Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.     

Study of artemisinin and sugar accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures

We studied the effect of genetic transformation on biologically active compound (artemisinin and its co-products (ART) as well as sugars) accumulation in Artemisia vulgaris and Artemisia dracunculus “hairy” root cultures. Glucose, fructose, sucrose, and mannitol were accumulated in A. vulgaris and A. dracunculus “hairy” root lines. Genetic transformation has led in some cases to the sugar content increasing or appearing of nonrelevant for the control plant carbohydrates. Sucrose content was 1.6 times higher in A. vulgaris “hairy” root lines. Fructose content was found to be 3.4 times higher in A. dracunculus “hairy” root cultures than in the control roots. The accumulation of mannitol was a special feature of the leaves of A. vulgaris and A. dracunculus control roots. A. vulgaris “hairy” root lines differed also in ART accumulation level. The increase of ART content up to 1.02?mg/g DW in comparison with the nontransformed roots (up to 0.687?mg/g DW) was observed. Thus, Agrobacterium rhizogenes-mediated genetic transformation can be used for obtaining of A. vulgaris and A. dracunculus “hairy” root culture produced ART and sugars in a higher amount than mother plants.     

Helminth protection against autoimmune diabetes in nonobese diabetic mice is independent of a type 2 immune shift and requires TGF-β

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.     

MPB2C, a microtubule-associated plant factor, is required for microtubular accumulation of tobacco mosaic virus movement protein in plants

Movement protein binding 2C (MPB2C) is a plant endogenous microtubule-associated protein previously identified as an interaction partner of tobacco (Nicotiana tabacum) mosaic virus movement protein (TMV-MP). In this work, the role of MPB2C in cell-to-cell transport of TMV-MP, viral spread of TMV, and subcellular localization of TMV-MP was examined. To this end, plants with reduced MPB2C levels were generated by a gene-silencing strategy. Local and systemic spread of TMV and cell-to-cell movement of TMV-MP were unimpaired in MPB2C-silenced plants as compared to nonsilenced plants, indicating that MPB2C is not required for intercellular transport of TMV-MP itself or spread of TMV. However, a clear change in subcellular distribution of TMV-MP characterized by a nearly complete loss of microtubular localization was observed in MPB2C-silenced plants. This result shows that the MPB2C is a central player in determining the complex subcellular localization of TMV-MP, in particular its microtubular accumulation, a phenomenon that has been frequently observed and whose role is still under discussion. Clearly, MPB2C mediated accumulation of TMV-MP at microtubules is not required for intercellular spread but may be a means to withdraw the TMV-MP from the cell-to-cell transport pathway.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Fe‐N‐C Oxygen Reduction Fuel Cell Catalyst Derived from Carbendazim: Synthesis,Structure, and Reactivity

New non‐PGM catalysts from the family of Fe‐N‐C pyrolyzed materials are reported. They are synthesized using a templating silica powder with iron nitrate and carbendazim (CBDZ) precursors (sacrificial support method). The synthesis involves high temperature pyrolysis, followed by etching of the sacrificial support (silica) and obtaining a “self‐supported” open frame morphology catalyst. Both the temperature of heat treatment and Fe to CBDZ ratio play a crucial role in the final catalytic activity in oxygen reduction reaction (ORR). Prepared materials have extremely high durability in RDE tests, ending up with more than 94% of initial activity (by E_1/2 value) after 10 000 cycles in an oxygen atmosphere, which is the result we report for the first time. Evaluation of these new M‐N‐C catalysts in a single membrane electrode assembly (MEA) has shown an exceptionally high open circuit voltage (OCV) of 1 V and the world's second best performance with no IR correction. MEA tests have shown high current density of 700 mA cm^‐2 at 0.6 V and 120 mA cm^‐2 at 0.8 V. In‐depth structure‐to‐property correlation presents an evidence that Fe‐N_x centers are the active sites playing a key role in oxygen reduction reaction.     

Adipocytokines and CD34 progenitor cells in Alzheimer's disease

Background

Alzheimer''s disease (AD) and atherosclerosis share common vascular risk factors such as arterial hypertension and hypercholesterolemia. Adipocytokines and CD34⁺ progenitor cells are associated with the progression and prognosis of atherosclerotic diseases. Their role in AD is not adequately elucidated.

Methods and Findings

In the present study, we measured in 41 patients with early AD and 37 age- and weight-matched healthy controls blood concentrations of adiponectin and leptin by enzyme linked immunoabsorbent assay and of CD34⁺ progenitor cells using flow cytometry. We found significantly lower plasma levels of leptin in AD patients compared with the controls, whereas plasma levels of adiponectin did not show any significant differences (AD vs. control (mean±SD): leptin:8.9±5.6 ng/mL vs.16.3±15.5 ng/mL;P = 0.038; adiponectin:18.5±18.1 µg/mL vs.16.7±8.9 µg/mL;P = 0.641). In contrast, circulating CD34⁺ cells were significantly upregulated in AD patients (mean absolute cell count±SD:253±51 vs. 203±37; P = 0.02) and showed an inverse correlation with plasma levels of leptin (r = −0.248; P = 0.037).In logistic regression analysis, decreased leptin concentration (P = 0.021) and increased number of CD34⁺ cells (P = 0.036) were both significantly associated with the presence of AD. According to multifactorial analysis of covariance, leptin serum levels were a significant independent predictor for the number of CD34⁺ cells (P = 0.002).

Conclusions

Our findings suggest that low plasma levels of leptin and increased numbers of CD34⁺ progenitor cells are both associated with AD. In addition, the results of our study provide first evidence that increased leptin plasma levels are associated with a reduced number of CD34⁺ progenitor cells in AD patients. These findings point towards a combined involvement of leptin and CD34⁺ progenitor cells in the pathogenesis of AD. Thus, plasma levels of leptin and circulating CD34⁺ progenitor cells could represent an important molecular link between atherosclerotic diseases and AD. Further studies should clarify the pathophysiological role of both adipocytokines and progenitor cells in AD and possible diagnostic and therapeutic applications.     

Rapid and asymmetric divergence of duplicate genes in the human gene coexpression network

Background  

While gene duplication is known to be one of the most common mechanisms of genome evolution, the fates of genes after duplication are still being debated. In particular, it is presently unknown whether most duplicate genes preserve (or subdivide) the functions of the parental gene or acquire new functions. One aspect of gene function, that is the expression profile in gene coexpression network, has been largely unexplored for duplicate genes.     

Coding region structural heterogeneity and turnover of transcription start sites contribute to divergence in expression between duplicate genes

Background  

Gene expression divergence is one manifestation of functional differences between duplicate genes. Although rapid accumulation of expression divergence between duplicate gene copies has been observed, the driving mechanisms behind this phenomenon have not been explored in detail.     

Increasing the efficiency of variance component quantitative trait loci analysis by using reduced-rank identity-by-descent matrices

Recent technological development in genetics has made large-scale marker genotyping fast and practicable, facilitating studies for detection of QTL in large general pedigrees. We developed a method that speeds up restricted maximum-likelihood (REML) algorithms for QTL analysis by simplifying the inversion of the variance-covariance matrix of the trait vector. The method was tested in an experimental chicken pedigree including 767 phenotyped individuals and 14 genotyped markers on chicken chromosome 1. The computation time in a chromosome scan covering 475 cM was reduced by 43% when the analysis was based on linkage only and by 72% when linkage disequilibrium information was included. The relative advantage of using our method increases with pedigree size, marker density, and linkage disequilibrium, indicating even greater improvements in the future.