SARM1 participates in axonal degeneration and mitochondrial dysfunction in prion disease.

Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss, axonal degeneration, and mitochondrial dysfunction. Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1. We found that depletion or dysfunctional mutation of SARM1 protected against NAD+ loss, axonal degeneration, and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126. NAD+ supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect. NAD+ supplementation in PrP106-126-incubated N2a cells, SARM1 depletion, and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival. Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP106-126 are partially dependent on SARM1 NADase activity. This pathway has potential as a therapeutic target in the early stages of prion disease.

Association of MDM4 Gene rs4245739 Polymorphism with the Risk and Clinical Characteristics of Colorectal Cancer in a Chinese Han Population.

BACKGROUND: Studies show that MDM4 may play a pivotal role in colorectal cancer (CRC). Recently, a host of studies suggest that MDM4 gene rs4245739 polymorphism may modify the risk of different cancers. METHODS: In this study, we were interested whether MDM4 gene rs4245739 polymorphism correlated with the risk and clinical characteristics of CRC. Logistic regression was adopted to estimate the association of rs4245739 polymorphism and CRC risk. RESULTS: We enrolled 444 CRC patients and 530 controls and found MDM4 gene rs4245739 polymorphism may decrease the risk of CRC. Stratified analyses uncovered that this variant was connected to a less risk of CRC in females, non-drinkers, non-smokers, and people under 60 years old. Additionally, rs4245739 polymorphism was related to TNM staging, pathological type, tumor size, and location of CRC. Furthermore, this polymorphism was significantly linked with the survival of CRC. CONCLUSION: Totally, this study suggests that MDM4 rs4245739 polymorphism is linked with the risk and clinical characteristics of CRC.

Optomechanical transistor: controlling the optical bistability in a photonic molecule.

We theoretically investigate the optical bistability in a composite photonic molecule cavity optomechanical system consisting of two whispering gallery mode microcavities, where one of the optical cavities is optomechanical with a high quality factor, and the other optical cavity is an auxiliary cavity with high cavity dissipation. By controlling the coupling strength J between the two cavities determined by their distance, the decay rate ratio δ of the two cavities, and the pump power P, the optical bistability can be controlled. Further, the transmission spectrum of the signal field can be efficiently attenuated or amplified, depending on the power of a second "gating" (pump) field P, and other parameters. Our study for photonic-molecule optomechanics systems may be a promising candidate for single-photon transistors and pave the way for potential applications in quantum information technologies.

PRAS40 alleviates neurotoxic prion peptide-induced apoptosis via mTOR-AKT signaling.

AIMS: The proline-rich Akt substrate of 40-kDa (PRAS40) protein is a direct inhibitor of mTORC1 and an interactive linker between the Akt and mTOR pathways. The mammalian target of rapamycin (mTOR) is considered to be a central regulator of cell growth and metabolism. Several investigations have demonstrated that abnormal mTOR activity may contribute to the pathogenesis of several neurodegenerative disorders and lead to cognitive deficits. METHODS: Here, we used the PrP peptide 106-126 (PrP106-126 ) in a cell model of prion diseases (also known as transmissible spongiform encephalopathies, TSEs) to investigate the mechanisms of mTOR-mediated cell death in prion diseases. RESULTS: We have shown that, upon stress caused by PrP106-126 , the mTOR pathway activates and contributes to cellular apoptosis. Moreover, we demonstrated that PRAS40 down-regulates mTOR hyperactivity under stress conditions and alleviates neurotoxic prion peptide-induced apoptosis. The effect of PRAS40 on apoptosis is likely due to an mTOR/Akt signaling. CONCLUSION: PRAS40 inhibits mTORC1 hyperactivation and plays a key role in protecting cells against neurotoxic prion peptide-induced apoptosis. Thus, PRAS40 is a potential therapeutic target for prion disease.

Overexpression of BAT3 alleviates prion protein fragment PrP106-126-induced neuronal apoptosis.

BACKGROUNDS AND AIMS: Prion diseases are a group of infectious neurodegenerative diseases characterized by neuronal death and degeneration. Human leukocyte antigen-B-associated transcript 3 (BAT3) is an important apoptosis regulator. We therefore investigated the interactions between BAT3 and prion protein and the potential role of BAT3 in PrP106-126-induced apoptosis. METHODS: BAT3 and prion protein were overexpressed in Hela, Neuro2A, or primary neuronal cells by transfection with BAT3-HA or PRNP-EGFP expression plasmids and their relationship studied by immunofluorescence and Western blotting. The effect of BAT3 on PrP106-126-induced cytotoxicity and apoptosis was detected by the CCK-8 assay and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The expression of cytochrome c and Bcl-2 was examined by Western blotting. RESULTS: BAT3 interacted with prion protein and enhanced PrP expression. After PrP106-126 peptide treated, BAT3 was transported from the nucleus to cytoplasm, increased cell viability, and protected neurons from PrP106-126-induced apoptosis through stabilizing the level of Bcl-2 protein and inhibiting the release of cytochrome c to cytoplasm. CONCLUSIONS: Our present data showed a novel molecular mechanism of PrP106-126-induced apoptotic process regulation through the overexpression of BAT3, which may be important for the basic regulatory mechanism of neuron survival in prion diseases and associated neurodegenerative diseases in vivo.

Neurotoxic effect of the complex of the ovine prion protein (OvPrP(C)) and RNA on the cultured rat cortical neurons.

Prion diseases are conformational diseases, many factors are involved in altering the conformation of prion, such as RNA, DNA, pH, and copper etc. However the neurotoxic mechanism of prion diseases is not clear yet. The aim of this study is to investigate the effect of the nucleoprotein complex of RNA and recombinant ovine prion protein (OvPrP(C)) on the cultured rat cortical neurons in vitro. Our previous study revealed that the nucleoprotein complex (OvPrP(C)-RNA) is characterized with high ß sheet conformation and proteinase K resistance. Here we found that the OvPrP(C)-RNA induced marked neuronal cell death by the MTT (3-(4,5-dimethyl-thiazole -2-yl)-2,5-diphenyl -tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and the neurotoxic effects were confirmed by testing the content of Bcl-2 Associated X protein (Bax) in the immunoprecipitation assay and Western blot assay. Compared to the control group, there is no significant difference of active Bax or total Bax after RNA alone treatment or OvPrP(C) alone treatment, but the OvPrP(C)-RNA induced significant increases of active Bax level, while the contents of total Bax had no obvious changes after OvPrP(C)-RNA treatment. The results suggested that OvPrP(C)-RNA is neurotoxic in vitro, which added further evidence to the current understanding of mechanism of cellular injury by RNA molecules for transformation of the PrP(C) to PrP(Sc).

Polymorphism of prion protein gene in sheep of Inner Mongolian, China.

Susceptibility to natural scrapie in sheep is associated with polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene. To assess the risk of scrapie in sheep raised in China, DNA from 30 sheep of two breeds was isolated, amplified and sequenced for the PrP gene. The ovine PrP gene was found to be highly homogenous. The genotype associated with high susceptibility to scrapie (VRQ) was absent, whereas that associated with the resistance (ARR) was present in 6.7% of sheep examined. ARK was also rare (6.7%). ARQ that is associated with an intermediate susceptibility was the genotype observed in the most of sheep examined (86.6%). These data suggest that Chinese sheep of Mongolian sheep breed are susceptible to scrapie.

PrP 106-126 altered PrP mRNA gene expression in mouse microglia BV-2 cells.

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126, and indicate that microglial cells might play a critical role in prion pathogenesis.

Variable levels of 37-kDa/67-kDa laminin receptor (RPSA) mRNA in ovine tissues: potential contribution to the regulatory processes of PrPSc propagation?

The 37-kDa laminin receptor precursor/67-kDa laminin receptor (LRP/LR, also known as ribosomal protein SA, RPSA) has been reported to be involved in cancer development and prion internalization. Previous studies have shown that the LRP/LR is expressed in a wide variety of tissues. In particular, expression of LRP/LR mRNA may be closely related to the degree of PrP(Sc) propagation. This study presents a detailed investigation of the LRP/LR mRNA expression levels in eleven normal ovine tissues. Using real-time quantitative PCR, the highest LRP/LR expression was found in neocortex (p < 0.05). Slightly lower levels were found in the heart and obex. Intermediate levels were seen in hippocampus, cerebellum, spleen, thalamus, mesenteric lymph node, and the lowest levels were present in liver, kidney, and lung. In general, the LRP/LR mRNA levels were much higher in neuronal tissues than in peripheral tissues. The observation that differences in LRP/LR mRNA expression levels are consistent with the corresponding variation in PrP(Sc) accumulation suggests that the 37-kDa/67-kDa laminin receptor may be involved in the regulation of PrP(Sc) propagation.

[Study on testing methods of Al content of polyimide hybrid films with Al2O3].

The inorganic particles hybrid polyimide films are newly emerging advanced materials with excellent corona-resistant and have been widely used in frequency control motor. The Al2O3/PI hybrid films with different Al2O3 contents were prepared by superfine aluminum power treated by coupling agent and polyamide acid (PAA). Qualitative analysis and quantitative analysis on the inorganic content to it were conducted using Fourier Transform Infrared Spectroscope (FTIR), X-ray photoelectron spectroscopy (XPS), Inductively coupled plasma spectrometry (ICP), Gravimetric method, Thermogravimetric Analysis (TG/air atmosphere). It was found that FTIR and XPS are good qualitative analysis methods. FTIR is used to inference possible components by analyzing the structure of material. The method has a lot of advantages such as easily operational, good repeatability, high accuracy and so on. XPS is mainly used to get information of elements contained in the material, it provides information about the core level binding energies and oxidation states of complexes. It can be used to identify the species and valence states of elements, measure the relative content of elements. The relative standard deviations (RSDs) of XPS and TG are too great to perform quantitative analysis, the RSDs of XPS are all above 5.0%, and TG's RSDs are also above 2.0%. So they can be only used as semiquantitative analysis methods. On the contrary, ICP and Gravimetric method are two excellent quantitative analysis methods, their RSDs are all below 1.0%. Moreover Gravimetric method only can be used to analyze single inorganic constituent complex material, although its measured value is closest approach to theoretical value. ICP is the most accurate method and it can be used to analyze multi inorganic components in complex material, this method proved to be easily operational, rapid, highly sensitive, and accurate, and can be adopted as the method of determining many elements simultaneously. So a method was got to analysis of inorganic constituent in complex material from the conclusion upward. Firstly, components in complex material are defined by using FTIR and XPS as qualitative analysis methods and then using the result of XPS as a reference, exactly quantitative analysis of inorganic constituent in complex material was performed by using Gravimetric method and ICP.

[Characterization of inorganic nano-alundum composite film of polyimide].

The key to the study on the regularity about the mechanical, thermology and electricities property of the inorganic nano-mingled organic composition thin film is to understand the incorporated quantity, the particle size and distribution of nano-inorganic matter in the membrane quickly and accurately. In the present paper, the chemical structure, surface morphology and the actual content of nano-Al2O3 of the nano Al2O3-composite film of polyimide were characterized by X-ray atomic fluorescent spectroscopy (XRF), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and atomic forced microscope (AFM). The results are that the organic phase of PI and the inorganic phase of Al2O3 formed a complex composite hybrid system of bond-to-bond pattern, the nano-Al2O3 particles in the film of PI are dispersed homogeneously, and the diameter of the particle is smaller than 50 nm; the weight content of Al2O3 is 7.9% by XRF. The approach we used is an effective way of analyzing the inorganic component of the organic composite film materials doped with the inorganic nano-phase materials with the merits of no pretreatment, no fed charge (for analysis of insulation materials), no-contagion, no destruction, high speed and high accuracy, etc.

Copper(II) inhibits in vitro conformational conversion of ovine prion protein triggered by low pH.

To gain insight into the conformational conversion of ovine prion protein (OvPrP(C)) at different pH values and/or in the presence of CuCl(2), the secondary structure of OvPrP(C) was analysed by circular dichroism (CD) spectroscopy. Copper treatment of OvPrP(C) under moderately acidic conditions (pH approximately 5.0-6.0) as well as physiological conditions (pH 7.4) also makes OvPrP(C) adopt protease-resistant and beta-sheet-rich conformation. However, under lower pH conditions (2.0-4.5) with copper treatment, OvPrP(C) gained higher alpha-helix structure. This study demonstrated that Cu(2+) can significantly modulate conformational conversion triggered by acidic pH, and this will provide therapeutic intervention approaches for prion diseases.

The quantification of prion gene expression in sheep using real-time RT-PCR.

Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its poorly explained role in organisms. Scrapie in sheep is the prototype of all prion diseases. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail. Herein we report on measurement of sheep PrP mRNA using absolute quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR). Total RNA was isolated from seven different regions of the central nervous system (CNS) and six peripheral organs of 18 sheep and PrP mRNA was quantified by real-time RT-PCR using an externally calibrated standard curve constructed with the recombinant PrP plasmid. The results showed that high levels of PrP mRNA were expressed in all seven regions of the brain examined, with obex and neocortex expressing the highest PrP, followed by cerebellum, spinal cord, hippocampi, conarium and thalamus, In peripheral organs examined, lymph node showed a level of PrP expression similar to that in overall brain, whereas spleen, heart, liver and lung showed moderate level of expression and kidney showed the lowest expression. Our study provided the first quantitative, tissue-specific data of PrP mRNA expression in sheep for further studies of pathogenesis of prion diseases.

Comparative analysis of the prion protein gene sequences in African lion.

The prion protein gene of African lion (Panthera Leo) was first cloned and polymorphisms screened. The results suggest that the prion protein gene of eight African lions is highly homogenous. The amino acid sequences of the prion protein (PrP) of all samples tested were identical. Four single nucleotide polymorphisms (C42T, C81A, C420T, T600C) in the prion protein gene (Prnp) of African lion were found, but no amino acid substitutions. Sequence analysis showed that the higher homology is observed to felis catus AF003087 (96.7%) and to sheep number M31313.1 (96.2%) Genbank accessed. With respect to all the mammalian prion protein sequences compared, the African lion prion protein sequence has three amino acid substitutions. The homology might in turn affect the potential intermolecular interactions critical for cross species transmission of prion disease.

[A preliminary approach of covering corrugator with rectangle periosteous flap in rhytidectomy].

OBJECTIVE: To evaluate the aesthetic outcome after application of rectangle periosteous flap to cover corrugator. METHODS: On the basis of regular rhytidectomy, a inter-eyebrow periosteous flap is applied to cover the fascia flap of corrugator by means of turnover. This manipulation helps to separate the skin from the muscle and prevent the re-adherence between the skin and muscle. RESULTS: The approach was applied on 15 cases. The follow-up ranged from 6 months to 1 year. The results were satisfactory. CONCLUSIONS: The method is effective in elimination of inter-eyebrow crease.

Development of a rapid real-time PCR assay for detection and quantification of four familiar species of Chlamydiaceae.

Chlamydiae are one of the causative agents of various diseases in animals and human beings, which include abortion, pneumonia, gastroenteritis, encephalomyelitis, conjunctivitis, arthritis and sexually transmitted diseases. Much work has been carried out to attempt to develop an efficient pathogen detection strategy. Here, we presented a Chlamydiaceae-specific 23S rRNA-based real-time PCR assay for simultaneous detection and quantification of four members of Chlamydiaceae family, C. trachomatis, C. psittaci, C. pneumoniae and C. pecorum, using SYBR Green and Lightcycler. The assay was characterized using plasmid constructs of the bacteria and verified on standard strains of all four species of the Chlamydiaceae and a large cohort of clinical samples collected from human and animals by comparison with fluorescence immunohistochemistry method. The results showed that the present real-time PCR assay was of high specificity and sensitivity. It was capable of detecting as few as 250fg of chlamydial DNA (equivalent to 10(-1)IFU) and was applicable to both liquid cultures and clinical samples. This assay may therefore offer a rapid, economic and reliable means for screening of the chlamydiaceae pathogens.

Quantification of prion gene expression in brain and peripheral organs of golden hamster by real-time RT-PCR.

Determination of tissue-specific expression of cellular prion protein (PrPc) is essential for understanding its poorly explained role in organisms. Herein we report on quantification of PrP mRNA in golden hamsters, a popular experimental model for studying mechanisms of transmissible spongiform encephalopathies (TSE), by real-time RT-PCR. Total RNA was isolated from four different regions of the brain and six peripheral organs of eight golden hamsters. PrP mRNA copy numbers were determined using absolute standard curve method with real-time quantitative PCR instrument. It was found that high mRNA levels were present in all four regions of the brain examined, including obex, neocortex, cerebellum, and thalamus. In peripheral organs examined, inguinal lymph node showed high level of the expression similar to that in overall brain; spleen, heart, liver, and lung showed moderate levels of the expression; and kidney showed the lowest expression. Our result is consistent with the potential involvement of different organs in prion diseases and offers essential data for further study of TSE mechanism in this animal model.

Comparative analysis of the prion protein open reading frame nucleotide sequences in peacock and parakeet.

The open reading frame of peacock and parakeet prion protein (PrP) genes was cloned and sequenced. The peacock and parakeet PrP genes consisted of 833 and 866 nucleotides encoding 266 and 277 amino acids, respectively (GenBank Accession numbers AY365065 and AY365066). Sequence analysis showed that the peacock and parakeet PrP genes had 93.67% homology to each other, 94.04% and 99.64% homology to the chicken PrP gene and 46.0% and 42.1% similarity to the mammalian PrP genes, respectively. The structural features of all known mammalian and avian PrPs, including N-terminal signal peptides, tandem repeats, conserved hydrophobic region, disulfide bridges and glycoinositol phospholipid anchor, were also found in peacock and parakeet PrPs. The parakeet and peacock PrPs, however, differed in the hexarepeat region, with the peacock having six and the parakeet having seven hexarepeats. The phylogenetic analysis showed that the PrP genes in 52 species were clustered into 2 distinct lineages, the avian and the mammalian. The peacock and parakeet PrP genes belonged to the same lineage but the peacock PrP was sub-classed with the pigeon PrP and the parakeet PrP was sub-classed with the duck and chicken PrPs. The present work added two more species data to the collection of the PrP genes and supported the previous findings that the PrP genes are highly conserved across species.

Altered expression of the prion gene in rat astrocyte and neuron cultures treated with prion peptide 106-126.

Neuronal degeneration and astrogliosis are hallmarks of prion disease. Synthetic prion protein (PrP) peptide 106-126 (PrP106-126) can induce death of neurons and proliferation of astrocytes in vitro and this neurotoxic effect depends on the expression of cellular PrP (PrPC) and is hence believed to be PrP(C) -mediated. To further elucidate the involvement of PrPC in PrP106-126-induced neurotoxicity, we determined the expression of PrP mRNA in primary culture of rat cortical neuron cells, cerebellar granule cells, and astrocytes following treatment with 50 microM of PrP106-126 scrambled PrP106-126 by quantitative real-time RT-PCR. As shown by MTT test, PrP106-126 induced significant death of neuron cells and marked proliferation of astrocytes after 10 days of treatment. Under the same treatment regimens, the level of PrP gene expression was significantly down-regulated in cortical neuron cell cultures and cerebellar granule cell cultures and was up-regulated in astrocyte cultures. The altered PrP gene expression occurred as early as 3 days after the treatment. After 10 days of treatment, while the cultured cortical neurons underwent further apoptosis, their expression of PrP gene started to recover gradually. These findings indicate that PrP 106-126 regulates transcription of the PrP gene and this activity is associated with its neurotoxicity in primary rat neuronal cultures.