Rapid and sensitive NMR method for osmolyte determination

We propose a rapid and sensitive method for osmolyte determination, based on one-dimensional and two-dimensional 1H NMR spectroscopy applied directly on culture of haloalkalophilic Halomonas pantelleriensis and acidothermophilic archaeon Sulfolobus solfataricus, without any extraction procedure. The osmoprotectants hydroxyectoine, ectoine, glutamate, glycine-betaine and treahalose can easily be quantified by integrating the peak areas with respect to an internal standard, and the concentrations evaluated with this method are in excellent agreement with the values previously reported. Furthermore, trace amount of osmoprotectants, often undetectable after extraction procedures, can also be evaluated.     

Ultrastructural analysis of the aberrant axoneme morphogenesis in thrips (Thysanoptera, Insecta)

Thrips spermiogenesis is characterized by unusual features in the differentiating spermatid cells. Three centrioles from which three individual short flagella are initially assembled, make the early spermatid a tri-flagellated cell. Successively, during spermatid maturation, the three basal bodies maintain a position close to the most anterior end of the elongating nucleus, so that the three axonemes are progressively incorporated in the spermatid cytoplasm, where they run in parallel to the main nuclear axis. Finally, the three axonemes amalgamate to form a microtubular bundle. The process starts with the formation of rifts at three specific points in each axonemal circumference, corresponding to sites 1,3,7 and leads to the formation of 9 microtubular rows of different length, i.e. 3 "dyads", 3 "triads" and 3 "tetrads". In the spermatozoon, the nucleus, the mitochondrion and the bundle of microtubules are arranged in a helicoidal pattern. The elongation of the spermatozoon is allowed by the deep anchorage of the spermatid to the cyst cell through a dense mass of material which, at the end of spermiogenesis, becomes a long anterior cylindrical structure. This bizarre "axoneme" does not show any trace of progressive movement but it is able to beat. According to the presence of dynein arms, sliding can take place only within each row and not between the rows. The possible molecular basis underlying the peculiar instability of thrips axonemes is discussed in light of the present knowledge on the organization of the axoneme in mutant organisms carrying alterations of the tubulin molecule.     

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure–activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K⁺ channel. Preferred compounds were subsequently evaluated for selectivity in an α₁-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> plantlet production of the endangered <Emphasis Type="Italic">Pinguicula vulgaris</Emphasis>

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free ¼MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.     

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.     

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.