High-resolution in vivo imaging of xylem-transported CO2 in leaves based on real-time 11C-tracing

Plant studies using the short-lived isotope C-11 to label photosynthate via atmospheric carbon dioxide (CO2), have greatly advanced our knowledge about the allocation of recent photosynthate from leaves to sinks. However, a second source for photosynthesis is CO2 in the transpiration stream, coming from respiration in plant tissues. Here, we use in vivo tracing of xylem-transported (CO2)-C-11 to increase our knowledge on whole plant carbon cycling.We developed a newmethod for in vivo tracing of xylem-transported CO2 in excised poplar leaves using C-11 in combination with positron emission tomography (PET) and autoradiography. To show the applicability of both measurement techniques in visualizing and quantifying CO2 transport dynamics, we administered the tracer via the cut petiole and manipulated the transport by excluding light or preventing transpiration. Irrespective of manipulation, some tracer was found in main and secondary veins, little of it was fixed in minor veins or mesophyll, while most of it diffused out the leaf. Transpiration, phloem loading and CO2 recycling were identified as mechanisms that could be responsible for the transport of internal CO2. Both C-11-PET and autoradiography can be successfully applied to study xylem-transported CO2, toward better understanding of leaf and plant carbon cycling, and its importance in different growing conditions

BAMMsat-on-BEXUS: A Technology and Operation Demonstration of a BioCubeSat Platform on a Stratospheric Balloon Flight Educational Program

This paper reports the current use of the REXUS/BEXUS educational program. The program allows university students across Europe to carry out scientific and technology experiments on research sounding rockets and balloons. BAMMsat-on-BEXUS (BoB) is an experiment from Cranfield University and University of Exeter performing a technology and operation demonstration of a bioCubeSat on a stratospheric balloon at an altitude of ~30km above the ground. BEXUS stands for Balloon Experiments for University Students and is realized under an agreement between the German Aerospace Centre (DLR), Swedish National Space Agency (SNSA), European Space Agency (ESA), and EuroLaunch. The term bioCubeSat could be used to refer to a nanosatellite in a CubeSat format with a biological experiment on-board. Over the last decade, a series of six bioCubeSats have been launched into orbit by NASA and a private company, SpacePharma, i.e., GeneSat, PharmaSat, O/OREOS, SporeSat, Dido-2, and EcAMSat. The BAMMsat concept (Bioscience, Astrobiology, Medicine and Material science on CubeSat) is a bioscience hardware platform which aims to advance the current state of the art technology, under development at Cranfield University, for application in LEO and beyond LEO. This generic platform can be flown as a free-flying CubeSat or hosted as a payload on a larger spacecraft. BAMMsat utilizes COTS sensors, actuators, and fluidic components to enable bioscience experiments by reproducing the features in a traditional laboratory into a miniaturized “laboratory.” It is designed to be compatible with the mass, volume, and power budget of a CubeSat payload and flexible for a broad range of applications and biological systems such as microorganisms, nematode worms, and mammalian cells cultures, including human cell cultures. The core features of BAMMsat are the ability to (i) house multiple samples, (ii) maintain samples in an appropriate local environment (ii) perturb sample fluidically, and (iv) monitor samples. BoB aims to perform a technology and operation demonstration of the BAMMsat bioCubeSat payload in an extreme environment such as the stratosphere. The experiment is to be flown on the BEXUS30 flight campaign in October 2020 from ESRANGE Space Centre, Sweden. The stratosphere can be used as an analog of some aspect of a relevant spaceflight physical environment such as reduced pressure (near-vacuum; ~11 mbar), and temperature (-50°C). The BEXUS flight campaign could also be used as an analog of pre-flight, flight and post-flight operation similar to orbital launch campaign. For bioscience experiments, the biological samples often imposed additional requirement during pre-flight to ensure its viability. BoB will house C. elegans in a 2U pressure vessel to demonstrate its functionality to provide a controlled thermal and fluidic environment with appropriate housekeeping control. This functionality reflects the hardware capability to maintain a viable biological sample. BoB has a 3U CubeSat form factor with 2U allocated for the BAMMsat hardware and 1U allocated as the BAMMsat-on-BEXUS bus. This paper reports progress at four months before flight campaign. The paper also discusses an overview of the experiment objectives and systems design, to build a representative CubeSat that is translatable into a free-flying orbital CubeSat

Possible charge inhomogeneities in the CuO2 planes of YBa2Cu3O6+x (x=0.25, 0.45, 0.65, 0.94) from pulsed neutron diffraction

The atomic pair distribution functions (PDF) of four powder samples of YBa2Cu3O6+x (x=0.25, 0.45, 0.65, 0.94) at 15 K have been measured by means of pulsed neutron diffraction. The PDF is modelled using a full-profile fitting approach to yield structural parameters. In contrast to earlier XAFS work we find no evidence of a split apical oxygen site. However, a slightly improved fit over the average crystallographic model results when the planar Cu(2) site is split along the z-direction. This is interpreted in terms of charge inhomogeneities in the CuO2 planes.Comment: 8 pages, 3 figure

Coalescent-based species delimitation in the sand lizards of the Liolaemus wiegmannii complex (Squamata: Liolaemidae)

Coalescent-based algorithms coupled with the access to genome-wide data have become powerful tools forassessing questions on recent or rapid diversification, as well as delineating species boundaries in the absence of reciprocal monophyly. In southern South America, the diversification of Liolaemus lizards during the Pleistocene is well documented and has been attributed to the climatic changes that characterized this recent period of time. Past climatic changes had harsh effects at extreme latitudes, including Patagonia, but habitat changes at intermediate latitudes of South America have also been recorded, including expansion of sand fields over northern Patagonia and Pampas). In this work, we apply a coalescent-based approach to study the diversification of the Liolaemus wiegmannii species complex, a morphologically conservative clade that inhabits sandy soils across northwest and south-central Argentina, and the south shores of Uruguay. Using four standard sequence markers (mitochondrial DNA and three nuclear loci) along with ddRADseq data we inferred species limits and a time calibrated species tree for the L. wiegmannii complex in order to evaluate the influence of Quaternary sand expansion/retraction cycles on diversification. We also evaluated the evolutionary independence of the recently described L. gardeli and inferred its phylogenetic position relative to L. wiegmannii. We find strong evidence for six allopatric candidate species within L. wiegmannii, which diversified during the Pleistocene. The Great Patagonian Glaciation (∼1 million years before present) likely split the species complex into two main groups: one composed of lineages associated with sub-Andean sedimentary formations, and the other mostly related to sand fields in the Pampas and northern Patagonia. We hypothesize that early speciation within L. wiegmannii was influenced by the expansion of sand dunes throughout central Argentina and Pampas. Finally, L. gardeli is supported as a distinct lineage nested within the L. wiegmannii complex.Fil: Villamil, Joaquín. Universidad de la República. Facultad de Ciencias; UruguayFil: Avila, Luciano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico para el Estudio de los Ecosistemas Continentales; ArgentinaFil: Morando, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico para el Estudio de los Ecosistemas Continentales; ArgentinaFil: Sites, Jack W.. University Brigham Young; Estados UnidosFil: Leaché, Adam D.. University of Washington; Estados UnidosFil: Maneyro, Raúl. Universidad de la República. Facultad de Ciencias; UruguayFil: Camargo Bentaberry, Arley. Universidad de la República; Urugua

Preparation of Calibration Standards of N1-H Paralytic Shellfish Toxin Analogues by Large-Scale Culture of Cyanobacterium Anabaena circinalis (TA04)

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ±0.1 μmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 μmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50–90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated

Small RNA profiling of low biomass samples: identification and removal of contaminants

Background Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, yet contamination with RNA is usually considered to be very unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids, such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands careful evaluation. Results Herein, we report on the presence of small RNA (sRNA) contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, thereby arguing in particular for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origin in blood plasma. To avoid artefacts in future experiments, we also devise several protocols for the removal of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using ‘ultra-clean’ extraction kits. Conclusion This is the first report on the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies. Keywords: RNA sequencing; Artefact removal; Exogenous RNA in human blood plasma; Contaminant RNA; Spin column

Constraining N2O emissions since 1940 using firn air isotope measurements in both hemispheres

N2O is currently the third most important anthropogenic greenhouse gas in terms of radiative forcing and its atmospheric mole fraction is rising steadily. To quantify the growth rate and its causes over the past decades, we performed a multi-site reconstruction of the atmospheric N2O mole fraction and isotopic composition using new and previously published firn air data collected from Greenland and Antarctica in combination with a firn diffusion and densification model. The multi-site reconstruction showed that while the global mean N2O mole fraction increased from (290±1)nmolmol-1 in 1940 to (322±1)nmolmol-1 in 2008, the isotopic composition of atmospheric N2O decreased by (-2.2±0.2)% for δ15Nav, (-1.0±0.3)% for δ18O, (-1.3±0.6)% for δ15Nα, and (-2.8±0.6)% for δ15Nβ over the same period. The detailed temporal evolution of the mole fraction and isotopic composition derived from the firn air model was then used in a two-box atmospheric model (comprising a stratospheric box and a tropospheric box) to infer changes in the isotopic source signature over time. The precise value of the source strength depends on the choice of the N2O lifetime, which we choose to fix at 123 years. The average isotopic composition over the investigated period is δ15Nav Combining double low line (-7.6±0.8)% (vs. air-N2), δ18O Combining double low line (32.2±0.2)% (vs. Vienna Standard Mean Ocean Water-VSMOW) for δ18O, δ15Nα Combining double low line (-3.0±1.9)% and δ15Nβ Combining double low line (-11.7±2.3)%. δ15Nav, and δ15Nβ show some temporal variability, while for the other signatures the error bars of the reconstruction are too large to retrieve reliable temporal changes. Possible processes that may explain trends in 15N are discussed. The 15N site preference (Combining double low line δ15Nα-δ15Nβ) provides evidence of a shift in emissions from denitrification to nitrification, although the uncertainty envelopes are large