Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury

Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modification. Therefore, it is necessary to quantitatively analyze the gene expression profile using proteomic techniques. In the present study, we established a rat model of closed brain injury using Marmarou＇s weight-drop device, and investigated hippocampal differential protein expression using two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry. A total of 364 protein peaks were detected on weak cation exchange-2 protein chips, including 37 differential protein peaks. 345 protein peaks were detected on immobilized metal affinity capture arrays-Cu, including 12 differential protein peaks Further examination of these differential proteins revealed that glucose-regulated protein and proteasome subunit alpha type 3 expression were significantly upregulated post-injury. These results indicate that brain injury can alter protein expression in the hippocampus, and that glucose-regulated protein and proteasome subunit alpha type 3 are closely associated with the occurrence and development of traumatic brain injury.

Effects of Different Extraction Methods on Antioxidant Activity of Inonotus obliquus Polysaccharides

[ Objective] This study aimed to explore the effects of different extraction methods on antioxidant activities of I. obliquus polysaccharides. [Method] Crude polysaccharides were extracted by using boiling ethanol precipitation method and ethanol-alkali extraction method; water-soluble and alkali-soluble polysac- charides were purified by using trichloroacetic acid method; antioxidant activities of I. obliquus polysaccharides were compared by Hydroxyl radical scavenging ex- periment and Superoxide anion radical scavenging experiment. ~ Result~ Yield of crude water-soluble polysaccharides, crude alkali-soluble polysaceharides, pure water-soluble polysaccharides and pure alkali-soluble polysaccharides were 14.0%, 27.7%, 7.0% and 6.2%, respectively. Hydroxyl radical scavenging effect of crude water-soluble polysaccharides and pure alkali-soluble polysaccharides had achieved the maximum with polysaccharide concentration of 0.9 and 0.06 mg/ml, and the scavenging rates were 54.3% and 10.9%, respectively; superoxide anion radical scavenging effect of crude water-soluble polysaccharides and pure water- soluble polysaceharides had achieved the maximum with polysaceharide concentration of 0. 1 and O. 07 mg/ml, and the scavenging rates were 24.2% and 27.2%, respectively. [ Conclusion] Antioxidanl activity of water-soluble polysaecharides was higher than that of alkali-soluble polysaccharides. 1. obliquus could be usedas a species of new efficient natural antioxidant medicinal fungi in application.

Exceptional-point-enhanced sensing in an all-fiber bending sensor

An exceptional-point(EP)enhanced fiber-optic bending sensor is reported.The sensor is implemented based on paritytime(PT)-symmetry using two coupled Fabry-Perot(FP)resonators consisting of three cascaded fiber Bragg gratings(FBGs)inscribed in an erbium-ytterbium co-doped fiber(EYDF).The EP is achieved by controlling the pumping power to manipulate the gain and loss of the gain and loss FP resonators.Once a bending force is applied to the gain FP resonator to make the operation of the system away from its EP,frequency splitting occurs,and the frequency spacing is a nonlinear function of the bending curvature,with an increased slope near the EP.Thus,by measuring the frequency spacing,the bending information is measured with increased sensitivity.To achieve high-speed and high-resolution interrogation,the optical spectral response of the sensor is converted to the microwave domain by implementing a dual-passband microwave-photonic filter(MPF),with the spacing between the two passbands equal to that of the frequency splitting.The proposed sensor is evaluated experimentally.A curvature sensing range from 0.28 to 2.74 m^(−1) is achieved with an accuracy of 7.56×10^(−4 )m^(−1 )and a sensitivity of 1.32 GHz/m^(−1),which is more than 4 times higher than those reported previously.

中性粒细胞“异质性”在缺血性卒中的病理生理作用

缺血性卒中发生后,中性粒细胞会第一时间做出反应参与血脑屏障破坏、脑水肿及出血转化等病理过程,因此抗中性粒细胞治疗,如抑制其激活、粘附以及抑制蛋白酶、细胞因子释放等都曾是缺血性卒中治疗的靶点和热点。基础研究虽然前景良好,但是在向临床应用转化时却困难重重。其中一个很重要的原因就是循环血中中性粒细胞自身的“异质性”(heterogeneity)特征,即其功能和作用机制都是动态变化的。深入研究并充分了解中性粒细胞“异质性”不仅可以让我们进一步理解缺血性卒中发生后中性粒细胞发挥的病理生理作用,还可以为解决缺血性卒中基础研究向临床应用转化的瓶颈难题提供理论依据,为缺血性卒中治疗另辟蹊径。目前研究表明,在缺血性卒中病程进展过程中,中性粒细胞在发育阶段、功能、表型蛋白、患者年龄和分布等方面都存在异质性,本综述拟从以上各方面进行详细阐述。

Acute high-altitude hypoxic brain injury Identification of ten differential proteins

Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain injury. However, little is currently known about the mechanisms responsible for mi- tochondrial dysfunction in hypobaric hypoxic brain damage. In this study, a rat model of hypobaric hypoxic brain injury was established to investigate the molecular mechanisms associated with mi- tochondrial dysfunction. As revealed by two-dimensional electrophoresis analysis, 16, 21, and 36 differential protein spots in cerebral mitochondria were observed at 6, 12, and 24 hours post-hypobaric hypoxia, respectively. Furthermore, ten protein spots selected from each hypobaric hypoxia subgroup were similarly regulated and were identified by mass spectrometry. These de- tected proteins included dihydropyrimidinase-related protein 2, creatine kinase B-type, is- ovaleryI-CoA dehydrogenase, elongation factor Ts, ATP synthase beta-subunit, 3-mercaptopyruvate sulfurtransferase, electron transfer flavoprotein alpha-subunit, Chain A of 2-enoyI-CoA hydratase, NADH dehydrogenase iron-sulfur protein 8 and tropomyosin beta chain. These ten proteins are all involved in the electron transport chain and the function of ATP synthase. Our findings indicate that hypobaric hypoxia can induce the differential expression of several cerebral mitochondrial proteins, which are involved in the regulation of mitochondrial energy production.

Underlying mechanism of protection from hypoxic injury seen with n-butanol extract of Potentilla anserine L. in hippocampal neurons

The alcohol and n-butanol extract of Potentilla anserine L. significantly protects myocardium from acute ischemic injury. However, its effects on rat hippocampal neurons and the mechanism of protection remain unclear. In this study, primary cultured hippocampal neurons from neonatal rats were incubated in 95% N2 and 5% CO2 for 4 hours. Results indicated that hypoxic injury decreased the viability of neurons, increased the expression levels of caspase-9 and caspase-3 mRNA, as well as cytochrome c, Caspase-9, and Caspase-3 protein. Pretreatment with 0.25, 0.062 5, 0.015 6 mg/mL n-butanol extract of Potentilla anserine L. led to a significant increase in cell viability. Expression levels of caspase-9 and caspase-3 mRNA, as well as cytochrome c, Caspase-9, and Caspase-3 protein, were attenuated. The neuroprotective effect of n-butanol extract of Potentilla anserine L. was equivalent to tanshinone IIA. Our data suggest that the n-butanol extract of Potentilla anserine L. could protect primary hippocampal neurons from hypoxic injury by deactivating mitochondrial cell death.

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott’s method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury.

Differential protein expression in rat cortical astrocytes following fluid percussion injury Two-dimensional gel electrophoresis and mass-spectrum detection

BACKGROUND： The Glasgow Coma Scale, computer tomography, and nuclear magnetic resonance imaging have been frequently used to diagnose brain injury. However, these methods do not accurately and quantitatively evaluate injury degree. However, proteomics displays some advantages. To date, there are few proteomics studies based on primary astrocyte cultures from a fluid percussion injury model. OBJECTIVE： To detect differential protein expression in rat cerebral cortical astrocytes following fluid percussion injury using two-dimensional gel electrophoresis and mass spectrum and to determine specific biological markers of brain injury. DESIGN, TIME AND SETTING： Complete, randomized grouping and proteomics experiments were performed at the Molecular Pathological Laboratory, Central Laboratory and Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard of Medical College of Chinese People＇s Armed Police Force from October 2007 to May 2008. MATERIALS： Inverted phase-contrast microscope was purchased from Olympus, Japan. PROTEAN IEF Cell isoelectric focusing electrophoresis system and PROTEAN II Xi-Cell vertical electrophoresis system were purchased from Bio-Rad, USA. Autofiex MALDI-TOF mass spectrometer was purchased from Bruker, Germany. METHODS： A total of 90 culture dishes, fully coated with Sprague Dawley rat cortical astrocytes, were randomly divided into control （n = 30） and injury （n = 60） groups. Astrocytes in the injury group were subjected to fluid percussion and subdivided into 4-hour （n = 30） and 48-hour injury （n = 30） groups. MAIN OUTCOME MEASURES： Cell morphology was observed using inverted phase-contrast microscopy. Cell total protein was extracted from each group, followed by two-dimensional gel electrophoresis and silver staining, and the differential protein expression was analyzed using PDQuest 7.0 software. Protein peptide mass fingerprinting of differential protein spots was obtained by matrix assisted laser desorption/ionization-time of flight mass spectrometry. The National Center for Biotechnology Information （NCBI） protein database was retrieved by Mascot to primarily identify protein type, Finally, differential protein expression was detected by Western blot analysis. RESULTS： Following fluid percussion injury, astrocytes displayed obvious swelling and increased intercellular space, with some cell detachment; the number of dead cells was significantly greater than the control group （P 〈 0.05）. Expression intensity of 114 protein spots was significantly greater in the injury group compared with the control group （P〈 0.05）; 9 of the 114 protein spots were identified and peptJde matching scores of 8 spots were 〉 61 （P 〈 0.05）. Protein types were identified and included cellular retinol binding protein, brain fatty acid binding protein 7, $100 calcium binding protein All, 60S acidic ribosomal protein P2, calponin 3, breast carcinoma amplified sequence 2 homolog, eukaryotic translation initiation factor 1A, and hypothetical protein LOC685814. Western blot detection revealed brain fatty acid binding protein 7 expression in cortical astrocytes, which increased with injury time compared with the control group （P 〈 0.05）. CONCLUSION： Results from this study showed morphological and proteomic changes in cortical astrocytes following fluid percussion injury. Brain fatty acid binding protein 7 was expressed in astrocytes and possibly played an important role in injury repair. Mass-spectrum identified differentially expressed proteins that correlated with cell metabolism regulation, signal transduction, and translation initiation, and could serve as specific biological markers of brain injury.

FFAR4 improves the senescence of tubular epithelial cells by AMPK/SirT3 signaling in acute kidney injury

Acute kidney injury(AKI)is a serious clinical complication with high morbidity and mortality rates.Despite substantial progress in understanding the mechanism of AKI,no effective therapy is available for treatment or prevention.We previously found that G protein-coupled receptor(GPCR)family member free fatty acid receptor 4(FFAR4)agonist TUG891 alleviated kidney dysfunction and tubular injury in AKI mice.However,the versatile role of FFAR4 in kidney has not been well characterized.In the study,the expression of FFAR4 was abnormally decreased in tubular epithelial cells(TECs)of cisplatin,cecal ligation/perforation and ischemia/reperfusion injury-induced AKI mice,respectively.Systemic and conditional TEC-specific knockout of FFAR4 aggravated renal function and pathological damage,whereas FFAR4 activation by TUG-891 alleviated the severity of disease in cisplatin-induced AKI mice.Notably,FFAR4,as a key determinant,was firstly explored to regulate cellular senescence both in injured kidneys of AKI mice and TECs,which was indicated by senescence-associatedβ-galactosidase(SA-β-gal)activity,marker protein p53,p21,Lamin B1,phospho-histone H2A.X,phospho-Rb expression,and secretory phenotype IL-6 level.Mechanistically,pharmacological activation and overexpression of FFAR4 reversed the decrease of aging-related SirT3 protein,where FFAR4 regulated SirT3 expression to exhibit anti-senescent effect via Gq subunit-mediated CaMKKβ/AMPK signaling in cisplatin-induced mice and TECs.These findings highlight the original role of tubular FFAR4 in cellular senescence via AMPK/SirT3 signaling and identify FFAR4 as a potential drug target against AKI.

Association of ABCB1 Gene Polymorphisms with Efficacy and Adverse Reaction to Risperidone or Paliperidone in Han Chinese Schizophrenic Patients

Dear editor, P-glycoprotein （P-gp, also known as ATP-binding cassette transport sub-family B member 1, ABCB1） is a potent ATP-dependent efflux pump for a wide variety of drugs. Although studies of its substrates are abundant [ 1, 2], and ABCB1 is a well-conserved gene, there is increasing evi- dence that its polymorphisms affect substrate specificity [3]. A previous study reported that the synonymous single nucleotide polymorphism （SNP） C3435T （rs1045642） affects the timing of co-translational folding and insertionof P-gp into the membrane,

Profiling of histone H3 trimethylation and distinct epigenetic pattern of chromosome Y in the transformed bronchial epithelial cells induced by consecutive arsenic treatment

To the Editor:Environmental arsenic is a known human carcinogen that is associated with cancers in lung,liver,skin,breast,bladder,kidney,prostate,etc.1 However,the underpinnings of ma-lignant transformation of the normal cells in response to environmental arsenic exposure remain poorly understood,in part due to mixed signaling pathways elicited by arsenic,especially the inorganic trivalent arsenic(As3þ).

Polarimetric parity-time symmetry in a photonic system

Parity-time(PT)symmetry has attracted intensive research interest in recent years.PT symmetry is conventionally implemented between two spatially distributed subspaces with identical localized eigenfrequencies and complementary gain and loss coefficients.The implementation is complicated.In this paper,we propose and demonstrate that PT symmetry can be implemented between two subspaces in a single spatial unit based on optical polarimetric diversity.By controlling the polarization states of light in the single spatial unit,the localized eigenfrequencies,gain,loss,and coupling coefficients of two polarimetric loops can be tuned,leading to PT symmetry breaking.As a demonstration,a fiber ring laser based on this concept supporting stable and single-mode lasing without using an ultranarrow bandpass filter is implemented.

Loss of mdig expression enhances DNA and histone methylation and metastasis of aggressive breast cancer

We previously reported that expression of an environmentally induced gene,mineral dust-induced gene(mdig),predicts overall survival in breast cancer patients.In the present report,we further demonstrate the differential roles of mdig between earlier-and later-stage breast cancers.In noncancerous breast,mdig is a proliferation factor for cell growth and cell motility.In breast cancer,however,higher levels of mdig negatively regulate the migration and invasion of cancer cells.Assessment of global DNA methylation,chromatin accessibility and H3K9me3 heterochromatin signature suggests that silencing mdig enhances DNA and histone methylation.Through immunostaining and data mining,we found that mdig is significantly upregulated in noninvasive and/or earlier-stage breast cancers.In contrast,in triple-negative and other invasive breast cancers,diminished mdig expression was noted,indicating that the loss of mdig expression could be an important feature of aggressive breast cancers.Taken together,our data suggest that mdig is a new biomarker that likely promotes tumor growth in the early stages of breast cancer while acting as a tumor suppressor to inhibit invasion and metastasis in later-stage tumors.

Genkwadane F-I,New Daphnane-type Diterpenes from the Flower Buds of Daphne genkwa Sieb.et Zucc.Exhibit Anti-tumor Activities via lnducing Apoptosis

of main observation and conclusion Genkwadane F-I(1-4),four new diterpenes,along with four known ones(5-8),were isolated from the flower buds of D.genkwa.The structures including absolute configurations of 1-4 were determined based on the analysis of NMR and ECD calculations.All compounds were evaluated for their cytotoxicities against MCF-7 and Hep3B cell lines,among which compounds 3 and 4 showed significant activities.Compound 4 exhibited the most potent activity with IC50 values of 1.33μM against MCF-7,and the IC50 values of compound 3 against MCF-7 and Hep3B were 5.54μM and 8.73μM;respectively.Further studies by Hoechst 33258 staining and Annexin V/PI analysis demonstrated that compounds 3 and 4 could induce apoptosis to exhibit anti-tumor activities.

Epigenetic connection between gut microbiota-derived short-chain fatty acids and chromatin histone modification in kidney diseases

Gut microbiota-derived metabolites influence host physiology and pathology,where short-chain fatty acids(SCFAs)are the main class of gut microbiota-derived metabolites.[1]SCFAs are fatty acids containing 1 to 6 carbon atoms,including acetate,propionate,butyrate,and pentanoate,which provide 6%to 10%of the body’s total daily energy and 60%to 70%of the colonic epithelial energy needed.[2]Anaerobic bacteria principally generate the SCFAs[1]in the intestine.Once produced,SCFAs are mainly absorbed in the colon(90%–95%),reach circulation,and play roles for distal organs and tissues.[2]