main tasks were to remove roads and technical infrastructure, prepare for natural recovery and remove undetonated ordnance. In total, 19 indicators were used to evaluate the restoration outcome, related to four overall restoration goals formulated by the Norwegian Parliament: nature protection, considerable nature benefit, safe civilian use, and restoration back to natural state. Despite an overall linear project cycle, a dynamic and adaptive process of planning, implementation and evaluation was performed at the individual site scale. A dynamic dialogue between all involved professions allowed for exchanging scientific and tacit knowledge, and continuous improvement of solutions. The study demonstrated the relevance of qualitative assessments combined with quantitative indicators – i.e., use of expert opinions and the continuous evaluation to feed back into planning and improving the implementation of restoration measures. A “Green training” procedure was developed, linking top-down formally defined settings of the project with bottom-up hand-on solutions. This procedure can be directly transferred to other large-scale mitigation and restoration projects. Demonstration sites like the one described here, are valuable to develop an expanded vision of restoration to meet the UN Sustainable Goals.     

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%–51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.     

Establishment of a novel testicular cell line from sterlet Acipenser ruthenus and evaluation of its applications

In this study, a cell line, designated as Acipenser ruthenus testis (ART), was successfully established from testis tissues of the sterlet Acipenser ruthenus and characterized by studying and comparing the expression of specific genes between the cell line and the parent gonad tissues. The results suggested that the developed ART cell line was composed of a mixture of germ cells and somatic cells. Ploidy analysis indicated that the cell line exhibited a high degree of genetic stability and that the cells remained in a good proliferating state after being subcultured to passage 80.     

A molecular hybrid producing simultaneously singlet oxygen and nitric oxide by single photon excitation with green light

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on “conventional” drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate ¹O₂ and NO with single photon excitation of biocompatible visible light, (ii) excellent ¹O₂ quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic ¹O₂ and NO.     

Pyranopyrazoles as efficient antimicrobial agents: Green,one pot and multicomponent approach

Innovative therapeutic heterocycles having precisely thiadiazolyl-pyranopyrazole fragments were prepared by using the ecofriendly synthetic route. Entire compounds formed in quantitative yields. All the composites tested for their antimicrobial effectiveness against four microbial, two beneficial fungi’s and accurately measured the minimum inhibitory concentrations (MIC and MBC/MFC), along with some initial structure activity relationships (SARs) also discussed. From the biological outcomes, the motif 6f provided an outstanding activity against all six pathogens. The possible presence of a nitro substituent on this composite may undoubtedly enhance the activity. In addition, amalgams 6d, 6g and 6l displayed promising antimicrobial results. This may be justified to the presence of electron capture group attached to the benzene ring, while the combinations 6j and 6k were zero effect towards all bacterial strains. The other compounds were excellent to low antimicrobial efficiency. The intriguing point was observed that all the active compounds had in common immense antibacterial effectiveness on Gram-negative bacteria than Gram-positive one and more antifungal activity on A. niger compare to other fungus. All things considered and suggested that this outstanding green synthetic approach is used to develop biological active compounds. On top of that, biological results confirmed that these biologically energetic motifs suitable for additional preclinical examine with the aim of standing novel innovative drugs.     

Macroscopic and Microscopic Morphological Features of Stromatolites Related To Activity of Eukaryote-Dominated Biofilms in an Acid Mine Drainage Environment: Biosignatures and Understanding Preservation of Stromatolites as Trace Fossils

Euglena-, diatom-, and algae-dominated biofilms are the principal producers of iron-rich biolaminates that result in biosedimentary structures, or stromatolites, in an acid mine drainage (AMD) environment in Indiana. These structures are considered trace fossils because they are produced by organism-sediment interactions and record physicochemical conditions of the environment. Our purpose was to link the biofilm types to specific micro- and micromorphological features and the physicochemical conditions under which they were formed. Analyses revealed that Euglena-dominated biofilm produced thin, porous microlaminae by trapping, binding, and relocating AMD precipitates as the biofilm kept pace with chemical sedimentation. More massive microlaminae were produced by high rates of chemical sedimentation brought on by increased discharge and dilution of acidity. Diatom- and algae-dominated biofilms produced thick, mm–cm-scale, porous, spongelike micro- to macrolaminae through oxygenic photosynthesis and/or metal uptake in extracellular polymeric substances, which promoted mineral precipitation on cell walls to create a rigid, porous structure. The variations in biolaminate textures within the stromatolites record seasonal changes in the microbial populations and physicochemical conditions of the AMD environment. These iron-rich stromatolites represent trace fossils that record morphological biosignatures of eukaryote-dominated microbial biofilms and may serve as appropriate proxies in the search for similar evidence of eukaryotic life in other iron-rich paleoenvironments, such as those on early Earth and Mars.