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Background: Replication factor C contains four active AAA+ ATPase sites. Results: Mutation of any one ATP binding site affects clamp loader activities. Conclusion: Although ATP sites may fill sequentially, ATP binding to all four sites is needed for RFC activity. Significance: This provides insight into the ATP-dependent conformational changes in RFC that drive the clamp loading reaction. Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.

Early chronic lead exposure continues to pose serious health risks for children, particularly those living in lower socioeconomic environments. This study examined effects on developing glomeruli in young C57BL/6J mice exposed to low (30 ppm), higher (330 ppm) or no lead via dams' drinking water from birth to sacrifice on post-natal day 28. Low-level lead exposed mice [BLL mean (SD); 3.19 (0.70) mu g/dL] had an increase in glomerular volume but no change in podocyte number compared to control mice [0.03 (0.01) mu g/dL]. Higher-level lead exposed mice [14.68 (2.74) mu g/dL] had no change in either glomerular volume or podocyte number. The increase in glomerular volume was explained by increases in glomerular capillary and mesangial volumes with no change in podocyte volume. Early chronic lead exposure yielding very low blood lead levels alters glomerular development in pre-adolescent animals. (C) 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Azimuthal dihadron correlations of charged particles have been measured in PbPb collisions at = 2.76TeV by the CMS collaboration, using data from the 2011 LHC heavy-ion run. The data set includes a sample of ultra-central (0-0.2% centrality) PbPb events collected using a trigger based on total transverse energy in the hadron forward calorimeters and the total multiplicity of pixel clusters in the silicon pixel tracker. A total of about 1.8 million ultra-central events were recorded, corresponding to an integrated luminosity of 120 mu b -aEuro parts per thousand 1. The observed correlations in ultra-central PbPb events are expected to be particularly sensitive to initial-state fluctuations. The single-particle anisotropy Fourier harmonics, from v (2) to v (6), are extracted as a function of particle transverse momentum. At higher transverse momentum, the v (2) harmonic becomes significantly smaller than the higher-order v (n) (n a parts per thousand yenaEuro parts per thousand 3). The p (T)-averaged v (2) and v (3) are found to be equal within 2%, while higher-order v (n) decrease as n increases. The breakdown of factorization of dihadron correlations into single-particle azimuthal anisotropies is observed. This effect is found to be most prominent in the ultra-central PbPb collisions, where the initial-state fluctuations play a dominant role. A comparison of the factorization data to hydrodynamic predictions with event-by-event fluctuating initial conditions is also presented.

In social species, the phenotype and fitness of an individual depend in part on the genotype of its social partners. However, how these indirect genetic effects affect genotype fitness in competitive situations is poorly understood in animal societies. We therefore studied phenotypic plasticity and fitness of two clones of the ant Cerapachys biroi in monoclonal and chimeric colonies. Here we show that, while clone B has lower fitness in isolation, surprisingly, it consistently outcompetes clone A in chimeras. The reason is that, in chimeras, clone B produces more individuals specializing in reproduction rather than cooperative tasks, behaving like a facultative social parasite. A cross-fostering experiment shows that the proportion of these individuals depends on intergenomic epistasis between larvae and nursing adults, explaining the flexible allocation strategy of clone B. Our results suggest that intergenomic epistasis can be the proximate mechanism for social parasitism in ants, revealing striking analogies between social insects and social microbes.

The effector activity of antibodies is dependent on engagement with Fc gamma receptors (Fc gamma Rs) and activation of the associated intracellular signaling pathways. Preclinical evaluation of therapeutic humanized or chimeric mAbs to study the interactions of their Fc regions with Fc gamma Rs is hampered by substantial structural and functional Fc gamma R diversity among species. In this report, we used mice expressing only human Fc gamma Rs to evaluate the contribution of Fc gamma R-mediated pathways to the neutralizing activity of an anti-anthrax toxin chimeric mAb. We observed that the protective activity of this mAb was highly dependent upon Fc gamma R engagement, with minimal protection against anthrax toxin observed in Fc gamma R-deficient mice following mAb administration. We generated anti-anthrax toxin mAbs with specific Fc domain variants with selectively enhanced affinity for particular human Fc gamma Rs and assessed their activity in Fc gamma R-humanized mice. We determined that Fc domain variants that were capable of selectively engaging activating Fc gamma Rs substantially enhanced the in vitro and in vivo activity of anthrax toxin-neutralizing antibodies. These findings indicate that the application of Fc domain engineering is a feasible strategy to enhance toxin-neutralizing activity and suggest that engineered antitoxin antibodies will have improved therapeutic efficacy.

The top-antitop quark (t (t) over bar) production cross section is measured in proton-proton collisions at root s = 8 TeV with the CMS experiment at the LHC, using a data sample corresponding to an integrated luminosity of 5.3 fb(-1). The measurement is performed by analysing events with a pair of electrons or muons, or one electron and one muon, and at least two jets, one of which is identified as originating from hadronisation of a bottom quark. The measured cross section is 239 +/- 2 (stat.) +/- 11 (syst.) +/- 6 (lum.) pb, for an assumed top-quark mass of 172.5 GeV, in agreement with the prediction of the standard model.

Despite 30 years of effort, there is no effective vaccine for HIV-1. However, antibodies can prevent HIV-1 infection in humanized mice and macaques when passively transferred. New single-cell-based methods have uncovered many broad and potent donor-derived antibodies, and structural studies have revealed the molecular bases for their activities. The new data suggest why such antibodies are difficult to elicit and inform HIV-1 vaccine development efforts. In addition to protecting against infection, the newly identified antibodies can suppress active infections in mice and macaques, suggesting they could be valuable additions to anti-HIV-1 therapies and to strategies to eradicate HIV-1 infection.

Background: Metabolic homeostasis is central to normal cardiac function. The molecular mechanisms underlying metabolic plasticity in the heart remain poorly understood. Results: Kruppel-like factor 15 (KLF15) is a direct and independent regulator of myocardial lipid flux. Conclusion: KLF15 is a core component of the transcriptional circuitry that governs cardiac metabolism. Significance: This work is the first to implicate the KLF transcription factor family in cardiac metabolism. The mammalian heart, the body's largest energy consumer, has evolved robust mechanisms to tightly couple fuel supply with energy demand across a wide range of physiologic and pathophysiologic states, yet, when compared with other organs, relatively little is known about the molecular machinery that directly governs metabolic plasticity in the heart. Although previous studies have defined Kruppel-like factor 15 (KLF15) as a transcriptional repressor of pathologic cardiac hypertrophy, a direct role for the KLF family in cardiac metabolism has not been previously established. We show in human heart samples that KLF15 is induced after birth and reduced in heart failure, a myocardial expression pattern that parallels reliance on lipid oxidation. Isolated working heart studies and unbiased transcriptomic profiling in Klf15-deficient hearts demonstrate that KLF15 is an essential regulator of lipid flux and metabolic homeostasis in the adult myocardium. An important mechanism by which KLF15 regulates its direct transcriptional targets is via interaction with p300 and recruitment of this critical co-activator to promoters. This study establishes KLF15 as a key regulator of myocardial lipid utilization and is the first to implicate the KLF transcription factor family in cardiac metabolism.

Hair follicles (HFs) undergo cyclical periods of growth, which are fueled by stem cells (SCs) at the base of the resting follicle. HF-SC formation occurs during HF development and requires transcription factor SOX9. Whether and how SOX9 functions in HF-SC maintenance remain unknown. By conditionally targeting Sox9 in adult HF-SCs, we show that SOX9 is essential for maintaining them. SOX9-deficient HF-SCs still transition from quiescence to proliferation and launch the subsequent hair cycle. However, once activated, bulge HF-SCs begin to differentiate into epidermal cells, which naturally lack SOX9. In addition, as HF-SC numbers dwindle, outer root sheath production is not sustained, and HF downgrowth arrests prematurely. Probing the mechanism, we used RNA sequencing (RNA-seq) to identify SOX9-dependent transcriptional changes and chromatin immunoprecipitation (ChIP) and deep sequencing (ChIP-seq) to identify SOX9-bound genes in HF-SCs. Intriguingly, a large cohort of SOX9-sensitive targets encode extracellular factors, most notably enhancers of Activin/pSMAD2 signaling. Moreover, compromising Activin signaling recapitulates SOX9-dependent defects, and Activin partially rescues them. Overall, our findings reveal roles for SOX9 in regulating adult HF-SC maintenance and suppressing epidermal differentiation in the niche. In addition, our studies expose a role for SCs in coordinating their own behavior in part through non-cell-autonomous signaling within the niche.

Ab-secreting cell (ASC) expansion and survival are important processes in optimizing vaccines and controlling autoimmunity. The microenvironment of the medullary cords is positioned to control these key processes. Previously, we imaged and characterized ASC differentiation and migration by intravital microscopy in the lymph node (LN) by transferring and activating B cells expressing yellow fluorescent protein only in the ASC compartment. In this study, we observed that yellow fluorescent protein(+) ASCs in the medullary cords migrated along myelomonocytic cells and arrested in contact with them. Acute ablation of myeloid cells using the human diphtheria receptor system (diphtheria toxin receptor [DTR]) expressed in Lysmd1-cre-positive cells increased ASC and Ab production by 2-fold. Increases in ASC numbers were associated with cell proliferation based on Ki-67 staining, rather than reduced apoptosis, or changes in egress from the LN. Using DTR-mediated ablation targeted to Ccr2-expressing myeloid cells also generated increases in ASCs. In contrast, neither the depletion of Gr-1-positive cells with an Ab nor the ablation of cells using a cd11c-DTR resulted in any change in ASCs. IL-6 cytokine signaling can enhance ASC production and has been implicated in dampening ASCs in lupus mouse models through myeloid cells. Using mixed bone marrow chimeras, we observed that IL-6 enhances ASC production, but IL-6 production was not required by myeloid cells to dampen ASCs in the LN. Inhibition of ASCs by these myeloid cells in the LN provides a new regulatory mechanism with implications for tuning Ab responses.