Adult neural stem/progenitor cells reduce NMDA-induced excitotoxicity via the novel neuroprotective peptide pentinin

Although the potential of adult neural stem cells to repair damage via cell replacement has been widely reported, the ability of endogenous stem cells to positively modulate damage is less well studied. We investigated whether medium conditioned by adult hippocampal stem/progenitor cells altered the extent of excitotoxic cell death in hippocampal slice cultures. Conditioned medium significantly reduced cell death following 24 h of exposure to 10 μM NMDA. Neuroprotection was greater in the dentate gyrus, a region neighboring the subgranular zone where stem/progenitor cells reside compared with pyramidal cells of the cornis ammonis. Using mass spectrometric analysis of the conditioned medium, we identified a pentameric peptide fragment that corresponded to residues 26–30 of the insulin B chain which we termed 'pentinin'. The peptide is a putative breakdown product of insulin, a constituent of the culture medium, and may be produced by insulin-degrading enzyme, an enzyme expressed by the stem/progenitor cells. In the presence of 100 pM of synthetic pentinin, the number of mature and immature neurons killed by NMDA-induced toxicity was significantly reduced in the dentate gyrus. These data suggest that progenitors in the subgranular zone may convert exogenous insulin into a peptide capable of protecting neighboring neurons from excitotoxic injury.     

High-Density Microwell Chip for Culture and Analysis of Stem Cells

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.     

Responses of eight boreal flat bug (Heteroptera: Aradidae) species to clear-cutting and forest fire

Boreal flat bugs include a high proportion of species that are considered negatively affected by forestry. Knowledge on the biology and habitat demands of individual species is generally limited. We examined the influence on flat bugs of stand-age and clear-cutting, comparing five classes of spruce stands. The five classes were: clear-cut, unthinned, and thinned (all three products of current clear-cutting forestry), mature managed and old-growth stands (these two had never been clear-cut). We also compared unburned and recently burned mature pine forest. Fire, but not stand age, had a pronounced effect on species richness and total abundance. Aradus depressus showed a significant association with older forest stands. Aradus betulae occurred only in clear-cuts and burned forest indicating that this species is favored by disturbance in general. Aradus lugubris, Aradus crenaticollis and Aradus brevicollis were found only in the burned forest. Aradus brevicollis has not previously been shown to be associated with fire.     

F-Spondin, expressed in somite regions avoided by neural crest cells, mediates inhibition of distinct somite domains to neural crest migration

Neural crest (NC) cells migrate exclusively into the rostral half of each sclerotome, where they avoid the dermomyotome and the paranotochordal sclerotome. F-spondin is expressed in these inhibitory regions and throughout the caudal halves. In vitro bioassays of NC spreading on substrates of rostral or caudal epithelial-half somites (RS or CS, respectively) revealed that NC cells adopt on RS a fibroblastic morphology, whereas on CS they fail to flatten. F-spondin inhibited flattening of NC cells on RS. Conversely, F-spondin antibodies prevented rounding up of NC cells on CS. Addition of F-spondin to trunk explants inhibited NC migration into the sclerotome, and treatment of embryos with anti-F-spondin antibodies yielded migration into otherwise inhibitory sites. Thus, somite-derived F-spondin is an inhibitory signal involved in patterning the segmental migration of NC cells and their topographical segregation within the RS.     

Expression of NADH-oxidases enhances ethylene productivity in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bacterial EFE

Ethylene is a major petrochemical for which biotechnological production methods are an attractive alternative. Here we use a system based on a bacterial ethylene forming enzyme (EFE) expressed in Saccharomyces cerevisiae. Metabolic modelling performed in a previous study identified re-oxidation of NADH as a factor limiting ethylene production in S. cerevisiae. In line with this, we here found that strains with multicopy plasmid expression of the heterologous oxidases nox and Aox1 led to significantly increased specific ethylene productivity, up 12 and 36%, respectively, compared to the control strain with empty plasmid. However the productivity and yield was only improved in the AOX expressing strain compared to that of the control strain. Both oxidase expressing strains also exhibited increased respiration rates compared to the reference strain, with specific oxygen consumption rates being roughly doubled in both strains. The AOX strain furthermore exhibited a significant increase in the EFE substrate 2-oxoglutarate formation compared to the reference strain, linking an improvement in ethylene production to both increased respiratory capacity and increased substrate availability, thereby corroborating our previous finding.     

Induced Variations in Brassinosteroid Genes Define Barley Height and Sturdiness,and Expand the Green Revolution Genetic Toolkit

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.The introduction of dwarfing genes to increase culm sturdiness of cereal crops was crucial for the first Green Revolution (Hedden, 2003). The culms of tall cereal crops were not strong enough to support the heavy spikes of high-yielding cultivars, especially under high-nitrogen conditions. As a result, plants fell over, a process known as lodging. This caused losses in yield and grain-quality issues attributable to fungal infections, mycotoxin contamination, and preharvest germination (Rajkumara, 2008). Today, a second Green Revolution is on its way, to revolutionize the agricultural sector and to ensure food production for a growing world population. Concurrently, global climate change is expected to cause more frequent occurrences of extreme weather conditions, including thunderstorms with torrential rain and strong winds, thus promoting cereal culm breakage (Porter and Semenov, 2005; National Climate Assessment Development Advisory Committee, 2013). Accordingly, plant architectures that resist lodging remain a major crop-improvement goal and identification of genes that regulate culm length is required to enhance the genetic toolbox in order to facilitate efficient marker-assisted breeding. The mutations and the corresponding genes that enabled the Green Revolution in wheat (Triticum aestivum) and rice (Oryza sativa) have been identified (Hedden, 2003). They all relate to gibberellin metabolism and signal transduction. It is now known that other plant hormones such as brassinosteroids are also involved in the regulation of plant height. Knowledge of the molecular mechanisms underlying the effects of the two hormones on cell elongation and division has mainly come from studies in Arabidopsis (Arabidopsis thaliana; Bai et al., 2012). Mutant-based breeding strategies to fine-tune brassinosteroid metabolism and signaling pathways could improve lodging behavior in modern crops (Vriet et al., 2012) such as barley (Hordeum vulgare), which is the fourth most abundant cereal in both area and tonnage harvested (http://faostat.fao.org).A short-culm phenotype in crops is often accompanied by other phenotypic changes. Depending on the penetrance of such pleiotropic characters, but also the parental background and different scientific traditions and expertise, short-culmed barley mutants were historically divided into groups, such as brachytic (brh), breviaristatum (ari), dense spike (dsp), erectoides (ert), semibrachytic (uzu), semidwarf (sdw), or slender dwarf (sld; Franckowiak and Lundqvist, 2012). Subsequent mutant characterization was limited to intragroup screens and very few allelism tests between mutants from different groups have been reported (Franckowiak and Lundqvist, 2012). Although the total number of short-culm barley mutants exceeds 500 (Franckowiak and Lundqvist, 2012), very few have been characterized at the DNA level (Helliwell et al., 2001; Jia et al., 2009; Chandler and Harding, 2013; Houston et al., 2013). One of the first identified haplotypes was uzu barley (Chono et al., 2003). The Uzu1 gene encodes the brassinosteroid hormone receptor and is orthologous to the BRASSINOSTEROID-INSENSITIVE1 (BRI1) gene of Arabidopsis, a crucial promoter of plant growth (Li and Chory, 1997). The uzu1.a allele has been used in East Asia for over a century and is presently distributed in winter barley cultivars in Japan, the Korean peninsula, and China (Saisho et al., 2004). Its agronomic importance comes from the short and sturdy culm that provides lodging resistance, and an upright plant architecture that tolerates dense planting.Today, more than 50 different brassinosteroids have been identified in plants (Bajguz and Tretyn, 2003). Most are intermediates of the complex biosynthetic pathway (Shimada et al., 2001). Approximately nine genes code for the enzymes that participate in the biosynthetic pathway from episterol to brassinolide (Supplemental Fig. S1). Brassinosteroid deficiency is caused by down-regulation of these genes, but it can also be associated with brassinosteroid signaling. The first protein in the signaling network is the brassinosteroid receptor encoded by BRI1 (Li and Chory, 1997; Kim and Wang, 2010). In this work, we show how to visually identify brassinosteroid-mutant barley plants and we describe more than 20 relevant mutations in four genes of the brassinosteroid biosynthesis and signaling pathways that can be used in marker-assisted breeding strategies.     

A human protein atlas for normal and cancer tissues based on antibody proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.     

Time-resolved multidimensional NMR with non-uniform sampling

Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D ¹H–¹⁵N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.     

Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.