Nucleoside Phosphonates. Development of Synthetic Methods and Reagents

Abstract

In this paper a short account of our recent research concerning development of new synthetic methods and new reagents for the preparation of DNA and RNA fragments and their analogues is given.

     

Biology and distribution of hemichordates (Enteropneusta) with emphasis on Harrimaniidae and description of Protoglossus bocki sp. nov. from Scandinavia

A new species of the genus Protoglossus is described from the west coast of the Scandinavian Peninsula. It lives buried in clay bottoms below the halocline where the salinity is at least 33–34 psu. Body small, estimated maximal length 1.5 cm. Collar broader than long, with a forward inclination. The thickest part is the collar region where it can be up to 1 mm in diameter. Proboscis colouration light pink to golden yellow; collar white with transversal yellow bands; branchial, hepatic and intestinal regions translucent pale yellow to golden yellow; brown intestine visible through the body wall. Proboscis groove extends through posterior half of proboscis. Nine to 17 pairs of gill openings, the size of the posteriormost successively smaller. It differs from the other European species, Protoglossus koehleri, in colouration, smaller size, fewer gill openings, body shape and proportions. It was sequenced (18S rRNA gene) and clustered within the family Harrimaniidae, with Saxipendium as its closest relative.     

Does Nocturnality Drive Binocular Vision? Octodontine Rodents as a Case Study

Binocular vision is a visual property that allows fine discrimination of in-depth distance (stereopsis), as well as enhanced light and contrast sensitivity. In mammals enhanced binocular vision is structurally associated with a large degree of frontal binocular overlap, the presence of a corresponding retinal specialization containing a fovea or an area centralis, and well-developed ipsilateral retinal projections to the lateral thalamus (GLd). We compared these visual traits in two visually active species of the genus Octodon that exhibit contrasting visual habits: the diurnal Octodon degus, and the nocturnal Octodon lunatus. The O. lunatus visual field has a prominent 100° frontal binocular overlap, much larger than the 50° of overlap found in O. degus. Cells in the retinal ganglion cell layer were 40% fewer in O. lunatus (180,000) than in O. degus (300,000). O. lunatus has a poorly developed visual streak, but a well developed area centralis, located centrally near the optic disk (peak density of 4,352 cells/mm²). O. degus has a highly developed visual streak, and an area centralis located more temporally (peak density of 6,384 cells/mm²). The volumes of the contralateral GLd and superior colliculus (SC) are 15% larger in O. degus compared to O. lunatus. However, the ipsilateral projections to GLd and SC are 500% larger in O. lunatus than in O. degus. Other retinorecipient structures related to ocular movements and circadian activity showed no statistical differences between species. Our findings strongly suggest that nocturnal visual behavior leads to an enhancement of the structures associated with binocular vision, at least in the case of these rodents. Expansion of the binocular visual field in nocturnal species may have a beneficial effect in light and contrast sensitivity, but not necessarily in stereopsis. We discuss whether these conclusions can be extended to other mammalian and non-mammalian amniotes.     

High-Throughput System for the Presentation of Secreted and Surface-Exposed Proteins from Gram-Positive Bacteria in Functional Metagenomics Studies

Complex microbial ecosystems are increasingly studied through the use of metagenomics approaches. Overwhelming amounts of DNA sequence data are generated to describe the ecosystems, and allow to search for correlations between gene occurrence and clinical (e.g. in studies of the gut microbiota), physico-chemical (e.g. in studies of soil or water environments), or other parameters. Observed correlations can then be used to formulate hypotheses concerning microbial gene functions in relation to the ecosystem studied. In this context, functional metagenomics studies aim to validate these hypotheses and to explore the mechanisms involved. One possible approach is to PCR amplify or chemically synthesize genes of interest and to express them in a suitable host in order to study their function. For bacterial genes, Escherichia coli is often used as the expression host but, depending on the origin and nature of the genes of interest and the test system used to evaluate their putative function, other expression systems may be preferable. In this study, we developed a system to evaluate the role of secreted and surface-exposed proteins from Gram-positive bacteria in the human gut microbiota in immune modulation. We chose to use a Gram-positive host bacterium, Bacillus subtilis, and modified it to provide an expression background that behaves neutral in a cell-based immune modulation assay, in vitro. We also adapted an E. coli – B. subtilis shuttle expression vector for use with the Gateway high-throughput cloning system. Finally, we demonstrate the functionality of this host-vector system through the cloning and expression of a flagellin-coding sequence, and show that the expression-clone elicits an inflammatory response in a human intestinal epithelial cell line. The expression host can easily be adapted to assure neutrality in other assay systems, allowing the use of the presented presentation system in functional metagenomics of the gut and other ecosystems.     

Mmi1, the Yeast Homologue of Mammalian TCTP,Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.     

Assessment of SLX4 Mutations in Hereditary Breast Cancers

Background

SLX4 encodes a DNA repair protein that regulates three structure-specific endonucleases and is necessary for resistance to DNA crosslinking agents, topoisomerase I and poly (ADP-ribose) polymerase (PARP) inhibitors. Recent studies have reported mutations in SLX4 in a new subtype of Fanconi anemia (FA), FA-P. Monoallelic defects in several FA genes are known to confer susceptibility to breast and ovarian cancers.

Methods and Results

To determine if SLX4 is involved in breast cancer susceptibility, we sequenced the entire SLX4 coding region in 738 (270 Jewish and 468 non-Jewish) breast cancer patients with 2 or more family members affected by breast cancer and no known BRCA1 or BRCA2 mutations. We found a novel nonsense (c.2469G>A, p.W823*) mutation in one patient. In addition, we also found 51 missense variants [13 novel, 23 rare (MAF<0.1%), and 15 common (MAF>1%)], of which 22 (5 novel and 17 rare) were predicted to be damaging by Polyphen2 (score = 0.65–1). We performed functional complementation studies using p.W823* and 5 SLX4 variants (4 novel and 1 rare) cDNAs in a human SLX4-null fibroblast cell line, RA3331. While wild type SLX4 and all the other variants fully rescued the sensitivity to mitomycin C (MMC), campthothecin (CPT), and PARP inhibitor (Olaparib) the p.W823* SLX4 mutant failed to do so.

Conclusion

Loss-of-function mutations in SLX4 may contribute to the development of breast cancer in very rare cases.     

RASSF1A Promoter Methylation Levels Positively Correlate with Estrogen Receptor Expression in Breast Cancer Patients

The aim of this study was to investigate the relationship between the promoter methylation in five cancer-associated genes and clinicopathologic features for identification of molecular markers of tumor metastatic potential and hormone therapy response efficiency in breast cancer. The methylation levels in paraffin-embedded tumor tissues, plasma, and blood cells from 151 sporadic breast cancer patients and blood samples of 50 controls were evaluated by quantitative multiplex methylation-specific polymerase chain reaction. DNA methylation of RAS-association domain family member 1 (RASSF1A), estrogen receptor 1 (ESR1), cadherin 1, type 1, E-cadherin (CDH1), TIMP metallopeptidase inhibitor 3 (TIMP3) and spleen tyrosine kinase (SYK) genes was detected in the tumors of 124, 19, 15, 15, and 6 patients with mean levels of 48.45%, 3.81%, 2.36%, 27.55%, and 10.81%, respectively. Plasma samples exhibited methylation in the same genes in 25, 10, 15, 17, and 3 patients with levels of 22.54%, 17.20%, 22.87%, 31.93%, and 27.42%, respectively. Cumulative methylation results confirmed different spectra in tumor and plasma samples. Simultaneous methylation in tumors and plasma were shown in less than 17% of patients. RASSF1A methylation levels in tumor samples statistically differ according to tumor size (P = .029), estrogen receptor (ER) and progesterone receptor (PR) status (P = .000 and P = .004), and immunohistochemical subtype (P = .000). Moreover, the positive correlation was found between RASSF1A methylation levels and percentage of cancer cells expressing ER and PR. The direct relationship between RASSF1A promoter methylation and expression of ER could aid the prognosis of hormonal therapy response.     

Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

An evaluation of analysis pipelines for DNA methylation profiling using the Illumina HumanMethylation450 BeadChip platform

The proper identification of differentially methylated CpGs is central in most epigenetic studies. The Illumina HumanMethylation450 BeadChip is widely used to quantify DNA methylation; nevertheless, the design of an appropriate analysis pipeline faces severe challenges due to the convolution of biological and technical variability and the presence of a signal bias between Infinium I and II probe design types. Despite recent attempts to investigate how to analyze DNA methylation data with such an array design, it has not been possible to perform a comprehensive comparison between different bioinformatics pipelines due to the lack of appropriate data sets having both large sample size and sufficient number of technical replicates. Here we perform such a comparative analysis, targeting the problems of reducing the technical variability, eliminating the probe design bias and reducing the batch effect by exploiting two unpublished data sets, which included technical replicates and were profiled for DNA methylation either on peripheral blood, monocytes or muscle biopsies. We evaluated the performance of different analysis pipelines and demonstrated that: (1) it is critical to correct for the probe design type, since the amplitude of the measured methylation change depends on the underlying chemistry; (2) the effect of different normalization schemes is mixed, and the most effective method in our hands were quantile normalization and Beta Mixture Quantile dilation (BMIQ); (3) it is beneficial to correct for batch effects. In conclusion, our comparative analysis using a comprehensive data set suggests an efficient pipeline for proper identification of differentially methylated CpGs using the Illumina 450K arrays.     

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

Background

Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations.

Methodology/Principal Findings

We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS.

Conclusions/Significance

MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive.