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.     

Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype

Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC) showing CpG island methylator phenotype (CIMP). Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APC^wt, TP53^mut, KRAS^mut; 3/8 marker methylated; HROC43: APC^mut, TP53^mut, KRAS^mut; 4/8 marker methylated; HROC60: APC^wt, TP53^mut, KRAS^wt; 4/8 marker methylated; HROC183: APC^mut, TP53^mut, KRAS^mut; 6/8 marker methylated). Cell lines were of epithelial origin (EpCAM⁺) with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan). Of note, most cell lines were sensitive towards “non-classical” CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib). This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo-) therapeutics helps bringing forward the concept of personalized tumor therapy.     

Nuclease-Assisted Suppression of Human DNA Background in Sepsis

Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C₀t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C₀t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.     

SUM1, an Apparent Positive Regulator of the Cryptic Mating-Type Loci in SACCHAROMYCES CEREVISIAE

The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MAT alpha HMRa mar1-1 is unable to mate because of the simultaneous expression of a and alpha information. A mutant of this strain was isolated that exhibits an alpha phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (suppressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and alpha information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.