Nucleosome Spacing Generated by ISWI and CHD1 Remodelers Is Constant Regardless of Nucleosome Density

Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the HAND-SANT-SLIDE (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.     

Comparing mortality in renal patients on hemodialysis versus peritoneal dialysis using a marginal structural model

When comparing the causal effect of peritoneal dialysis (PD) and hemodialysis (HD) treatment on lowering mortality in renal patients, using observational data, it is necessary to adjust for different forms of confounding and informative censoring. Both the type of dialysis treatment that is started with and mortality are affected by baseline covariates. Longitudinal and baseline variables can affect both the probability of switching from one type of dialysis to the other, and mortality. Longitudinal and baseline variables can also affect the probability of receiving a kidney transplant, possibly causing informative censoring. Adjusting for longitudinal variables by including them as covariates in a regression model potentially causes bias, for instance by losing a possible indirect effect of dialysis on mortality via these longitudinal variables. Instead, we fitted a marginal structural model (MSM) to estimate the causal effect of dialysis type, adjusted for confounding and informative censoring. We used the MSM to compare the hazard of death as well as cumulative survival between the potential treatment trajectories "always PD" and "always HD" over time, conditional on age and diabetes mellitus status. We used inverse probability weighting (IPW) to fit the MSM.     

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.     

Functional and molecular analysis of the double-positive stage-specific CD8 enhancer E8III during thymocyte development

Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8(III) enhancer, which directs expression in double-positive (DP) thymocytes, and E8(II), which is active in DP cells and CD8(+) T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8(III) enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8(III) in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8(II) is also deleted. These results indicate that E8(III) is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development.     

Calcium,Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.     

Crystal Structure of the ATPase Domain of the Human AAA+ Protein Paraplegin/SPG7

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations.

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A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.