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.

     

Stereochemistry of Internucleotide Bond Formation by the H-Phosphonate Method. 1. Synthesis and 31P Nmr Analysis of 16 Diribonulceoside (3′-5′)-H-Phosphonates and the Corresponding Phosphorothioates

Sixteen diribonucleoside (3′-5′)-H-phosphonates were synthesized via condensation of the protected ribonucleoside 3′-H-phosphonates with nucleosides, and the influence of a nucleoside sequence on the observed stereoselectivity was analyzed. ₃₁P NMR spectroscopy was used to evaluate a relationship between chemical shift and absolute configuration at the phosphorous center of the H-phosphonate diesters as well as of the corresponding phosphorothioate diesters. Although for the most cases such correlation was found, there was however several exceptions to the rule where the relative positions of resonances arising from R _P and S _P diastereomers were reversed.     

The Effect of Proteasome Inhibition on the Generation of the Human Leukocyte Antigen (HLA) Peptidome

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.The repertoires and levels of peptides, presented by the major histocompatibility complex (MHC)¹ class I molecules at the cells'' surface, are modulated by multiple factors. These include the rates of synthesis and degradation of their source proteins, the transport efficacy of the peptides through the transporter associated with antigen processing (TAP) into the endoplasmic reticulum (ER), their subsequent processing and loading onto the MHC molecules within the ER, and the rates of transport of the MHC molecules with their peptide cargo to the cell surface. The off-rates of the presented peptides, the residence time of the MHC complexes at the cell surface, and their retrograde transport back into the cytoplasm, influence, as well, the presented peptidomes (reviewed in (1)). Even though significant portions of the MHC class I peptidomes are thought to be derived from newly synthesized proteins, including misfolded proteins, defective ribosome products (DRiPs), and short lived proteins (SLiPs), most of the MHC peptidome is assumed to originate from long-lived proteins, which completed their functional cellular roles or became defective (retirees), (reviewed in (2)).The main protease, supplying the MHC peptidome production pipeline, is thought to be the proteasome (3). It is also responsible for generation of the final carboxyl termini of the MHC peptides (4), (reviewed in (5)). The final trimming of the n-termini of the peptides, within the endoplasmic reticulum (ER), is thought to be performed by amino peptidases, such as ERAP1/ERAAP, which fit the peptides into their binding groove on the MHC molecules (6) (reviewed in (7)). Nonproteasomal proteolytic pathways were also suggested as possible contributors to the MHC peptidome, including proteolysis by the ER resident Signal peptide peptidase (8, 9), the cytoplasmic proteases Insulin degrading enzyme (10), Tripeptidyl peptidase (11–13), and a number of proteases within the endolysosome pathway (reviewed recently in (14–17)). In contrast to the mostly cytoplasmic and ER production of the MHC class I peptidome, the class II peptidome is produced in a special compartment, associated with the endolysosome pathway (18–20). This pathway is also thought to participate in the cross presentation of class I peptides, derived from proteins up-taken by professional antigen presenting cells (21), (reviewed in (15–17, 22)).The centrality of the proteasomes in the generation of the MHC peptidome was deduced mostly from the observed change in presentation levels of small numbers of selected peptides, following proteasome inhibition (3, 23). Even the location of some of the genes encoding the catalytic subunits of the immunoproteasome (LMP2 and LMP7) (24) within the MHC class II genomic locus, was suggested to support the involvement of the proteasome in the generation of the MHC class I peptidome (25). Similar conclusions were deduced from alterations in peptide presentation, following expression of the catalytic subunits of the immunoproteasome (26), (reviewed in (5)). Yet, although most of the reports indicated reductions in presentation of selected peptides by proteasome inhibition (3, 27–29), others have observed only limited, and sometimes even opposite effects (23, 30–32).The matter is further complicated by the indirect effects of proteasome inhibition used for such studies on the arrest of protein synthesis by the cells (33–35), on the transport rates of the MHC molecules to the cell surface, and on their retrograde transport back to the vesicular system (36) (reviewed in (37)). Proteasome inhibition likely causes shortage of free ubiquitin, reduced supply of free amino acids, and induces an ER unfolded protein response (UPR), which signals the cells to block most (but not all) cellular protein synthesis (reviewed in (38)). Because a significant portion of the MHC peptidome originates from degradation of DRiPs and SLiPs (reviewed in (2)), arrest of new protein synthesis should influence the presentation of their derived MHC peptides. Taken together, these arguments may suggest that merely following the changes in the presentation levels of the MHC molecules, or even of specific MHC peptides, after proteasome inhibition, does not provide the full picture for deducing the relative contribution of the proteasomal pathway to the production of the MHC peptidome (reviewed in (7)).MHC peptidome analysis can be performed relatively easily by modern capillary chromatography combined with mass spectrometry (reviewed in (39)). The peptides are recovered from immunoaffinity purified MHC molecules after detergent solubilization of the cells (40, 41), from soluble MHC molecules secreted to the cells'' growth medium (42, 43) or from patients'' plasma (44). The purified peptides pools are resolved by capillary chromatography and the individual peptides are identified and quantified by tandem mass spectrometry (40), (reviewed in (45–47)). In cultured cells, quantitative analysis can also be followed by metabolic incorporation of stable isotope labeled amino acids (SILAC) (48). Furthermore, the rates of de novo synthesis of both MHC peptides and their proteins of origin can be followed using the dynamic-SILAC proteomics approach (49) with its further adaptation to HLA peptidomics (50–52).This study attempts to define the relative contribution of the proteasomes to the production of HLA class I peptidome by simultaneously following the effects of proteasome inhibitors, epoxomicin and bortezomib (Velcade), on the rates of de novo synthesis of both the HLA class I peptidome and the cellular proteome of the same MCF-7 human breast cancer cultured cells. The proteasome inhibitors did not reduce the levels of HLA presentations, yet affected the rates of production of both the HLA peptidome and cellular proteome, mostly decreasing, but also increasing some of the synthesis rates of the HLA peptides and cellular proteins. Thus, we suggest that the degree of contribution of the proteasomal pathway to the production of the HLA-I peptidome should be re-evaluated in accordance with their effects on the entire HLA class-I peptidome, while taking into consideration the inhibitors'' effects on the synthesis (and degradation) rates of the source proteins of each of the studied HLA peptides.     

Plant species richness–productivity relationships in a low-productive boreal region

Local species richness–productivity (SR–P) relationship is usually reported as unimodal if long productivity gradients are sampled. However, it tends to be monotonically increasing in low-productive environments due to the decreasing part of the SR–P curve being truncated. Previous work indicated that this can hold true for forest herb layers, because of an upper bound on productivity caused mainly by canopy shading. Here, we ask whether the same pattern exists in a region with an upper bound on productivity caused by a harsh climate. We sampled herbaceous vegetation of boreal forests and grasslands in a low-productive region of central Yakutia (NE Siberia) with dry and winter-cool continental climate. We collected data on species composition, herb-layer productivity (aboveground herbaceous biomass), soil chemistry and light availability. We applied regression models to discriminate between monotonically increasing, decreasing and unimodal responses of herb-layer species richness to measured variables and analysed trends in the species-pool size and beta diversity along the productivity gradient. Our expectation of the monotonically increasing SR–P relationship was confirmed for neither forest herb layers nor grasslands. In the forest herb layers, no relationship was detected. In grasslands, the relationship was unimodal with species richness decline starting at much lower productivity levels than in more productive temperate grasslands. Potential causes for this decline are either limitation of local species richness by the species pool, which contains few species adapted to more productive habitats, or competitive exclusion, which can become an important control of species richness under lower levels of productivity than is the case in temperate grasslands.     

Interferon Induced IFIT Family Genes in Host Antiviral Defense

Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IFN-stimulated genes. This family contains a cluster of duplicated loci. Most mammals have IFIT1, IFIT2, IFIT3 and IFIT5; however, bird, marsupial, frog and fish have only IFIT5. Regardless of species, IFIT5 is always adjacent to SLC16A12. IFIT family genes are predominantly induced by type I and type III interferons and are regulated by the pattern recognition and the JAK-STAT signaling pathway. IFIT family proteins are involved in many processes in response to viral infection. However, some viruses can escape the antiviral functions of the IFIT family by suppressing IFIT family genes expression or methylation of 5'' cap of viral molecules. In addition, the variants of IFIT family genes could significantly influence the outcome of hepatitis C virus (HCV) therapy. We believe that our current review provides a comprehensive picture for the community to understand the structure and function of IFIT family genes in response to pathogens in human, as well as in animals.     

Systematic biases in DNA copy number originate from isolation procedures

Background

The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal.

Results

While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing.

Conclusion

These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses.     

Publication Bias in Recent Meta-Analyses

Introduction

Positive results have a greater chance of being published and outcomes that are statistically significant have a greater chance of being fully reported. One consequence of research underreporting is that it may influence the sample of studies that is available for a meta-analysis. Smaller studies are often characterized by larger effects in published meta-analyses, which can be possibly explained by publication bias. We investigated the association between the statistical significance of the results and the probability of being included in recent meta-analyses.

Methods

For meta-analyses of clinical trials, we defined the relative risk as the ratio of the probability of including statistically significant results favoring the treatment to the probability of including other results. For meta-analyses of other studies, we defined the relative risk as the ratio of the probability of including biologically plausible statistically significant results to the probability of including other results. We applied a Bayesian selection model for meta-analyses that included at least 30 studies and were published in four major general medical journals (BMJ, JAMA, Lancet, and PLOS Medicine) between 2008 and 2012.

Results

We identified 49 meta-analyses. The estimate of the relative risk was greater than one in 42 meta-analyses, greater than two in 16 meta-analyses, greater than three in eight meta-analyses, and greater than five in four meta-analyses. In 10 out of 28 meta-analyses of clinical trials, there was strong evidence that statistically significant results favoring the treatment were more likely to be included. In 4 out of 19 meta-analyses of observational studies, there was strong evidence that plausible statistically significant outcomes had a higher probability of being included.

Conclusions

Publication bias was present in a substantial proportion of large meta-analyses that were recently published in four major medical journals.     

Predictive Factors of Late Venous Aortocoronary Graft Failure: Ultrastructural Studies

Background

Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients.

Methods

The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure.

Results

In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm² vs. 2.02 mm²) and tunica media area (1.09 mm² vs. 0.93 mm²) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development.

Conclusions

Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.     

Epigenetic Alterations at Genomic Loci Modified by Gene Targeting in Arabidopsis thaliana

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown.