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Find video protocols related to scientific articles indexed in Pubmed.
[Influencing factors for lymphocyte subsets in children aged 0-6 years.]
Zhongguo Dang Dai Er Ke Za Zhi
PUBLISHED: 11-20-2014
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To investigate the influencing factors for lymphocyte subsets in children 0 to 6 years of age.
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Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.
J Theor Comput Chem
PUBLISHED: 11-18-2014
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Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-? both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-?. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-? SH2.
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Specific recognition of guanines in non-duplex regions of nucleic acids with potassium tungstate and hydrogen peroxide.
Nucleic Acids Res.
PUBLISHED: 10-31-2014
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Structural features of nucleic acids have become an integral part of current biomedical research. Highly selective and readily performed methods with little toxicity that target guanosines in non-duplex nucleic acids are needed, which led us to search for an effective agent for guanosine sequencing. Treatment of DNA or RNA with potassium tungstate and hydrogen peroxide produced damaged guanosines in DNA or RNA sequences. The damaged guanosines in non-duplex DNA could be cleaved by hot piperidine. Similarly, damaged guanosines in non-duplex RNA could be cleaved by aniline acetate. We could identify structural features of nucleic acid using this strategy instead of dimethyl sulphate and Ribonuclease T1.
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Sensitive detection of DNA methyltransferase activity based on exonuclease-mediated target recycling.
Anal. Chem.
PUBLISHED: 10-30-2014
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DNA methylation plays vital roles in various biological processes in both prokaryotes and eukaryotes. In bacteria, modification of adenine at N6 can protect bacterial DNA against cleavage by restriction enzymes, and bacterial DNA adenine methyltransferases are essential for bacterial virulence and viability. DNA adenine methyltransferase (DAM) targets the sequence of 5'-GATC-3' and can convert adenine into N(6)-methyladenine (m(6)A). Because mammals do not methylate DNA at adenine, bacterial DAM represents an excellent candidate for antibiotic development. Here, we developed an exonuclease III-aided target recycling strategy to sensitively assay activity of DAM. In this method, a hairpin probe labeled with a donor fluorophore (FAM) at the 5' end and a quencher (BHQ) close to the 3' end (FQ probe) was employed as reporter. Another hairpin substrate containing sequence of GATC was used as the methylation substrate of DAM. Once the hairpin substrate was methylated by DAM, it could be recognized and cleaved by Dpn I, which allows the release of a single-stranded oligodeoxynucleotide (ssODN). The ssODN can then hybridize to the 3' protruding terminus of FQ probe, which subsequently triggers the exonuclease III-mediated target recycling reaction and therefore can significantly improve the detection sensitivity of DAM. The exonuclease-mediated target recycling strategy is extremely sensitive and as low as 0.01 U/mL DAM can be distinctly determined. Using this developed method, we evaluated DAM activity in different growth stages of E. coli cells, and we also demonstrated that the assay has the potential to screen suitable inhibitor drugs for DAM for disease(s) treatment.
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?-catenin stabilization in gonadotropes impairs follicle-stimulating hormone synthesis in male mice in vivo.
Endocrinology
PUBLISHED: 10-25-2014
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Though classically considered a WNT signaling intermediary, CTNNB1 (?-catenin) can also mediate GnRH induction of gonadotropin ? subunit (Fshb and Lhb) transcription in the murine gonadotrope-like cell line L?T2. Here, we assessed CTNNB1's role in gonadotropin synthesis in vivo. We used a Cre/lox approach to introduce both gain- and loss-of-function mutations in the murine Ctnnb1 gene in gonadotrope cells. Gonadotropin production and fertility were normal in Ctnnb1 knockout mice. Similarly, females harboring a deletion of exon 3 of Ctnnb1, which stabilizes the resulting CTNNB1 protein, showed normal fertility and gonadotropin synthesis. Interestingly, males with the activating CTNNB1-?exon 3 mutation exhibited 50% reductions in FSH synthesis and secretion, without a corresponding change in LH. This selective regulation of FSH suggested an alteration in the activin/inhibin/follistatin system. Indeed, CTNNB1-?exon 3 males showed a 60% increase in serum inhibin B levels and, in culture, their pituitaries exhibited a greater sensitivity to exogenous inhibin than controls. At the same time, pituitary, but not testicular, follistatin (Fst) expression was increased significantly in these mice. Castration normalized FSH levels in CTNNB1-?exon 3 males to those seen in castrated controls. Paradoxically, pituitaries from CTNNB1-?exon 3 males exhibited greater basal and activin-stimulated FSH synthesis in vitro. Similarly, CTNNB1-?exon 3 overexpression potentiated activin A-induced murine Fshb promoter activity in L?T2 cells. Together, these results indicate that CTNNB1 is dispensable for gonadotropin synthesis in vivo. However, sustained CTNNB1 signaling potentiates activin-induced Fshb expression in gonadotropes, but this effect is overcome in vivo by enhanced inhibin feedback sensitivity and Fst expression.
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Relationship Between 18F-FDG Accumulation and Lactate Dehydrogenase A Expression in Lung Adenocarcinomas.
J. Nucl. Med.
PUBLISHED: 10-23-2014
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(18)F-FDG PET has been widely used in the management of malignant tumors. Lactate dehydrogenase A (LDHA) plays an important role in the development, invasion, and metastasis of malignancies. However, the relationship between (18)F-FDG accumulation and LDHA expression has not been investigated.
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Unexpected Reconstruction of the ?-Boron (111) Surface.
Phys. Rev. Lett.
PUBLISHED: 10-21-2014
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We report a novel reconstruction of the ?-boron (111) surface, discovered using ab initio evolutionary structure prediction, and show that this unexpected neat structure has a much lower energy than the recently proposed (111)-I_{R,(a)} surface. In this reconstruction, all single interstitial boron atoms bridge neighboring B_{12} icosahedra by polar covalent bonds, and this satisfies the electron counting rule, leading to the reconstruction-induced metal-semiconductor transition. The peculiar charge transfer between the interstitial atoms and the icosahedra plays an important role in stabilizing the surface.
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Amino-Modified Tetraphenylethene Derivatives as Nucleic Acid Stain: Relationship between the Structure and Sensitivity.
ACS Appl Mater Interfaces
PUBLISHED: 10-13-2014
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A series of new amino-functionalized tetraphenylethene (TPE) derivatives were designed and synthesized to study the effect of molecular structures on the detection of nucleic acid. Contrastive studies revealed that the number of binding groups, the length of hydrophobic linking arm and the configuration of TPE molecule all play important roles on the sensitivity of the probes in nucleic acid detection. Z-TPE3 with two binding amino groups, long linking arms, and cis configuration was found to be the most sensitive dye in both solution and gel matrix. Z-TPE3 is able to stain dsDNA with the lowest amount of 1 ng and exclusively stain 40 ng of short oligonucleotide with only 10 nt. This work is of important significance for the further design of TPE probes as biosensors with higher sensitivity.
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Differentiation of human amniotic epithelial cells into Schwann?like cells via indirect co?culture with Schwann cells in vitro.
Mol Med Rep
PUBLISHED: 09-24-2014
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Human amniotic epithelial cells (hAECs) exhibit multi?lineage differentiation ability. The present study investigated the possibility that hAECs possess the potential to differentiate into Schwann?like cells using an in vitro indirect co?culture approach. hAECs were isolated via enzymatic digestion, and immunocytochemistry and flow cytometry were performed to identify the hAECs. The hAECs were co?cultured with Schwann cells (SCs) to differentiate the hAECs into Schwann?like cells via induced proximity. The expression of typical S?100 SC markers in the co?cultured hAECs was determined via immunocytochemistry. For the functional experiments, reverse transcription quantitative polymerase chain reaction (RT?qPCR) was performed to measure the expression levels of nerve growth factor (NGF), brain?derived neurotrophic factor (BDNF) and glial cell?derived neurotrophic factor (GDNF) mRNA. In addition, neurite outgrowth was measured in PC12 cells following co?culture with the differentiated hAECs. Subsequent to co?culture with SCs for 21 days, the hAECs exhibited spindle?like morphology. The immunocytochemistry results revealed that the co?cultured hAECs expressed S?100, indicating differentiation into Schwann?like cells. RT?qPCR revealed that NGF, BDNF and GDNF expression was upregulated upon differentiation. The average axon length of the PC12 cells increased from 21.32±5.45 to 51.32±8.56 µm subsequent to co?culture with the differentiated hAECs. These results demonstrate that this indirect co?culture microenvironment induced the hAECs to differentiate into Schwann?like cells that exhibited the morphological, phenotypic and functional characteristics of SCs. Therefore, the use of differentiated hAECs that exhibit the characteristics of SCs provides a promising alternative to the present techniques used for peripheral nerve regeneration.
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Anthocyanin-rich fractions from red raspberries attenuate inflammation in both RAW264.7 macrophages and a mouse model of colitis.
Sci Rep
PUBLISHED: 08-29-2014
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Edible berries have a broad spectrum of biomedical functions, including improving immune responses and reducing risk for chronic diseases. In this study, the anti-inflammatory activities of crude extracts (CEs), anthocyanin-rich fractions (ARFs), and des-anthocyanin fractions (DAFs) from seven berries were evaluated based on their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)/IFN-?-activated RAW264.7 macrophages. ARFs from red raspberries (RR-ARFs) exhibited the highest efficiency in suppressing NO synthesis. The anti-inflammatory properties were also demonstrated by reducing the expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1?) and IL-6 in RAW264.7 cells. The luciferase reporter assay demonstrated that the activities of NF-?B and AP-1 signaling pathways were significantly suppressed by RR-ARFs. Further studies showed that RR-ARFs decreased the phosphorylation of IKK, I?B?, p65 and JNK and the nuclear translocation of p65 in LPS/IFN-?-stimulated RAW264.7 cells. In a mouse colitis model, dextran sulfate sodium (DSS)-induced weight loss and histological damage were significantly ameliorated by RR-ARFs treatment. Taken together, our results indicate that RR-ARFs attenuate inflammation both in vitro and in vivo primarily by inhibiting the activation of NF-?B and MAPKs. The anti-inflammatory of RR-ARFs could be harnessed and applied in animal agriculture, drug and food industries.
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Flexible graphene electrode-based organic photovoltaics with record-high efficiency.
Nano Lett.
PUBLISHED: 08-28-2014
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Advancements in the field of flexible high-efficiency solar cells and other optoelectronic devices will strongly depend on the development of electrode materials with good conductivity and flexibility. To address chemical and mechanical instability of currently used indium tin oxide (ITO), graphene has been suggested as a promising flexible transparent electrode but challenges remain in achieving high efficiency of graphene-based polymer solar cells (PSCs) compared to their ITO-based counterparts. Here we demonstrate graphene anode- and cathode-based flexible PSCs with record-high power conversion efficiencies of 6.1 and 7.1%, respectively. The high efficiencies were achieved via thermal treatment of MoO3 electron blocking layer and direct deposition of ZnO electron transporting layer on graphene. We also demonstrate graphene-based flexible PSCs on polyethylene naphthalate substrates and show the device stability under different bending conditions. Our work paves a way to fully graphene electrode-based flexible solar cells using a simple and reproducible process.
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Hydrogen Sulfide Alleviates Diabetic Nephropathy in a Streptozotocin-induced Diabetic Rat Model.
J. Biol. Chem.
PUBLISHED: 08-27-2014
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Accumulating evidence has demonstrated that hydrogen sulfide (H2S) plays critical roles in the pathogenesis of chronic kidney diseases. This study was designed to investigate whether H2S has protective effects against diabetic nephropathy. Diabetic rats were induced by intraperitoneal injection of streptozotocin and administrated with H2S donor NaHS for 12 weeks. Rat glomerular mesangial cells were pretreated with NaHS or MAPK inhibitors (U0126, SP600125, and SB203580) prior to high glucose exposure, and cell proliferation was determined. Our findings suggest that H2S can improve renal function and attenuate glomerular basement membrane thickening, mesangial matrix deposition, and renal interstitial fibrosis in diabetic rats. H2S was found to reduce high glucose-induced oxidative stress by activating the Nrf2 antioxidant pathway and to exert anti-inflammatory effects by inhibiting NF-?B signaling. In addition, H2S reduced high glucose-induced mesangial cell proliferation by blockade of MAPK signaling pathways. Moreover, H2S was also found to inhibit the renin-angiotensin system in diabetic kidney. In conclusion, our study demonstrates that H2S alleviates the development of diabetic nephropathy by attenuating oxidative stress and inflammation, reducing mesangial cell proliferation, and inhibiting renin-angiotensin system activity.
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[Effects of hippocampal stimulus on ?? subunit of extrasynaptic GABA(A) receptor in kainic acid-induced epileptic rats].
Zhonghua Yi Xue Za Zhi
PUBLISHED: 08-27-2014
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To observe the effects of electrical hippocampal stimulation of ?? subunit of extrasynaptic GABA(A) receptor in kainic acid-induced epileptic rats, explore the optimal therapeutic parameters and elucidate the possible mechanism of electrical stimulation for hippocampal epilepsy.
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[The effects of prone position ventilation combined with recruitment maneuvers on outcomes in patients with severe acute respiratory distress syndrome].
Zhonghua Nei Ke Za Zhi
PUBLISHED: 08-23-2014
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To evaluate the effects of prone position ventilation combined with recruitment maneuvers (RM) on clinical outcomes in patients with severe acute respiratory distress syndrome (ARDS).
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A novel hybrid baculovirus-adeno-associated viral vector-mediated radionuclide reporter gene imaging system for stem cells transplantation monitoring.
Appl. Microbiol. Biotechnol.
PUBLISHED: 08-22-2014
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Hybrid baculovirus-adeno-associated virus (BV-AAV) containing enhanced green fluorescent protein (eGFP) reporter gene or human sodium-iodide symporter (hNIS) reporter gene flanked by inverted terminal repeats (ITRs) derived from AAV (BV-CMV-eGFP-ITR and BV-CMV-hNIS-ITR) were constructed and used to investigate the feasibility of using hybrid BV-AAV transgenic vector to mediate hNIS reporter gene imaging for monitoring bone marrow-derived mesenchymal stem cells (BM-MSCs) transplantation therapy as a novel biotechnological platform in radionuclide reporter gene imaging. The results showed that the infection efficiency of BV-CMV-eGFP-ITR in BM-MSCs reached 84.25?±?1.38 %, and there were no obvious adverse effects on BM-MSCs. The (125)I(-) and (99m)TcO4 (-) uptake assays showed that the radionuclide accumulation induced by BA-AAV-mediated hNIS was highly efficient in infected BM-MSCs. Furthermore, there was a robust correlation between the infected BM-MSCs cell number and the (125)I(-) accumulation amount (R (2)?=?0.9026). The micro-SPECT/CT imaging showed that BV-CMV-hNIS-ITR-infected BM-MSCs accumulated radioiodine efficiently in vivo, exhibiting obvious radiotracer accumulation in transplantation sites. Further quantitative analysis revealed that 30 min might be the optimal imaging time point. Moreover, the revealed high target/individual organ background ratios also supported the feasibility of BV-AAV-mediated hNIS reporter gene imaging for monitoring BM-MSCs transplantation in most of commonly used transplantation sites, thus highlighting this promise biotechnological platform in radionuclide reporter gene imaging for stem cell transplantation therapy.
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[Quantitative analysis of the built-up area expansion in Su-Xi-Chang region, China].
Ying Yong Sheng Tai Xue Bao
PUBLISHED: 08-19-2014
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Based on RS-derived maps and socio-economic statistics, this paper analyzed the spatiotemporal dynamic pattern and driving mechanism of built-up area expansion in Su-Xi-Chang region from 1990 to 2010. 3S-based spatial analysis techniques were used, landscape expansion indices were calculated, and multiple stepwise regression models were also used. In the past 20 years, the built-up area had experienced rapid-moderate-accelerating expansion stages, which was strongly affected by the national development strategies and policies regarding land use. During the study period, the built-up area had increased by 2218.9 km2 mainly due to the encroaching from paddy field, dryland and artificial ponds. From 1990-1995, the pattern of built-up area expansion was dominated by the infilling and edge-expansion type; from 1995-2000, the outlying-type had overplayed infilling and edge-expansion types due to policy restrictions on the latter; after 2000, the outlying-type growth had decreased by a large extent, whereas the infilling mode had increased dramatically, which resulted in the spatially compact pattern of the newly built-up area. The increase of urban population and the boom of regional economy were the major driving forces of built-up area expansion. The study implied that improvements were urgently needed in land management system and high-efficiency use of cropland. Promoting the compact development of built-up area was also crucial for striving toward regional sustainability.
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Adaptive, convergent origins of the pygmy phenotype in African rainforest hunter-gatherers.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-18-2014
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The evolutionary history of the human pygmy phenotype (small body size), a characteristic of African and Southeast Asian rainforest hunter-gatherers, is largely unknown. Here we use a genome-wide admixture mapping analysis to identify 16 genomic regions that are significantly associated with the pygmy phenotype in the Batwa, a rainforest hunter-gatherer population from Uganda (east central Africa). The identified genomic regions have multiple attributes that provide supporting evidence of genuine association with the pygmy phenotype, including enrichments for SNPs previously associated with stature variation in Europeans and for genes with growth hormone receptor and regulation functions. To test adaptive evolutionary hypotheses, we computed the haplotype-based integrated haplotype score (iHS) statistic and the level of population differentiation (FST) between the Batwa and their agricultural neighbors, the Bakiga, for each genomic SNP. Both |iHS| and FST values were significantly higher for SNPs within the Batwa pygmy phenotype-associated regions than the remainder of the genome, a signature of polygenic adaptation. In contrast, when we expanded our analysis to include Baka rainforest hunter-gatherers from Cameroon and Gabon (west central Africa) and Nzebi and Nzime neighboring agriculturalists, we did not observe elevated |iHS| or FST values in these genomic regions. Together, these results suggest adaptive and at least partially convergent origins of the pygmy phenotype even within Africa, supporting the hypothesis that small body size confers a selective advantage for tropical rainforest hunter-gatherers but raising questions about the antiquity of this behavior.
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Effects of cryopreservation at -80 degrees C on the formulation and pathogenicity of the obligate aphid pathogen Pandora nouryi.
Pol. J. Microbiol.
PUBLISHED: 08-14-2014
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Cryopreservation at -80 degrees C is an alternative to liquid nitrogen storage for Entomophthorales. However, detailed studies about its effects on fungal pathogenicity and formulation are very limited. In the present study, the obligate aphid pathogen Pandora nouryi was formulated as mycelia grown on millet-gel granules after preservation as primary spores at -80 degrees C for 3-18 months, although its ability to produce infectious conidia gradually diminished. The sporulation capacity of this granular formulation was reduced to 18.5 x 10(4) conidia/mg after 18 months of storage, which was still higher than that of mycotized aphids. The half-decline time of sporulation capacity was computed as 13.6 months. The infectivity to the green peach aphid Myzus persicae had no significant decline in 12 months. The ability to yield resting spores within host carcasses remained unchanged, and the probability of resting spore formation increased with the conidial concentrations that infect aphids. Therefore, cryopreservation at -80 degrees C exerted a marginal impact on formulation and pathogenicity of P. nouryi and can substitute for costly liquid nitrogen storage in routine laboratory studies. The potential of the formulation in aphid biocontrol can be maintained although there is a risk of losing fungal sporulation ability in long-term preservation.
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General rules for the arrangements and gating motions of pore-lining helices in homomeric ion channels.
Nat Commun
PUBLISHED: 08-08-2014
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The pore-lining helix (PLH) bundles are central to the function of all ion channels, as their conformational rearrangements dictate channel gating. Here we explore all plausible oligomeric arrangements of the PLH bundles within homomeric ion channels by building models using generic restraints. In particular, the distance between two neighbouring PLHs was bounded both below and above in order to avoid steric clash and allow proper packing. The resulting models provide a theoretical representation of the accessible space for oligomeric arrangements. While the represented space is confined, it encompasses nearly all the ion channel PLH bundles for which the structures are currently known. For a multitude of channels, gating models suggested by paths within the confined accessible space are in qualitative agreement with those established in previous structural and computational studies.
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Antiinflammatory effects of orientin-2"-O-galactopyranoside on lipopolysaccharide-stimulated microglia.
Biol. Pharm. Bull.
PUBLISHED: 08-05-2014
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Microglia activation-mediated neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and human immunodeficiency virus (HIV)-associated dementia. Inhibition of microglia activation may alleviate neurodegeneration under neuroinflammatory conditions. In the present study, we compared three flavone C-glycosides extracted from Trollius chinensis BUNGE using a cell-based assay to evaluate their antiinflammatory effects on microglial cells. The results showed that orientin-2"-O-galactopyranoside (OGA) significantly inhibited the production of nitric oxide and tumor necrosis factor (TNF)-? in lipopolysaccharide (LPS)-stimulated microglial cells. OGA also markedly inhibited the LPS-induced expression of TNF-?, interleukin-1?, inducible nitric oxide (NO) synthase, and cyclooxygenase-2, which was accompanied by suppression of the activation of nuclear factor (NF)-?B and the extracellular signal-regulated kinase (ERK) signal pathway. In addition, OGA decreased LPS-induced reactive oxygen species generation, which appears to be related to the activation of the NF-E2-related factor2 (NRF2)/ heme oxygenase-1 (HO-1) pathway in BV-2 microglial cells. Furthermore, OGA reduced the cytotoxicity of activated microglia toward HT-22 neuroblastoma cells in a co-culture system. Taken together, the present study demonstrated that the induction of HO-1-mediated inhibition of the NF-?B and ERK pathways contributes significantly to the antineuroinflammatory and neuroprotective effects elicited by OGA.
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Synthesis of isoprenoid bisphosphonate ethers through C-P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase.
Beilstein J Org Chem
PUBLISHED: 07-18-2014
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A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 ?M against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 ?M concentration.
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Formation of DNA:RNA Hybrid G-Quadruplexes of Two G-Quartet Layers in Transcription: Expansion of the Prevalence and Diversity of G-Quadruplexes in Genomes.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 07-09-2014
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G-quadruplexes are implicated in important cellular processes. Previous studies mostly focused on intramolecular G-quadruplexes of three or more G-quartets. Those composed of two G-quartets were only shown to form in single-stranded oligonucleotides. On the basis of electrophoresis, DMS footprinting, fluorescence labeling, and photo-cross-linking, we detected the formation of DNA:RNA hybrid G-quadruplexes (HQs) of two G-quartets during the transcription of DNA duplexes. These HQs have a lifetime on the minute scale and are stabilized by a stabilizing ligand. They are far shorter-lived than the HQs of three G-quartets, which last for hours. The occurrence of putative formation motifs of such HQs shows a transcription-dependent strand-biased selection, thus supporting their formation and function in genomes. They are present in almost all human genes in large amounts. We speculate that the two-G-quartet HQs may be a distinct type of G-quadruplexes that may play a role in timely responsive processes and for purposes of fine-tuning.
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Both protein dynamics and ligand concentration can shift the binding mechanism between conformational selection and induced fit.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-30-2014
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This study aimed to shed light on the long debate over whether conformational selection (CS) or induced fit (IF) is the governing mechanism for protein-ligand binding. The main difference between the two scenarios is whether the conformational transition of the protein from the unbound form to the bound form occurs before or after encountering the ligand. Here we introduce the IF fraction (i.e., the fraction of binding events achieved via IF), to quantify the binding mechanism. Using simulations of a model protein-ligand system, we demonstrate that both the rate of the conformational transition and the concentration of ligand molecules can affect the IF fraction. CS dominates at slow conformational transition and low ligand concentration. An increase in either quantity results in a higher IF fraction. Despite the many-body nature of the system and the involvement of multiple, disparate types of dynamics (i.e., ligand diffusion, protein conformational transition, and binding reaction), the overall binding kinetics over wide ranges of parameters can be fit to a single exponential, with the apparent rate constant exhibiting a linear dependence on ligand concentration. The present study may guide future kinetics experiments and dynamics simulations in determining the IF fraction.
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[Infect of pingshen decoction on serum HGF, Cys C and TGF-beta1 diabetic nephropathy in early stage].
Zhongguo Zhong Yao Za Zhi
PUBLISHED: 06-25-2014
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Study the serum level of HGF, Cys C and TGF-beta1 in type 2 diabetic nephropathy (DN), the infect of Pingshen decoction on those index. Selected 69 cases of 2 type DN and randomly divided into therapy group (36 cases) and control group (33 cases). The therapy group were treated with Pingshen decoction 1 dose/d, bid po. The control group were treated with NephritisShu tablet, 6 tablet, tid po. 8 weeks was a course. Before and after treatment, we examine the serum level of HGF, Cys C and TGF-beta1 by ELISA and immunonephelometry, and compare with 30 cases of healthy control group. The study demonstrates that before treatment, the serum level of HGF in both groups were significantly lower than healthy control group (P < 0.01), but Cys C, TGF-beta1 were significantly higher (P < 0.01). After treatment, the serum level of HGF of both groups were increased. The serum level of HGF of therapy group were significantly higher than of control group (P < 0.01), but the serum level of Cys C and TGF-beta1 were significantly lower than control group (P < 0.01). The serum level of HGF was correlated negatively with Cys C,TGF-beta1. In control group, the UAER, urine beta2-MG and quantity of 24-hour urine protein were significantly decreased after treatment (P < 0.01). The index of urine of therapy group were significantly lower than control group (P < 0.01). Results indicate that test of serum level of HGF and Cys C,TGF-beta1 of diabetic nephropathy have important clinical significance. Pingshen decoction can effectively intervene in the serum level of HGF and Cys C, TGF-beta1 and index of urine.
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Etifoxine promotes glia-derived neurite outgrowth in vitro and in vivo.
J Reconstr Microsurg
PUBLISHED: 06-23-2014
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?Peripheral nerve injuries usually require a graft to facilitate axonal regeneration into the distal nerve stump. Acellular nerve grafts are good candidates for nerve repair, but clinical outcomes from grafts are not always satisfactory. Etifoxine is a ligand of the 18-kDa translocator protein (TSPO) and has been demonstrated to serve multiple functions in nervous system.
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Highly exothermic and superhydrophobic Mg/fluorocarbon core/shell nanoenergetic arrays.
ACS Appl Mater Interfaces
PUBLISHED: 06-23-2014
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Mg/fluorocarbon core/shell nanoenergetic arrays are prepared onto silicon substrate, with Mg nanorods as the core and fluorocarbon as the shell. Mg nanorods are deposited by the glancing angle deposition technique, and the fluorocarbon layer is then prepared as a shell to encase the Mg nanorods by the magnetron sputtering deposition process. Scanning electron microscopy and transmission electron microscopy show the core/shell structure of the Mg/fluorocarbon arrays. X-ray energy-dispersive spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy are used to characterize the structural composition of the Mg/fluorocarbon. It is found that the as-prepared fluorocarbon layer consists of shorter molecular chains compared to that of bulk polytetrafluoroethylene, which is proven beneficial to the low onset reaction temperature of Mg/fluorocarbon. Water contact angle test demonstrates the superhydrophobicity of the Mg/fluorocarbon arrays, and a static contact angle as high as 162° is achieved. Thermal analysis shows that the Mg/fluorocarbon material exhibits a very low onset reaction temperature of about 270 °C as well as an ultrahigh heat of reaction approaching 9 kJ/g. A preliminary combustion test reveals rapid combustion wave propagation, and a convective mechanism is adopted to explain the combustion behaviors.
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Ginkgo Biloba Extract (EGb 761) Promotes Peripheral Nerve Regeneration and Neovascularization After Acellular Nerve Allografts in a Rat Model.
Cell. Mol. Neurobiol.
PUBLISHED: 06-16-2014
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This study aimed to investigate whether or not ginkgo biloba extract (EGb 761) enhances peripheral nerve regeneration and vascularization after repair using acellular nerve allografts (ANA). Seventy-two Sprague-Dawley rats were randomly divided into three experimental groups: a unilateral 15-mm sciatic nerve defect was created and repaired with an autologous graft (autograft group); the same defect was repaired with an 18 mm ANA with an i.p. injection of normal saline for 10 days (saline group); and in the final group, the same defect was repaired with an 18 mm ANA with an i.p. injection of EGb 761 for 10 days (EGb 761 group). Axon outgrowth and vascularization were evaluated by immunocytochemistry 14 days post-implantation. The expression of genes associated with angiogenesis was analyzed by real-time polymerase chain reaction (PCR) seven days post-implantation. Compared with the saline group, rats in the EGb 761 group significantly increased the number of myelinated fibers and the average diameter of the nerves within the graft. There is no significant difference between the EGb 761 group and the autograft group. The expression of CD34 and NF200 was significantly higher in the EGb 761 group than in the saline group. Additionally, EGb 761 treatment increased the expression of several angiogenesis-related genes, including Vegf, SOX18, Prom 1, and IL-6. In conclusion, ANA repair with EGb 761 treatment demonstrates effects on peripheral nerve regeneration and vascularization that are equal to those of autologous graft repair, and that are superior to ANA repair alone.
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The role of IMP dehydrogenase 2 in Inauhzin-induced ribosomal stress.
Elife
PUBLISHED: 04-13-2014
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The 'ribosomal stress (RS)-p53 pathway' is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. Inosine monophosphate (IMP) dehydrogenase 2 (IMPDH2) is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ suppresses cancer cell growth by dually targeting SIRT1 and IMPDH2.
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Brain CD47 expression in a swine model of intracerebral hemorrhage.
Brain Res.
PUBLISHED: 04-11-2014
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CD47 contributes to neuronal death, inflammation and angiogenesis after brain ischemia. The role of CD47 in intracerebral hemorrhage (ICH) has not been investigated and the current study examined brain CD47 expression in a pig ICH model. Pigs received a blood injection or needle insertion into the right frontal lobe and were euthanized at different times to examine CD47 expression. Pigs were also treated with an iron chelator, deferoxamine, (50mg/kg, i.m.) or vehicle and killed at day-3 to examine the effects on CD47. ICH resulted in upregulation of brain CD47 in both white and gray matter by both immunohistochemistry and Western blot. A time-course showed ICH-induced CD47 upregulation from 4h to day-14, with a peak at day-3. CD47 positive cells were neurons, microglia/macrophage and oliogodendrocytes. Brain CD47 levels were lower in the ipsilateral white and gray matter in pigs which had deferoxamine treatment. In conclusion, CD47 expression was increased in the perihematomal white and gray matter after ICH. Deferoxamine and iron may modulate CD47 expression.
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Uveitis and T cell lymphoma: a rare but notable relationship.
Med. Oncol.
PUBLISHED: 03-25-2014
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Uveitis is an autoimmune disease of the eye that refers to a number of intraocular inflammatory conditions. We identified three rare cases of T cell lymphoma diagnosed following an initial presentation of uveitis. This study summarized the clinical features of these rare cases and evaluated the association between uveitis and lymphoma. We analyzed the etiology of uveitis diagnosed in the Department of Pediatrics and Internal Medicine at our hospital. Five patients were diagnosed with uveitis and lymphoma. We present case reports of three patients who were diagnosed with T cell lymphoma following an initial presentation of uveitis. From January 1985 to June 2013, there were 80 cases of uveitis and 1,900 cases of lymphoma in the Department of Pediatrics and Internal Medicine at our hospital. We found five patients that were diagnosed with uveitis and lymphoma; of these, three had a definite pathologic diagnosis of T cell lymphoma. The odds ratio (OR) was 6.08 (95 % confidence interval [CI] of 1.93-14.94, P = 0.002) for the five patients with uveitis and lymphoma, suggesting a significant association. The OR was 3.67 (95 % CI of 1.16-11.65, P = 0.053) for the three patients with uveitis and T cell lymphoma. Uveitis may be a rare presentation of malignant disease, such as lymphoma. Ophthalmologists and general practitioners should be familiar with the differential diagnosis of uveitis.
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Proximal gastrectomy with jejunal interposition and TGRY anastomosis for proximal gastric cancer.
World J. Gastroenterol.
PUBLISHED: 03-09-2014
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To compare the short-term outcomes of patients who underwent proximal gastrectomy with jejunal interposition (PGJI) with those undergoing total gastrectomy with Roux-en-Y anastomosis (TGRY).
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Further Development of the FFT-based Method for Atomistic Modeling of Protein Folding and Binding under Crowding: Optimization of Accuracy and Speed.
J Chem Theory Comput
PUBLISHED: 03-04-2014
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Recently, we (Qin, S.; Zhou, H. X. J. Chem. Theory Comput. 2013, 9, 4633-4643) developed the FFT-based method for Modeling Atomistic Proteins-crowder interactions, henceforth FMAP. Given its potential wide use for calculating effects of crowding on protein folding and binding free energies, here we aimed to optimize the accuracy and speed of FMAP. FMAP is based on expressing protein-crowder interactions as correlation functions and evaluating the latter via fast Fourier transform (FFT). The numerical accuracy of FFT improves as the grid spacing for discretizing space is reduced, but at increasing computational cost. We sought to speed up FMAP calculations by using a relatively coarse grid spacing of 0.6 Å and then correcting for discretization errors. This strategy was tested for different types of interactions (hard-core repulsion, nonpolar attraction, and electrostatic interaction) and over a wide range of protein-crowder systems. We were able to correct for the numerical errors on hard-core repulsion and nonpolar attraction by an 8% inflation of atomic hard-core radii and on electrostatic interaction by a 5% inflation of the magnitudes of protein atomic charges. The corrected results have higher accuracy and enjoy a speedup of more than 100-fold over those obtained using a fine grid spacing of 0.15 Å. With this optimization of accuracy and speed, FMAP may become a practical tool for realistic modeling of protein folding and binding in cell-like environments.
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Folic acid-conjugated iron oxide porous nanorods loaded with doxorubicin for targeted drug delivery.
Colloids Surf B Biointerfaces
PUBLISHED: 02-24-2014
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Iron oxide porous nanorods (IOPNR) with lengths ranging from 40nm to 60nm and pore diameters ranging from 5nm to 10nm were prepared, and further modified with NH2-PEG-FA (FA-PEG-IOPNR) for ligand targeting and modified with NH2-PEG-OCH3 (PEG-IOPNR) as a control. Instead of chemical bonding, doxorubicin (DOX), a low water solubility anticancer drug, was loaded in the pores of the modified IOPNR because of their porous structure and high porosity. The release of DOX in acidic PBS solution (pH 5.3) was faster than that in neutral (pH 7.4) solution. The analysis results from TEM, inductively coupled plasma emission spectroscopy, confocal laser scanning microscopy, and flow cytometry analyses indicated that the presence of FA on the surface of the nanorods increase the cellular uptake of nanorods in the case of HeLa cells, a folate receptor (FR)-positive cell line. In contrast, for COS 7 cells, a FR-negative cell line, FA ligand on the surface of the nanorods showed no effect on the cellular uptake. MTT assay indicated that the cytotoxicity of DOX loaded in FA-PEG-IOPNR to HeLa cells was higher than that of DOX in PEG-IOPNR. In the case of COS 7 cells, no significant difference between the cytotoxicity of DOX loaded in FA-PEG-IOPNR and PEG-IOPNR was found. These results suggested that FA-PEG-IOPNR had the potential for target delivery of chemotherapeutic into cancer cells.
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Crystal structure prediction and its application in Earth and materials sciences.
Top Curr Chem
PUBLISHED: 02-18-2014
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Evolutionary algorithms, based on physically motivated forms of variation operators and local optimization, proved to be a powerful approach in determining the crystal structure of materials. This review summarized the recent progress of the USPEX method as a tool for crystal structure prediction. In particular, we highlight the methodology in (1) prediction of molecular crystal structures and (2) variable-composition structure predictions, and their applications to a series of systems, including Mg(BH4)2, Xe-O, Mg-O compounds, etc. We demonstrate that this method has a wide field of applications in both computational materials design and studies of matter at extreme conditions.
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Factors predicting sensory and motor recovery after the repair of upper limb peripheral nerve injuries.
Neural Regen Res
PUBLISHED: 02-12-2014
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To investigate the factors associated with sensory and motor recovery after the repair of upper limb peripheral nerve injuries.
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DNA nanomachines as evolved molecular beacons for in vitro and in vivo detection.
Talanta
PUBLISHED: 01-29-2014
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Modern biosensors require high sensitivity, great signal enhancement and extensive applicability for detection and diagnostic purposes. Traditional molecular beacons (MBs) do not meet these requirements because of the lack of signal amplification. The current amplification pathways using enzymes, DNAzymes and nanoparticles are usually quite sophisticated and are limited to specific applications. Herein, we developed simple biosensors based on the structure of kissing-hairpin. Through hybridization amplification of these nanomachines, the evolved MBs could greatly enhance the detected signals (approximately 10-fold higher than the signals generated by traditional molecular beacons), reduce the sensing limits for targets and, remarkably, distinguish single-base mismatches specifically for nucleic acid detection. In addition, these new MBs can be directly applied in living cells. By introducing aptamer sequences, these novel sensors can also detect proteins and small molecules. These properties were exemplified by the detection of both the ?-actin gene and thrombin. The simplicity, sensitivity and flexibility of these devices make them appropriate for more expansive applications.
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Baseline HBsAg predicts response to pegylated interferon-?2b in HBeAg-positive chronic hepatitis B patients.
World J. Gastroenterol.
PUBLISHED: 01-16-2014
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To evaluate the predictive effect of baseline hepatitis B surface antigen (HBsAg) on response to pegylated interferon (PEG-IFN)-?2b in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients.
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Mechanical coupling maintains the fidelity of NMDA receptor-mediated currents.
Nat. Neurosci.
PUBLISHED: 01-10-2014
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The fidelity of integration of pre- and postsynaptic activity by NMDA receptors (NMDARs) requires a match between agonist binding and ion channel opening. To address how agonist binding is transduced into pore opening in NMDARs, we manipulated the coupling between the ligand-binding domain (LBD) and the ion channel by inserting residues in a linker between them. We found that a single residue insertion markedly attenuated the ability of NMDARs to convert a glutamate transient into a functional response. This was largely a result of a decreased likelihood of the channel opening and remaining open. Computational and thermodynamic analyses suggest that insertions prevent the agonist-bound LBD from effectively pulling on pore lining elements, thereby destabilizing pore opening. Furthermore, this pulling energy was more prominent in the GluN2 subunit. We conclude that an efficient NMDAR-mediated synaptic response relies on a mechanical coupling between the LBD and the ion channel.
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Theoretical frameworks for multiscale modeling and simulation.
Curr. Opin. Struct. Biol.
PUBLISHED: 01-10-2014
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Biomolecular systems have been modeled at a variety of scales, ranging from explicit treatment of electrons and nuclei to continuum description of bulk deformation or velocity. Many challenges of interfacing between scales have been overcome. Multiple models at different scales have been used to study the same system or calculate the same property (e.g., channel conductance). Accurate modeling of biochemical processes under in vivo conditions and the bridging of molecular and subcellular scales will likely soon become reality.
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Development and validation of an LC-MS/MS method for determination of the L-type voltage-gated calcium channel/NMDA receptor antagonist NGP1-01 in mouse serum.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 01-07-2014
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NGP1-01 (8-benzylamino-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane) is a heterocyclic cage compound with multifunctional calcium channel blocking activity that has been demonstrated to be neuroprotective in several neurodegenerative models. A sensitive internal standard LC-MS/MS method was developed and validated to quantify NGP1-01 in mouse serum. The internal standard (IS) was 8-(2-phenylethylamino)-8,11-oxapentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane. Sample preparation involved a protein precipitation procedure by addition of acetonitrile. Chromatographic separation was carried out on a Phenomenex Kinetex phenyl-hexyl column (100 mm×2.1mm, 2.6 ?m) employing a gradient (45% isocratic 3 min, 45-95% linear gradient 6 min, 95% isocratic 3 min) of an elution mobile phase of 5mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5mM ammonium acetate in 2% acetonitrile. Detection was achieved by a QTrap 5500 mass spectrometer (AB Sciex) employing electrospray ionization in the positive mode with multiple-reaction-monitoring (MRM) for NGP1-01 (m/z 266?91) and IS (m/z 280?105). The method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method had a linear range of at least 0.5-50 ng/mL with a correlation coefficient 0.999. The intra-assay and inter-assay precisions (%CV) were equal to or within the range of 1.0-4.3% and the accuracies (% relative error) equal to or within -2.5% to 3.4%. The analyte was stable for at least 2 months at -20°C, for at least 8h at room temperature and for at least three freeze-thaw cycles. The extraction recovery was 94.9 to 105.0%, with a %CV ? 9.5%. The technique was found to be free of any matrix effects as determined by experiments involving five different lots of mouse serum. Cross-talk interferences were not present. Two different gradient slope chromatography runs were done on dosed mouse serum samples to assess a possible positive error in peak area determination from in-source fragmentation of metabolites generating the same MRM transitions as the parent drug or IS. No such interference was found in the NGP1-01 peak, while a minor interference was identified in the IS peak. The optimized method was applied to the measurement of NGP1-01 in serum of dosed mice.
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Design rules for selective binding of nuclear localization signals to minor site of importin ?.
PLoS ONE
PUBLISHED: 01-01-2014
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Selectivity is a critical issue in molecular recognition. However, design rules that underlie selectivity are often not well understood. Here, we studied five classical nuclear localization signals (NLSs) that contain the motif KRx(W/F/Y)xxAF and selectively bind to the minor site of importin ?. The selectivity for the minor site is dissected by building structural models for the NLS-importin ? complexes and analyzing the positive design and negative design in the NLSs. In our models, the KR residues of the motif occupy the P1' and P2' pockets of importin ?, respectively, forming hydrogen-bonding and cation-? interactions. The aromatic residue at the P4' position plays dual roles in the selectivity for the minor site: by forming ?-stacking with W357 of importin ? to reinforce the minor-site binding; and by clashing with the P5 pocket in the major binding site. The F residue at the P8' position occupies a deep pocket, providing additional stabilization. The P7' position sits on a saddle next to the P8' pocket and hence requires a small residue; the A residue fulfills this requirement. The principal ideas behind these blind predictions turn out to be correct in an evaluation against subsequently available X-ray structures for the NLS-importin ? complexes, but some details are incorrect. These results illustrate that the selectivity for the minor site can be achieved via a variety of design rules.
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Systematic Investigations of Different Cytosine Modifications on CpG Dinucleotide Sequences: The Effects on the B-Z Transition.
J. Am. Chem. Soc.
PUBLISHED: 12-27-2013
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We have first demonstrated the distinctive effects of three newly reported epigenetic modifications, including 5hmC, 5fC, and 5caC, on B-Z transition of CpG dinucleotide DNAs. We have performed detailed assays and compared their effects. We further studied the regulation of B-Z transition of CpG dinucleotide dodecamers by alternating oxidation and alternating reduction.
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Effects of Macromolecular Crowding on the Conformational Ensembles of Disordered Proteins.
J Phys Chem Lett
PUBLISHED: 12-07-2013
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Due to their conformational malleability, intrinsically disordered proteins (IDPs) are particularly susceptible to influences of crowded cellular environments. Here we report a computational study of the effects of macromolecular crowding on the conformational ensemble of a coarse-grained IDP model, by using two approaches. In one, the IDP is simulated along with the crowders; in the other, crowder-free simulations are postprocessed to predict the conformational ensembles under crowding. We found significant decreases in the radius of gyration of the IDP under crowding, and suggest repulsive interactions with crowders as a common cause for chain compaction in a number of experimental studies. The postprocessing approach accurately reproduced the conformational ensembles of the IDP in the direct simulations here, and holds enormous potential for realistic modeling of IDPs under crowding, by permitting thorough conformation sampling for the proteins even when they and the crowders are both represented at the all-atom level.
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An FFT-based method for modeling protein folding and binding under crowding: benchmarking on ellipsoidal and all-atom crowders.
J Chem Theory Comput
PUBLISHED: 11-05-2013
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It is now well recognized that macromolecular crowding can exert significant effects on protein folding and binding stability. In order to calculate such effects in direct simulations of proteins mixed with bystander macromolecules, the latter (referred to as crowders) are usually modeled as spheres and the proteins represented at a coarse-grained level. Our recently developed postprocessing approach allows the proteins to be represented at the all-atom level but, for computational efficiency, has only been implemented for spherical crowders. Modeling crowder molecules in cellular environments and in vitro experiments as spheres may distort their effects on protein stability. Here we present a new method that is capable for treating aspherical crowders. The idea, borrowed from protein-protein docking, is to calculate the excess chemical potential of the proteins in crowded solution by fast Fourier transform (FFT). As the first application, we studied the effects of ellipsoidal crowders on the folding and binding free energies of all-atom proteins, and found, in agreement with previous direct simulations with coarse-grained protein models, that the aspherical crowders exert greater stabilization effects than spherical crowders of the same volume. Moreover, as demonstrated here, the FFT-based method has the important property that its computational cost does not increase strongly even when the level of details in representing the crowders is increased all the way to all-atom, thus significantly accelerating realistic modeling of protein folding and binding in cell-like environments.
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Functional and structural alterations of peritoneum and secretion of fibrotic cytokines in rats caused by high glucose peritoneal dialysis solutions.
Ren Fail
PUBLISHED: 10-24-2013
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Abstract Objective: To determine functional and structural alterations of peritoneum and fibrotic cytokines expression in peritoneal dialysis (PD) rats. Methods: 28 Sprague-Dawley (S-D) rats were randomly divided into four groups and dialyzed with various solutions daily for four weeks: (1) no solution (CON group), (2) 0.9% Saline solution (NS group), (3) 1.5% Dianeal (LG group), (4) 4.25% Dianeal (HG group). Peritoneal equilibration tests, ultrafiltration function and effluent protein quantification were measured. Peritoneum morphology was studied and immunohistochemistry were performed for detection of transforming growth factor ?1 (TGF-?1), connective tissue growth factor (CTGF), and fibronectin (FN) proteins. Reverse transcriptional-polymerase chain reaction was used to analyze the expression of TGF-?1, CTGF mRNA. Results: Administration of 4.25% Dianeal caused functional and structural changes of peritoneum, including protein loss through the transport process, decrease of peritoneal solute transport rate and ultrafiltration capacity. The collagen of peritoneum in the HG group was thicker than the other groups. The levels of CTGF, TGF-?1, and FN proteins were significantly the highest in the HG group, followed by the LG group. The liner correlation analysis showed positive correlations between the levels of CTGF, TGF-?1, and FN proteins and the collagen thickness. The expression of TGF-?1 and CTGF mRNA in the HG group were significantly higher than those in the other groups and were indicated positive correlation. Conclusion: Using high glucose peritoneal dialysis solutions in rats may not only lead to processing of peritoneal fibrosis, which is promoted by ectopic expression of TGF-?1, but also increase the expression of CTGF. CTGF is an important fibrotic media of peritoneal fibrosis in PD rats.
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An L-DNA G-quadruplex: application for peroxidase DNAzyme.
Nucleosides Nucleotides Nucleic Acids
PUBLISHED: 10-22-2013
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L-DNA is the mirror-image form of natural D-DNA. We demonstrate that one left-handed G-rich sequence can form an L-DNA intramolecular G-quadruplex. Further investigation revealed that a DNAzyme formed by an L-nucleotide G-quadruplex exhibited peroxidase catalytic efficiency. The enhancement of the color change of the oxygenation product ABTS(•-) caused by L-nucleotide G-quadruplex formation could be clearly observed with naked eyes. This research provides a new concept for the application of the L-DNA peroxidase DNAzyme complex in nuclease-containing biological systems.
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Quantification of free sialic acid in human plasma through a robust quinoxalinone derivatization and LC-MS/MS using isotope-labeled standard calibration.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 10-04-2013
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We report an accurate quantification of free sialic acid (SA) in human plasma using LC-MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC-MS/MS analysis of free SA. In addition, the use of (13)C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring (MRM) mode for sensitive and selective detection. We have achieved a lower limit of quantification (LLOQ) of 20ng/mL and a total running time of 4.2min, which is the most sensitive and quick measurement for free SA in biomatrices.
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Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-30-2013
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P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergent-solubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATP-bound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membrane-embedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.
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5-Methyldeoxycytidine enhances the substrate activity of DNA polymerase.
Chem. Commun. (Camb.)
PUBLISHED: 09-18-2013
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Here, we first demonstrated that 5-MedCTP could be incorporated into the synthetic DNA template by the exonuclease deficient Klenow fragment with a much higher efficiency than dCTP and 5-hydroxymethyl-dCTP. Further, we first conducted a comparable study of primer extension reaction using templates containing deoxycytidine (dC) or 5-methyldeoxycytidine (5-mdC) for incorporating different triphosphates. Based on our findings, 5-methyldeoxycytidine could enhance the substrate activity of the Klenow fragment (exo-) and this feature could potentially be used in DNA methylation analysis.
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Systematic investigation of DNAs with modified cytosines under hot alkali treatment.
Chem. Commun. (Camb.)
PUBLISHED: 09-16-2013
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We have conducted the first systematic investigation of DNAs with modified cytosines, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), under hot alkali treatment.
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Discrimination between 5-hydroxymethylcytosine and 5-methylcytosine in DNA by selective chemical labeling.
Bioorg. Med. Chem. Lett.
PUBLISHED: 09-15-2013
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Detection of DNA damage has been greatly improved following the development of equipment and techniques, however, discrimination between 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) is still a thorny problem. In the present study, an approach to oxidize and selective label (Ox-Labeling) 5-hmC in native DNA has been reported, which conveniently distinguishes 5-hmC from 5-mC using simple and effective processes.
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Molecular Structure of RADA16-I Designer Self-Assembling Peptide Nanofibers.
ACS Nano
PUBLISHED: 09-05-2013
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The designer self-assembling peptide RADA16-I forms nanofiber matrices which have shown great promise for regenerative medicine and three-dimensional cell culture. The RADA16-I amino acid sequence has a ?-strand-promoting alternating hydrophobic/charged motif, but arrangement of ?-strands into the nanofiber structure has not been previously determined. Here we present a structural model of RADA16-I nanofibers, based on solid-state NMR measurements on samples with different schemes for (13)C isotopic labeling. NMR peak positions and line widths indicate an ordered structure composed of ?-strands. The NMR data show that the nanofibers are composed of two stacked ?-sheets stabilized by a hydrophobic core formed by alanine side chains, consistent with previous proposals. However, the previously proposed antiparallel ?-sheet structure is ruled out by measured (13)C-(13)C dipolar couplings. Instead, neighboring ?-strands within ?-sheets are parallel, with a registry shift that allows cross-strand staggering of oppositely charged arginine and aspartate side chains. The resulting structural model is compared to nanofiber dimensions observed via images taken by transmission electron microscopy and atomic force microscopy. Multiple NMR peaks for each alanine side chain were observed and could be attributed to multiple configurations of side chain packing within a single scheme for intermolecular packing.
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Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium tuberculosis Virulence Factor: A Structural Study.
J. Mol. Biol.
PUBLISHED: 08-20-2013
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MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC.
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A quantitative LC-MS/MS method for determination of thiazolidinedione mitoNEET ligand NL-1 in mouse serum suitable for pharmacokinetic studies.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 08-14-2013
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Thiazolidinedione (TZD) compounds have shown promise as antidiabetic, antibiotics, antifungal and neuroprotective agents. The mitochondrial effect of a novel mitoNEET ligand, NL-1 {5-[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]-1,3-thiazolidine-2,4-dione}, and other TZD compounds, is a newly proposed mechanism for the neuroprotective action of these TZD compounds. In this work, a sensitive LC-MS/MS assay has been developed and validated for quantification of NL-1 in mouse serum. Sample preparation involved an acetonitrile protein precipitation procedure with addition of an internal standard NL-2 {5-[(4-hydroxy-3,5-dimethyl-phenyl)methyl]thiazolidine-2,4-dione}. LC-MS/MS analysis utilized a Columbus C-18 HPLC column (2mm×50mm, 5?m). Chromatography employed a multiple step gradient program that featured a steep linear gradient (25-95% in 0.5min) of 15?M ammonium acetate (additive for eliminating carry-over) in 2% methanol mixing with increasing proportions of 100% methanol. The HPLC was interfaced to a QTrap 5500 mass spectrometer (AB Sciex) equipped with an electrospray ionization source used in a negative ionization mode. Multiple reaction monitoring (MRM) of m/z 334?263 for NL-1 and m/z 250?179 for NL-2 was done. The method had a linear range of at least 1-100ng/mL in serum. The intra-assay and inter-assay percent coefficient of variation (%CV) were less than 4% and accuracies (%RE) ranged from -2.7% to 2.0%. The analytical procedure gave 96-115% absolute extraction recovery of NL-1. The relative matrix effect was measured and found to be insignificant. The analyte in serum was confirmed to be stable during storage and treatment. The method is suitable for pharmacokinetic (PK) studies of the parent drug NL-1 based on the preliminary serum results from dosed NL-1 mouse studies.
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Ab initio calculations and validation of the pH-dependent structures of the His37-Trp41 quartet, the heart of acid activation and proton conductance in the M2 protein of Influenza A virus.
Chem Sci
PUBLISHED: 08-10-2013
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The M2 protein of Influenza A virus forms a homotetrameric proton channel activated by low pH. The His37-Trp41 quartet is the heart of acid activation and proton conductance, but the functional mechanism is still controversial. We carried out ab initio calculations to model the pH-dependent structures of the His37-Trp41 quartet. In our model at neutral pH, the four His37 residues are configured into a pair of dimers; in each dimer, a proton is shared between N?1 on one residue and N?2 on the other, and, under the restraint of the backbone, the two imidazole rings are nearly parallel, in contrast to a perpendicular arrangement for a free imidazole-imidazolium dimer. Within each dimer the +1 charge is highly delocalized, contributing to its stabilization in a low dielectric environment. The N?1-H-N?2 strong hydrogen bonds result in significantly downfield shifted N?1 and N?2 chemical shifts (at 169.7 and 167.6 ppm, respectively), in good agreement with experiments. In our model at acidic pH (where the channel becomes activated), a third proton binds to an imidazole-imidazolium dimer; the imidazole rings rotate away (each by ~55°) from each other, destroying the dimer structure. The two imidazoliums are stabilized by hydrogen bonds with water molecules and a cation-? interaction with Trp41. The Raman spectra calculated for the His37-Trp41 quartet at neutral and acidic pH are in agreement with experiments. Our calculations support an activation and conductance mechanism in which a hydronium ion from the N-terminal side passes a proton to an imidazole-imidazolium dimer; when the Trp41 gate is open, relaying of a proton onto a water molecule from the C-terminal side then allows the imidazole-imidazolium dimer to reform and be ready for the next round of proton conductance.
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Folding free energy surfaces of three small proteins under crowding: validation of the postprocessing method by direct simulation.
Phys Biol
PUBLISHED: 08-06-2013
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We have developed a postprocessing method for modeling biochemical processes such as protein folding under crowded conditions (Qin and Zhou 2009 Biophys. J. 97 12-19). In contrast to the direct simulation approach, in which the protein undergoing folding is simulated along with crowders, the postprocessing method requires only the folding simulation without crowders. The influence of the crowders is then obtained by taking conformations from the crowder-free simulation and calculating the free energies of transferring to the crowders. This postprocessing yields the folding free energy surface of the protein under crowding. Here the postprocessing results for the folding of three small proteins under repulsive crowding are validated by those obtained previously by the direct simulation approach (Mittal and Best 2010 Biophys. J. 98 315-20). This validation confirms the accuracy of the postprocessing approach and highlights its distinct advantages in modeling biochemical processes under cell-like crowded conditions, such as enabling an atomistic representation of the test proteins.
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A novel combined bisulfite UDG assay for selective 5-methylcytosine detection.
Talanta
PUBLISHED: 08-05-2013
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DNA modification, a significant epigenetic event, largely affects genes binding with the transcription factors and some other DNA binding proteins. Among DNA modifications, methylation, especially cytosine methylation is of great importance and attracts extensive studies as it leads to the silence of tumor-suppressor gene expression. In this work, a novel combined bisulfite Uracil-DNA glycosylase (UDG) assay has been developed on the basis of bisulfite modification to generate uracil from cytosine, subsequent UDG-mediated uracil elimination and ultimate DNA cleavage in alkaline condition. This strategy can be used to selectively detect exact number and loci of 5-methylcytosine residues regardless of sequence context. Moreover, it provides linear quantitative results of DNA methylation level across a wide range.
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In Situ Synthesis of CuO and Cu Nanostructures with Promising Electrochemical and Wettability Properties.
Small
PUBLISHED: 08-02-2013
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A strategy is presented for the in situ synthesis of single crystalline CuO nanorods and 3D CuO nanostructures, ultra-long Cu nanowires and Cu nanoparticles at relatively low temperature onto various substrates (Si, SiO2 , ITO, FTO, porous nickel, carbon cotton, etc.) by one-step thermal heating of copper foam in static air and inert gas, respectively. The density, particle sizes and morphologies of the synthesized nanostructures can be effectively controlled by simply tailoring the experimental parameters. A compressive stress based and subsequent structural rearrangements mechanism is proposed to explain the formation of the nanostructures. The as-prepared CuO nanostructures demonstrate promising electrochemical properties as the anode materials in lithium-ion batteries and also reversible wettability. Moreover, this strategy can be used to conveniently integrate these nanostructures with other nanostructures (ZnO nanorods, Co3 O4 nanowires and nanowalls, TiO2 nanotubes, and Si nanowires) to achieve various hybrid hierarchical (CuO-ZnO, CuO-Co3 O4 , CuO-TiO2 , CuO-Si) nanocomposites with promising properties. This strategy has the potential to provide the nano society with a general way to achieve a variety of nanostructures.
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Combining central venous-to-arterial partial pressure of carbon dioxide difference and central venous oxygen saturation to guide resuscitation in septic shock.
J Crit Care
PUBLISHED: 07-05-2013
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Central venous oxygen saturation (Scvo2) is a useful therapeutic target when treating septic shock. We hypothesized that combining Scvo2 and central venous-to-arterial partial pressure of carbon dioxide difference (?Pco2) may provide additional information about survival.
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Blind prediction of interfacial water positions in CAPRI.
Proteins
PUBLISHED: 06-24-2013
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We report the first assessment of blind predictions of water positions at protein-protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions-20 groups submitted a total of 195 models-were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models-a very good docking performance per se-only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein-water interactions and their role in stabilizing protein complexes.Proteins 2013. © 2013 Wiley Periodicals, Inc.
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Simulation and Modeling of Crowding Effects on the Thermodynamic and Kinetic Properties of Proteins with Atomic Details.
Biophys Rev
PUBLISHED: 05-28-2013
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Recent experimental studies of protein folding and binding under crowded solutions suggest that crowding agents exert subtle influences on the thermodynamic and kinetic properties of the proteins. While some of the crowding effects can be understood qualitatively from simple models of the proteins, quantitative rationalization of these effects requires an atomistic representation of the protein molecules in modeling their interactions with crowders. A computational approach, known as postprocessing, has opened the door for atomistic modeling of crowding effects. This review summarizes the applications of the postprocessing approach for studying crowding effects on the thermodynamics and kinetics of protein folding, conformational transition, and binding. The integration of atomistic modeling with experiments in crowded solutions promises new insight into biochemical processes in cellular environments.
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Activation of signaling receptors: do ligands bind to receptor monomer, dimer, or both?
BMC Biophys
PUBLISHED: 05-23-2013
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A recent study by Dietz et al. using single-molecule fluorescence microscopy techniques demonstrates that, in the absence of the ligand InlB, the MET receptor exists as both a monomer and a dimer on the cell membrane, and addition of the ligand leads to increased MET dimerization. Under the crowded conditions of the cell membrane, dimer formation may be a common phenomenon for cell surface receptors. Ligand binding to both monomeric and dimeric receptors may provide parallel routes to receptor activation.
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Using the concept of transient complex for affinity predictions in CAPRI rounds 20-27 and beyond.
Proteins
PUBLISHED: 05-22-2013
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Predictions of protein-protein binders and binding affinities have traditionally focused on features pertaining to the native complexes. In developing a computational method for predicting protein-protein association rate constants, we introduced the concept of transient complex after mapping the interaction energy surface. The transient complex is located at the outer boundary of the bound-state energy well, having near-native separation and relative orientation between the subunits but not yet formed most of the short-range native interactions. We found that the width of the binding funnel and the electrostatic interaction energy of the transient complex are among the features predictive of binders and binding affinities. These ideas were very promising for the five affinity-related targets (T43-45, 55, and 56) of CAPRI rounds 20-27. For T43, we ranked the single crystallographic complex as number 1 and were one of only two groups that clearly identified that complex as a true binder; for T44, we ranked the only design with measurable binding affinity as number 4. For the nine docking targets, continuing on our success in previous CAPRI rounds, we produced 10 medium-quality models for T47 and acceptable models for T48 and T49. We conclude that the interaction energy landscape and the transient complex in particular will complement existing features in leading to better prediction of binding affinities. Proteins 2013; 81:2229-2236. © 2013 Wiley Periodicals, Inc.
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Modeling protein association mechanisms and kinetics.
Curr. Opin. Struct. Biol.
PUBLISHED: 05-03-2013
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Substantial advances have been made in modeling protein association mechanisms and in calculating association rate constants (ka). We now have a clear understanding of the physical factors underlying the wide range of experimental ka values. Half of the association problem, where ka is limited by diffusion, is perhaps solved, and for the other half, where conformational changes become rate-limiting, a number of promising methods are being developed for ka calculations. Notably, the binding kinetics of disordered proteins are receiving growing attention, with dock-and-coalesce emerging as a general mechanism. Progress too has been made in the modeling of protein association kinetics under conditions mimicking the heterogeneous, crowded environments of cells, an endeavor that should ultimately lead to a better understanding of cellular functions.
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[Impacts on the appetite regulating factors of infantile anorexia treated with acupuncture at Sifeng (EX-UE 10)].
Zhongguo Zhen Jiu
PUBLISHED: 04-30-2013
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To explore the dynamic change and clinical efficacy of acupuncture at Sifeng (EX-UE 10) on appetite regulating factors in the serum of infantile anorexia.
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A small GTPase?like protein fragment of Mycoplasma promotes tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.
Mol Med Rep
PUBLISHED: 04-01-2013
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The Mycoplasma genus comprises a group of microbes that cause persistent infection in humans and its role in promoting tumor development has long been a concern. Although mixtures of components isolated from Mycoplasma have been shown to activate host Rho family small GTPases and Stat3, no individual factor with this activity has been reported. In the current study, a conserved small GTPase-like protein fragment (SGLP) from Mycoplasma pulmonis chromosome partition protein, Smc, was identified as a virulence factor. SGLP was observed to interact with Rac1 and Stat3. The wild?type (wt) SGLP, which contains a WxxxE motif, induced activation of Rac1 and phosphorylation of Stat3 at the tyrosine?705 residue, while the SGLP mutant containing a mutation from WxxxE to AxxxA did not exert the same effects. Moreover, SGLP?induced Stat3 phosphorylation was observed to be dependent upon Rac1 activity. Furthermore, wt SGLP was observed to promote cell migration and increase bromodeoxyuridine incorporation in HeLa cells and the SGLP mutant did not elicit these effects in HeLa cells. In conclusion, the current observations suggest that SGLP is an important virulence factor of Mycoplasma, which contributes to tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.
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An ab initio study on the transition paths from graphite to diamond under pressure.
J Phys Condens Matter
PUBLISHED: 03-11-2013
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We calculate and compare the transition paths from graphite to two types of diamond using the variable cell nudged elastic band method. For the phase transition from graphite to cubic diamond, we analyze in detail how the ? bonds transit to the ? bonds in an electronic structure. Meanwhile, a new transition path with a lower energy barrier for the transformation from graphite to hexagonal diamond is discovered. The path has its own peculiar sp(2)-sp(3) bonding configurations, serving as a transition state. Further calculation suggests that the sp(2)-sp(3) transition state represents an expected general phenomenon for cold-compressed graphite.
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The effect of microdosimetric 12C6+ heavy ion irradiation and Mg2+ on canthaxanthin production in a novel strain of Dietzia natronolimnaea.
BMC Microbiol.
PUBLISHED: 03-10-2013
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Dietzia natronolimnaea is one of the most important bacterial bioresources for high efficiency canthaxanthin production. It produces the robust and stable pigment canthaxanthin, which is of special interest for the development of integrated biorefineries. Mutagenesis employing 12C6+ irradiation is a novel technique commonly used to improve microorganism productivity. This study presents a promising route to obtaining the highest feasible levels of biomass dry weight (BDW), and total canthaxanthin by using a microdosimetric model of 12C6+ irradiation mutation in combination with the optimization of nutrient medium components.
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Community-wide evaluation of methods for predicting the effect of mutations on protein-protein interactions.
Proteins
PUBLISHED: 03-04-2013
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Community-wide blind prediction experiments such as CAPRI and CASP provide an objective measure of the current state of predictive methodology. Here we describe a community-wide assessment of methods to predict the effects of mutations on protein-protein interactions. Twenty-two groups predicted the effects of comprehensive saturation mutagenesis for two designed influenza hemagglutinin binders and the results were compared with experimental yeast display enrichment data obtained using deep sequencing. The most successful methods explicitly considered the effects of mutation on monomer stability in addition to binding affinity, carried out explicit side-chain sampling and backbone relaxation, evaluated packing, electrostatic, and solvation effects, and correctly identified around a third of the beneficial mutations. Much room for improvement remains for even the best techniques, and large-scale fitness landscapes should continue to provide an excellent test bed for continued evaluation of both existing and new prediction methodologies.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.