The rockefeller university

As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.

Anti-Mullerian hormone (AMH) is a member of the transforming growth factor-beta superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pretreatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

The expression of extracellular matrix protein periostin (POSTN) was attenuated in Med1(-/-) mouse embryonic fibroblasts (MEFs), which exhibited a decreased capability to support hematopoietic progenitor cells (HPCs) in vitro. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1(+/+) MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN antibody, the growth of BM cells and number of long-term culture-initiating cells (LTC-ICs) were attenuated. When BM cells were cocultured with Med1(-/-) MEFs in the presence of recombinant POSTN, the growth of BM cells and the number of LTC-ICs were restored. Moreover, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the growth and cobblestone formation, a leukemic stem cell feature, of stromal cell dependent MB-1 myeloblastoma cells. POSTN was expressed both in BM cells and variably in different BM stromal cells. Expression in the latter cells was increased by physical interaction with hematopoietic cells. The receptor for POSTN, integrin alpha v beta 3, was expressed abundantly in BM stromal cells. The addition of recombinant POSTN to BM stromal cells induced intracellular signaling downstream of integrin alpha v beta 3. These results suggest that stromal cell POSTN supports both normal HPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner. (C) 2016 Elsevier Inc. All rights reserved.

The level of activity of many animals including humans rises and falls with a period of similar to 24 hours. The intrinsic biological oscillator that gives rise to this circadian oscillation is driven by a molecular feedback loop with an approximately 24 hour cycle period and is influenced by the environment, most notably the light: dark cycle. In addition to the circadian oscillations, behavior of many animals is influenced by multiple oscillations occurring at fasterultradian-time scales. These ultradian oscillations are also thought to be driven by feedback loops. While many studies have focused on identifying such ultradian oscillations, less is known about how the ultradian behavioral oscillations interact with each other and with the circadian oscillation. Decoding the coupling among the various physiological oscillators may be important for understanding how they conspire together to regulate the normal activity levels, as well in disease states in which such rhythmic fluctuations in behavior may be disrupted. Here, we use a wavelet-based cross-frequency analysis to show that different oscillations identified in spontaneous mouse behavior are coupled such that the amplitude of oscillations occurring at higher frequencies are modulated by the phase of the slower oscillations. The patterns of these interactions are different among different individuals. Yet this variability is not random. Differences in the pattern of interactions are confined to a low dimensional subspace where different patterns of interactions formclusters. These clusters expose the differences among individuals-males and females are preferentially segregated into different clusters. These sex-specific features of spontaneous behavior were not apparent in the spectra. Thus, our methodology reveals novel aspects of the structure of spontaneous animal behavior that are not observable using conventional methodology.

We explore the momentum analyticity of the static transverse polarization tensor of a 2+1 dimensional holographic superconductor in its normal phase, aiming at finding the holographic counterpart of the singularities underlying the Friedel oscillations of an ordinary field theory. We prove that the polarization tensor is a meromorphic function with an infinite number of poles located on the complex momentum plane off real axis. With the aid of the WKB approximation these poles are found to lies asymptotically along two straight lines parallel to the imaginary axis for a large momentum magnitude. The similarity between the holographic Green's function and that of an weakly coupled ordinary field theory (e.g., 2+1 dimensional QED) regarding the location of the momentum singularities offers further support to the validity of the gauge/gravity duality.

Voltage-gated potassium (K-v) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo-electron microscopy structure of mammalian K(v)10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K-v structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K-v channels and provides a template to further understand the gating properties of Eag1 and related channels.

Arabidopsis HSI2 (HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE 2) which carries a EAR (ERF-associated amphiphilic repression) motif acts as a repressor of seed maturation genes and lipid biosynthesis, whereas MEDIATOR (MED) is a conserved multiprotein complex linking DNA-bound transcription factors to RNA polymerase II transcription machinery. How HSI2 executes its repressive function through MED is hitherto unknown. Here, we show that HSI2 and its homolog, HSI2-lik (HSL1), are able to form homo-and heterocomplexes. Both factors bind to the TRAP240 domain of MED13, a subunit of the MED CDK8 module. Mutant alleles of the med13 mutant show elevated seed maturation gene expression and increased lipid accumulation in cotyledons; in contrast, HSI2- or MED13-overexpressing plants display the opposite phenotypes. The overexpression phenotypes of HSI2 and MED13 are abolished in med13 and hsi2 hsl1, respectively, indicating that HSI2 and MED13 together are required for these functions. The HSI2 C-terminal region interacts with HDA6, whose overexpression also reduces seed maturation gene expression and lipid accumulation. Moreover, HSI2, MED13 and HDA6 bind to the proximal promoter and 50-coding regions of seed maturation genes. Taken together, our results suggest that HSI2 recruits MED13 and HDA6 to suppress directly a subset of seed maturation genes post-germination.