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Genotoxicity assessment of pulp and paper mill effluent before and after bacterial degradation using Allium cepa test.Friday, December 02, 2016
Haq I, Kumar S, Raj A, Lohani M, Satyanarayana GN,
Chemosphere. 29-Nov-2016
A lignin peroxidases-producing Serratia liquefaciens was used for bioremediation of pulp and paper (P&P) mill effluent. The treatment led to reduction of chemical oxygen demand (COD), colour, lignin and phenolic content by 84%, 72%, 61% and 95%, respectively. The effluent detoxification was studied by genotoxicity assays using Allium cepa L. (onion) root tip cells. Genotoxicity studies included measuring mitotic index (MI), chromosomal aberrations (CA) and nuclear abnormalities (NA) in root tip cells following treatment with 25, 50, 75 and 100% (v/v) of effluent. The root tip cells grown in untreated effluent showed a significant decrease in MI from 69% (control) to 32%, 27%, 22% and 11% at 25%, 50%, 75% and 100% effluent concentration, respectively. This indicated that the untreated effluent was highly cytotoxic in nature. Further, root tip cells, when treated with different concentrations of effluent showed various CA and NA including c-mitosis, stickiness, chromosome loss, chromosome break, anaphase bridge, multipolar anaphase, vagrant chromosomes, micronucleated and binucleated cells. The MI observed in root tip cells grown in bacterial treated effluents at similar concentrations (25, 50, 75 and 100% v/v) showed an increase of 33%, 36%, 42% and 66%. CA showed a substantial decrease and in some instances, complete absence of CA was also observed. The findings suggest that S. liquefaciens culture could be a potential bacterial culture for bioremediation of P&P mill effluent, as it is effective in substantial lowering of pollutants load as well as reduces the cytotoxic and genotoxic effects of effluent.
Structural optimization of diphenylpyrimidine derivatives (DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors with improved activity toward B leukemia cell lines.Friday, December 02, 2016
Zhao D, Huang S, Qu M, Wang C, Liu Z, Li Z, Peng J, Liu K, Li Y, Ma X, Shu X,
European journal of medicinal chemistry. 23-Nov-2016
A new series of diphenylpyrimidine derivatives (DPPYs) bearing various aniline side chains at the C-2 position of pyrimidine core were synthesized as potent BTK inhibitors. Most of these inhibitors displayed improved activity against B leukemia cell lines compared with lead compound spebrutinib. Subsequent studies showed that the peculiar inhibitor 7j, with IC50 values of 10.5 μM against Ramos cells and 19.1 μM against Raji cells, also displayed slightly higher inhibitory ability than the novel agent ibrutinib. Moreover, compound 7j is not sensitive to normal cells PBMC, indicating low cell cytotoxicity. In addition, flow cytometry analysis indicated that 7j significantly induced the apoptosis of Ramos cells, and arrested the cell cycle at the G0/G1 phase. These explorations provided new clues to discover pyrimidine scaffold as more effective BTK inhibitors.
Biomarker analysis of American toad (Anaxyrus americanus) and grey tree frog (Hyla versicolor) tadpoles following exposure to atrazine.Friday, December 02, 2016
Snyder MN, Henderson WM, Glinski DA, Purucker ST,
Aquatic toxicology (Amsterdam, Netherlands). 21-Nov-2016
The objective of the current study was to use a biomarker-based approach to investigate the influence of atrazine exposure on American toad (Anaxyrus americanus) and grey tree frog (Hyla versicolor) tadpoles. Atrazine is one of the most frequently detected herbicides in environmental matrices throughout the United States. In surface waters, it has been found at concentrations from 0.04-2859μg/L and thus presents a likely exposure scenario for non-target species such as amphibians. Studies have examined the effect of atrazine on the metamorphic parameters of amphibians, however, the data are often contradictory. Gosner stage 22-24 tadpoles were exposed to 0 (control), 10, 50, 250 or 1250μg/L of atrazine for 48h. Endogenous polar metabolites were extracted and analyzed using gas chromatography coupled with mass spectrometry. Statistical analyses of the acquired spectra with machine learning classification models demonstrated identifiable changes in the metabolomic profiles between exposed and control tadpoles. Support vector machine models with recursive feature elimination created a more efficient, non-parametric data analysis and increased interpretability of metabolomic profiles. Biochemical fluxes observed in the exposed groups of both A. americanus and H. versicolor displayed perturbations in a number of classes of biological macromolecules including fatty acids, amino acids, purine nucleosides, pyrimidines, and mono- and di-saccharides. Metabolomic pathway analyses are consistent with findings of other studies demonstrating disruption of amino acid and energy metabolism from atrazine exposure to non-target species.
Hyperferritinemia is a potential marker of chronic chikungunya: A retrospective study on the Island of Curaçao during the 2014-2015 outbreak.Friday, December 02, 2016
Anfasa F, Provacia L, GeurtsvanKessel C, Wever R, Gerstenbluth I, Osterhaus AD, Martina BE,
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 18-Nov-2016
The results showed that the presence of an Asian genotype of CHIKV in Curaçao for the first time. Moreover, we found an association between ferritin levels with chronic arthralgia.
Advances in Zika Virus Research: Stem Cell Models, Challenges, and Opportunities.Friday, December 02, 2016
Ming GL, Tang H, Song H,
Cell stem cell. 01-Dec-2016
The re-emergence of Zika virus (ZIKV) and its suspected link with various disorders in newborns and adults led the World Health Organization to declare a global health emergency. In response, the stem cell field quickly established platforms for modeling ZIKV exposure using human pluripotent stem cell-derived neural progenitors and brain organoids, fetal tissues, and animal models. These efforts provided significant insight into cellular targets, pathogenesis, and underlying biological mechanisms of ZIKV infection as well as platforms for drug testing. Here we review the remarkable progress in stem cell-based ZIKV research and discuss current challenges and future opportunities.
Cardiac complications associated with the influenza viruses A subtype H7N9 or pandemic H1N1 in critically ill patients under intensive care.Friday, December 02, 2016
Wang J, Xu H, Yang X, Zhao D, Liu S, Sun X, Huang JA, Guo Q,
The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases. 29-Nov-2016
Critically ill hospitalized H7N9 patients experienced a higher rate of cardiac complications than did patients with 2009 pH1N1 infections, with the exception of pericardial effusion. This study may help in the prevention, identification, and treatment of influenza-induced cardiac complications in both pH1N1 and H7N9 infections.
Tumor Interferon Signaling Regulates a Multigenic Resistance Program to Immune Checkpoint Blockade.Friday, December 02, 2016
Benci JL, Xu B, Qiu Y, Wu TJ, Dada H, Twyman-Saint Victor C, Cucolo L, Lee DS, Pauken KE, Huang AC, Gangadhar TC, Amaravadi RK, Schuchter LM, Feldman MD, Ishwaran H, Vonderheide RH, Maity A, Wherry EJ, Minn AJ,
Cell. 01-Dec-2016
Therapeutic blocking of the PD1 pathway results in significant tumor responses, but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB) and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.
Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D, and xenograft models.Friday, December 02, 2016
Agrawal K, Das V, Otmar M, Krečmerová M, Džubák P, Hajdúch M,
Cytometry. Part A : the journal of the International Society for Analytical Cytology. 02-Dec-2016
Aberrant DNA methylation that results in silencing of genes has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is elusive due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and synthetic molecules that can alter methylation patterns exist, however, their usefulness in epigenetic therapy remains unknown. This underlines the need for effective tumor models for large-scale screening of drug candidates with potent hypomethylation activity. In this study, we present the development of a cell-based DNA demethylation detection system, which is amenable for high content screening of epigenetic drugs in two-dimensional and three-dimensional cell culture models. Additionally, the detection system also supports the in vivo monitoring of demethylation efficacy of potential lead compounds from in vitro screens in tumor xenografts. The described detection system not only permits the continuous monitoring of demethylation but also of the induced cytostatic/cytotoxic drug effects in live cells, as a function of time. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene, which is silenced on account of promoter hypermethylation in human colorectal cancer. The described work will provide the researchers with an efficient tool for epigenetic drug screens on a high throughput platform and would therefore benefit academic and industrial drug discovery. © 2016 International Society for Advancement of Cytometry.
Copper Oxide Nanoparticles Impact Several Toxicological Endpoints and Cause Neurodegeneration in Caenorhabditis elegans.Friday, December 02, 2016
Mashock MJ, Zanon T, Kappell AD, Petrella LN, Andersen EC, Hristova KR,
PloS one. 2016
Engineered nanoparticles are becoming increasingly incorporated into technology and consumer products. In 2014, over 300 tons of copper oxide nanoparticles were manufactured in the United States. The increased production of nanoparticles raises concerns regarding the potential introduction into the environment or human exposure. Copper oxide nanoparticles commonly release copper ions into solutions, which contribute to their toxicity. We quantified the inhibitory effects of both copper oxide nanoparticles and copper sulfate on C. elegans toxicological endpoints to elucidate their biological effects. Several toxicological endpoints were analyzed in C. elegans, including nematode reproduction, feeding behavior, and average body length. We examined three wild C. elegans isolates together with the Bristol N2 laboratory strain to explore the influence of different genotypic backgrounds on the physiological response to copper challenge. All strains exhibited greater sensitivity to copper oxide nanoparticles compared to copper sulfate, as indicated by reduction of average body length and feeding behavior. Reproduction was significantly reduced only at the highest copper dose, though still more pronounced with copper oxide nanoparticles compared to copper sulfate treatment. Furthermore, we investigated the effects of copper oxide nanoparticles and copper sulfate on neurons, cells with known vulnerability to heavy metal toxicity. Degeneration of dopaminergic neurons was observed in up to 10% of the population after copper oxide nanoparticle exposure. Additionally, mutants in the divalent-metal transporters, smf-1 or smf-2, showed increased tolerance to copper exposure, implicating both transporters in copper-induced neurodegeneration. These results highlight the complex nature of CuO nanoparticle toxicity, in which a nanoparticle-specific effect was observed in some traits (average body length, feeding behavior) and a copper ion specific effect was observed for other traits (neurodegeneration, response to stress).
Chemical and Cellular Assays Combined with In Vitro Digestion to Determine the Antioxidant Activity of Flavonoids from Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Leaves.Friday, December 02, 2016
Zhang Y, Chen S, Wei C, Gong H, Li L, Ye X,
PloS one. 2016
Oxidative stress is highly associated with the development of cardiovascular diseases and cancer and has drawn great attention. Natural products suggest a potential role in prevention of these disorders. The aim of this study was to investigate the antioxidant and anti-cancer properties of Chinese bayberry leaves (Myrica rubra Sieb. et Zucc.) flavonoids (BLF) comprehensively through the combination of in vitro digestion, chemical and cellular antioxidant assays. Based on the LC/MS data, the major flavonoids of BLF were myricitrin and quercetin 3-rhamnoside. BLF owned strong chemical and cellular antioxidant activity (CAA) with its CAA value at 4253.884 ± 435.366 μmol of QE/100 g DW. After the in vitro digestion, the total flavonoids content, myricitrin and quercetin 3-rhamnoside decreased significantly (P < 0.05). Lower levels of the total flavonoid content and cellular uptake of myricitrin and quercetin 3-rhamnoside might contribute to the lower CAA value of digested BLF (DBLF). However, DBLF still owns considerable chemical antioxidant activities and CAA compared with many plants. Furthermore, both BLF and DBLF exhibited dose-dependent relationship against HepG2 proliferation. Taken together, BLF has a great potential to be developed as a natural antioxidant for promoting public health.
The Therapeutic Response of Gastrointestinal Stromal Tumors to Imatinib Treatment Assessed by Intravoxel Incoherent Motion Diffusion-Weighted Magnetic Resonance Imaging with Histopathological Correlation.Friday, December 02, 2016
Pan F, Den J, Zhang C, Wang H, Cheng J, Wu W, Hong N, Wang Y,
PloS one. 2016
IVIM measurements may serve as more sensitive imaging biomarkers than ADC when assessing GIST response to Imatinib as early as one day after treatment.
Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis.Friday, December 02, 2016
Jiang F, Huang W, Wang Y, Tian P, Chen X, Liang Z,
PloS one. 2016
Nucleic acid amplification testing combined with acid-fast staining in lung biopsy tissues can lead to early and accurate diagnosis in patients with smear-negative pulmonary tuberculosis. For patients with positive histological AFB and negative tuberculous PCR results in lung tissue, NTM infection should be suspected and could be identified by specific probe assays or 16S rRNA sequencing.
Molecular Markers of Radiation Induced Attenuation in Intrahepatic Plasmodium falciparum Parasites.Friday, December 02, 2016
Oakley MS, Verma N, Zheng H, Anantharaman V, Takeda K, Gao Y, Myers TG, Pham PT, Mahajan B, Kumar N, Sangweme D, Tripathi AK, Mlambo G, Aravind L, Kumar S,
PloS one. 2016
Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.
Inhibition of cancer antioxidant defense by natural compounds.Friday, December 02, 2016
Sznarkowska A, Kostecka A, Meller K, Bielawski KP,
Oncotarget. 30-Nov-2016
All classic, non-surgical anticancer approaches like chemotherapy, radiotherapy or photodynamic therapy kill cancer cells by inducing severe oxidative stress. Even tough chemo- and radiotherapy are still a gold standard in cancer treatment, the identification of non-toxic compounds that enhance their selectivity, would allow for lowering their doses, reduce side effects and risk of second cancers. Many natural products have the ability to sensitize cancer cells to oxidative stress induced by chemo- and radiotherapy by limiting antioxidant capacity of cancer cells. Blocking antioxidant defense in tumors decreases their ability to balance oxidative insult and results in cell death. Though one should bear in mind that the same natural compound often exerts both anti-oxidant and pro-oxidant properties, depending on concentration used, cell type, exposure time and environmental conditions. Here we present a comprehensive overview of natural products that inhibit major antioxidant defense mechanisms in cancer cells and discuss their potential in clinical application.
MiR-146a polymorphism correlates with lung cancer risk in Chinese nonsmoking females.Friday, December 02, 2016
Yin Z, Cui Z, Ren Y, Xia L, Li H, Zhou B,
Oncotarget. 30-Nov-2016
This study provides evidence that the common rs2910164 polymorphism in miR-146a strongly correlates with lung cancer risk in nonsmoking females in northeast China. The genotypes of miR-146a rs2910164 were determined in 1131 patients with lung cancer and 1003 healthy control subjects. Tissue samples were used to evaluate the association between miRNA expression and lung cancer risk as well as the correlation between rs2910164 genotypes and miR-146a expression. The secondary structures of the wild-type and variant miR-146a sequences were predicted, and luciferase-based target assays were used to test whether miR-146a bound to tumor necrosis factor receptor associated factor 6 (TRAF6) mRNA. Individuals carrying heterozygous CG genotype of miR-146a rs2910164 had less risk of lung cancer than those carrying homozygous wild CC genotype (OR = 0.76, 95% CI = 0.60-0.98, P = 0.032). We found no significant association between miR-146a expression and lung cancer risk. MiR-146a expression differed in those carrying the CC genotype as compared with the CG or the GG genotype (P = 0.032 and 0.001), and the secondary structure of the C allele differed slightly from the G allele. Significantly lower levels of luciferase activity were observed when the TRAF6 3'UTR was cotransfected with miR-146a-3p carrying the rs2910164 C allele (P = 0.001). Thus, miR-146a rs2910164 polymorphism may influence susceptibility to lung cancer in Chinese nonsmoking females through targeting TRAF6.
KRAS exon 2 codon 13 mutation is associated with a better prognosis than codon 12 mutation following lung metastasectomy in colorectal cancer.Friday, December 02, 2016
Renaud S, Guerrera F, Seitlinger J, Costardi L, Schaeffer M, Romain B, Mossetti C, Claire-Voegeli A, Filosso PL, Legrain M, Ruffini E, Falcoz PE, Oliaro A, Massard G,
Oncotarget. 29-Nov-2016
KRAS exon 2 codon 13 mutations, compared to codon 12 mutations, seem to be associated with better outcomes following lung metastasectomy in CRC. Prospective multicenter studies are necessary to fully understand the prognostic value of KRAS mutations in the lung metastases of CRC.
Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility: results from a large-scale collaboration.Friday, December 02, 2016
Permuth JB, Reid B, Earp M, Chen YA, Monteiro AN, Chen Z, Aocs Study Group Chenevix-Trench G, Fasching PA, Beckmann MW, Lambrechts D, Vanderstichele A, Van Niewenhuyse E, Vergote I, Rossing MA, Doherty JA, Chang-Claude J, Moysich K, Odunsi K, Goodman MT, Shvetsov YB, Wilkens LR, Thompson PJ, Dörk T, Bogdanova N, Butzow R, Nevanlinna H, Pelttari L, Leminen A, Modugno F, Edwards RP, Ness RB, Kelley J, Heitz F, Karlan B, Lester J, Kjaer SK, Jensen A, Giles G, Hildebrandt M, Liang D, Lu KH, Wu X, Levine DA, Bisogna M, Berchuck A, Cramer DW, Terry KL, Tworoger SS, Poole EM, Bandera EV, Fridley B, Cunningham J, Winham SJ, Olson SH, Orlow I, Bjorge L, Kiemeney LA, Massuger L, Pejovic T, Moffitt M, Le N, Cook LS, Brooks-Wilson A, Kelemen LE, Gronwald J, Lubinski J, Wentzensen N, Brinton LA, Lissowska J, Yang H, Hogdall E, Hogdall C, Lundvall L, Pharoah PD, Song H, Campbell I, Eccles D, McNeish I, Whittemore A, McGuire V, Sieh W, Rothstein J, Phelan CM, Risch H, Narod S, McLaughlin J, Anton-Culver H, Ziogas A, Menon U, Gayther S, Ramus SJ, Gentry-Maharaj A, Pearce CL, Wu AH, Kupryjanczyk J, Dansonka-Mieszkowska A, Schildkraut JM, Cheng JQ, Goode EL, Sellers TA,
Oncotarget. 12-Jul-2016
RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1. Unconditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI), with adjustment for European ancestry. We conducted gene-level analyses using the Admixture Maximum Likelihood (AML) test and the Sequence-Kernel Association test for common and rare variants (SKAT-CR). Association analysis revealed top risk-associated SNP rs77027562 (OR (95% CI)= 1.39 (1.17-1.64), P=1.0x10-4) in ADAR3 and rs185455523 in SND1 (OR (95% CI)= 0.68 (0.56-0.83), P=2.0x10-4). When restricting to serous histology (n=6,500), the magnitude of association strengthened for rs185455523 (OR=0.60, P=1.0x10-4). Gene-level analyses revealed that variation in ADAR was associated (P<0.05) with EOC susceptibility, with PAML=0.022 and PSKAT-CR=0.020. Expression quantitative trait locus analysis in EOC tissue revealed significant associations (P<0.05) with ADAR expression for several SNPs in ADAR, including rs1127313 (G/A), a SNP in the 3' untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing.
High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing.Friday, December 02, 2016
Moffitt JR, Hao J, Bambah-Mukku D, Lu T, Dulac C, Zhuang X,
Proceedings of the National Academy of Sciences of the United States of America. 22-Nov-2016
Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background-from off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.
Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.Friday, December 02, 2016
Bai F, Morcos F, Cheng RR, Jiang H, Onuchic JN,
Proceedings of the National Academy of Sciences of the United States of America. 29-Nov-2016
Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.
Urban park soil microbiomes are a rich reservoir of natural product biosynthetic diversity.Friday, December 02, 2016
Charlop-Powers Z, Pregitzer CC, Lemetre C, Ternei MA, Maniko J, Hover BM, Calle PY, McGuire KL, Garbarino J, Forgione HM, Charlop-Powers S, Brady SF,
Proceedings of the National Academy of Sciences of the United States of America. 28-Nov-2016
Numerous therapeutically relevant small molecules have been identified from the screening of natural products (NPs) produced by environmental bacteria. These discovery efforts have principally focused on culturing bacteria from natural environments rich in biodiversity. We sought to assess the biosynthetic capacity of urban soil environments using a phylogenetic analysis of conserved NP biosynthetic genes amplified directly from DNA isolated from New York City park soils. By sequencing genes involved in the biosynthesis of nonribosomal peptides and polyketides, we found that urban park soil microbiomes are both rich in biosynthetic diversity and distinct from nonurban samples in their biosynthetic gene composition. A comparison of sequences derived from New York City parks to genes involved in the biosynthesis of biomedically important NPs produced by bacteria originally collected from natural environments around the world suggests that bacteria producing these same families of clinically important antibiotics, antifungals, and anticancer agents are actually present in the soils of New York City. The identification of new bacterial NPs often centers on the systematic exploration of bacteria present in natural environments. Here, we find that the soil microbiomes found in large cities likely hold similar promise as rich unexplored sources of clinically relevant NPs.
Enhanced limonene production in cyanobacteria reveals photosynthesis limitations.Friday, December 02, 2016
Wang X, Liu W, Xin C, Zheng Y, Cheng Y, Sun S, Li R, Zhu XG, Dai SY, Rentzepis PM, Yuan JS,
Proceedings of the National Academy of Sciences of the United States of America. 23-Nov-2016
Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.
An α/β-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization.Friday, December 02, 2016
Zheng Q, Wang S, Duan P, Liao R, Chen D, Liu W,
Proceedings of the National Academy of Sciences of the United States of America. 23-Nov-2016
Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6'-fluoro-7', 8'-epoxy-TSR, a key intermediate in the preparation of the analog 6'-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/β-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6' fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.
Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.Friday, December 02, 2016
Shimizu M, Noguchi Y, Sakiyama Y, Kawakami H, Katayama T, Takada S,
Proceedings of the National Academy of Sciences of the United States of America. 29-Nov-2016
Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.
Optical magnetic detection of single-neuron action potentials using quantum defects in diamond.Friday, December 02, 2016
Barry JF, Turner MJ, Schloss JM, Glenn DR, Song Y, Lukin MD, Park H, Walsworth RL,
Proceedings of the National Academy of Sciences of the United States of America. 22-Nov-2016
Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution-e.g., magnetic resonance imaging methods and magnetoencephalography-or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector.
Control of HIV-1 gene expression by SR proteins.Friday, December 02, 2016
Mahiet C, Swanson CM,
Biochemical Society transactions. 15-Oct-2016
Cellular proteins are required for all steps of human immunodeficiency virus type 1 (HIV-1) gene expression including transcription, splicing, 3'-end formation/polyadenylation, nuclear export and translation. SR proteins are a family of cellular RNA-binding proteins that regulate and functionally integrate multiple steps of gene expression. Specific SR proteins are best characterised for regulating HIV-1 RNA splicing by binding specific locations in the viral RNA, though recently they have also been shown to control transcription, 3'-end formation, and translation. Due to their importance in regulating HIV-1 gene expression, SR proteins and their regulatory factors are potential antiviral drug targets.
Regulatory mechanisms of phosphatase of regenerating liver (PRL)-3.Friday, December 02, 2016
Rubio T, Köhn M,
Biochemical Society transactions. 15-Oct-2016
The phosphatase of regenerating liver (PRL)-3 is overexpressed in many human cancer types and tumor metastases when compared with healthy tissues. Different pathways and mechanisms have been suggested to modulate PRL-3 expression levels and activity, giving some valuable insights but still leaving an incomplete picture. Investigating these mechanisms could provide new targets for therapeutic drug development. Here, we present an updated overview and summarize recent findings concerning the different PRL-3 expression regulatory mechanisms and posttranslational modifications suggested to modulate the activity, localization, or stability of this phosphatase.
Tolerance and Efficacy with Simultaneous Use of Two Monoclonal Antibodies for a Patient with Hypereosinophilic Syndrome and Ulcerative Colitis.Friday, December 02, 2016
Patel B, Butterfield JH, Weiler CR, Kane SV,
Monoclonal antibodies in immunodiagnosis and immunotherapy. 02-Dec-2016
The development and utilization of monoclonal antibodies (mAbs) have been of great interest in all fields of medicine. A substantial increase in the production and development of mAbs has occurred because these biologic agents are proving to be effective and less toxic given their targeted mechanism of action. However, data are limited on coadministration of two or more mAbs. With the increasing availability of mAbs and the comorbidities of some patients, assessment is needed of the ability to safely use multiple mAbs for an individual patient. Although the efficacy of coadministered mAbs may be inferred from their specific targets, we could find no literature reporting such a finding. Herein, we report our experience using two different classes of mAbs to treat hypereosinophilic syndrome and ulcerative colitis in a single patient.
Neutrophil Elastase Activity is Associated with Exacerbations and Lung Function Decline in Bronchiectasis.Friday, December 02, 2016
Chalmers JD, Moffitt KL, Suarez-Cuartin G, Sibila O, Finch S, Furrie E, Dicker A, Wrobel K, Elborn JS, Walker B, Martin SL, Marshall SE, Huang JT, Fardon TC,
American journal of respiratory and critical care medicine. 02-Dec-2016
Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.
Proteomic profiling identifies N-acetylmuramoyl-l-alanine amidase as a novel biomarker of sepsis.Friday, December 02, 2016
da Silva FP, Cataldi TR, de Lima TM, Starzynski PN, Barbeiro HV, Labate MT, CéMachado MC, de Souza HP, Labate CA,
Biomarkers in medicine. 02-Dec-2016
This is the first description of N-acetylmuramoyl-l-alanine amidase as a biomarker that can be used alone or in conjunction with other biomarkers to facilitate the diagnosis of sepsis in the critically ill.
Determining Functional Aptamer-Protein Interaction by Biolayer Interferometry.Friday, December 02, 2016
Lou X, Egli M, Yang X,
Current protocols in nucleic acid chemistry. 01-Dec-2016
Short single-stranded nucleic acids called aptamers are widely being explored as recognition molecules of high affinity and specificity for binding a wide range of target molecules, particularly protein targets. In biolayer interferometry (BLI), a simple Dip-and-Read approach in which the aptamer-coated biosensors are dipped into microplate wells is used to study the interactions between an aptamer and its target protein. Here we describe the protocol for the analysis of the interaction between a well-characterized anti-thrombin RNA aptamer with thrombin (Basic Protocol). We also report on the protocol for the affinity screening of a panel of anti-thrombin RNA aptamers with a single phosphorodithioate (PS2) modification, whereby the position of the modification along the RNA backbone is varied systematically (Support Protocol). The PS2 modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphodiester backbone linkage. The PS2-modified RNAs are nuclease resistant and several in vitro and in vivo assays have demonstrated their biological activity. For example, combining the PS2 with the 2'-OMe modification affords increased loading of modified small interfering RNA (siRNA) duplexes into the RNA-induced silencing complex (RISC) as well as enhanced gene-silencing antitumor activity. © 2016 by John Wiley & Sons, Inc.
Rolling Circle Amplification with Chemically Modified Nucleoside Triphosphates.Friday, December 02, 2016
Hollenstein M, Damha MJ,
Current protocols in nucleic acid chemistry. 01-Dec-2016
Modified nucleoside triphosphates (dN*TPs) represent facile and versatile precursors for the introduction of chemical diversity into nucleic acids. While dN*TPs have been utilized in a plethora of practical applications, very little attention has been devoted to the assessment of their compatibility with isothermal amplification strategies. In this context, rolling circle amplification (RCA) is a wide-spread enzymatic replication method in which small single-stranded DNA (ssDNA) circles serve as templates in primer extension reactions yielding very long, ssDNA products. RCA is a pivotal tool for the generation of biosensor and diagnostic devices and is currently evaluated for its usefulness to create novel drug delivery systems. This unit describes the experimental procedures for the synthesis of modified RCA products using dN*TPs bearing chemical alterations at any possible location of the nucleosidic scaffold. Two ligation methods are presented for the generation of the DNA nanocircles that serve as templates for RCA, followed by a description of the RCA method itself and an assessment of the nuclease resistance of the ensuing products. © 2016 by John Wiley & Sons, Inc.
Carboplatin-Complexed and cRGD-Conjugated Unimolecular Nanoparticles for Targeted Ovarian Cancer Therapy.Friday, December 02, 2016
Wang Y, Wang L, Chen G, Gong S,
Macromolecular bioscience. 01-Dec-2016
Platinum-based chemotherapy has been widely used to treat cancers including ovarian cancer; however, it suffers from dose-limiting toxicity. Judiciously designed drug nanocarriers can enhance the anticancer efficacy of platinum-based chemotherapy while reducing its systemic toxicity. Herein the authors report a stable and water-soluble unimolecular nanoparticle constructed from a hydrophilic multi-arm star block copolymer poly(amidoamine)-b-poly(aspartic acid)-b-poly(ethylene glycol) (PAMAM-PAsp-PEG) conjugated with both cRGD (cyclo(Arg-Gly-Asp-D-Phe-Cys) peptide and cyanine5 (Cy5) fluorescent dye as a platinum-based drug nanocarrier for targeted ovarian cancer therapy. Carboplatin is complexed to the poly(aspartic acid) inner shell via pH-responsive ion-dipole interactions between carboplatin and the carboxylate groups of poly(aspartic acid). Based on flow cytometry and confocal laser scanning microscopy analyses, cRGD-conjugated unimolecular nanoparticles exhibit much higher cellular uptake by ovarian cancer cells overexpressing αv β3 integrin than nontargeted (i.e., cRGD-lacking) ones. Carboplatin-complexed cRGD-conjugated nanoparticles also exhibit higher cytotoxicity than nontargeted nanoparticles as well as free carboplatin, while empty unimolecular nanoparticles show no cytotoxicity. These results indicate that stable unimolecular nanoparticles made of individual hydrophilic multi-arm star block copolymer molecules conjugate with tumor-targeting ligands and dyes (i.e., PAMAM-PAsp-PEG-cRGD/Cy5) are promising nanocarriers for platinum-based anticancer drugs for targeted cancer therapy.
The prey's scent - Volatile organic compound mediated interactions between soil bacteria and their protist predators.Friday, December 02, 2016
Schulz-Bohm K, Geisen S, Wubs ER, Song C, de Boer W, Garbeva P,
The ISME journal. 02-Dec-2016
Protists are major predators of bacteria in soils. However, it remains unknown how protists sense their prey in this highly complex environment. Here, we investigated whether volatile organic compounds (VOCs) of six phylogenetic distinct soil bacteria affect the performance of three different soil protists and how that relates to direct feeding interactions. We observed that most bacteria affected protist activity by VOCs. However, the response of protists to the VOCs was strongly dependent on both the bacterial and protist interacting partner. Stimulation of protist activity by volatiles and in direct trophic interaction assays often coincided, suggesting that VOCs serve as signals for protists to sense suitable prey. Furthermore, bacterial terpene synthase mutants lost the ability to affect protists, indicating that terpenes represent key components of VOC-mediated communication. Overall, we demonstrate that volatiles are directly involved in protist-bacterial predator-prey interactions.The ISME Journal advance online publication, 2 December 2016; doi:10.1038/ismej.2016.144.
Detecting biomarkers of secondhand marijuana smoke in young children.Friday, December 02, 2016
Wilson KM, Torok MR, Wei B, Wang L, Robinson M, Sosnoff CS, Blount BC,
Pediatric research. 02-Dec-2016
Metabolites of marijuana smoke can be detected in children; in this cohort, 16% were exposed. Detectable COOH-THC is more common in children with tobacco smoke exposure. More research is needed to assess the health impacts of marijuana smoke exposure on children and inform public health policy.
Extraction of Plant-based Capsules for Microencapsulation Applications.Friday, December 02, 2016
Potroz MG, Mundargi RC, Park JH, Tan EL, Cho NJ,
Journal of visualized experiments : JoVE. 09-Nov-2016
Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.
Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform.Friday, December 02, 2016
Hanson C, Israelsen ND, Sieverts M, Vargis E,
Journal of visualized experiments : JoVE. 10-Nov-2016
Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available. Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags(1). Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.
Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase.Friday, December 02, 2016
Romeo E, Pontis S, Ponzano S, Bonezzi F, Migliore M, Di Martino S, Summa M, Piomelli D,
Journal of visualized experiments : JoVE. 23-Nov-2016
Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme's active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.
Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses.Friday, December 02, 2016
Ahn JJ, Selvanantham T, Zhang MA, Mallevaey T, Dunn SE,
Journal of visualized experiments : JoVE. 16-Nov-2016
L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen(1-3). In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection(4). The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.
Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy.Friday, December 02, 2016
Villanueva-Cabello TM, Martinez-Duncker I,
Journal of visualized experiments : JoVE. 08-Nov-2016
The methods described herein for activation of naïve CD4(+) T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4(+) T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during naïve CD4(+) T cell activation. Also, real-time PCR experiments using <40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.
Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks.Friday, December 02, 2016
Mukherjee A, Vasquez KM,
Journal of visualized experiments : JoVE. 10-Nov-2016
High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5'-TA-3' is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5' end to a 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.
Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins.Friday, December 02, 2016
O'Brien J, Hayder H, Peng C,
Journal of visualized experiments : JoVE. 17-Nov-2016
The National Institute of Health's ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.
High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity.Friday, December 02, 2016
Chiaraviglio L, Kang YS, Kirby JE,
Journal of visualized experiments : JoVE. 16-Nov-2016
Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1(st) generation assay) or fluorescent protein reporters (2(nd) generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.
Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b.Friday, December 02, 2016
Han Y, Lyman KA, Clutter M, Schiltz GE, Ismail QA, Cheng X, Luan CH, Chetkovich DM,
Journal of visualized experiments : JoVE. 11-Nov-2016
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows 'hit' compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that 'hit' compounds are not artifactual.
Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10.Friday, December 02, 2016
Süring K, Bach S, Höflich C, Straff W,
Journal of visualized experiments : JoVE. 19-Nov-2016
Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions. So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm.
Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays.Friday, December 02, 2016
Naylor J, Rossi A, Brankin C, Hornigold DC,
Journal of visualized experiments : JoVE. 10-Nov-2016
As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists(1). Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).
A spectral interior CT by a framelet-based reconstruction algorithm.Friday, December 02, 2016
Wang Y, Wang G, Mao S, Cong W, Ji Z, Cai JF, Ye Y,
Journal of X-ray science and technology. 22-Nov-2016
Reducing radiation dose is an important goal in medical computed tomography (CT), for which interior tomography is an effective approach. There have been interior reconstruction algorithms for monochromatic CT, but in reality, X-ray sources are polychromatic. Using a polychromatic acquisition model and motivated by framelet-based image processing algorithms, in this paper, we propose an interior reconstruction algorithm to obtain an image with spectral information assuming only one scan with a current energy-integrating detector. This algorithm is a new nonlinear iterative method by minimizing a special functional under a polychromatic acquisition model for X-ray CT, where the attenuation coefficients are energy-dependent. Experimental results validate that our algorithm can effectively reduce the beam-hardening artifacts and metal artifacts. It also produces color overlays which are useful in tumor identification and quantification.
PINK1, Parkin, and Mitochondrial Quality Control: What can we Learn about Parkinson's Disease Pathobiology?Friday, December 02, 2016
Truban D, Hou X, Caulfield TR, Fiesel FC, Springer W,
Journal of Parkinson's disease. 30-Nov-2016
The first clinical description of Parkinson's disease (PD) will embrace its two century anniversary in 2017. For the past 30 years, mitochondrial dysfunction has been hypothesized to play a central role in the pathobiology of this devastating neurodegenerative disease. The identifications of mutations in genes encoding PINK1 (PTEN-induced kinase 1) and Parkin (E3 ubiquitin ligase) in familial PD and their functional association with mitochondrial quality control provided further support to this hypothesis. Recent research focused mainly on their key involvement in the clearance of damaged mitochondria, a process known as mitophagy. It has become evident that there are many other aspects of this complex regulated, multifaceted pathway that provides neuroprotection. As such, numerous additional factors that impact PINK1/Parkin have already been identified including genes involved in other forms of PD. A great pathogenic overlap amongst different forms of familial, environmental and even sporadic disease is emerging that potentially converges at the level of mitochondrial quality control. Tremendous efforts now seek to further detail the roles and exploit PINK1 and Parkin, their upstream regulators and downstream signaling pathways for future translation. This review summarizes the latest findings on PINK1/Parkin-directed mitochondrial quality control, its integration and cross-talk with other disease factors and pathways as well as the implications for idiopathic PD. In addition, we highlight novel avenues for the development of biomarkers and disease-modifying therapies that are based on a detailed understanding of the PINK1/Parkin pathway.
The Clinical Use of Cerebrospinal Fluid Biomarkers for Alzheimer's Disease Diagnosis: The Italian Selfie.Friday, December 02, 2016
Sancesario GM, Toniolo S, Chiasserini D, Di Santo SG, Zegeer J, Bernardi G, Musicco M, Caltagirone C, Parnetti L, Bernardini S,
Journal of Alzheimer's disease : JAD. 28-Nov-2016
Although the use of cerebrospinal fluid (CSF) amyloid β1-42 (Aβ42), tau (T-tau), and phosphorylated tau (p-tau181) gives added diagnostic and prognostic values, the diffusion is still limited in clinical practice and only a restricted number of patients receive an integrated clinico-biological diagnosis. By a survey, we aimed to do a "selfie" of the use and diffusion of CSF biomarkers of dementia in Italy, the standardization of pre-analytical procedures, the harmonization of ranges, and the participation to Quality Control programs. An online questionnaire was sent to the members of SIBioC and SINdem-ITALPLANED and to main neurological clinics all over Italy. In Italy, 25 laboratories provide biomarkers analysis in addition to a network of 15 neighboring hospitals. In sum, 40 neurological centers require CSF analyses. 7/20 regions (35%) lack CSF laboratories. Standardization of pre-analytical procedures is present in 62.02% of the laboratories; only 56.00% of the laboratories participate in International Quality Control. There is no harmonization of cut-offs. In Italy, the use of CSF biomarkers is still limited in clinical practice. Standardization and harmonization of normal ranges are needed. To optimize and expand the use of CSF biomarkers, a cost-benefit analysis should be promoted by scientific societies and national health services.
Proteomics Analysis of Blood Serums from Alzheimer's Disease Patients Using iTRAQ Labeling Technology.Friday, December 02, 2016
Shen L, Liao L, Chen C, Guo Y, Song D, Wang Y, Chen Y, Zhang K, Ying M, Li S, Liu Q, Ni J,
Journal of Alzheimer's disease : JAD. 01-Dec-2016
Alzheimer' disease (AD) is the most common form of dementia affecting up to 6% of the population over the age of 65. In order to discover differentially expressed proteins that might serve as potential biomarkers, the serums from AD patients and healthy controls were compared and analyzed using the proteomics approach of isobaric tagging for relative and absolute quantitation (iTRAQ). For the first time, AD biomarkers in serums are investigated in the Han Chinese population using iTRAQ labeled proteomics strategy. Twenty-two differentially expressed proteins were identified and out of which nine proteins were further validated with more sample test. Another three proteins that have been reported in the literature to be potentially associated with AD were also investigated for alteration in expression level. Functions of those proteins were mainly related to the following processes: amyloid-β (Aβ) metabolism, cholesterol transport, complement and coagulation cascades, immune response, inflammation, hemostasis, hyaluronan metabolism, and oxidative stress. These results support current views on the molecular mechanism of AD. For the first time, differential expression of zinc-alpha-2-glycoprotein (AZGP1), fibulin-1 (FBLN1), platelet basic protein (PPBP), thrombospondin-1 (THBS1), S100 calcium-binding protein A8 (S100A8), and S100 calcium-binding protein A9 (S100A9) were detected in the serums of AD patients compared with healthy controls. These proteins might play a role in AD pathophysiology and serve as potential biomarkers for AD diagnosis. Specifically, our results strengthened the crucial role of Aβ metabolism and blood coagulation in AD pathogenesis and proteins related to these two processes may be used as peripheral blood biomarkers for AD.
Shape-Attributes of Brain Structures as Biomarkers for Alzheimer's Disease.Friday, December 02, 2016
Glozman T, Solomon J, Pestilli F, Guibas L,
Journal of Alzheimer's disease : JAD. 26-Nov-2016
We describe a fully automatic framework for classification of two types of dementia based on the differences in the shape of brain structures. We consider Alzheimer's disease (AD), mild cognitive impairment of individuals who converted to AD within 18 months (MCIc), and normal controls (NC). Our approach uses statistical learning and a feature space consisting of projection-based shape descriptors, allowing for canonical representation of brain regions. Our framework automatically identifies the structures most affected by the disease. We evaluate our results by comparing to other methods using a standardized data set of 375 adults available from the Alzheimer's Disease Neuroimaging Initiative (ADNI). Our framework is sensitive to identifying the onset of Alzheimer's disease, achieving up to 88.13% accuracy in classifying MCIc versus NC, outperforming previous methods.
Autotaxin is Related to Metabolic Dysfunction and Predicts Alzheimer's Disease Outcomes.Friday, December 02, 2016
McLimans KE, Willette AA,
Journal of Alzheimer's disease : JAD. 01-Dec-2016
These results are comparable to previous reports using insulin resistance. CSF autotaxin may be a useful dysmetabolism biomarker for examining AD outcomes and risk.
The Correlation between Inflammatory Biomarkers and Polygenic Risk Score in Alzheimer's Disease.Friday, December 02, 2016
Morgan AR, Touchard S, O'Hagan C, Sims R, Majounie E, Escott-Price V, Jones L, Williams J, Morgan BP,
Journal of Alzheimer's disease : JAD. 26-Nov-2016
Plasma biomarkers to aid the early diagnosis of Alzheimer's disease (AD) or to monitor disease progression have long been sought and continue to be widely studied. Biomarkers that correlate with AD polygenic risk score, a measure of the polygenic architecture of the disease and highly predictive of AD status, would be excellent candidates. Therefore, we undertook a preliminary study to assess the association of plasma inflammatory biomarkers with an overall AD polygenic risk score as well as with an inflammation-specific AD polygenic risk score in a sample set of 93 AD cases. We measured five complement biomarkers [complement receptor 1 (CR1), clusterin, complement component 9 (C9), C1 inhibitor (C1inh), terminal complement complex (TCC)] and the benchmark inflammatory marker C-reactive protein (CRP). Plasma clusterin level showed an association with overall AD polygenic risk score, while clusterin, C1inh, and CRP levels each displayed some association with the inflammatory-specific AD polygenic risk score. The results suggest that elevated plasma levels of inflammatory biomarkers, including complement proteins, associate with polygenic risk scores in AD, further strengthening the link between genetic and biomarker disease predictors and indicating a potential role for these markers in disease prediction and patient stratification in AD.
Alzheimer's Disease Histological and Behavioral Manifestations in Transgenic Mice Correlate with Specific Gut Microbiome State.Friday, December 02, 2016
Shen L, Liu L, Ji HF,
Journal of Alzheimer's disease : JAD. 28-Nov-2016
Alzheimer's disease (AD) is a neurodegenerative brain disease and is the most common form of dementia. In recent years, many studies indicated the association of gut microbiota changes with metabolic diseases. However, the gut microbiota of AD has not been investigated. The present study aims to compare the gut microbiota in APP/PS1 transgenic mice of AD and C57/Bl6 wild-type (WT) mice by pyrosequencing the V3 and V4 regions of the bacterial 16S ribosomal RNA genes. The 3-, 6-, and 8-month-old APP/PS1 and WT mice were used to explore the effects of age on the gut microbiota. First, the results indicated that impaired spatial learning and memory appeared in 6-month-old APP/PS1 mice and was further aggravated in the 8-month-old group, which was consistent with immunohistochemical studies of amyloid plaque. Second, AD histological and behavioral manifestations in the APP/PS1 mice were found to be correlated with a specific gut microbiome state. Third, the microbiota diversity of APP/PS1 mice decreased with increased age. Fourth, further inspection showed that the abundance of Helicobacteraceae and Desulfovibrionaceae at the family level and Odoribacter and Helicobacter at the genus level increased significantly in APP/PS1 mice than in WT mice, while Prevotella abundance in WT mice was significantly higher than in APP/PS1 mice. More human studies are warranted to explore the potential of gut microbiota as diagnostic biomarkers or therapeutic target for AD.
Decreased Expression of hsa-miR-4274 in Cerebrospinal Fluid of Normal Pressure Hydrocephalus Mimics with Parkinsonian Syndromes.Friday, December 02, 2016
Jurjević I, Miyajima M, Ogino I, Akiba C, Nakajima M, Kondo A, Kikkawa M, Kanai M, Hattori N, Arai H,
Journal of Alzheimer's disease : JAD. 28-Nov-2016
Levels of CSF hsa-miR-4274 can differentiate PS from patients with iNPH, AD, and NC. This may be clinically useful for diagnostic purposes and predicting shunt treatment responses.
Challenges in Screening and Recruitment for a Neuroimaging Study in Cognitively Impaired Geriatric Inpatients.Friday, December 02, 2016
Apostolova I, Lange C, Roberts A, Igel HJ, Mäurer A, Liese S, Estrella M, Prasad V, Stechl E, Lämmler G, Steinhagen-Thiessen E, Buchert R,
Journal of Alzheimer's disease : JAD. 28-Nov-2016
Enrollment for neuroimaging studies presents considerable additional challenges in acutely hospitalized geriatric patients compared to outpatient settings. Low rate of approaching potential candidates by attending geriatricians and a high rate of screen failures have to be anticipated in the study design.
The Effects of Gene Mutations on Default Mode Network in Familial Alzheimer's Disease.Friday, December 02, 2016
Li X, Westman E, Thordardottir S, Ståhlbom AK, Almkvist O, Blennow K, Wahlund LO, Graff C,
Journal of Alzheimer's disease : JAD. 29-Nov-2016
Familial Alzheimer's disease (FAD) mutations have very high penetrance but age at onset and rate of disease progression differ. Neuroimaging and cerebrospinal fluid (CSF) examinations in mutation carriers (MCs) may provide an opportunity to identify early biomarkers that can be used to track disease progression from presymptomatic to the dementia stages of disease. The default mode network (DMN) is a resting state neuronal network composed of regions known to associate with amyloid deposition in AD. We hypothesized that functional connectivity in the DMN might change at pre-clinical stages in FAD MCs and correlate with changes in CSF biomarkers as a consequence of AD brain pathology. To test the hypothesis, we compared the functional connectivity in DMN between pre-MCs/MCs and non-carriers (NCs). No significant differences between pre-MCs and NCs were observed. When comparing all MCs with NCs, significant decreased functional connectivity in the right inferior parietal lobule, right precuneus, and left posterior cingulate cortex were found. We also found statistically significant correlations between CSF amyloid-β 42 and tau protein levels and average Z-score, a resting-state functional MRI measurement reflecting the degree of the correlation between a given voxel's time courses and the time courses corresponding to DMN, from the region with statistical difference. The observed disruption of DMN and pathological levels of AD CSF-biomarkers in FAD MCs are similar to the changes described in sporadic AD, which give further support that amyloid and tau pathology impairs neuronal and synaptic function.
Tau Platelets Correlate with Regional Brain Atrophy in Patients with Alzheimer's Disease.Friday, December 02, 2016
Slachevsky A, Guzmán-Martínez L, Delgado C, Reyes P, Farías GA, Muñoz-Neira C, Bravo E, Farías M, Flores P, Garrido C, Becker JT, López OL, Maccioni RB,
Journal of Alzheimer's disease : JAD. 26-Nov-2016
This exploratory study showed that HMW/LMW tau ratio is significantly higher in AD patients than control subjects, and that it is associated with specific brain regions atrophy. Determination of peripheral markers of AD pathology can help understanding the pathophysiology of neurodegeneration in AD.
A Metal-Free Method for Producing MRI Contrast at Amyloid-β.Friday, December 02, 2016
Hilt S, Tang T, Walton JH, Budamagunta M, Maezawa I, Kálai T, Hideg K, Singh V, Wulff H, Gong Q, Jin LW, Louie A, Voss JC,
Journal of Alzheimer's disease : JAD. 26-Nov-2016
Alzheimer's disease (AD) is characterized by depositions of the amyloid-β (Aβ) peptide in the brain. The disease process develops over decades, with substantial neurological loss occurring before a clinical diagnosis of dementia can be rendered. It is therefore imperative to develop methods that permit early detection and monitoring of disease progression. In addition, the multifactorial pathogenesis of AD has identified several potential avenues for AD intervention. Thus, evaluation of therapeutic candidates over lengthy trial periods also demands a practical, noninvasive method for measuring Aβ in the brain. Magnetic resonance imaging (MRI) is the obvious choice for such measurements, but contrast enhancement for Aβ has only been achieved using Gd(III)-based agents. There is great interest in gadolinium-free methods to image the brain. In this study, we provide the first demonstration that a nitroxide-based small-molecule produces MRI contrast in brain specimens with elevated levels of Aβ. The molecule is comprised of a  fluorene (a molecule with high affinity for Aβ) and a nitroxide spin label (a paramagnetic MRI contrast species). Labeling of brain specimens with the spin-labeled fluorene produces negative contrast in samples from AD model mice whereas no negative contrast is seen in specimens harvested from wild-type mice. Injection of spin-labeled fluorene into live mice resulted in good brain penetration, with the compound able to generate contrast 24-h post injection. These results provide a proof of concept method that can be used for early, noninvasive, gadolinium-free detection of amyloid plaques by MRI.
The Brain's Structural Connectome Mediates the Relationship between Regional Neuroimaging Biomarkers in Alzheimer's Disease.Friday, December 02, 2016
Pandya S, Kuceyeski A, Raj A,
Journal of Alzheimer's disease : JAD. 28-Nov-2016
Alzheimer's disease (AD), one of the most common causes of dementia in adults, is a progressive neurodegenerative disorder exhibiting well-defined neuropathological hallmarks. It is known that disease pathology involves misfolded amyloid-β (Aβ) and tau proteins, and exhibits a relatively stereotyped progression over decades. The relationship between AD neuropathological hallmarks (Aβ, hypometabolism, and tau proteins) and imaging biomarkers (MRI, AV-45/FDG-PET) is not fully understood. In addition, biomarker pathologies are oftentimes discordant, wherein it may show varying levels of abnormality across brain regions. Evidence based on recent elucidation of trans-neuronal "prion-like" transmission and other available data already suggests that disease spread follows the brain's fiber connectivity network. Thereby, the brain's connectome information can be used to predict the process of disease spread in AD. A recently established mathematical model of AD pathology spread using a connectome-based network diffusion model was successful in encapsulating neurodegenerative progression. Motivated by these network-based findings, the current study explores whether and how network connectivity mediates the interactions between various AD biomarkers. We hypothesized that the structural connectivity matrix will mediate the cross-sectional association between regional AD-associated hypometabolism and Aβ deposition. Given recent reports of inherent or lifetime activity of brain regions as strong predictors of Aβ deposition in patients, we also tested whether healthy metabolism exerts a network-mediated effect on Aβ deposition and hypometabolism in AD patients. We found that regional Aβ deposition is best predicted by a linear combination of both regional healthy local metabolism and connectome-mediated regional healthy metabolism.
Immunoglobulin superfamily genes are novel prognostic biomarkers for breast cancer.Friday, December 02, 2016
Li Y, Guo M, Fu Z, Wang P, Zhang Y, Gao Y, Yue M, Ning S, Li D,
Oncotarget. 29-Nov-2016
Breast cancer progression is associated with dysregulated expression of the immunoglobulin superfamily (IgSF) genes that are involved in cell-cell recognition, binding and adhesion. Despite widespread evidence that many IgSF genes could serve as effective biomarkers, this potential has not been realized because the studies have focused mostly on individual genes and not the entire network. To gain a global perspective of the IgSF-related biomarkers, we constructed an IgSF-directed neighbor network (IDNN) and an IgSF-directed driver network (IDDN) by integrating multiple levels of data, including IgSF genes, breast cancer driver genes, protein-protein interaction (PPI) networks and gene expression profiling data. Our study shows that IgSF genes in the PPI network have important topological features related to cancer. Most IgSF genes are either cancer driver genes themselves or associated with them. We also identified a 21-gene IgSF network module with enriched mutations that are associated with overall survival based on 450 breast cancer patient samples extracted from The Cancer Genome Atlas (TCGA) and multiple independent microarray validation datasets. These results highlight the potential of IgSF genes as novel diagnostic, prognostic and therapeutic targets for breast cancer.
SAHA and/or MG132 reverse the aggressive phenotypes of glioma cells: An in vitro and vivo study.Friday, December 02, 2016
Yang XF, Zhao ZJ, Liu JJ, Yang XH, Gao Y, Zhao S, Shi S, Huang KQ, Zheng HC,
Oncotarget. 29-Nov-2016
To elucidate the anti-tumor effects and molecular mechanisms of SAHA (a histone deacetylase inhibitor) and MG132 (a proteasome inhibitor) on the aggressive phenotypes of glioma cells, we treated U87 and U251 cells with SAHA or/and MG132, and detected phenotypes' assays with phenotype-related molecules examined. It was found that SAHA or/and MG132 treatment suppressed proliferation in both concentration- and time-dependent manners, inhibited energy metabolism, migration, invasion and lamellipodia formation, and induced G2 arrest and apoptosis in the glioma cells. The treatment with SAHA increased the expression of acetyl-histones 3 and 4, which were recruited to the promoters of p21, p27, Cyclin D1, c-myc and Nanog to down-regulate their transcriptional levels. Expression of acetyl-histones 3 and 4 was higher in gliomas than normal brain tissues. Both drugs' exposure suppressed tumor growth in nude mice by inducing apoptosis and inhibiting proliferation, but increased serum aminotransferase and creatinine. These results indicated that SAHA and/or MG132 may suppress the aggressive phenotypes of glioma cells. They might be employed to treat the glioma if both hepatic and renal injuries are prevented.
Advances in Capillary Electrophoresis and the Implications for Drug Discovery.Friday, December 02, 2016
Ouimet CM, D'Amico CI, Kennedy RT,
Expert opinion on drug discovery. 02-Dec-2016
Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery. Areas Covered: This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described. Expert opinion: Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.
Optimization of a textile dye degradation in a recirculating fluidized-bed reactor using magnetite/S2O8(2-) process.Friday, December 02, 2016
Aghdasinia H, Arehjani P, Vahid B, Khataee A,
Environmental technology. 02-Dec-2016
Optimization of Acid Orange 7 (AO7) treatment using heterogeneous Fenton-like method in a recirculating fluidized-bed reactor (FBR) was investigated by central composite design (CCD). Natural magnetite (NM) as Fenton-like catalyst was characterized using scanning electron microscopy (SEM). Nonlinear CCD model was obtained for prediction of dye degradation as a function of experimental variables such as peroxydisulfate concentration (0.1-0.5 mmol/L), initial AO7 concentration (5-25 mg/L), pH (3-9) and NM dosage (0.25-1.25 g/L) after 105 min of treatment. The calculated results by the model were in consistent with the experimental results (R(2)= 0.959). Furthermore, the model is suitable to estimate the optimum operational conditions and determine the effects of the parameters for maximum AO7 degradation. Eventually, gas chromatography-mass spectroscopy (GC-MS) was used for recognition of the dye degradation by-products.
Validation of antibody reagents for mucin analysis in chronic inflammatory airway diseases.Friday, December 02, 2016
Krause T, Röckendorf N, Gaede KI, Ramaker K, Sinnecker H, Frey A,
mAbs. 02-Dec-2016
In chronic inflammatory airway diseases, mucins display disease-related alterations in quantity, composition and glycosylation. This opens the possibility to diagnose and monitor inflammatory airway disorders and their exacerbation based on mucin properties. For such an approach to be reasonably versatile and diagnostically meaningful, the mucin of interest must be captured in a reliable, patient-independent way. To identify appropriate mucin-specific reagents, we tested anti-mucin antibodies on mucin-content-standardized, human bronchoalveolar lavage fluid samples in immunoblot assays. All commercially available monoclonal antibodies against the major airway mucin MUC5AC were screened, except for those with known specificity for carbohydrates, as glycosylation patterns are not mucin-specific. Our results indicated considerable inter-patient and inter-antibody variability in mucin recognition for all antibodies and samples tested. The best results in terms of signal strength and reproducibility were obtained with antibodies Mg-31, O.N.457 and 45M1. Additional epitope mapping experiments revealed that only one of the antibodies with superior binding to MUC5AC recognized linear peptide epitopes on the protein backbone.
An update on the use of cerebrospinal fluid analysis as a diagnostic tool in multiple sclerosis.Friday, December 02, 2016
Gastaldi M, Zardini E, Franciotta D,
Expert review of molecular diagnostics. 02-Dec-2016
Intrathecal B-lymphocyte activation is a hallmark of multiple sclerosis (MS), a multi-factorial inflammatory-demyelinating disease of the central nervous system. Such activation has a counterpart in the cerebrospinal fluid (CSF) oligoclonal IgG bands (OCB), whose diagnostic role in MS has been downgraded within the current McDonald's criteria. With a theoretico-practical approach, the authors review the physiopathological basis of the CSF dynamics, and the state-of-the-art of routine CSF analysis and CSF biomarkers in MS. Areas covered: The authors discuss pros and cons of CSF analysis, including critical evaluations of both well-established, and promising diagnostic and prognostic laboratory tools. New acquisitions on the CSF and cerebral interstitial fluid dynamics are also presented. The authors searched the PubMed database for English-language articles reported between January 2010 and June 2016, using the key words 'multiple sclerosis', 'cerebrospinal fluid', 'oligoclonal bands'. Reference lists of relevant articles were scanned for additional studies. Expert commentary: The availability of performing high-quality, routine CSF tests in specialized laboratories, the emerging potential of novel CSF biomarkers, and the trend for early treatments should induce a reappraisal of CSF analysis for diagnostic and prognostic purposes in MS. Further procedural and methodological improvements seem to be necessary in both research and translational diagnostic CSF settings.
Phase II Study of Dasatinib in Previously Treated Patients with Advanced Non-Small Cell Lung Cancer.Friday, December 02, 2016
Kelley MJ, Jha G, Shoemaker D, Herndon JE, Gu L, Barry WT, Crawford J, Ready N,
Cancer investigation. 02-Dec-2016
The Src pathway in activated in about one-third of non-small cell lung cancer (NSCLC) tumors. Dasatinib has Src-inhibitor activity. We examined the activity of dasatinib in 37 patients with advanced, previously treated NSCLC. Among the 29 patients who underwent pre-treatment biopsy for RNA biomarker analysis, 25 were treated with dasatinib 70 mg twice daily. There were no responses. Five patients discontinued treatment due to toxicity. Three patients had minor biopsy-related pneumothoraces. Given the lack of responses, no biomarkers were analyzed. Dasatinib 70 mg twice daily does not have activity nor is it well tolerated in unselected patients with advanced stage, previously treated NSCLC.
Upregulation of T-cell Immunoglobulin and Mucin-Domain Containing-3 (Tim-3) in Monocytes/Macrophages Associates with Gastric Cancer Progression.Friday, December 02, 2016
Wang Z, Yin N, Zhang Z, Zhang Y, Zhang G, Chen W,
Immunological investigations. 02-Dec-2016
T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) is an important immune regulatory molecule in cancer immune system. However, expression and function of Tim-3 in monocytes/macrophages in cancer progression mainly remain unclear. In this study, we analyzed Tim-3 levels in peripheral blood mononuclear cells (PBMCs) from 62 gastric cancer patients and 45 healthy controls using flow cytometry and then associated Tim-3 levels with clinical pathological data from patients. We found Tim-3 level was significantly upregulated in monocytes from gastric cancer patients compared with those from healthy controls, and that upregulated Tim-3 levels associated with depth of tumor invasion and tumor lymph node metastasis and advanced clinical stages of gastric cancer patients. Furthermore, tumor-bearing mouse experiments revealed that Tim-3 level on monocytes/macrophages associated with xenograft formation and growth. In addition, culture of monocytes from healthy controls with gastric cancer cell-conditioned medium upregulated Tim-3 expression, but IL-10, TNF-α, IFN-γ, or GM-CSF treatment or T-bet, Eomes, and T-bet/Eomes double gene knockout did not affect Tim-3 levels in blood monocytes/macrophages from human or mouse, respectively. Gal-9/Tim-3 signal was able to significantly stimulate monocyte to secrete IL-6, IL-8, and IL-10, but not IL-1β, IL-12p70, or TNF-α in presence of LPS. In conclusion, our study demonstrated that Tim-3 expressed by monocyte/macrophages might be an important mechanism in gastric cancer progression.
Tools for outcome prediction in patients with community acquired pneumonia.Friday, December 02, 2016
Khan F, Owens MB, Restrepo M, Povoa P, Martin-Loeches I,
Expert review of clinical pharmacology. 02-Dec-2016
Community-acquired pneumonia (CAP) is one of the most common causes of mortality world-wide. The mortality rate of patients with CAP is influenced by the severity of the disease, treatment failure and the requirement for hospitalization and/or intensive care unit (ICU) management, all of which may be predicted by biomarkers and clinical scoring systems. Areas Covered: We review the recent literature examining the efficacy of established and newly-developed clinical scores, biological and inflammatory markers such as C-Reactive protein (CRP), procalcitonin (PCT) and Interleukin-6 (IL-6), whether used alone or in conjunction with clinical severity scores to assess the severity of CAP, predict treatment failure, guide acute in-hospital or ICU admission and predict mortality. Expert Commentary: The early prediction of treatment failure using clinical scores and biomarkers plays a developing role in improving survival of patients with CAP by identifying high-risk patients requiring hospitalization or ICU admission; and may enable more efficient allocation of resources. However, it is likely that combinations of scoring systems and biomarkers will be of greater use than individual markers. Further larger studies are needed to corroborate the additive value of these markers to clinical prediction scores to provide a safer and more effective assessment tool for clinicians.
Synthesis and Cytotoxicity of Dendritic Platinum Nanoparticles with HEK-293 Cells.Friday, December 02, 2016
Shim K, Kim J, Heo YU, Jiang B, Li C, Shahabuddin M, Wu KC, Hossain MS, Yamauchi Y, Kim JH,
Chemistry, an Asian journal. 02-Dec-2016
Dendritic platinum nanoparticles (DPNs) have been synthesized from l-ascorbic acid and an amphiphilic non-ionic surfactant (Brij-58) via a sonochemical method. The particle size and shape of the DPNs could be tuned by changing the reduction temperature, resulting in a uniform DPN with a size of 23 nm or 60 nm. The facets of DPNs have been studied by high-resolution transmission electron microscopy. The cytotoxicity of DPNs has been investigated using human embryonic kidney cells (HEK-293), and the biological adaptability exhibited by DPNs has opened a pathway to biomedical applications such as drug-delivery systems, photothermal treatment, and biosensors.
Relationship between serum cytokines receptors and matrix metalloproteinase 9 levels and coronary artery disease.Friday, December 02, 2016
Mirhafez SR, Avan A, Tajfard M, Mohammadi S, Moohebati M, Fallah A, Ghazavi H, Savadi H, Ghayour Mobarhan M,
Journal of clinical laboratory analysis. 02-Dec-2016
We demonstrate the significant correlation of MMP-9 with CAD with sensitivity of 95.45%, suggesting its role as a biomarker in CAD patients. Further studies in larger population - preferably multicenter setting - are warranted to explore the functional role of this marker in coronary artery disease.
Investigation and intervention of autophagy to guide cancer treatment with nanogels.Friday, December 02, 2016
Zhang X, Liang X, Gu J, Chang D, Zhang J, Chen Z, Ye Y, Wang C, Tao W, Zeng X, Liu G, Zhang Y, Mei L, Gu Z,
Nanoscale. 02-Dec-2016
Cancer cells use autophagy to resist poor survival environmental conditions such as low PH, poor nutrients as well as chemical therapy. Nanogels have been used as efficient chemical drug carriers for cancer treatment. However, the effect of nanogels on autophagy is still unknown. Here, we used Rab proteins as the marker of multiple trafficking vesicles in endocytosis and LC3 as the marker of autophagy to investigate the intracellular trafficking network of Rhodamine B (Rho)-labeled nanogels. The nanogels were internalized by the cells through multiple protein dependent endocytosis and micropinocytosis. After inception by the cells, the nanogels were transported into multiple Rab positive vesicles including early endosomes (EEs), late endosomes (LEs), recycling endosomes (REs) and lipid droplets. Finally, these Rab positive vesicles were transported to lysosome. In addition, GLUT4 exocytosis vesicles could transport the nanogels out of the cells. Moreover, nanogels could induce autophagy and be sequestered in autophagosomes. The crosstalk between autophagosomes and Rab positive vesicles were investigated, we found that autophagosomes may receive nanogels through multiple Rab positive vesicles. Co-delivery of autophagy inhibitors such as chloroquine (CQ) and the chemotherapeutic drug doxorubicin (DOX) by nanogels blocked the autophagy induced by DOX greatly decreasing both of the volume and weight of the tumors in mice tumor models. Investigation and intervention of the autophagy pathway could provide a new method to improve the therapeutic effect of anticancer nanogels.
Heterogeneous PEGylation of diamond nanoparticles.Friday, December 02, 2016
Barnard AS,
Nanoscale. 02-Dec-2016
Coating the surfaces of inorganic nanoparticles with polyethylene glycol (PEG) is an important step in the development of many nanoparticle-based drug delivery systems. The efficiency with which drug molecules can be loaded on to nanoparticle surfaces is contingent on the concentration, distribution and stability of the PEG coating. In this study the distribution and relative stability of PEG on diamond nanoparticles is predicted, for clean and passivated surface structures, in 3D. This is an ideal exemplar, since PEGylated diamond nanoparticles are already being trialed as carriers for doxorubicin (DOX). The results show that PEGylation is favorable near the {100} facets regardless of surface reconstructions or pre-treatment, but pre-treatment is required to increase the probability of stable and homogeneous PEGylation on other facets.
Human antibody 3E1 targets the HA stem region of H1N1 and H5N6 influenza A viruses.Friday, December 02, 2016
Wang W, Sun X, Li Y, Su J, Ling Z, Zhang T, Wang F, Zhang H, Chen H, Ding J, Sun B,
Nature communications. 02-Dec-2016
As influenza A viruses remain a major threat to human health worldwide, the discovery of broadly neutralizing monoclonal antibodies that recognize conserved epitopes would facilitate the development of antibody-based therapeutic strategies. Here we report that a VH4-4-encoded human mAb named 3E1 could neutralize H1 and H5 subtype viruses in vitro and protect mice against the H1N1 and H5N6 viruses by inhibiting the low pH-induced conformational rearrangement of haemagglutinin (HA), hence blocking membrane fusion. The crystal structures of 3E1 Fab in complex with HA of two H1N1 strains reveal that 3E1, with both heavy and light chains, binds to a conserved epitope of the HA stem region, comprising parts of the fusion peptide, the F subdomain and the outermost β-strand preceding helix A. Altogether, these data suggest the potential of 3E1 as a therapeutic drug against H1 and H5 subtype viruses.
Disentangling the roles of cholesterol and CD59 in intermedilysin pore formation.Friday, December 02, 2016
Boyd CM, Parsons ES, Smith RA, Seddon JM, Ces O, Bubeck D,
Scientific reports. 02-Dec-2016
The plasma membrane provides an essential barrier, shielding a cell from the pressures of its external environment. Pore-forming proteins, deployed by both hosts and pathogens alike, breach this barrier to lyse target cells. Intermedilysin is a cholesterol-dependent cytolysin that requires the human immune receptor CD59, in addition to cholesterol, to form giant β-barrel pores in host membranes. Here we integrate biochemical assays with electron microscopy and atomic force microscopy to distinguish the roles of these two receptors in mediating structural transitions of pore formation. CD59 is required for the specific coordination of intermedilysin (ILY) monomers and for triggering collapse of an oligomeric prepore. Movement of Domain 2 with respect to Domain 3 of ILY is essential for forming a late prepore intermediate that releases CD59, while the role of cholesterol may be limited to insertion of the transmembrane segments. Together these data define a structural timeline for ILY pore formation and suggest a mechanism that is relevant to understanding other pore-forming toxins that also require CD59.
High-throughput chinmedomics-based prediction of effective components and targets from herbal medicine AS1350.Friday, December 02, 2016
Liu Q, Zhang A, Wang L, Yan G, Zhao H, Sun H, Zou S, Han J, Ma CW, Kong L, Zhou X, Nan Y, Wang X,
Scientific reports. 02-Dec-2016
This work was designed to explore the effective components and targets of herbal medicine AS1350 and its effect on "Kidney-Yang Deficiency Syndrome" (KYDS) based on a chinmedomics strategy which is capable of directly discovering and predicting the effective components, and potential targets, of herbal medicine. Serum samples were analysed by UPLC-MS combined with pattern recognition analysis to identify the biomarkers related to the therapeutic effects. Interestingly, the effectiveness of AS1350 against KYDS was proved by the chinmedomics method and regulated the biomarkers and targeting of metabolic disorders. Some 48 marker metabolites associated with alpha-linolenic acid metabolism, fatty acid metabolism, sphingolipids metabolism, phospholipid metabolism, steroid hormone biosynthesis, and amino acid metabolism were identified. The correlation coefficient between the constituents in vivo and the changes of marker metabolites were calculated by PCMS software and the potential effective constituents of AS1350 were also confirmed. By using chinmedomics technology, the components in AS1350 protecting against KYDS by re-balancing metabolic disorders of fatty acid metabolism, lipid metabolism, steroid hormone biosynthesis, etc. were deduced. These data indicated that the phenotypic characterisations of AS1350 altering the metabolic signatures of KYDS were multi-component, multi-pathway, multi-target, and overall regulation in nature.
BHX Inhibits the Wnt Signaling Pathway by Suppressing β-catenin Transcription in the Nucleus.Friday, December 02, 2016
Ding F, Wang M, Du Y, Du S, Zhu Z, Yan Z,
Scientific reports. 02-Dec-2016
BHX (N-(4-hydroxybenzyl)-1,3,4-triphenyl-4,5-dihydro-1H-pyrazole-5-carboxamide), a Wnt signaling pathway inhibitor, effectively inhibits tumor cell growth, but the underlying mechanism is unclear. Thus, we aim to investigate the effects and associated mechanism of BHX action on A549 and MCF-7 cell lines. In our study, MTT(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and xenograft model assay indicated that cell growth was inhibited by BHX at a range of concentrations in vitro and in vivo. The expression of β-catenin and Wnt signaling pathway downstream target genes were decreased evidently under BHX treatment. Flow cytometry also revealed that BHX treatment significantly induced G1 arrest. Further analysis showed that BHX lowered the transcriptional level of β-catenin. In conclusion, BHX inhibited the nuclear synthesis of β-catenin, thereby suppressing the Wnt signaling pathway and further inhibiting tumor growth and proliferation.
Prostate Cancer Patients-Negative Biopsy Controls Discrimination by Untargeted Metabolomics Analysis of Urine by LC-QTOF: Upstream Information on Other Omics.Friday, December 02, 2016
Fernández-Peralbo MA, Gómez-Gómez E, Calderón-Santiago M, Carrasco-Valiente J, Ruiz-García J, Requena-Tapia MJ, Luque de Castro MD, Priego-Capote F,
Scientific reports. 02-Dec-2016
The existing clinical biomarkers for prostate cancer (PCa) diagnosis are far from ideal (e.g., the prostate specific antigen (PSA) serum level suffers from lack of specificity, providing frequent false positives leading to over-diagnosis). A key step in the search for minimum invasive tests to complement or replace PSA should be supported on the changes experienced by the biochemical pathways in PCa patients as compared to negative biopsy control individuals. In this research a comprehensive global analysis by LC-QTOF was applied to urine from 62 patients with a clinically significant PCa and 42 healthy individuals, both groups confirmed by biopsy. An unpaired t-test (p-value < 0.05) provided 28 significant metabolites tentatively identified in urine, used to develop a partial least squares discriminant analysis (PLS-DA) model characterized by 88.4 and 92.9% of sensitivity and specificity, respectively. Among the 28 significant metabolites 27 were present at lower concentrations in PCa patients than in control individuals, while only one reported higher concentrations in PCa patients. The connection among the biochemical pathways in which they are involved (DNA methylation, epigenetic marks on histones and RNA cap methylation) could explain the concentration changes with PCa and supports, once again, the role of metabolomics in upstream processes.
Glial pannexin1 contributes to tactile hypersensitivity in a mouse model of orofacial pain.Friday, December 02, 2016
Hanstein R, Hanani M, Scemes E, Spray DC,
Scientific reports. 02-Dec-2016
Drug studies in animal models have implicated pannexin1 (Panx1) in various types of pain, including trigeminal hypersensitivity, neuropathic pain and migraine. However, the tested drugs have limited specificity and efficacy so that direct evidence for Panx1 contribution to pain has been lacking. We here show that tactile hypersensitivity is markedly attenuated by deletion of Panx1 in a mouse model of chronic orofacial pain; in this model, trigeminal ganglion Panx1 expression and function are markedly enhanced. Targeted deletion of Panx1 in GFAP-positive glia or in neurons revealed distinct effects. Panx1 deletion in GFAP-positive glia cells prevented hypersensitivity completely, whereas deletion of neuronal Panx1 reduced baseline sensitivity and the duration of hypersensitivity. In trigeminal ganglia with genetically encoded Ca(2+) indicator in GFAP-positive glia or in neurons, both cell populations were found to be hyperactive and hyper-responsive to ATP. These novel findings reveal unique roles for GFAP-positive glial and neuronal Panx1 and describe new chronic pain targets for cell-type specific intervention in this often intractable disease.
Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.Friday, December 02, 2016
Barahona E, Jiménez-Vicente E, Rubio LM,
Scientific reports. 02-Dec-2016
When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.
Elevated cerebral spinal fluid biomarkers in children with mucopolysaccharidosis I-H.Friday, December 02, 2016
Raymond GV, Pasquali M, Polgreen LE, Dickson PI, Miller WP, Orchard PJ, Lund TC,
Scientific reports. 02-Dec-2016
Mucopolysaccharidosis (MPS) type-IH is a lysosomal storage disease that results from mutations in the IDUA gene causing the accumulation of glycosaminoglycans (GAGs). Historically, children with the severe phenotype, MPS-IH (Hurler syndrome) develop progressive neurodegeneration with death in the first decade due to cardio-pulmonary complications. New data suggest that inflammation may play a role in MPS pathophysiology. To date there is almost no information on the pathophysiologic changes within the cerebral spinal fluid (CSF) of these patients. We evaluated the CSF of 25 consecutive patients with MPS-IH. While CSF glucose and total protein were within the normal range, we found a significantly mean elevated CSF opening pressure at 24 cm H2O (range 14-37 cm H2O). We observed a 3-fold elevation in CSF heparan sulfate and a 3-8 fold increase in MPS-IH specific non-reducing ends, I0S0 and I0S6. Cytokine analyses in CSF of children with MPS-IH showed significantly elevated inflammatory markers including: MCP-1 SDF-1a, IL-Ra, MIP-1b, IL-8, and VEGF in comparison to unaffected children. This is the largest report of CSF characteristics in children with MPS-IH. Identification of key biomarkers may provide further insight into the inflammatory-mediated mechanisms related to MPS diseases and perhaps lead to improved targeted therapies.
A comprehensive map of molecular drug targets.Friday, December 02, 2016
Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B, Hersey A, Oprea TI, Overington JP,
Nature reviews. Drug discovery. 02-Dec-2016
The success of mechanism-based drug discovery depends on the definition of the drug target. This definition becomes even more important as we try to link drug response to genetic variation, understand stratified clinical efficacy and safety, rationalize the differences between drugs in the same therapeutic class and predict drug utility in patient subgroups. However, drug targets are often poorly defined in the literature, both for launched drugs and for potential therapeutic agents in discovery and development. Here, we present an updated comprehensive map of molecular targets of approved drugs. We curate a total of 893 human and pathogen-derived biomolecules through which 1,578 US FDA-approved drugs act. These biomolecules include 667 human-genome-derived proteins targeted by drugs for human disease. Analysis of these drug targets indicates the continued dominance of privileged target families across disease areas, but also the growth of novel first-in-class mechanisms, particularly in oncology. We explore the relationships between bioactivity class and clinical success, as well as the presence of orthologues between human and animal models and between pathogen and human genomes. Through the collaboration of three independent teams, we highlight some of the ongoing challenges in accurately defining the targets of molecular therapeutics and present conventions for deconvoluting the complexities of molecular pharmacology and drug efficacy.
Noncontact and Wide-Field Characterization of the Absorption and Scattering Properties of Apple Fruit Using Spatial-Frequency Domain Imaging.Friday, December 02, 2016
Hu D, Fu X, He X, Ying Y,
Scientific reports. 02-Dec-2016
Spatial-frequency domain imaging (SFDI), as a noncontact, low-cost and wide-field optical imaging technique, offers great potential for agro-product safety and quality assessment through optical absorption (μa) and scattering (μ) property measurements. In this study, a laboratory-based SFDI system was constructed and developed for optical property measurement of fruits and vegetables. The system utilized a digital light projector to generate structured, periodic light patterns and illuminate test samples. The diffuse reflected light was captured by a charge coupled device (CCD) camera with the resolution of 1280 × 960 pixels. Three wavelengths (460, 527, and 630 nm) were selected for image acquisition using bandpass filters in the system. The μa and μ were calculated in a region of interest (ROI, 200 × 300 pixels) via nonlinear least-square fitting. Performance of the system was demonstrated through optical property measurement of 'Redstar' apples. Results showed that the system was able to acquire spatial-frequency domain images for demodulation and calculation of the μa and μ. The calculated μa of apple tissue experiencing internal browning (IB) were much higher than healthy apple tissue, indicating that the SFDI technique had potential for IB tissue characterization.
Multiparameter mechanical and morphometric screening of cells.Friday, December 02, 2016
Masaeli M, Gupta D, O'Byrne S, Tse HT, Gossett DR, Tseng P, Utada AS, Jung HJ, Young S, Clark AT, Di Carlo D,
Scientific reports. 02-Dec-2016
We introduce a label-free method to rapidly phenotype and classify cells purely based on physical properties. We extract 15 biophysical parameters from cells as they deform in a microfluidic stretching flow field via high-speed microscopy and apply machine-learning approaches to discriminate different cell types and states. When employing the full 15 dimensional dataset, the technique robustly classifies individual cells based on their pluripotency, with accuracy above 95%. Rheological and morphological properties of cells while deforming were critical for this classification. We also show the application of this method in accurate classifying cells based on their viability, drug screening and detecting populations of malignant cells in mixed samples. We show that some of the extracted parameters are not linearly independent, and in fact we reach maximum classification accuracy by using only a subset of parameters. However, the informative subsets could vary depending on cell types in the sample. This work shows the utility of an assay purely based on intrinsic biophysical properties of cells to identify changes in cell state. In addition to a label-free alternative to flow cytometry in certain applications, this work, also can provide novel intracellular metrics that would not be feasible with labeled approaches (i.e. flow cytometry).
Paper-based CRP Monitoring Devices.Friday, December 02, 2016
Lin SC, Tseng CY, Lai PL, Hsu MY, Chu SY, Tseng FG, Cheng CM,
Scientific reports. 02-Dec-2016
Here, we discuss the development of a paper-based diagnostic device that is inexpensive, portable, easy-to-use, robust, and capable of running simultaneous tests to monitor a relevant inflammatory protein for clinical diagnoses i.e. C-reactive protein (CRP). In this study, we first attempted to make a paper-based diagnostic device via the wax printing method, a process that was used in previous studies. This device has two distinct advantages: 1) reduced manufacturing and assay costs and operation duration via using wax printing method to define hydrophobic boundaries (for fluidic devices or general POC devices); and, 2) the hydrophilicity of filter paper, which is used to purify and chromatographically correct interference caused by whole blood components with a tiny amount of blood sample (only 5 μL). Diagnosis was based on serum stain length retained inside the paper channels of our device. This is a balanced function between surface tension and chromatographic force following immune reactions (CRP assays) with a paper-embedded biomarker.
Inflammatory markers associated with abdominal aortic aneurysm.Friday, December 02, 2016
Lindberg S, Zarrouk M, Holst J, Gottsäter A,
European cytokine network. 01-Sep-2016
Several inflammatory biomarkers were significantly elevated and correlated with aortic diameter among 65-year old men with AAA at ultrasound screening. IL-6, homocysteine and use of antihypertensive medication remained elevated in the logistic regression model, together with known risk markers for AAA such as smoking and signs of atherosclerosis.
LL-37, HNP-1, and HBD2/3 modulate the secretion of cytokines TNF-α, IL-6, IFN-γ, IL-10 and MMP1 in human primary cell cultures.Friday, December 02, 2016
Medina Santos CE, López Hurtado CN, Rivas Santiago B, Gonzalez-Amaro R, Cataño Cañizales YG, Martínez Fierro ML, Enciso-Moreno JA, García Hernández MH,
European cytokine network. 01-Sep-2016
The aim of this study was to evaluate the effects of the LL-37, HNP-1 and HBD2/3 peptides on cytokine and MMP production in human polymorphonuclear cells, mononuclear cells and chondrocytes. The levels of cytokines in supernatants from mononuclear and polymorphonuclear cell cultures were measured with a cytometric bead array by flow cytometry. Likewise, the levels of metalloproteinase/MMP-1, 3, and 13 were measured in supernatants from chondrocyte cultures using an ELISA. The expression of RANKL on lymphocytes was analyzed by flow cytometry. We observed increased levels of TNF-α, IL-6 and IL-10 in mononuclear and polymorphonuclear cell cultures stimulated with HBD-2/3. We also observed increased levels of IFN-γ, IL-10, and IL-6 in mononuclear cell cultures stimulated with HNP-1, and increased IL-6 levels were observed in polymorphonuclear cell cultures exposed to HNP-1. We also found that the MMP-1 level increased in the chondrocyte cultures stimulated with HBD-3, whereas the MMP-1 level was decreased in cultures exposed to LL-37. The present report is the first study to determine that HNP-1and HBD2/3 promote the secretion of pro-inflammatory cytokines by polymorphonuclear and mononuclear cells and the secretion of MMP by chondrocytes, whereas LL-37 diminishes MMP1 secretion. Our results suggest that HBD-2/3 and HNP1 might play a pathological role in rheumatoid arthritis, while LL-37 might have a protective role.
Methoxyflavones from New Lingzhi Medicinal Mushroom, Ganoderma lingzhi (Agaricomycetes).Friday, December 02, 2016
Shimizu K, Amen YM, Kaifuchi S,
International journal of medicinal mushrooms. 2016
Ganoderma lingzhi is one of the most famous medicinal fungi in the world. It has been used in folk medicine, especially in East Asian countries. It is also a white-rot fungus with strong wood degradation ability, especially against lignin. Different classes of bioactive natural products have been reported in Ganoderma, including triterpenes, polysaccharides, sterols, and peptides. The triterpenes and polysaccharides are the primary bioactive compounds of Ganoderma. We report for the first time the presence of 3 methoxyflavones as minor constituents in G. linghzi. The 3 compounds were identified based on different spectroscopic techniques, including 1- and 2-dimensional nuclear magnetic resonance (1H-1H correlation spectroscopy, heteronuclear single quantum coherence, and heteronuclear multiple bond correlation) and mass spectrometry (high-resolution electrospray ionization mass spectrometry). Our report provides an approach to a possible biosynthetic pathway for biosynthetic genes in the mushrooms. Another great possibility is that these compounds may exist or be formed through degradation of the components in the woody substrate, such as lignin, and then subsequently translocate to the fruiting bodies.
Molecular Pathology of Malignant Transformation of Oral Submucous Fibrosis.Friday, December 02, 2016
Chattopadhyay A, Ray JG,
Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer. 2016
Oral submucous fibrosis (OSF) is prevalent mostly in Southeast Asia, particularly in the Indian subcontinent. Chewing betel nuts and betel leaves, with or without tobacco, has been associated with OSF. Betel quid contents including guvacine, arecoline, guvacoline, arecaidine, and chavibetol are considered to play an important part in the occurrence of OSF. Transformation of OSF to squamous cell carcinoma (SCC) is variable, but up to 13% conversion of OSF to SCC has been reported. Various genetic and molecular mechanisms impact the malignant transformation of OSF, causing changes in the cell cycle, DNA, keratinocytes, and keratin; tumor-cell proliferation and survival; angiogenesis; fibrosis through epithelial-mesenchymal transitions (EMTs), and tissue hypoxia. All are reviewed here, including potential biomarkers for malignant transformation of OSF. These interactions are not fully understood, but a critical mass of knowledge is building up to ultimately allow the understanding of all mechanisms involved.
Addition of Zeolites to Improve the Functional Characteristics of the Hen of the Wood or Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).Friday, December 02, 2016
Vunduk J, Klaus A, Kozarski M, Dordevic R, Miladinovic Z, Jovanovic L, Niksic M, Van Griensven LJ,
International journal of medicinal mushrooms. 2016
Kaolinite and the modified natural zeolite minazel plus (M+) were used as supplements in substrate used for the production of the medicinal mushroom Grifola frondosa. Growth stimulation, expressed as yield and biological efficiency, was observed when M+ (1%) was added. The production cycle was shortened by half as a result of the zeolites' ion-exchange ability, stimulation of enzyme activity, and water retain capacity. Inductively coupled plasma optical emission spectrometry of fruiting bodies showed the absence of heavy metals (arsenic, cadmium, and lead), whereas the concentration of calcium increased greatly and the concentrations of iron, magnesium, and zinc increased moderately under the influence of M+. Solid-state nuclear magnetic resonance showed a positive impact on the β-glucan ratio, which could have been caused by the epimerization reaction stimulated by zeolites. The functionality of the mushroom was evaluated through several antioxidant activity assays, and in all cases a positive effect was established: M+ was statistically more effective in comparison with kaolinite. A strong correlation was established between the antioxidative activity of cultivated fruiting bodies and the tested compounds (total phenolic compounds, carbohydrates, and minerals).
Pharmacological Properties of Biocompounds from Spores of the Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes): A Review.Friday, December 02, 2016
Soccol CR, Bissoqui LY, Rodrigues C, Rubel R, Sella SR, Leifa F, de Souza Vandenberghe LP, Soccol VT,
International journal of medicinal mushrooms. 2016
Ganoderma lucidum is a well-known representative of mushrooms that have been used in traditional Chinese medicine for centuries. New discoveries related to this medicinal mushroom and its biological properties are frequently reported. However, only recently have scientists started to pay special attention to G. lucidum spores. This is in part because of the recent development of methods for breaking the spore wall and extracting biocompounds from the spore. Although some research groups are working with G. lucidum spores, data in the literature are still limited, and the methods used have not been systematized. This review therefore describes the main advances in techniques for breaking the spore wall and extracting biocompounds from the spore. In addition, the major active components identified and their biological properties, such as neurological activity and antiaging and cell-protective effects, are investigated because these are of importance for potential drug development.
Gene Therapy for Rheumatoid Arthritis.Friday, December 02, 2016
Liu S, Maeyama K,
Critical reviews in immunology. 2016
With the aim of controlling disease relapse and bone deformation of individual joints, the application of gene therapy in rheumatoid arthritis (RA) has slowly progressed on a trial-and-error basis. Several new therapeutic targets have been identified in preclinical studies in animal models, although a limited number of gene-based clinical trials have been conducted. In this article, we summarize the status of gene therapy for RA by addressing issues related to innovating drug development. More disease- and target-specific preclinical tests are required to overcome the insufficient information regarding pharmacokinetics and toxicokinetics, which are related to safety issues in the field of RA gene therapy.
Submicron Emulsions and Their Applications in Oral Delivery.Friday, December 02, 2016
Mundada V, Patel M, Sawant K,
Critical reviews in therapeutic drug carrier systems. 2016
A "submicron emulsion" is an isotropic mixture of drug, lipids, and surfactants, usually with hydrophilic cosolvents and with droplet diameters ranging from 10 to 500 nm. Submicron emulsions are of increasing interest in medicine due to their kinetic stability, high solubilizing capacity, and tiny globule size. Because of these properties, they have been applied in various fields, such as personal care, cosmetics, health care, pharmaceuticals, and agrochemicals. Submicron emulsions are by far the most advanced nanoparticulate systems for the systemic delivery of biologically active agents for controlled drug delivery and targeting. They are designed mainly for pharmaceutical formulations suitable for various routes of administration like parenteral, ocular, transdermal, and oral. This review article describes the marked potential of submicron emulsions for oral drug delivery owing to their numerous advantages like reduced first pass metabolism, inhibition of P-glycoprotein efflux system, and enhanced absorption via intestinal lymphatic pathway. To overcome the limitations of liquid dosage forms, submicron emulsions can be formulated into solid dosage forms such as solid self-emulsifying systems. This article covers various types of submicron emulsions like microemulsion, nanoemulsion, and self-emulsifying drug delivery system (SEDDS), and their potential pharmaceutical applications in oral delivery with emphasis on their advantages, limitations, and advancements.
Review Article: Fabricated Microparticles: An Innovative Method to Minimize the Side Effects of NSAIDs in Arthritis.Friday, December 02, 2016
Abadi SS, Moin A, Veerabhadrappa GH,
Critical reviews in therapeutic drug carrier systems. 2016
Microparticles are polymeric bodies ranging 1-1000 µm that constitute a variety of forms such as microcapsules, microspheres, microcages, microshells, microrods, biosensors microparticles, radiolabeled microparticles, and so forth. This review focuses on general microparticles, mainly microcapsules and microspheres. Nonsteriodal anti-inflammatory drugs (NSAIDs) are one of the mostcommonly prescribed medications in the world. Most of the NSAIDs available have severe side effects. With increased awareness of NSAID-induced gastrointestinal (GI) side effects, safety has become a priority in treatment of arthritis and other inflammatory diseases with NSAIDs. A trend in NSAID development has been to improve therapeutic efficacy while reducing the severity of GI side effects by altering dosage through modified release to optimize drug delivery. One such approach is the use of fabricated microparticles such as microcapsules and microspheres as carriers of drugs. Microparticles provide delivery of macromolecules and micromolecules via different routes and effectively control the release profile of such drugs. Microcapsules and microspheres are compatible with most natural and synthetic polymers and can be used for several routes of administration, including parenteral, oral, nasal, intra-ocular, topical, and the like. Because of greater stability and multiple manufacturing techniques, microspheres and microcapsules are preferred as drug carriers over other colloidal drug delivery systems. Microparticles provide effective protection of the encapsulated agent against degradation by enzymatic activities, controlled and confined delivery of drugs from a few hours to months, and ingenious administration compared to alternative forms of controlled-release parenteral dosages, such as macro-sized implants. This comprehensive overview of fabricated microparticles describes microencapsulation technologies to produce microparticles for targeted therapy of arthritis and other inflammatory diseases which provide constant and prolonged therapeutic effects that reduce dosing frequency and thereby minimize potential adverse effects of NSAIDs such as GI irritation and insufficient patient compliance. The present review describes the latest developments in microparticulate drug delivery systems and the best alternatives for safe and effective microcapsular systems in a controlled manner for the delivery of NSAIDs.
Oral Squamous Cell Carcinoma: Current Treatment Strategies and Nanotechnology-Based Approaches for Prevention and Therapy.Friday, December 02, 2016
Gharat SA, Momin M, Bhavsar C,
Critical reviews in therapeutic drug carrier systems. 2016
Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer; it involves damage to oral epithelial cells due to accumulation of multiple genetic mutations in the cells. OSCC remains major cause of morbidity and mortality in patients with head and neck cancers. Tobacco, smoking, alcohol consumption alone or with chewing tobacco, and betel quid are potential carcinogens contributing to the high occurrence of OSCC. Current treatment modalities for OSCC like chemoradiotherapy, surgery, EGFR inhibitors and COX-2 inhibitors, and photodynamic therapy have led to the major problems related to non-specific cell death. Nanoengineered systems offer solutions to these problems that not only minimize the major drawbacks of nonspecific cell death but also maximize the efficacy of the cancer therapeutic agents. Various efficacious nanotechnology-based carrier systems are being widely investigated for their potential in OSCC treatment: polymeric nanoparticles, polymeric micelles, nanoemulsions and layered nanoemulsions, nanoliposomes, solid lipid nanoparticles and nanolipid carriers, cyclodextrin complexes, hydrogels, metallic nanoparticles, nanocarbon tubes, and receptor mediated drug delivery systems. We highlight the etiology, line of the treatment and chemopreventive measures related to OSCC. We focus on data available in the research carried out worldwide in past 15 years related to the management of OSCC.
Overview on Therapeutic Applications of Microparticulate Drug Delivery Systems.Friday, December 02, 2016
Bale S, Khurana A, Reddy AS, Singh M, Godugu C,
Critical reviews in therapeutic drug carrier systems. 2016
Research in novel drug delivery systems is being explored competitively in order to attain maximum therapeutic effect while minimizing the adverse effects. Despite several advancements in pharmaceutical formulations, one of the major challenges still persisting is sustained drug release. Microencapsulation enacts as an intelligent approach with a strong therapeutic impact and is in demand globally in medical technology due to its specific and attractive properties, including biocompatibility, stability, target specificity, uniform encapsulation, better compliance, and controlled and sustained release patterns that are responsible for diminishing the toxicity and dosage frequency. Microparticles are successful delivery systems that encapsulate both water-insoluble and sparingly water-soluble agents to elicit their efficacy with a great potential attributed to their unique properties: particle size, shape, structure, drug loading, entrapment efficiency, porosity, and release profile. Several marketed microparticle-based formulations are available, including risperidone, buserelin, and octreotide acetate, and some of them are in clinical trials. The present review highlights the detailed therapeutic applications of microparticles with advances from the last decade to treat various disease conditions, including cancer, diabetes, cardiovascular diseases, and neurological disorders, as well as for vaccine delivery, ocular and pulmonary delivery, gene transfer, etc., and exemplifies the future perspectives in these aspects. One day in the future, microparticle-based formulations may become broadly researched in drug delivery systems.
Retrospective use of PBPK Modelling to Understand a Clinical Drug-Drug Interaction between Dextromethorphan and GSK1034702.Friday, December 02, 2016
Hobbs MJ, Bloomer J, Dear G,
Xenobiotica; the fate of foreign compounds in biological systems. 02-Dec-2016
The DDI observed with repeat administration of GSK1034702 (5 mg) can be attributed to metabolism dependent inhibition of CYP2D6. Further in vitro data were generated and several potential mechanisms proposed to explain the interaction observed following a single dose of GSK1034702.
The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin.Friday, December 02, 2016
Reslan M, Kayser V,
Pharmaceutical development and technology. 02-Dec-2016
Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium Oxide (D2O), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of D2O on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that D2O enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T, however the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with D2O can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.
Performance of current measurement system in poloidal field power supply for Experimental Advanced Superconducting Tokamak.Friday, December 02, 2016
Liu DM, Li J, Wan BN, Lu Z, Wang LS, Jiang L, Lu CH, Huang J,
The Review of scientific instruments. Nov-2016
As one of the core subsystems of the Experimental Advanced Superconducting Tokamak (EAST), the poloidal field power system supplies energy to EAST's superconducting coils. To measure the converter current in the poloidal field power system, a current measurement system has been designed. The proposed measurement system is composed of a Rogowski coil and a newly designed integrator. The results of the resistor-inductor-capacitor discharge test and the converter equal current test show that the current measurement system provides good reliability and stability, and the maximum error of the proposed system is less than 1%.
Investigation on quench initiation and propagation characteristics of GdBCO coil co-wound with a stainless steel tape as turn-to-turn metallic insulation.Friday, December 02, 2016
Kim YG, Song JB, Choi YH, Yang DG, Kim SG, Lee HG,
The Review of scientific instruments. Nov-2016
This paper investigates the quench initiation and propagation characteristics of a metallic insulation (MI) coil by conducting thermal quench tests for a GdBCO single-pancake coil co-wound with a stainless steel tape as the turn-to-turn MI. The test results confirmed that the MI coil exhibited superior thermal and electrical stabilities compared to the conventional coils co-wound with organic insulation material because the operating current could flow along the radial direction due to the existence of a turn-to-turn contact when a local hot spot was generated. The results of the quench test at a heater current (Ih) of 12, 13, and 14 A indicate that the MI coil possesses a self-protecting characteristic resulting from the "current bypass" through the turn-to-turn contact. However, the test coil was not self-protecting at Ih = 15 A because the Joule heat energy generated by the radial current flow was not completely dissipated due to the characteristic resistance of the metallic insulation tape and the non-superconducting materials, including the substrate, stabilizer, and buffer layers within the high-temperature superconductor (HTS) tape. Even though the MI coil possesses superior thermal and electrical stability relative to those of conventional HTS coils co-wound with an organic material as turn-to-turn insulation, it is essential to consider the critical role of the Joule heat energy resulting from the operating current and stored magnetic energy as well as the characteristic resistances in order to further develop self-protective 2G HTS magnets.
Electromagnetic interference reduction design of alternating integrator for EAST.Friday, December 02, 2016
Liu DM, Wan BN, Li J, Wang Y, Shen B, Gong XZ, He YG,
The Review of scientific instruments. Nov-2016
An alternating integrator has been designed for the Experimental Advanced Superconducting Tokamak that is intended for long pulse operation of up to 1000 s. The electromagnetic operating environment for the device is so complex that it could affect the performance of the integrator. The new integrator system is carefully designed and actualized based on specific reduced electromagnetic interference requirements, which were formulated based on consideration of processing of the input signals, the isolation properties, and the circuit board layout and grounding. The developed integrator shows excellent electromagnetic compatibility and low-drift properties.
Source: NCBI - Disclaimer and Copyright notice
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