The Performance of Alfalfa Synthetics in the First and Advanced Generations

During alfalfa breeding investigations conducted at the Nebraska Agricultural Experiment Station, numerous superior clones were selected and tested as clones, and in polycross progeny tests. Information was needed on the performance of synthetic varieties in the first and advanced generations, on the optimum number of clones to include in a synthetic variety, and on parent-progeny relationships. Clones with high general combining ability for forage yield as measured by polycross progeny tests, and in certain instances specific combining ability based on single-cross tests, were intercrossed in various ways to produce synthetic varieties. A group of synthetics varying in number of parents from 2 to 6 clones, having in some instances certain clones as common parents, was tested initially in the first generation of synthesis (referred to as Syn-1 from here on), later in the Syn-1 versus the Syn-2, and in some instances in the Syn-1, Syn-2, and Syn-3, and ultimately in the Syn-1,-2,-3, and -4 generations. The purposes of this bulletin are to report (1) comparative results obtained in yield trials involving the Syn-1,-2,-3, and -4 generations of 5 two-clone and 14 multiple-clone synthetics at Lincoln, Nebraska, and Ithaca, New York, and (2) parent-progeny relationships

Genome-wide identification and functional analysis of Apobec-1-mediated C-to-U RNA editing in mouse small intestine and liver

BackgroundRNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoprotein B mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1-mediated C-to-U RNA editing remain incompletely explored.ResultsDeep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and Apobec-1-deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs, all within 3' untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites are unique to liver. Changes in RNA editing lead to corresponding changes in intestinal mRNA and protein levels for 11 genes. Analysis of RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression reveals that a subset of targets identified in wild-type mice are restored in Apobec-1-deficient mouse intestine and liver following Apobec-1 rescue. We find distinctive polysome profiles for several RNA editing targets and demonstrate novel exonic editing sites in nuclear preparations from intestine but not hepatic apolipoprotein B RNA. RNA editing is validated using cell-free extracts from wild-type but not Apobec-1-deficient mice, demonstrating that Apobec-1 is required.ConclusionsThese studies define selective, tissue-specific targets of Apobec-1-dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific

Renal stone detection using a low kilo-voltage paediatric CT protocol – A porcine phantom study

yesIntroduction: Reducing tube voltage is an effective dose saving method in computed tomography (CT) assuming tube current is not concurrently increased. Recent innovations in scanner technology now enable CT tube voltage reduction to 70 kV thereby increasing opportunities for dose reduction in paediatric patients, but it is unclear if the increased image noise associated with 70 kV impacts on ability to visualise renal stones accurately. The purpose was to assess detectability of nephrolithiasis using a bespoke paediatric phantom and low kV, non-contrast CT and to assess inter-observer agreement. Methods: Forty-two renal stones of different size and chemical composition were inserted into porcine kidneys and positioned in a bespoke, water-filled phantom mimicking a 9-year-old child weighing approximately 33kg. The phantom was scanned using 120 and 70 kV CT protocols, and the detectability of the stones was assessed by three radiologists. Absolute agreement and Fleiss’ kappa regarding detectability were assessed. Results: The mean diameter of renal stones as measured physically was 4.24 mm ranging from 1 to 11 mm. Four stones were missed by at least one observer. One observer had a sensitivity of 93 and 95% at 70 and 120 kV, respectively, while the sensitivity for observers 2 and 3 was 98% at both kV levels. Specificity was 100% across readers and kV levels. Absolute agreement between the readers at 70 kV was 92% (kappa = 0.86) and 98% (kappa = 0.96) at 120 kV indicating a strong agreement at both kV levels. Conclusions: The results suggest that lowering the kV does not affect the detection rate of renal stones and may be a useful dose reduction strategy for assessment of nephrolithiasis in children

Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds

Mining gene functional networks to improve mass-spectrometry-based protein identification

Motivation: High-throughput protein identification experiments based on tandem mass spectrometry (MS/MS) often suffer from low sensitivity and low-confidence protein identifications. In a typical shotgun proteomics experiment, it is assumed that all proteins are equally likely to be present. However, there is often other evidence to suggest that a protein is present and confidence in individual protein identification can be updated accordingly

Integrating shotgun proteomics and mRNA expression data to improve protein identification

Motivation: Tandem mass spectrometry (MS/MS) offers fast and reliable characterization of complex protein mixtures, but suffers from low sensitivity in protein identification. In a typical shotgun proteomics experiment, it is assumed that all proteins are equally likely to be present. However, there is often other information available, e.g. the probability of a protein's presence is likely to correlate with its mRNA concentration

The plant LINC complex at the nuclear envelope

Significant advances in understanding the plant nuclear envelope have been made over the past few years; indeed, knowledge of the protein network at the nuclear envelope is rapidly growing. One such network, the linker of nucleoskeleton and cytoskeleton (LINC) complex, is known in animals to connect chromatin to the cytoskeleton through the nuclear envelope. The LINC complex is made of Sad1/Unc84 (SUN) and Klarsicht/Anc1/Syne1 homology (KASH) proteins which have been recently characterized in plants. SUN proteins are located within the inner nuclear membrane, while the KASH proteins are included into the outer nuclear membrane. SUN and KASH domains interact and bridge the two nuclear membranes. In Arabidopsis, KASH proteins also interact with the tryptophan-proline-proline (WPP) domain-interacting tail-anchored protein 1 (WIT1), associated with the nuclear pore complex and with myosin XI-i which directly interacts with the actin cytoskeleton. Although evidence for a plant LINC complex connecting the nucleus to the cytoskeleton is growing, its interaction with chromatin is still unknown, but knowledge gained from animal models strongly suggests its existence in plants. Possible functions of the plant LINC complex in cell division, nuclear shape, and chromatin organization are discussed

Insight into the Assembly Properties and Functional Organisation of the Magnetotactic Bacterial Actin-like Homolog, MamK

Magnetotactic bacteria (MTB) synthesize magnetosomes, which are intracellular vesicles comprising a magnetic particle. A series of magnetosomes arrange themselves in chains to form a magnetic dipole that enables the cell to orient itself along the Earth’s magnetic field. MamK, an actin-like homolog of MreB has been identified as a central component in this organisation. Gene deletion, fluorescence microscopy and in vitro studies have yielded mechanistic differences in the filament assembly of MamK with other bacterial cytoskeletal proteins within the cell. With little or no information on the structural and behavioural characteristics of MamK outside the cell, the mamK gene from Magnetospirillium gryphiswaldense was cloned and expressed to better understand the differences in the cytoskeletal properties with its bacterial homologues MreB and acitin. Despite the low sequence identity shared between MamK and MreB (22%) and actin (18%), the behaviour of MamK monitored by light scattering broadly mirrored that of its bacterial cousin MreB primarily in terms of its pH, salt, divalent metal-ion and temperature dependency. The broad size variability of MamK filaments revealed by light scattering studies was supported by transmission electron microscopy (TEM) imaging. Filament morphology however, indicated that MamK conformed to linearly orientated filaments that appeared to be distinctly dissimilar compared to MreB suggesting functional differences between these homologues. The presence of a nucleotide binding domain common to actin-like proteins was demonstrated by its ability to function both as an ATPase and GTPase. Circular dichroism and structural homology modelling showed that MamK adopts a protein fold that is consistent with the ‘classical’ actin family architecture but with notable structural differences within the smaller domains, the active site region and the overall surface electrostatic potential