Transcriptomes of human primary skin fibroblasts of healthy individuals reveal age-associated mRNAs and long noncoding RNAs.

Changes in the transcriptomes of human tissues with advancing age are poorly cataloged. Here, we sought to identify the coding and long noncoding RNAs present in cultured primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22-89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Using high-throughput RNA sequencing and a linear regression model, we identified 1437 coding RNAs (mRNAs) and 1177 linear and circular long noncoding (lncRNAs) that were differentially abundant as a function of age. Gene set enrichment analysis (GSEA) revealed select transcription factors implicated in coordinating the transcription of subsets of differentially abundant mRNAs, while long noncoding RNA enrichment analysis (LncSEA) identified RNA-binding proteins predicted to participate in the age-associated lncRNA profiles. In summary, we report age-associated changes in the global transcriptome, coding and noncoding, from healthy human skin fibroblasts and propose that these transcripts may serve as biomarkers and therapeutic targets in aging skin.

Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter.

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

LncRNA OIP5-AS1-directed miR-7 degradation promotes MYMX production during human myogenesis.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.

Reactive Grand-Canonical Monte Carlo Simulations for Modeling Hydration of MgCl2.

Thermochemical heat-storage applications, based on the reversible endo-/exothermic hydration reaction of salts, are intensively investigated to search for compact heat-storage devices. To achieve a truly valuable storage system, progressively complex salts are investigated. For these salts, the equilibrium temperature and pressure conditions are not always easy to predict. However, these conditions are crucial for the design of thermochemical heat-storage systems. A biased grand-canonical Monte Carlo (GCMC) tool is developed, enabling the study of equilibrium conditions at the molecular level. The GCMC algorithm is combined with reactive force field molecular dynamics (ReaxFF), which allows bond formation within the simulation. The Weeks-Chandler-Andersen (WCA) potential is used to scan multiple trial positions for the GCMC algorithm at a small cost. The most promising trial positions can be selected for recomputation with the more expensive ReaxFF. The developed WCA-ReaxFF-GCMC tool was used to study the hydration of MgCl2·nH2O. The simulation results show a good agreement with experimental and thermodynamic equilibriums for multiple hydration levels. The hydration shows that water, present at the surface of crystalline salt, deforms the surface layers and promotes further hydration of these deformed layers. Additionally, the WCA-ReaxFF-GCMC algorithm can be used to study other, non-TCM-related, reactive sorption processes.

Improved Pd/CeO2 Catalysts for Low-Temperature NO Reduction: Activation of CeO2 Lattice Oxygen by Fe Doping.

Developing better three-way catalysts with improved low-temperature performance is essential for cold start emission control. Density functional theory in combination with microkinetics simulations is used to predict reactivity of CO/NO/H2 mixtures on a small Pd cluster on CeO2(111). At low temperatures, N2O formation occurs via a N2O2 dimer over metallic Pd3. Part of the N2O intermediate product re-oxidizes Pd, limiting NO conversion and requiring rich conditions to obtain high N2 selectivity. High N2 selectivity at elevated temperatures is due to N2O decomposition on oxygen vacancies. Doping CeO2 by Fe is predicted to lead to more oxygen vacancies and a higher N2 selectivity, which is validated by the lower onset of N2 formation for a Pd catalyst supported on Fe-doped CeO2 prepared by flame spray pyrolysis. Activating ceria surface oxygen by transition metal doping is a promising strategy to improve the performance of three-way catalysts.

AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production.

Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.

Interaction of OIP5-AS1 with MEF2C mRNA promotes myogenic gene expression.

Long noncoding (lnc)RNAs potently regulate gene expression programs in physiology and disease. Here, we describe a key function for lncRNA OIP5-AS1 in myogenesis, the process whereby myoblasts differentiate into myotubes during muscle development and muscle regeneration after injury. In human myoblasts, OIP5-AS1 levels increased robustly early in myogenesis, and its loss attenuated myogenic differentiation and potently reduced the levels of the myogenic transcription factor MEF2C. This effect relied upon the partial complementarity of OIP5-AS1 with MEF2C mRNA and the presence of HuR, an RNA-binding protein (RBP) with affinity for both transcripts. Remarkably, HuR binding to MEF2C mRNA, which stabilized MEF2C mRNA and increased MEF2C abundance, was lost after OIP5-AS1 silencing, suggesting that OIP5-AS1 might serve as a scaffold to enhance HuR binding to MEF2C mRNA, in turn increasing MEF2C production. These results highlight a mechanism whereby a lncRNA promotes myogenesis by enhancing the interaction of an RBP and a myogenic mRNA.

Hepatic HuR modulates lipid homeostasis in response to high-fat diet.

Lipid transport and ATP synthesis are critical for the progression of non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) forms complexes with NAFLD-relevant transcripts. It associates with intron 24 of Apob pre-mRNA, with the 3'UTR of Uqcrb, and with the 5'UTR of Ndufb6 mRNA, thereby regulating the splicing of Apob mRNA and the translation of UQCRB and NDUFB6. Hepatocyte-specific HuR knockout reduces the expression of APOB, UQCRB, and NDUFB6 in mice, reducing liver lipid transport and ATP synthesis, and aggravating high-fat diet (HFD)-induced NAFLD. Adenovirus-mediated re-expression of HuR in hepatocytes rescues the effect of HuR knockout in HFD-induced NAFLD. Our findings highlight a critical role of HuR in regulating lipid transport and ATP synthesis.

circSamd4 represses myogenic transcriptional activity of PUR proteins.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

Linear Activation Energy-Reaction Energy Relations for LaBO3 (B = Mn, Fe, Co, Ni) Supported Single-Atom Platinum Group Metal Catalysts for CO Oxidation.

Single-atom catalysts are at the center of attention of the heterogeneous catalysis community because they exhibit unique electronic structures distinct from nanoparticulate forms, resulting in very different catalytic performance combined with increased usage of often costly transition metals. Proper selection of a support that can stably keep the metal in a high dispersion is crucial. Here, we employ spin-polarized density functional theory and microkinetics simulations to identify optimum LaBO3 (B = Mn, Fe, Co, Ni) supported catalysts dispersing platinum group metals as atoms on their surface. We identify a strong correlation between the CO adsorption energy and the d-band center of the doped metal atom. These CO adsorption strength differences are explained in terms of the electronic structure. In general, Pd-doped surfaces exhibit substantially lower activation barriers for CO2 formation than the Rh- and Pt-doped surfaces. Strong Brønsted-Evans-Polanyi correlations are found for CO oxidation on these single-atom catalysts, providing a tool to predict promising compositions. Microkinetics simulations show that Pd-doped LaCoO3 is the most active catalyst for low-temperature CO oxidation. Moderate CO adsorption strength and low reaction barriers explain the high activity of this composition. Our approach provides guidelines for the design of highly active and cost-effective perovskite supported single-atom catalysts.

Water-gas-shift reaction on reduced gold-substituted Ce1-xO2(111) surfaces: the role of Au charge.

Density functional theory is employed to investigate the role of Au charge in the water-gas-shift (WGS) reaction on a CeO2(111) surface with a cerium atom replaced by a gold atom. The oxidation state of the gold atom, varied between +3 and -1, is controlled by altering the number and configuration of oxygen vacancies. The findings indicate that Au3+ and Au- are not catalytically active for the WGS reaction because of a high energy barrier of +1.54 eV required to dissociate water and +1.40 eV to produce H2 and CO2, respectively. However, when Au is in a modest oxidation state of +1, the overall reaction barrier for the WGS reaction via the carboxyl mechanism is reduced to 0.79-0.98 eV. It therefore appears that Au species with an oxidation state of +1 play a significant role in the WGS reaction at low temperatures (T < ∼550 K).

The charge states of Au on gold-substituted Ce1-xO2(111) surfaces with multiple oxygen vacancies.

The surface structures and the charge on Au for a gold atom adsorbed on the cerium vacancy of the (111) surface of CeO2 in the presence of oxygen vacancies are investigated via density functional theory calculations. Without oxygen vacancies, the Au atom adsorbed on a Ce vacancy of CeO2(111) is highly positively charged. By systematically exploring oxygen vacancy configurations, it is found that not only does the Au charge have a trend of decreasing when the number of oxygen vacancies increases, but this charge also can be significantly changed by adjusting the locations of the oxygen vacancies without altering the vacancy number. Consequently, by arranging the oxygen vacancy configuration, the Au can exist in both positive and negative states in the case of three oxygen vacancies. Detailed analyses show that the variation in the Au charge with oxygen vacancies can be attributed to the distribution of the leftover electrons after the oxygen vacancies were created, which, in turn, depends on the vacancy number and locations of the vacancies. These results suggest a potential method to adjust the oxidation states of Au atoms absorbed on ceria. In addition, the implications of the findings for experimental results are also discussed.

Improved infrared-sensing running wheel systems with an effective exercise activity indicator.

This paper describes an infrared-sensing running wheel (ISRW) system for the quantitative measurement of effective exercise activity in rats. The ISRW system provides superior exercise training compared with commercially available traditional animal running platforms. Four infrared (IR) light-emitting diode/detector pairs embedded around the rim of the wheel detect the rat's real-time position; the acrylic wheel has a diameter of 55 cm and a thickness of 15 cm, that is, it is larger and thicker than traditional exercise wheels, and it is equipped with a rubber track. The acrylic wheel hangs virtually frictionless, and a DC motor with an axially mounted rubber wheel, which has a diameter of 10 cm, drives the acrylic wheel from the outer edge. The system can automatically train rats to run persistently. The proposed system can determine effective exercise activity (EEA), with the IR sensors (which are connected to a conventional PC) recording the rat exercise behavior. A prototype of the system was verified by a hospital research group performing ischemic stroke experiments on rats by considering middle cerebral artery occlusion. The experimental data demonstrated that the proposed system provides greater neuroprotection in an animal stroke model compared with a conventional treadmill and a motorized running wheel for a given exercise intensity. The quantitative exercise effectiveness indicator showed a 92% correlation between an increase in the EEA and a decrease in the infarct volume. This indicator can be used as a noninvasive and objective reference in clinical animal exercise experiments.

Therapeutic evaluation of etanercept in a model of traumatic brain injury.

Antagonism of tumor necrosis factor-alpha with etanercept has proved to be effective in the treatment of spinal cord injury and centrally endotoxin-induced brain injury. However, etanercept may offer promise as therapy for traumatic brain injury (TBI). In this study, anesthetized rats, immediately after the onset of TBI, were divided into two major groups and given the vehicle solution (1 mL/kg of body weight) or etanercept (5 mg/kg of body weight) intraperitoneally once per 12 h for consecutive 3 days. Etanercept caused attenuation of TBI-induced cerebral ischemia (e.g., increased cellular levels of glutamate and lactate-to-pyruvate ratio), damage (e.g., increased cellular levels of glycerol) and contusion and motor and cognitive function deficits. TBI-induced neuronal apoptosis (e.g., increased numbers of terminal deoxynucleotidyl transferase αUTP nick-end labeling and neuronal-specific nuclear protein double-positive cells), glial apoptosis (e.g., increased numbers of terminal deoxynucleotidyl transferase αUTP nick-end labeling and glial fibrillary acidic protein double-positive cells), astrocytic (e.g., increased numbers of glial fibrillary acidic protein positive cells) and microglial (e.g., increased numbers of ionized calcium-binding adapter molecule 1-positive cells) activation and activated inflammation (e.g., increased levels of tumor necrosis factor-alpha, interleukin-1ß and interleukin-6) were all significantly reduced by etanercept treatment. These findings suggest that etanercept may improve outcomes of TBI by penetrating into the cerebrospinal fluid in rats.

A force plate measurement system to assess hindlimb weight support of spinal cord injured rats.

This paper describes a force plate system for quantitative measurement of the hindlimb weight support of rats. The system is built around a microcontroller and uses strain gauges to measure individually the weight applied by each limb and also the general hindquarters of the rat. The sum of weights on the individual force plates adds up to the total weight of the rat. Mathematical comparison of the weights of the different force plates allows calculation of the weight percentage of the hindquarters (W%HQ=(hindquarters weight/total weight)x100%). When hindlimb impairment is high, the W%HQ is high and vise versa, allowing hindlimb weight support to be evaluated by the W%HQ. An actual laboratory embodiment is demonstrated and real experiments are performed on spinal cord damaged rats. W%HQ results are compared with Basso, Beattie, Bresnahan (BBB) locomotor behavioural test results on the same rats at approximately the same time. When a rat is placed in the correct position of the test chamber, the user can use a local keypad/LCD display (standalone mode) or the PC keyboard/display to control the system and access the current data. Comparing our results with those of the BBB method confirms the proposed hardware and W%HQ metric represent very well the recovery of a rat after spinal cord injury. Medical investigators report that under actual use, the presented system is stable, accurate and easy to use. Additional advantages of the presented force plate system include stand-alone capability, non-dependence on subjective human judgement and quantitative results.

An inclined plane system with microcontroller to determine limb motor function of laboratory animals.

This study describes a high-accuracy inclined plane test system for quantitative measurement of the limb motor function of laboratory rats. The system is built around a microcontroller and uses a stepping motor to drive a ball screw, which changes the angle of the inclined plane. Any of the seven inclination speeds can be selected by the user. Two infrared (IR) LED/detector pairs function as interrupt sensors for objective determination of the moment that the rat loses its grip on the textured flooring of the starting area and slips down the plane. Inclination angle at the moment of IR interrupt (i.e. rat slip) is recorded. A liquid crystal display module shows the inclination speed and the inclination angle. The system can function as a stand alone device but a RS232 port allows connection to a personal computer (PC), so data can be sent directly to hard disk for storage and analysis. Experiments can be controlled by a local keypad or by the connected PC. Advantages of the presented system include easy operation, high accuracy, non-dependence on human observation for determination of slip angle, stand-alone capability, low cost and easy modification of the controlling software for different types of experiments. A fully functional prototype of the system is described. The prototype was used experimentally by a hospital group testing traumatic brain injury experiments, and some of their results are presented for system verification. It is found that the system is stable, accurate and easily used by investigators.