Description: The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a...

Description: The molecular basis underlying the physiologically well-defined orexigenic function of glucocorticoid (Gc) is unclear. Brain-specific homeobox factor (Bsx) is a positive regulator of the orexigenic neuropeptide, agouti-related peptide (AgRP), in AgRP neurons of the hypothalamic arcuate nucleus. Here, we show that in response to fasting-elevated Gc levels, Gc receptor (GR) and Bsx synergize to direct activation of AgRP transcription. This synergy is dictated by unique sequence features in a novel Gc response element in AgRP (AgRP-GRE). In contrast to AgRP-GRE, Bsx suppresses transactivation directed by many conventional GREs, functioning as a gene context-dependent modulator of GR actions or a target selector for GR. Consistent with this finding, AgRP-GRE drives fasting-dependent activation of a target gene specifically in GR + Bsx + AgRP neurons. These results define AgRP as a common orexigenic target gene of GR and Bsx and provide an opportunity to identify their additional common targets, facilitating our understanding of the molecular basis underlying the orexigenic activity of Gc and Bsx.

Description: Nanoparticles are known to have physical properties different from their bulk analogs, a characteristic that bears wide technological potential. Detailed knowledge of the atomic structures of diverse metastable/stable polymorphs in alumina nanoparticles with varying particle size is essential to understand their macroscopic properties and the behavior of their phase transitions. In this study, we report high-resolution solid-state 27 Al 2D triple-quantum (3Q) magic-angle spinning (MAS) and 1D MAS nuclear magnetic resonance (NMR) spectra for alumina nanoparticles with varying temperature and particle size with an aim to explore the nature of phase transitions in alumina nanoparticles. Although the 27 Al MAS NMR spectra of alumina nanoparticles cannot fully resolve all the crystallographically distinct Al sites for metastable aluminas such as -, -, and -Al 2 O 3 , the simulation of 27 Al MAS NMR spectra collected at different magnetic fields following the Czjzek model allows us to obtain the quantitative fractions of alumina polymorphs in nanoparticles and the NMR characteristics of their Al sites. The 27 Al 3QMAS NMR spectra resolved crystallographically distinct [6] Al and [4] Al sites in (,)- and -Al 2 O 3 in the isotropic dimension for the first time. The fraction of -Al 2 O 3 gradually increases up to ~1473 K at the expense of a decrease in (,)-Al 2 O 3 . The onset of formation of α-Al 2 O 3 from metastable aluminas is observed above ~1493 K. Several phase transitions in alumina nanoparticles observed in the current study include, , -〉 -Al 2 O 3 , , -〉 α-Al 2 O 3 , and -〉 α-Al 2 O 3 . Although the phase transition , -〉 -Al 2 O 3 occurs gradually with increasing annealing temperature from 873 to 1473 K, the phase transitions , -〉 α-Al 2 O 3 and -〉 α-Al 2 O 3 occur dramatically within a narrow temperature range between 1473 and 1573 K. The observed difference in temperature range (gradual vs. dramatic) for phase transition , -〉 -Al 2 O 3 and ,, -〉 α-Al 2 O 3 originates from the different structural disorder in the metastable aluminas (i.e., -, -, -) and α-Al 2 O 3 . The effect of particle size on the phase transition (,) -〉 -Al 2 O 3 between 298 and ~1473 K is not observed significantly. On the other hand, the transition temperature for ,, -〉 α-Al 2 O 3 , where the 50% for alumina is α-Al 2 O 3 , apparently increases as the particle size increases (as evidenced by TEM observation), indicating a larger energy penalty for phase transitions into α-Al 2 O 3 in larger alumina nanoparticles. This could be due to higher surface energy of -Al 2 O 3 than that of α-Al 2 O 3 and/or the fact that the transition from -Al 2 O 3 to α-Al 2 O 3 is kinetically favored for smaller nanoparticles. The mechanistic details of phase transitions among alumina polymorphs provided in the current study yield insights into the nature of the phase transition mechanisms for other oxide nanoparticles ubiquitous in the Earth’s surface environment.

Description: Despite efforts to understand the amorphization mechanisms of zeolites upon heating and subsequent dehydration, little is known about the extent of Si-Al disorder and topological variations in both crystalline and amorphous phases during amorphization. In this study, we investigated the atomic structure and the extent of configurational disorder (e.g., Si-Al ordering) in Na-zeolite A and other dehydrated phases during their temperature-induced amorphization using multi-nuclear solid-state NMR. We also report the first multi-nuclear ( 17 O, 29 Si, and 27 Al) NMR spectra of the intermediate amorphous phases. 29 Si MAS NMR results confirm the prevalence of amorphous phases up to ~1073 K and variation in Q-species for the crystalline phases. The 27 Al quadrupolar coupling constant of the [4] Al peak in Na-zeolite A and the intermediate amorphous phases increase with increasing temperature, which suggests an increase in the topological disorder associated with the structural distortion around [4] Al. 2D 17 O 3QMAS NMR spectra resolve the crystallographically distinct Si-O-Al sites in Na-zeolite A and three types of oxygen linkages namely, Si-O-Al, Si-O-Si, and Al-O-Al in the intermediate amorphous phases, which provides an unambiguous experimental evidence for an increase in the Si-Al disorder during the amorphization of zeolite. The detailed structural changes in Na-zeolite A and other dehydrated phases at various temperatures provide insights into the structural changes of other aluminosilicates during amorphization, thereby highlighting the changes in Si-Al ordering.

Description: Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

Description: Purpose: To examine the associations between intracranial artery calcifications (IACs) and coronary artery calcifications (CACs) in patients with ischemic stroke and to assess the predictive value of IAC for asymptomatic coronary artery disease (CAD). Materials and Methods: This retrospective study, approved by an institutional review board that waived the need for informed consent, included 314 consecutive patients who had acute ischemic stroke and who underwent both brain and coronary computed tomography (CT) within 1 month of stroke. IAC was quantified semiautomatically by calculating both Agatston scores (area of calcification multiplied by a weighted value assigned to its highest Hounsfield unit) and volumes on thin-section unenhanced images and was correlated with coronary calcium scores and volumes. Quartiles were created for IAC scores and were used for logistic regression analysis. An optimal IAC score cutoff value was determined and used to predict the presence of asymptomatic CAD. Independent factors for asymptomatic CAD were assessed by using multiple logistic regression analysis. Receiver operating characteristic curve analysis was performed to evaluate the added value of IAC scores for prediction of asymptomatic CAD. Results: IAC and CAC were significantly correlated for both Agatston scores and volumes ( R = 0.665 and 0.663, respectively; P 〈 .001). A graded association was found between IAC scores and presence of asymptomatic CAD. Both IAC scores of 120.11 or greater (odds ratio [OR], 2.57; 95% confidence interval [CI]: 1.45, 4.55) and diabetes mellitus (OR, 4.23; 95% CI: 2.42, 7.4) were independent predictors for asymptomatic CAD. Adding the IAC score to analytic models significantly improved the ability to predict asymptomatic CAD. Conclusion: The IAC scores quantified by using unenhanced CT correlate significantly with coronary calcium scores and may serve as an independent predictor of asymptomatic CAD in patients with ischemic stroke. © RSNA, 2013

Description: In the basolateral membrane of proximal-tubule cells, NBCe1-A (SLC4A4, variant A), operating with an apparent Na + :HCO 3 – stoichiometry of 1:3, contributes to the reclamation of HCO 3 – from the glomerular filtrate, thereby preventing whole body acidosis. Others have reported that NBCe1-like activity in human, rabbit, and rat renal preparations is substantially influenced by lithium, sulfite, oxalate, and harmaline. These data were taken as evidence for the presence of distinct Na + and CO 3 2– binding sites in NBCe1-A, favoring a model of 1 Na + :1 HCO 3 – :1 CO 3 2– . Here, we reexamine these findings by expressing human or rabbit NBCe1-A clones in Xenopus oocytes. In oocytes, NBCe1-A exhibits a 1:2 stoichiometry and could operate in one of five thermodynamically equivalent transport modes: 1 ) cotransport of Na + + 2 HCO 3 – , 2 ) cotransport of Na + + CO 3 2– , 3 ) transport of NaCO 3 – , 4 ) exchange of Na + + HCO 3 – for H + , or 5 ) HCO 3 – -activated exchange of Na + for 2 H + . In contrast to the behavior of NBCe1-like activity in renal preparations, we find that cloned NBCe1-A is only slightly stimulated by Li + , not at all influenced by sulfite or oxalate, and only weakly inhibited by harmaline. These negative data do not uniquely support any of the five models above. In addition, we find that NBCe1-A mediates a small amount of Na + -independent NO 3 – transport and that NBCe1-A is somewhat inhibited by extracellular benzamil. We suggest that the features of NBCe1-like activity in renal preparations are influenced by yet-to-be-identified renal factors. Thus the actual ionic substrates of NBCe1 remain to be identified.

Description: The epithelial-mesenchymal transition (EMT) is a novel mechanism that promotes renal fibrosis. Transforming growth factor-β (TGF-β), angiotensin II, aldosterone, high glucose, and urinary albumin are well-known causes of EMT and renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed EMT induced by the above agents in tubular epithelial cells. All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. Exposure of tubular cells to TGF-β (10 ng/ml), angiotensin II (1 μM), aldosterone (100 nM), high glucose (30 mM), and albumin (5 mg/ml) for 5 days induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ROS and NADPH oxidase 4 (Nox4) expression were increased, and antioxidants such as tiron and N -acetylcysteine inhibited EMT induction. Metformin (the best known clinical activator of AMPK) suppressed EMT induction through inhibition of ROS via induction of heme oxygenase-1 and endogenous antioxidant thioredoxin. An AMPK inhibitor (compound C) and AMPK small interfering RNA blocked the effect of metformin, and another AMPK activator [5-aminoimidazole-4-carboxamide-1β riboside (AICAR)] exerted the same effects as metformin. In conclusion, AMPK activation might be beneficial in attenuating the tubulointerstitial fibrosis induced by TGF-β, angiotensin II, aldosterone, high glucose, and urinary albumin.

Description: Osteoclast differentiation is regulated by transcriptional, post-transcriptional, and post-translational mechanisms. MicroRNAs are fundamental post-transcriptional regulators of gene expression. The function of the miR-29 (a/b/c) family in cells of the osteoclast lineage is not well understood. In primary cultures of mouse bone marrow-derived macrophages, inhibition of miR-29a, -29b, or -29c diminished formation of TRAP (tartrate-resistant acid phosphatase-positive) multinucleated osteoclasts, and the osteoclasts were smaller. Quantitative RT-PCR showed that all miR-29 family members increased during osteoclast differentiation, in concert with mRNAs for the osteoclast markers Trap (Acp5) and cathepsin K. Similar regulation was observed in the monocytic cell line RAW264.7. In stably transduced RAW264.7 cell lines expressing an inducible miR-29 competitive inhibitor (sponge construct), miR-29 knockdown impaired osteoclastic commitment and migration of pre-osteoclasts. However, miR-29 knockdown did not affect cell viability, actin ring formation, or apoptosis in mature osteoclasts. To better understand how miR-29 regulates osteoclast function, we validated miR-29 target genes using Luciferase 3′-UTR reporter assays and specific miR-29 inhibitors. We demonstrated that miR-29 negatively regulates RNAs critical for cytoskeletal organization, including Cdc42 (cell division control protein 42) and Srgap2 (SLIT-ROBO Rho GTPase-activating protein 2). Moreover, miR-29 targets RNAs associated with the macrophage lineage: Gpr85 (G protein-coupled receptor 85), Nfia (nuclear factor I/A), and Cd93. In addition, Calcr (calcitonin receptor), which regulates osteoclast survival and resorption, is a novel miR-29 target. Thus, miR-29 is a positive regulator of osteoclast formation and targets RNAs important for cytoskeletal organization, commitment, and osteoclast function. We hypothesize that miR-29 controls the tempo and amplitude of osteoclast differentiation.

Description: The successful synthesis of a transcript by RNA polymerase II (RNAPII) is a multistage process with distinct rate-limiting steps that can vary depending on the particular gene. A growing number of genes in a variety of organisms are regulated at steps after the recruitment of RNAPII. The best-characterized Saccharomyces cerevisiae gene regulated in this manner is CYC1 . This gene has high occupancy of RNAPII under non-inducing conditions, defining it as a poised gene. Here, we find that subunits of the head module of Mediator, Med18 and Med20, and Med19 are required for activation of transcription at the CYC1 promoter in response to environmental cues. These subunits of Mediator are required at the preloaded promoter for normal levels of recruitment and activity of the general transcription factor TFIIH. Strikingly, these Mediator components are dispensable for activation by the same activator at a different gene, which lacks a preloaded polymerase in the promoter region. Based on these results and other studies, we speculate that Mediator plays an essential role in triggering an inactive polymerase at CYC1 into a productively elongating form.