Electron microscopy of DNA.helicase-I complexes in the act of strand separation

Electron microscopy was used to characterize the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase I. Linear DNA with 5'-protruding strands as well as single-stranded gaps was incubated, under unwinding assay conditions, with the helicase. E. coli single-stranded-DNA-binding protein (SSB) was added to order the denatured DNA. Up to 70% of the sites of SSB-complexed DNA were observed as forks. The position of the strand-separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site. The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules. A mutant helicase I (helicase I del29) which, unlike the wild-type enzyme, fails to show cooperative DNA-binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB. Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules. SSB displaces helicase I from single-stranded DNA but fails to do so from a fork or a single-strand/double-strand junction. The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands. Helicase I unwinds in the 5'-3' direction of the bound strand. Observations so far indicate that the enzyme exploits the single strand at the initial DNA-binding site for orienting its action, and not the complementary, completely base-paired strand.     

Mind the gaps: overlooking inaccessible regions confounds statistical testing in genome analysis

Background

The current versions of reference genome assemblies still contain gaps represented by stretches of Ns. Since high throughput sequencing reads cannot be mapped to those gap regions, the regions are depleted of experimental data. Moreover, several technology platforms assay a targeted portion of the genomic sequence, meaning that regions from the unassayed portion of the genomic sequence cannot be detected in those experiments. We here refer to all such regions as inaccessible regions, and hypothesize that ignoring these regions in the null model may increase false findings in statistical testing of colocalization of genomic features.

Results

Our explorative analyses confirm that the genomic regions in public genomic tracks intersect very little with assembly gaps of human reference genomes (hg19 and hg38). The little intersection was observed only at the beginning and end portions of the gap regions. Further, we simulated a set of synthetic tracks by matching the properties of real genomic tracks in a way that nullified any true association between them. This allowed us to test our hypothesis that not avoiding inaccessible regions (as represented by assembly gaps) in the null model would result in spurious inflation of statistical significance. We contrasted the distributions of test statistics and p-values of Monte Carlo-based permutation tests that either avoided or did not avoid assembly gaps in the null model when testing colocalization between a pair of tracks. We observed that the statistical tests that did not account for assembly gaps in the null model resulted in a distribution of the test statistic that is shifted to the right and a distribution of p-values that is shifted to the left (indicating inflated significance). We observed a similar level of inflated significance in hg19 and hg38, despite assembly gaps covering a smaller proportion of the latter reference genome.

Conclusion

We provide empirical evidence demonstrating that inaccessible regions, even when covering only a few percentages of the genome, can lead to a substantial amount of false findings if not accounted for in statistical colocalization analysis.

     

Artificial carbohydrate antigens: a block synthesis of a linear, tetrasaccharide repeating-unit of the Shigella flexneri variant Y polysaccharide

Glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl bromide gave methyl 2,4-di-O-benzoyl-3-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl) -alpha-L-rhamnopyranoside (4) in 93% yield. Conversion of 4 into the corresponding glycosyl bromide was accomplished with dibromomethyl methyl ether. Under Koenigs-Knorr conditions, this bromide reacted with 8-(methoxycarbonyl)octyl 2-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glycopyranosyl)- 3,4-di-O- benzyl-alpha-L-rhamnopyranoside, to provide the protected tetrasaccharide in 91% yield. Removal of blocking groups gave 8-(methoxycarbonyl)octyl O-alpha-L-rhamnopyranosyl-(1---- 3)-O-alpha-L-rhamnopyranosyl-(1---- 3)-O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1----2)-alpha-L- rhamnopyranoside. Together with previously synthesized tetrasaccharides of the Shigella flexneri Y O-antigen, this oligosaccharide has been used to study the conformation of O-antigens and to assist in the selection of S. flexneri, variant Y, specific monoclonal antibodies.     

Accommodating pathway information in expression quantitative trait locus analysis

The availability of high-throughput genotyping technologies and microarray assays has allowed researchers to consider pursuing investigations whose ultimate goal is the identification of genetic variations that influence levels of gene expression, e.g., "expression quantitative trait locus" or "eQTL" mapping studies. However, the large number of genes whose expression levels can be tested for association with genetic variations in such studies can create both statistical and biological interpretive problems. We consider the integrated analysis of eQTL mapping data that incorporates pathway, function, and disease process information. The goal of this analysis is to determine if compelling patterns emerge from the data that are consistent with the notion that perturbations in the molecular physiologic environment induced by genetic variations implicate the expression patterns of multiple genes via genetic network relationships or feedback mechanisms. We apply available genetic network and pathway analysis software, as well as a novel regression analysis technique, to carry out the proposed studies. We also consider extensions of the proposed strategies and areas of future research.     

Cellular stress leads to the formation of membraneless stress assemblies in eukaryotic cells

In cells at steady state, two forms of cell compartmentalization coexist: membrane‐bound organelles and phase‐separated membraneless organelles that are present in both the nucleus and the cytoplasm. Strikingly, cellular stress is a strong inducer of the reversible membraneless compartments referred to as stress assemblies. Stress assemblies play key roles in survival during cell stress and in thriving of cells upon stress relief. The two best studied stress assemblies are the RNA‐based processing‐bodies (P‐bodies) and stress granules that form in response to oxidative, endoplasmic reticulum (ER), osmotic and nutrient stress as well as many others. Interestingly, P‐bodies and stress granules are heterogeneous with respect to both the pathways that lead to their formation and their protein and RNA content. Furthermore, in yeast and Drosophila, nutrient stress also leads to the formation of many other types of prosurvival cytoplasmic stress assemblies, such as metabolic enzymes foci, proteasome storage granules, EIF2B bodies, U‐bodies and Sec bodies, some of which are not RNA‐based. Nutrient stress leads to a drop in cytoplasmic pH, which combined with posttranslational modifications of granule contents, induces phase separation.     

Effects of extracorporeal shock waves (ESW) on human bone marrow cell cultures]

INTRODUCTION: Extracorporeal shockwave therapy (ESWT) is a new method of treating insertion tendopathy and pseudo-arthrosis, the clinical importance of which cannot yet be definitively assessed, and the underlying mechanisms of which are still unclear. AIM: To develop an experimental set-up enabling the standardised application of ESWT to human bone marrow cell culture and the determination of the effect of ESWT. MATERIAL AND METHODS: After 14 days incubation, human bone marrow cell cultures were subjected to ESWT using 200/500 pulses at an energy flux densities ED + of 0.03, 0.04, 0.07, 0.11 and 0.25 millijoule/mm2. Samples were obtained for LDH measurement 15 minutes, 4 h and 18 h after ESWT. Transmission light microscopy was carried out before and after ESWT to determine cell numbers and for morphological analysis. RESULTS: Gaps in the cellular tissue first appear at an energy of 0.01 millijoule/mm2. At energies of 0.25 millijoule/mm2, morphologically altered cells, thinned out cellular tissue with a cell-free focal zone are found. At low energy levels, defects have been repaired ca. 1 week after ESWT. No significant increase in LDH was detected at any of the energy levels applied. CONCLUSION: Increasing energy and higher pulse frequency is associated with an increase in the size and number of holes in cellular tissue and in cell separation. Regeneration capability (regrowth, sprouting, normal cell form) decreases as the energy level increases. Changes can be detected even at the lowest energy flux densities, which up until now had been assumed to have no effect on cell morphology or number. The standardised application of ESWT to human bone marrow cell cultures provides reproducible results that can be controlled by a placebo ESWT application.