Instability of the Octarepeat Region of the Human Prion Protein Gene

Prion diseases are a family of unique fatal transmissible neurodegenerative diseases that affect humans and many animals. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans, accounting for 85–90% of all human prion cases, and exhibits a high degree of diversity in phenotypes. The etiology of sCJD remains to be elucidated. The human prion protein gene has an octapeptide repeat region (octarepeats) that normally contains 5 repeats of 24–27 bp (1 nonapeptide and 4 octapeptide coding sequences). An increase of the octarepeat numbers to six or more or a decrease of the octarepeat number to three is linked to genetic prion diseases with heterogeneous phenotypes in humans. Here we report that the human octarepeat region is prone to either contraction or expansion when subjected to PCR amplification in vitro using Taq or Pwo polymerase and when replicated in wild type E. coli cells. Octarepeat insertion mutants were even less stable, and the mutation rate for the wild type octarepeats was much higher when replicated in DNA mismatch repair-deficient E.coli cells. All observed octarepeat mutants resulting from DNA replication in E.coli were contained in head-to-head plasmid dimers and DNA mfold analysis (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form) indicates that both DNA strands of the octarepeat region would likely form multiple stable hairpin structures, suggesting that the octarepeat sequence may form stable hairpin structures during DNA replication or repair to cause octarepeat instability. These results provide the first evidence supporting a somatic octarepeat mutation-based model for human sCJD etiology: 1) the instability of the octarepeat region leads to accumulation of somatic octarepeat mutations in brain cells during development and aging, 2) this instability is augmented by compromised DNA mismatch repair in aged cells, and 3) eventually some of the octarepeat mutation-containing brain cells start spontaneous de novo prion formation and replication to initiate sCJD

Extreme Dynamic Responses of MW-Level Wind Turbine Tower in the Strong Typhoon Considering Wind-Rain Loads

The damage and collapse accidents of wind turbines during violent typhoons and rainstorms have increased in recent years. To determine the dynamic response characteristics of high-power wind turbines under extreme conditions, wind load and rain load are simulated. The typhoon average wind velocity and fluctuating wind velocity are simulated by the unstable wind profile and harmony superposition method. The raindrop size distribution is simulated by the M-P spectrum, and the rain load is calculated according to the momentum theorem. A finite element model is established to study the aerodynamic responses of a wind turbine under random typhoon load and typhoon-rain loads. The maximum displacements and accelerations at the tower top and the maximum von Mises stresses at the tower bottom are calculated and compared after considering various combinations of wind direction deflections and rainfall intensities. The results indicate that instantaneous wind direction deflection has a substantial impact on the dynamic responses of wind turbines, and after introducing the effect of rain, the dynamic responses increase up to 13.7% with increasing rainfall intensities. This study has significant implications for analysing collapse accidents of wind turbines and for optimising the design of wind turbines under extreme typhoon conditions

Melanocortin receptor-4 mediates the anorectic effect induced by the nucleus tractus solitarius injection of Glucagon-like Peptide-2 in fasted rats

Glucagon-like peptide-2 (GLP-2) is secreted from enteroendocrine L-type cells of the gut and also released from preproglucagonergic (PPG) neurons in the nucleus tractus solitarius (NTS) and adjacent medial reticular nucleus of the brain stem. The neurons in the NTS express GLP-2, and the neurons send extensive projections to the hypothalamus. Recent studies show that the intracerebroventricular administration of GLP-2 significantly suppresses food intake in animals and some evidence suggest that the melanocortin receptor-4 (MC4-R) signaling in the hypothalamus is required for intracerebroventricular GLP-2-mediated inhibition of feeding. There is proopiomelanocortin (POMC) positive neurons expressing MC4-R in the NTS. Suppression of MC4-R expressing neurons in the brain stem inhibits gastric emptying. In this study, we tested the effects of NTS GLP-2R activation and blockade on feeding behavior and evaluated the endogenous melanocortin system's role in the NTS in mediating effects of GLP-2 on feeding behavior in fed and fasted rats. Our results demonstrated that microinjection of GLP-2 into the NTS suppressed food intake in fasted-refeeding rats but did not affect food intake in free-feeding rats, and this inhibition was blocked by pretreatment of either Exendin (9–39) or SHU 9119, suggesting the GLP-2 system in the NTS exerts an inhibitory action on food intake. MC4-R mediates this action in the NTS

GLP-2 decreases food intake in the dorsomedial hypothalamic nucleus (DMH) through Exendin (9-39) in male Sprague-Dawley (SD) rats

Glucagon-like peptide 2 (GLP-2), a member of Glucagon peptide family involved in regulating energy metabolism, can be produced and secreted by preproglucagonergic (PPG) neurons in the brain. GLP-2 reduces food intake but at which brain sites GLP-2 exerts its feeding-suppress effects are still unclear. In this study, we used the stereological microinjection technique and behavioral test to examine the functions of locally delivered GLP-2 into DMH on feeding behavior. We compared effects of different concentration of GLP-2 on the food intake behavior in free-feeding rats and fasted-refeeding rats. We found that GLP-2 inhibited food intake in fasted rats after a short-term intervention in a dose-dependent manner. Importantly, the effects of locally delivered GLP-2 can be blocked by specific GLP-1 receptor antagonist Exendin(9-39), but not the melanocortin-4 receptor antagonist SHU9119, indicating the involvement of specificity of GLP-2 signaling in regulating the feeding behavior. Taken together, our data revealed that GLP-2 peptide pharmacologically inhibited food intake in DMH and this effect could be blocked functionally by Exendin(9-39)

Test and Simulation Analysis of Soybean Seed Throwing Process

In order to analyze the effect of different factors on the bouncing and rolling distance of soybeans at the time of seed throwing, tests and discrete element method (DEM) are employed to analyze test soil and three representative soybean varieties. The parameters between soybean seed particles and soil particles are calibrated by means of a piling test and simulation. A seed throwing test apparatus is improved to analyze the effects of seed throwing height, soil plane inclination angle and collision orientation on the bouncing and rolling distance of soybean seeds. The effect of relative seed throwing speed on the bouncing and rolling distance of soybean seeds is analyzed using a computer vision seeding test bench. On this basis, the above-mentioned test procedure is simulated and compared with the test results. The results showed that the bouncing distance of the soybean seed particles was not significant. The rolling distance had a certain randomness when the seed throwing height was different. When the inclination of the soil plane became larger, the rolling distance increased. When the sphericity of the soybean seed particles was high, the effect of different collision orientations was not obvious. If the sphericity was low, the rolling distance was the shortest when colliding in the horizontal orientation and the longest when colliding in the vertical orientation. The larger the relative seed throwing speed, the larger the rolling distance of the soybean seed particles

Fatigue Life Evaluation of Wind Turbine Tower Based on Measured Data

Fatigue life is a crucial design factor which dictates the safe operation of the wind turbines, but it is influenced by uncertain factors such as environmental loads, analytical models, material properties, and manufacturing methods. In this study, a 1.5 MW wind turbine was monitored in operation to understand the fatigue mechanism and enhance wind turbine design. The influence of different operating conditions on fatigue damage was analyzed by correlating strain monitoring data with supervisory control and data acquisition (SCADA) data. Furthermore, a fatigue evaluation method based on measured strain data was proposed. Fatigue damage increases with the increase of wind and rotation speed. More than 50% of the damage occurred at the rated rotation speed state, the corresponding wind speed was greater than the rated wind speed and the pitch control system was active. The findings of this study provide insights for investigating the real fatigue state of similar wind turbine towers and improving the return on investment by closely estimating their service life

Instability of octarepeats during DNA replication in DH5α cells.

<p>(A) Mutant clones from replication of pOct5 in DH5α cells. pOct5 was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with <i>Sac</i>II and <i>Spe</i>I and separated on a 2% agarose gel. Shown are plasmid DNAs from 3 colonies that produced two octarepeat bands of equal molar ratio. (B) Mutant clones from replication of pOct11b in DH5α cells. pOct11b was transformed into DH5α. Plasmid DNAs were prepared from the resulting colonies, digested with <i>Sac</i>II and <i>Spe</i>I and separated on a 2% agarose gel. Shown are plasmid DNAs from 4 colonies that produced two octarepeat bands of equal molar ratio. (C–D) Unusual mutant clones from replication of pOct11b in DH5α cells. Shown are plasmid DNAs from two pOct11b-transformed DH5α colonies that produced 2–3 octarepeat bands upon digestion with <i>Sac</i>II and <i>Spe</i>I, of which the template-sized band is much stronger than the mutant bands (C). The unequal molar ratio of the octarepeat bands suggests the presence in these colonies of mixed plasmid DNA species where each species produced one of the octarepeat bands. Re-transformation of these plasmid DNAs into DH5α cells resulted in separation of the mixed plasmid DNA species and produced colonies that each contained only one plasmid DNA species as confirmed by restriction analysis and sequencing (D). For all panels, the octarepeat sequence is indicated above each lane, the arrowhead points to the band whose sequence is shown above the lane, and the black box marks the template-sized Oct5 or Oct11 band from a non-mutant clone. Rep. No., number of repeats; M,100-bp DNA Ladder.</p

Examination of Bone Marrow Mesenchymal Stem Cells Seeded onto Poly(3-hydroxybutyrate-co-3-hydroxybutyrate) Biological Materials for Myocardial Patch

The implantation of the Bone Marrow Mesenchymal Stem Cells (BMSCs) into the heart has been reported to be effective for the treatment of myocardial infarction; however, the methods most suitable for supporting stem cell growth in a myocardial patch, still remain unknown. We used a new polymer material composed of poly(3-hydroxybutyrate-co-3-hydroxybutyrate) [P(3HB-co-3HB)] co-cultured with BMSCs to create a myocardial patch. The BMSCs were obtained from healthy male BSL-C57 mice. The cells were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. The cells were seeded for 24 hours onto the P(3HB-co-3HB) biological material films (n=8). Cell-biomaterial constructs were fixed and analyzed using different methods. BMSCs were CD34-, CD45-, CD90+ (low) and CD73+. The cells were stained with anti-cardiac troponin T (cTnT) and anti-connexin 43 (CX43) antibodies after 5-azacytidine treatment. Scanning electron microscopy revealed that the morphology of the BMSC was normal and that cell numbers were more abundant on the P(3HB-co-3HB) material surfaces. The growth curve of the BMSCs on the biomaterial patches showed the P(3HB-co-3HB) material enhanced good stem cell growth. Owing to its excellent biocompatibility and biodegradability properties, in particular its porosity, the P(3HB-co-3HB) is hailed as an optimal material to support myocardial cell growth and to create a myocardial patch in patients with myocardial infarction