Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

Material properties of films from enzymatically tailored arabinoxylans

Rye arabinoxylan, with an initial arabinose to xylose (Ara/Xyl) ratio of 0.50, was enzymatically modified with alpha-L-arabinofuranosidase. Different enzyme dosages were used to prepare arabinoxylan samples with a gradient of arabinose content varying from Ara/Xyl ratio 0.50 to 0.20. The degree of polymerization of the arabinoxylans was not affected by the enzymatic treatment, as detected with SEC-MALLS. Arabinoxylan samples with an Ara/Xyl ratio of 0.30 and below agglomerated in a water solution as seen by changes in light scattering. All samples, however, formed cohesive films upon drying, without addition of external plasticizers. The film from untreated arabinoxylan was completely amorphous; whereas films of the enzyme-treated arabinoxylans were semicrystalline with an increasing degree of crystallinity with decreasing arabinose content as determined by WAXS. Oxygen permeability measurements of the films showed that decreased arabinose content also resulted in lower oxygen permeability of the films. All films were strong and relatively stiff, but showed variations in strain at break. The moderately debranched film with an Ara/Xyl ratio of 0.37 had highest strain at break among all the films tested, yet was stiff and strong. This material also exhibited yielding and had stress/strain behavior similar to synthetic semicrystalline polymers, with a tendency to strain-induced crystallization. Such a combination of mechanical properties combined with oxygen barrier properties is very attractive for packaging applications.     

Cultivar-related differences in the distribution of cell-wall-bound thionins in compatible and incompatible interactions between barley and powdery mildew

Leaf-specific thionins of barley (Hordeum vulgare L.) have been identified as a novel class of cell-wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The distribution of these polypeptides has been studied in the host-pathogen system of barley and Erisyphe graminis DC.f.sp. hordei Marchal (powdery mildew). Immunogold-labelling of thionins in several barley cultivars indicates that resistance or susceptibility may be attributed to the presence or absence of thionins at the penetration site in walls and papillae of epidermal leaf cells.All of the leaf-specific thionin genes are confined to the distal end of the short arm of chromosome 6 of barley. None of the genes for cultivarspecific resistance to powdery mildew which have previously been mapped on barley chromosomes are found close to this locus.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

The whereabouts of an ancient wanderer: global phylogeography of the solitary ascidian Styela plicata

Genetic tools have greatly aided in tracing the sources and colonization history of introduced species. However, recurrent introductions and repeated shuffling of populations may have blurred some of the genetic signals left by ancient introductions. Styela plicata is a solitary ascidian distributed worldwide. Although its origin remains unclear, this species is believed to have spread worldwide by travelling on ship's hulls. The goals of this study were to infer the genetic structure and global phylogeography of S. plicata and to look for present-day and historical genetic patterns. Two genetic markers were used: a fragment of the mitochondrial gene Cytochrome Oxidase subunit I (COI) and a fragment of the nuclear gene Adenine Nucleotide Transporter/ADP-ATP Translocase (ANT). A total of 368 individuals for COI and 315 for ANT were sequenced from 17 locations worldwide. The levels of gene diversity were moderate for COI to high for ANT. The Mediterranean populations showed the least diversity and allelic richness for both markers, while the Indian, Atlantic and Pacific Oceans had the highest gene and nucleotide diversities. Network and phylogenetic analyses with COI and ANT revealed two groups of alleles separated by 15 and 4 mutational steps, respectively. The existence of different lineages suggested an ancient population split. However, the geographic distributions of these groups did not show any consistent pattern, indicating different phylogeographic histories for each gene. Genetic divergence was significant for many population-pairs irrespective of the geographic distance among them. Stochastic introduction events are reflected in the uneven distribution of COI and ANT allele frequencies and groups among many populations. Our results confirmed that S. plicata has been present in all studied oceans for a long time, and that recurrent colonization events and occasional shuffling among populations have determined the actual genetic structure of this species.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species‐specific detection in environmental DNA

Targeted species‐specific and community‐wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R² = 0.81, p < .001, biofouling R² = 0.68, p < .001); however, qPCR copy numbers were on average 125‐fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Alpha-methylated derivatives of 2-arachidonoyl glycerol: synthesis, CB1 receptor activity, and enzymatic stability

Alpha-methylated analogues of the endogenous cannabinoid, 2-arachidonoyl glycerol (2-AG), were synthesized aiming to the improved enzymatic stability of 2-AG. In addition, the CB1 activity properties of fluoro derivatives of 2-AG were studied. The CB1 receptor activity was determined by the [35S]GTPgammaS binding assay, and the enzymatic stability of alpha-methylated analogues was determined in rat cerebellar membranes. The results indicate that even if the alpha-methylated 2-AG derivatives are slightly weaker CB1 receptor agonists than 2-AG, they are clearly more stable than 2-AG. In addition, the results showed that the replacement of the hydroxyl group(s) of 2-AG by fluorine does not improve the CB1 activity of 2-AG.