Characterization of BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei.

PubMed

Campos, Cristine G; Borst, Luke; Cotter, Peggy A

2013-04-01

Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3' to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

Enhancing the oral bioavailability of biochanin A by encapsulation in mixed micelles containing Pluronic F127 and Plasdone S630

PubMed Central

Wu, Xiaoyan; Ge, Weihong; Shao, Tengfei; Wu, Weijun; Hou, Jian; Cui, Li; Wang, Jing; Zhang, Zhenghai

2017-01-01

Biochanin A (BCA), a natural dietary isoflavone, has been reported to show anticancer activities. However, its low biological availability and poor aqueous solubility limit its usefulness as a chemotherapeutic agent. We developed BCA-loaded micelles with Pluronic F127 and Plasdone S630 (BCA-FS). The optimized, spherical-shaped BCA-FS was obtained at a ratio of 1:1 (F127:S630). The particle size was 25.17±1.2 nm, and the zeta potential was −10.9±0.24 mV. BCA solubility in water increased to 5.0 mg/mL after encapsulation, and the drug-loading efficiency was 5.88%±0.76%. In vitro release experiments showed a delayed release of BCA from the mixed micelles. Furthermore, the BCA absorption permeability across a Caco-2 cell monolayer from the apical side to the basolateral side increased by 54% in BCA-FS. A pharmacokinetics evaluation showed a 2.16-fold increase in the relative oral bioavailability of BCA-FS compared with raw BCA, indicating that the mixed micelles may promote absorption in the gastrointestinal tract. A gastrointestinal safety assay was used to assess the reliability and safety of BCA-FS. On the basis of these findings, we conclude that this simple nanomicelle system could be leveraged to deliver BCA and other hydrophobic drugs. PMID:28260893

Enhancing the oral bioavailability of biochanin A by encapsulation in mixed micelles containing Pluronic F127 and Plasdone S630.

PubMed

Wu, Xiaoyan; Ge, Weihong; Shao, Tengfei; Wu, Weijun; Hou, Jian; Cui, Li; Wang, Jing; Zhang, Zhenghai

2017-01-01

Biochanin A (BCA), a natural dietary isoflavone, has been reported to show anticancer activities. However, its low biological availability and poor aqueous solubility limit its usefulness as a chemotherapeutic agent. We developed BCA-loaded micelles with Pluronic F127 and Plasdone S630 (BCA-FS). The optimized, spherical-shaped BCA-FS was obtained at a ratio of 1:1 (F127:S630). The particle size was 25.17±1.2 nm, and the zeta potential was -10.9±0.24 mV. BCA solubility in water increased to 5.0 mg/mL after encapsulation, and the drug-loading efficiency was 5.88%±0.76%. In vitro release experiments showed a delayed release of BCA from the mixed micelles. Furthermore, the BCA absorption permeability across a Caco-2 cell monolayer from the apical side to the basolateral side increased by 54% in BCA-FS. A pharmacokinetics evaluation showed a 2.16-fold increase in the relative oral bioavailability of BCA-FS compared with raw BCA, indicating that the mixed micelles may promote absorption in the gastrointestinal tract. A gastrointestinal safety assay was used to assess the reliability and safety of BCA-FS. On the basis of these findings, we conclude that this simple nanomicelle system could be leveraged to deliver BCA and other hydrophobic drugs.

A Novel Function of the Fe65 Neuronal Adaptor in Estrogen Receptor Action in Breast Cancer Cells*

PubMed Central

Sun, Yuefeng; Kasiappan, Ravi; Tang, Jinfu; Webb, Panida L.; Quarni, Waise; Zhang, Xiaohong; Bai, Wenlong

2014-01-01

Fe65 is a multidomain adaptor with established functions in neuronal cells and neurodegeneration diseases. It binds to the C terminus of the Aβ amyloid precursor protein and is involved in regulating gene transcription. The present studies show that Fe65 is expressed in breast cancer (BCa) cells and acts as an ERα transcriptional coregulator that is recruited by 17β-estradiol to the promoters of estrogen target genes. Deletion analyses mapped the ERα binding domain to the phosphotyrosine binding domain 2 (PTB2). Ectopic Fe65 increased the transcriptional activity of the ERα in a PTB2-dependent manner in reporter assays. Fe65 knockdown decreased, whereas its stable expression increased the transcriptional activity of endogenous ERα in BCa cells and the ability of estrogens to stimulate target gene expression, ERα, and coactivator recruitment to target gene promoters and cell growth. Furthermore, Fe65 expression decreased the antagonistic activity of tamoxifen (TAM), suggesting a role for Fe65 in TAM resistance. Overall, the studies define a novel role for the neuronal adaptor in estrogen actions in BCa cells. PMID:24619425

Denaturation of proteins by SDS and tetraalkylammonium dodecyl sulfates.

PubMed

Lee, Andrew; Tang, Sindy K Y; Mace, Charles R; Whitesides, George M

2011-09-20

This article describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C(+); C(+) = Na(+) and tetra-n-alkylammonium, NR(4)(+), where R = Me, Et, Pr, and Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of anionic surfactant dodecylsulfate (DS(-)). An analysis of the denaturation of BCA in solutions of Na(+)DS(-) and NR(4)(+)DS(-) (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS(-) (BCA(D)-DS(-)(n,sat)) are indistinguishable and independent of the cation below the critical micellar concentration (cmc) and independent of the total concentration of DS(-) above the cmc. At concentrations of C(+)DS(-) above the cmc, BCA denatured at rates that depended on the cation; the rates decreased by a factor >10(4) in the order of Na(+) ≈ NMe(4)(+) > NEt(4)(+) > NPr(4)(+) > NBu(4)(+), which is the same order as the values of the cmc (which decrease from 4.0 mM for Na(+)DS(-) to 0.9 mM for NBu(4)(+)DS(-) in Tris-Gly buffer). The relationship between the cmc values and the rates of formation of BCA(D)-DS(-)(n,sat()) suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS(-) rather than with micelles of (C(+)DS(-))(n). A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na(+)DS(-) and NR(4)(+)DS(-) observed with BCA was not general. Instead, the influence of NR(4)(+) on the association of DS(-) with these proteins depended on the protein. The selection of the cation contributed to the properties (including the composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS(-). These results suggest that the variation in the behavior of NR(4)(+)DS(-) with changes in R may be exploited in methods used to analyze and separate mixtures of proteins. © 2011 American Chemical Society

Naked DNA Immunization of Prevention of Prostate Cancer in a Dunning Rat Prostate Tumor Model

DTIC Science & Technology

2006-06-01

is performed by use of the BCA protein test. 6 Bioburden assay (for DNA of a final concentration not more than 1 mg/ml); 1 colonies following United...the policy of the Government, and no official endorsement should be inferred. For purpose of this article , information includes news releases... articles , manuscripts, brochures, advertisements, still and motion pictures, speeches, trade association proceedings etc. ABSTRACT The ability of

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS).

PubMed

Schlegel, Benjamin J; Nowell, Victoria J; Parreira, Valeria R; Soltes, Glenn; Prescott, John F

2012-10-01

This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature.

Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS)

PubMed Central

Schlegel, Benjamin J.; Nowell, Victoria J.; Parreira, Valeria R.; Soltes, Glenn; Prescott, John F.

2012-01-01

This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature. PMID:23543949

Silica surface characterization as a function of formation and surface treatment using traditional methods and proteins as surface probes

NASA Astrophysics Data System (ADS)

Korwin-Edson, Michelle Lynn

Previous works have shown that cells proliferate differently depending on the chemistry of the glass on which they are growing. Since proteins form the bonds between cells and glass, the hypothesis of this study is that proteins can distinguish between surface chemical variations of glass. This theory was examined through the use of various silica forms, a few select proteins, four surface treatment procedures, and a variety of characterization techniques. The silica forms include amorphous slides, cane, fiber, microspheres, fumed silica and quartz crystal terminals. The proteins selected were human serum albumin, mouse Immunoglobulin G, streptavidin, antimouse IgG, and biotin. The surface treatments utilized to bring about chemical variation on the silica surface were HF acid etching, ethanol cleaning, water plasma treatments, and 1000°C heat treatments. The characterization techniques encompassed both traditional material techniques and biological methods. The techniques studied were atomic force microscopy (AFM), chemical force microscopy (CFM), glancing incidence X-ray analysis (GIXA), fluorescence spectrometry, polyacrylamide gel electrophoresis (SDS-PAGE), and bicinchoninic acid (BCA) assay. It was the main goal of this project to determine the feasibility of these techniques in utilizing proteins as glass surface probes. Proteins were adsorbed to all of the various forms and the binding ability was studied by either stripping off the protein and quantifying them, or by deductive reasoning through the use of "depleted" protein solutions. Fluorimetry and BCA assay both utilized the depleted solutions, but the high error associated with this protocol was prohibitive. SDS-PAGE with streptavidin was very difficult due to staining problems, however the IgG proteins were able to be quantified with some success. GIXA showed that the protein layer thickness is monolayer in nature, which agrees well with the AFM fluid tapping data on protein height, but in addition showed features on the order of ten protein agglomerations. CFM is by far the most promising technique for utilizing proteins as surface probes. Functionalized tips of -COOH, streptavidin and -CH3 are able to discern between surface treatments, but not forms. A general conclusion is that adhesion forces are greatest for -COOH, then streptavidin, and least for -CH3.

A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis.

PubMed

Keith, D; Hong, B; Christensen, M

1997-05-01

A quick, simple, and efficient extraction technique was developed for the removal of protein from soft hydrophilic contact lenses. An extraction solvent consisting of a 50:50 mix of 0.2% trifluoroacetic acid and acetonitrile was used to remove protein from in vitro laboratory-deposited and human-worn contact lenses. The protein removed was analyzed using HPLC, bicinchoninic acid (BCA) analysis, and SDS-PAGE gel electreophoresis. Extraction efficiency for lysozyme from laboratory-deposited Group IV lenses was determined to be approximately 100%. Group IV human-worn contact lenses were extracted and analyzed for lysozyme by HPLC and total protein by bicinchoninic acid (BCA) analysis. Groups I, II, III, and IV contact lenses deposited with an artificial tear protein solution and human-worn lenses were extracted and analyzed by SDS-PAGE gel electreophoresis and micro-BCA. The ACN/TFA procedure offers a simple, quick, and efficient extraction technique for removal of protein from contact lenses for subsequent analysis.

Denaturation of Proteins by SDS and by Tetra-alkylammonium Dodecyl Sulfates

PubMed Central

Lee, Andrew; Tang, Sindy K. Y.; Mace, Charles R.

2011-01-01

This paper describes the use of capillary electrophoresis (CE) to examine the influence of different cations (C+; C+ = Na+ and tetra-n-alkylammonium, NR4 +, where R = Me, Et, Pr, Bu) on the rates of denaturation of bovine carbonic anhydrase II (BCA) in the presence of the anionic surfactant dodecylsulfate (DS−). Analysis of the denaturation of BCA in solutions of Na+DS− and NR4 +DS− (in Tris-Gly buffer) indicated that the rates of formation of complexes of denatured BCA with DS− (BCAD-DS−n,sat) are indistinguishable and independent of the cation below the critical micellar concentration (cmc), and independent of the total concentration of DS− above the cmc. At concentrations of C+DS− above the cmc, BCA denatured with rates that depended on the cation; the rates decreased by a factor > 104, in the order Na+ ~ NMe4 + > NEt4 + > NPr4 + > NBu4 + – the same order as the values of cmc (which decrease from 4.0 mM for Na+DS− to 0.9 mM for NBu4 +DS− in Tris-Gly buffer). The relationship between values of cmc and rates of formation of BCAD-DS−n,sat suggested that the kinetics of denaturation of BCA involve the association of this protein with monomeric DS−, rather than with micelles of (C+DS−)n. A less-detailed survey of seven other proteins (α-lactalbumin, β-lactoglobulin A, β-lactoglobulin B, carboxypeptidase B, creatine phosphokinase, myoglobin, and ubiquitin) showed that the difference between Na+DS− and NR4 +DS− observed with BCA was not general. Instead, the influence of NR4 + on the association of DS− with these proteins depended on the protein. The selection of cation contributed to the properties (including composition, electrophoretic mobility, and partitioning behavior in aqueous two-phase systems) of aggregates of denatured protein and DS−. These results suggest that variation in the behavior of NR4 +DS− with changes in R may be exploited in methods for analyzing and separating mixtures of proteins. PMID:21834533

Silibinin downregulates MMP2 expression via Jak2/STAT3 pathway and inhibits the migration and invasive potential in MDA-MB-231 cells.

PubMed

Byun, Hyo Joo; Darvin, Pramod; Kang, Dong Young; Sp, Nipin; Joung, Youn Hee; Park, Jong Hwan; Kim, Sun Jin; Yang, Young Mok

2017-06-01

Worldwide, breast cancer (BCa) is the most common cancer in women. Among its subtypes, triple-negative breast cancer (TNBC) is an aggressive form associated with diminished survival. TNBCs are characterized by their absence, or minimal expression, of the estrogen and progesterone receptors, as well as the human epidermal growth factor receptor 2 (i.e. ER-/-, PR-/-, Her2-/Low). Consequently, treatment for this subtype of BCa remains problematic. Silibinin, a derivative of the flavonoid silymarin, is reported to have anticancer activities against hepatic and non-small cell lung cancers. We hypothesized that silibinin might inhibit cell-extracellular matrix interactions via the regulation, expression, and activation of STAT3 in TNBCs, which could directly inhibit metastasis in silibinin-treated BCa cells. Using proliferation assays, we found that exposure to silibinin at a concentration of 200 µM inhibited the proliferation of breast cancer (BCa) cells; this concentration also inhibited phosphorylation of STAT3 and its principal upstream kinase, Jak2. Furthermore, we found that silibinin inhibited the nuclear translocation of STAT3, as well as its binding to the MMP2 gene promoter. The ability of silibinin to inhibit metastasis was further studied using an in vitro invasion assay. The results confirm the role of STAT3 as a critical mediator in the invasive potential of BCa cells, and STAT3 knock-down resulted in inhibition of invasion. The invasion ability of silibinin-treated BCa cells was studied in detail with the expression of MMP2. Prevention of STAT3 activation also resulted in the inhibition of MMP2 expression. Use of a small interfering RNA to knock down STAT3 (siSTAT3) allowed us to confirm the role of STAT3 in regulating MMP2 expression, as well as the mechanism of action of silibinin in inhibiting MMP2. Taken together, we found that silibinin inhibits the Jak2/STAT3/MMP2 signaling pathway, and inhibits the proliferation, migration, and invasion of triple-negative BCa cells.

Development of a Method for Evaluating Floor Dry-Cleanability from Wheat Flour in the Food Industry.

PubMed

Barreca, F; Cardinali, G D; Borgese, E; Russo, M

2017-04-01

Many productive processes are characterized by inadequate protocols of sanitation that increase the possibility of proliferation of microbial contaminants, especially on surfaces. The use of this method for evaluating the degree of floor cleanability in agri-food companies is important not only to reduce the risk of contamination of products, but also to provide companies with a tool to identify critical issues. The method is based on the usage of bicinchoninic acid assay (BCA) in a solution at a 1:50 ratio of Cu 2+ /BCA, which is ideal for detecting the amount of proteins contained in wheat flour residues on industrial flooring. Spectrophotometric analysis allowed identifying maximum absorbance values at 562 nm for different protein concentrations, although the construction of a regression function led to the definition of the intervals of evaluation corresponding to different degrees of cleanliness from residues of wheat flour. The results of the absorbance curves, obtained by applying the proposed evaluation method to 6 tiles commonly used in agri-food buildings, showed the clear persistence of food material on 2 tiles with surface relief. In particular, such tiles showed a higher presence of proteins, with a level of contamination 440% higher. Furthermore, a robotic system was designed to standardize the cleaning method commonly employed in agri-food companies to remove solid particles from flooring. © 2017 Institute of Food Technologists®.

Exosomes from adriamycin-resistant breast cancer cells transmit drug resistance partly by delivering miR-222.

PubMed

Yu, Dan-Dan; Wu, Ying; Zhang, Xiao-Hui; Lv, Meng-Meng; Chen, Wei-Xian; Chen, Xiu; Yang, Su-Jin; Shen, Hongyu; Zhong, Shan-Liang; Tang, Jin-Hai; Zhao, Jian-Hua

2016-03-01

Breast cancer (BCa) is one of the major deadly cancers in women. However, treatment of BCa is still hindered by the acquired-drug resistance. It is increasingly reported that exosomes take part in the development, metastasis, and drug resistance of BCa. However, the specific role of exosomes in drug resistance of BCa is poorly understood. In this study, we investigate whether exosomes transmit drug resistance through delivering miR-222. We established an adriamycin-resistant variant of Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line (MCF-7/Adr) from a drug-sensitive variant (MCF-7/S). Exosomes were isolated from cell supernatant by ultracentrifugation. Cell viability was assessed by MTT assay and apoptosis assay. Individual miR-222 molecules in BCa cells were detected by fluorescence in situ hybridization (FISH). Then, FISH was combined with locked nucleic acid probes and enzyme-labeled fluorescence (LNA-ELF-FISH). Individual miR-222 could be detected as bright photostable fluorescent spots and then the quantity of miR-222 per cell could be counted. Stained exosomes were taken in by the receipt cells. MCF-7/S acquired drug resistance after co-culture with exosomes from MCF-7/Adr (A/exo) but did not after co-culture with exosomes from MCF-7/S (S/exo). The quantity of miR-222 in A/exo-treated MCF-7/S was significantly greater than in S/exo-treated MCF-7/S. MCF-7/S transfected with miR-222 mimics acquired adriamycin resistance while MCF-7/S transfected with miR-222 inhibitors lost resistance. In conclusion, exosomes are effective in transmitting drug resistance and the delivery of miR-222 via exosomes may be a mechanism.

Molecular analysis of urothelial cancer cell lines for modeling tumor biology and drug response.

PubMed

Nickerson, M L; Witte, N; Im, K M; Turan, S; Owens, C; Misner, K; Tsang, S X; Cai, Z; Wu, S; Dean, M; Costello, J C; Theodorescu, D

2017-01-05

The utility of tumor-derived cell lines is dependent on their ability to recapitulate underlying genomic aberrations and primary tumor biology. Here, we sequenced the exomes of 25 bladder cancer (BCa) cell lines and compared mutations, copy number alterations (CNAs), gene expression and drug response to BCa patient profiles in The Cancer Genome Atlas (TCGA). We observed a mutation pattern associated with altered CpGs and APOBEC-family cytosine deaminases similar to mutation signatures derived from somatic alterations in muscle-invasive (MI) primary tumors, highlighting a major mechanism(s) contributing to cancer-associated alterations in the BCa cell line exomes. Non-silent sequence alterations were confirmed in 76 cancer-associated genes, including mutations that likely activate oncogenes TERT and PIK3CA, and alter chromatin-associated proteins (MLL3, ARID1A, CHD6 and KDM6A) and established BCa genes (TP53, RB1, CDKN2A and TSC1). We identified alterations in signaling pathways and proteins with related functions, including the PI3K/mTOR pathway, altered in 60% of lines; BRCA DNA repair, 44%; and SYNE1-SYNE2, 60%. Homozygous deletions of chromosome 9p21 are known to target the cell cycle regulators CDKN2A and CDKN2B. This loci was commonly lost in BCa cell lines and we show the deletions extended to the polyamine enzyme methylthioadenosine (MTA) phosphorylase (MTAP) in 36% of lines, transcription factor DMRTA1 (27%) and antiviral interferon epsilon (IFNE, 19%). Overall, the BCa cell line genomic aberrations were concordant with those found in BCa patient tumors. We used gene expression and copy number data to infer pathway activities for cell lines, then used the inferred pathway activities to build a predictive model of cisplatin response. When applied to platinum-treated patients gathered from TCGA, the model predicted treatment-specific response. Together, these data and analysis represent a valuable community resource to model basic tumor biology and to study the pharmacogenomics of BCa.

Microvolume protein concentration determination using the NanoDrop 2000c spectrophotometer.

PubMed

Desjardins, Philippe; Hansen, Joel B; Allen, Michael

2009-11-04

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 microL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

PubMed

Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

2015-01-01

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.

Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Lee, Jiho; Chang, Jeong Ho

2014-12-01

This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively.

Liquid chromatography-mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: application to interaction study with biochanin A (an isoflavone).

PubMed

Singh, Sheelendra Pratap; Wahajuddin; Ali, Mushir M; Kohli, Kanchan; Jain, Girish Kumar

2011-10-01

Tamoxifen is the agent of choice for the treatment of estrogen receptor-positive breast cancer. Tamoxifen is a substrate of P-glycoprotein (P-gp) and microsomal cytochrome P450 (CYP) 3A, and biochanin A (BCA) is an inhibitor of P-gp and CYP3A. Hence, it could be expected that BCA would affect the pharmacokinetics of tamoxifen. In the present study we have developed and validated a simple, sensitive and specific LC-ESI-MS/MS method for the simultaneous quantification of tamoxifen and its metabolite 4-hydroxytamoxifen with 100 μL rat plasma using centchroman as an internal standard (IS). Tamoxifen, 4-hydroxytamoxifen and IS were separated on a Supelco Discovery C18 (4.6 mm × 50 mm, 5.0 μm) column under isocratic condition using 0.0 1M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) as a mobile phase. The mobile phase was delivered at a flow rate of 0.8 mL/min. The method was proved to be accurate and precise at linearity range of 0.78-200 ng/mL with a correlation coefficient (r) of ≥ 0.996. The intra- and inter-day assay precision ranged from 1.89 to 8.54% and 3.97 to 10.26%, respectively; and intra- and inter-day assay accuracy was between 87.63 and 109.06% and 96 and 103.89%, respectively for both the analytes. The method was successfully applied to study the effect of oral co-administration of BCA (an isoflavone) on the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen in female rats. The coadministration of BCA caused no significant changes in the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen. However, the peak plasma concentration (C(max)) of 4-hydroxytamoxifen in BCA pretreated rats was significantly (P<0.05) lower than those from control group. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of angiogenesis by leflunomide via targeting the soluble ephrin-A1/EphA2 system in bladder cancer.

PubMed

Chu, Maolin; Zhang, Chunying

2018-01-24

Angiogenesis plays an important role in bladder cancer (BCa). The immunosuppressive drug leflunomide has attracted worldwide attention. However, the effects of leflunomide on angiogenesis in cancer remain unclear. Here, we report the increased expression of soluble ephrin-A1 (sEphrin-A1) in supernatants of BCa cell lines (RT4, T24, and TCCSUP) co-cultured with human umbilical vein endothelial cells (HUVECs) compared with that in immortalized uroepithelial cells (SV-HUC-1) co-cultured with HUVECs. sEphrin-A1 is released from BCa cells as a monomeric protein that is a functional form of the ligand. The co-culture supernatants containing sEphrin-A1 caused the internalization and down-regulation of EphA2 on endothelial cells and dramatic functional activation of HUVECs. This sEphrin-A1/EphA2 system is mainly functional in regulating angiogenesis in BCa tissue. We showed that leflunomide (LEF) inhibited angiogenesis in a N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced bladder carcinogenesis model and a tumor xenograft model, as well as in BCa cell and HUVEC co-culture systems, via significant inhibition of the sEphrin-A1/EphA2 system. Ephrin-A1 overexpression could partially reverse LEF-induced suppression of angiogenesis and subsequent tumor growth inhibition. Thus, LEF has a significant anti-angiogenesis effect on BCa cells and BCa tissue via its inhibition of the functional angiogenic sEphrin-A1/EphA2 system and may have potential for treating BCa beyond immunosuppressive therapy.

Proteomic Approach for Extracting Cytoplasmic Proteins from Streptococcus sanguinis using Mass Spectrometry

PubMed Central

El-Rami, Fadi; Nelson, Kristina; Xu, Ping

2017-01-01

Streptococcus sanguinis is a commensal and early colonizer of oral cavity as well as an opportunistic pathogen of infectious endocarditis. Extracting the soluble proteome of this bacterium provides deep insights about the physiological dynamic changes under different growth and stress conditions, thus defining “proteomic signatures” as targets for therapeutic intervention. In this protocol, we describe an experimentally verified approach to extract maximal cytoplasmic proteins from Streptococcus sanguinis SK36 strain. A combination of procedures was adopted that broke the thick cell wall barrier and minimized denaturation of the intracellular proteome, using optimized buffers and a sonication step. Extracted proteome was quantitated using Pierce BCA Protein Quantitation assay and protein bands were macroscopically assessed by Coomassie Blue staining. Finally, a high resolution detection of the extracted proteins was conducted through Synapt G2Si mass spectrometer, followed by label-free relative quantification via Progenesis QI. In conclusion, this pipeline for proteomic extraction and analysis of soluble proteins provides a fundamental tool in deciphering the biological complexity of Streptococcus sanguinis. PMID:29152022

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

PubMed Central

Desjardins, Philippe; Hansen, Joel B.; Allen, Michael

2009-01-01

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 μL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay. PMID:19890248

Importin-11 overexpression promotes the migration, invasion, and progression of bladder cancer associated with the deregulation of CDKN1A and THBS1.

PubMed

Zhao, Junjie; Shi, Lei; Zeng, Shuxiong; Ma, Chong; Xu, Weidong; Zhang, Zhensheng; Liu, Qingzuo; Zhang, Peng; Sun, Yinghao; Xu, Chuanliang

2018-06-01

We recently determined that a novel oncogene, IPO11 from 5q12, participates in bladder cancer (BCa) progression. However, the biological function of IPO11 and the molecular mechanisms through which it contributes to BCa progression remain unclear. The aim of this study was to investigate the role of IPO11 in BCa aggressiveness and elucidate the molecular mechanisms underlying its effects in BCa. The mRNA expression levels of IPO11 in BIU-87, RT4, UMUC3, EJ, 5637, T24, J82, and HT-1376 cell lines were determined using quantitative real-time polymerase chain reaction. Expression of importin-11 was detected in 134 formalin-fixed and paraffin-embedded (FFPE) BCa tissues and 10 paired nonneoplastic bladder tissue specimens by immunohistochemistry. The copy number of IPO11 was examined in 25 FFPE BCa specimens using fluorescent in situ hybridization. The effects of IPO11 on migration, invasion, and cell proliferation were investigated in EJ and 5637 cell lines using RNA interference. Potential molecular mechanisms were investigated using whole transcriptome sequencing and bioinformatic approaches in EJ cells and IPO11-silenced EJ cells and verified using quantitative real-time polymerase chain reaction. Endogenous IPO11 mRNA was highly expressed in 6 invasive BCa cell lines (EJ, HT-1376, UMUC3, 5637, J82, and T24) but had a low expression in the noninvasive BCa cell line BIU-87 and the papillary BCa cell line RT4. Immunohistochemical staining revealed that 87 (64.9%) of 134 FFPE BCa tissues displayed importin-11 overexpression. Moreover, importin-11 overexpression was positively associated with increased tumor stages and tumor grades, lymphatic invasion, and lymph node metastasis. Furthermore, importin-11 overexpression was detected in 100% (14/14) of BCa tissues with IPO11 amplification, and IPO11 amplification was not observed in 2 additional BCa tissues with importin-11 overexpression. Small interfering RNA-mediated knockdown of IPO11 is sufficient to inhibit the motility and invasiveness of EJ and 5637 cells. IPO11 knockdown also inhibited cell proliferation in EJ cells, whereas this was not observed in 5637 cells or the in vivo experiments. Using whole transcriptome sequencing, we found that 22 genes (including IPO11) were differentially expressed in IPO11-silenced EJ cells compared with wild-type EJ cells, 4 of which were upregulated, and 18 of which were downregulated. KEGG pathway enrichment analysis of the significantly differentially expressed genes showed that the proteoglycans in cancer pathway (pathway Id: hsa05205) was most significantly enriched among 10 genetically altered pathways and referred to 6 significantly altered genes (CDKN1A, HBEGF, PTK2, THBS1, CCNG2, and EGR1). The next 3 most significantly enriched pathways in order were the p53, ErbB, and BCa pathways. CDKN1A and THBS1 were the most 2 frequently covered genes and were involved in 9 and 6 pathways, respectively. They were also 2 key proteins in the BCa pathway (pathway Id: hsa05219) that were downregulated in IPO11-knockdown EJ cells compared with wild-type EJ cells. Importin-11 overexpression can promote BCa cell invasiveness, probably associated with the deregulation of CDKN1A and THBS1 primarily through the activation of the proteoglycans in cancer pathway and the classical BCa pathway. Importin-11 may be a useful target through which the progression of noninvasive BCa to invasive BCa can be blocked. Copyright © 2018 Elsevier Inc. All rights reserved.

Nanohardness, corrosion and protein adsorption properties of CuAlO2 films deposited on 316L stainless steel for biomedical applications

NASA Astrophysics Data System (ADS)

Chang, Shih-Hang; Chen, Jian-Zhang; Hsiao, Sou-Hui; Lin, Guan-Wei

2014-01-01

This study preliminarily assesses the biomedical applications of CuAlO2 coatings according to nanoindentation, electrochemical, and protein adsorption tests. Nanoindentation results revealed that the surface hardness of 316L stainless steel increased markedly after coating with CuAlO2 films. Electrochemical tests of corrosion potential, breakdown potential, and corrosion current density showed that the corrosion resistance properties of 316L stainless steel are considerably improved by CuAlO2 coatings. Bicinchoninic acid (BCA) protein assay results revealed that the protein adsorption behavior of 316L stainless steel did not exhibit notable differences with or without CuAlO2 coatings. A CuAlO2 coating of 100 nm thickness improved the surface nanohardness and corrosion resistance ability of 316L stainless steel. CuAlO2 is a potential candidate for biomaterial coating applications, particularly for surface modification of fine, delicate implants.

Bovine serum albumin release from novel chitosan-fluoro-aluminosilicate glass ionomer cement: stability and cytotoxicity studies.

PubMed

Limapornvanich, Araya; Jitpukdeebodintra, Suwanna; Hengtrakool, Chanothai; Kedjarune-Leggat, Ureporn

2009-09-01

This study aimed to evaluate the effect of adding chitosan (CS) to conventional glass ionomer cement (GIC) on protein release and its cytotoxicity. Bovine serum albumin (BSA) was used as the released protein from two glass ionomer formulations. One (GIC+BSA) contained fluoro-aluminosilicate glass mixed with BSA, and another (GIC:CS+BSA) used a similar glass and BSA with 20% chitosan. Six disc specimens per group (10mm in diameter, 2mm in height) were prepared and placed in phosphate buffer saline, which was replaced at various times over 2 weeks. The released protein was determined by a BCA assay. Cytotoxicity of the extracts from these materials for 1, 2 and 7 days to dental pulp cells was evaluated using MTT assay. The GIC:CS+BSA released a burst of BSA in the first 6h, and slowly released at different rates over the 2 weeks. GIC+BSA showed a similar result, but protein could not be detected at the 12h. The protein release rate of GIC:CS+BSA was significantly greater than GIC+BSA (P<0.01); nearly three times higher. The released BSA had the same molecular weight as evaluated by SDS-PAGE. From the MTT assay, the percentages of viable cells were significantly different and can be arranged as: GIC:CS+BSA>GIC:CS>GI+BSA>GI and the cytotoxicity was increased by time of extraction. Chitosan added in glass ionomer cement can prolong release of BSA as well as not increasing the toxicity to pulp cells. This material may be useful for protein delivery.

Development of a new microtiter plate format for clinically relevant assays.

PubMed

Piletska, Elena V; Piletsky, Stanislav S; Whitcombe, Michael J; Chianella, Iva; Piletsky, Sergey A

2012-02-21

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.

Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

PubMed

Mil-Homens, Dalila; Pinto, Sandra N; Matos, Rute G; Arraiano, Cecília; Fialho, Arsenio M

2017-04-01

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence. © 2016 John Wiley & Sons Ltd.

Enamel matrix derivative enhances tissue formation around scaffolds used for tissue engineering of ligaments.

PubMed

Messenger, Michael P; Raïf, El M; Seedhom, Bahaa B; Brookes, Steven J

2010-02-01

The following in vitro translational study investigated whether enamel matrix derivative (EMD), an approved biomimetic treatment for periodontal disease (Emdogain) and hard-to-heal wounds (Xelma), enhanced synovial cell colonization and protein synthesis around a scaffold used clinically for in situ tissue engineering of the torn anterior cruciate ligament (ACL). Synovial cells were enzymatically extracted from bovine synovium and dynamically seeded onto polyethylene terephthalate (PET) scaffolds. The cells were cultured in low-serum medium (0.5% FBS) for 4 weeks with either a single administration of EMD at the start of the 4 week period or multiple administrations of EMD at regular intervals throughout the 4 weeks. Samples were harvested and evaluated using the Hoechst DNA assay, BCA protein assay, cresolphthalein complexone calcium assay, SDS-PAGE, ELISA and electron microscopy. A significant increase in cell number (DNA) (p < 0.01), protein content (p < 0.01) and TGFbeta1 synthesis (p < 0.01) was observed with multiple administrations of EMD. Additionally, SDS-PAGE showed an increase in high molecular weight proteins, characteristic of the fibril-forming collagens. Electron microscopy supported these findings, showing that scaffolds treated with multiple administrations of EMD were heavily coated with cells and extracellular matrix (ECM) that enveloped the fibres. Multiple administrations of EMD to synovial cell-seeded scaffolds enhanced the formation of tissue in vitro. Additionally, it was shown that EMD enhanced TGFbeta1 synthesis of synovial cells, suggesting a potential mode of action for EMD's capacity to stimulate tissue regeneration.

Exosomes decrease sensitivity of breast cancer cells to adriamycin by delivering microRNAs.

PubMed

Mao, Ling; Li, Jian; Chen, Wei-Xian; Cai, Yan-Qin; Yu, Dan-Dan; Zhong, Shan-Liang; Zhao, Jian-Hua; Zhou, Jian-Wei; Tang, Jin-Hai

2016-04-01

While adriamycin (adr) offers improvement in survival for breast cancer (BCa) patients, unfortunately, drug resistance is almost inevitable. Mounting evidence suggests that exosomes act as a vehicle for genetic cargo and constantly shuttle biologically active molecules including microRNAs (miRNAs) between heterogeneous populations of tumor cells, engendering a resistance-promoting niche for cancer progression. Our recent study showed that exosomes from docetaxel-resistance BCa cells could modulate chemosensitivity by delivering miRNAs. Herein, we expand on our previous finding and explore the relevance of exosome-mediated miRNA delivery in resistance transmission of adr-resistant BCa sublines. We now demonstrated the selective packing of miRNAs within the exosomes (A/exo) derived from adr-resistant BCa cells. The highly expressed miRNAs in A/exo were significantly increased in recipient fluorescent sensitive cells (GFP-S) after A/exo incorporation. Gene ontology analysis of predicted targets showed that the top 30 most abundant miRNAs in A/exo were involved in crucial biological processes. Moreover, A/exo not only loaded miRNAs for its production and release but also carried miRNAs associated with Wnt signaling pathway. Furthermore, A/exo co-culture assays indicated that miRNA-containing A/exo was able to increase the overall resistance of GFP-S to adr exposure and regulate gene levels in GFP-S. Our results reinforce our earlier reports that adr-resistant BCa cells could manipulate a more deleterious microenvironment and transmit resistance capacity through altering gene expressions in sensitive cells by transferring specific miRNAs contained within exosomes.

High-throughput assay for optimising microbial biological control agent production and delivery

USDA-ARS?s Scientific Manuscript database

Lack of technologies to produce and deliver effective biological control agents (BCAs) is a major barrier to their commercialization. A myriad of variables associated with BCA cultivation, formulation, drying, storage, and reconstitution processes complicates agent quality maximization. An efficie...

Effects of Pesticide Treatments on Nutrient Levels in Worker Honey Bees (Apis mellifera).

PubMed

Feazel-Orr, Haley K; Catalfamo, Katelyn M; Brewster, Carlyle C; Fell, Richard D; Anderson, Troy D; Traver, Brenna E

2016-03-01

Honey bee colony loss continues to be an issue and no factor has been singled out as to the cause. In this study, we sought to determine whether two beekeeper-applied pesticide products, tau-fluvalinate and Fumagilin-B(®), and one agrochemical, chlorothalonil, impact the nutrient levels in honey bee workers in a natural colony environment. Treatments were performed in-hive and at three different periods (fall, spring, and summer) over the course of one year. Bees were sampled both at pre-treatment and two and four weeks post-treatment, weighed, and their protein and carbohydrate levels were determined using BCA and anthrone based biochemical assays, respectively. We report that, based on the pesticide concentrations tested, no significant negative impact of the pesticide products was observed on wet weight, protein levels, or carbohydrate levels of bees from treated colonies compared with bees from untreated control colonies.

Effects of Pesticide Treatments on Nutrient Levels in Worker Honey Bees (Apis mellifera)

PubMed Central

Feazel-Orr, Haley K.; Catalfamo, Katelyn M.; Brewster, Carlyle C.; Fell, Richard D.; Anderson, Troy D.; Traver, Brenna E.

2016-01-01

Honey bee colony loss continues to be an issue and no factor has been singled out as to the cause. In this study, we sought to determine whether two beekeeper-applied pesticide products, tau-fluvalinate and Fumagilin-B®, and one agrochemical, chlorothalonil, impact the nutrient levels in honey bee workers in a natural colony environment. Treatments were performed in-hive and at three different periods (fall, spring, and summer) over the course of one year. Bees were sampled both at pre-treatment and two and four weeks post-treatment, weighed, and their protein and carbohydrate levels were determined using BCA and anthrone based biochemical assays, respectively. We report that, based on the pesticide concentrations tested, no significant negative impact of the pesticide products was observed on wet weight, protein levels, or carbohydrate levels of bees from treated colonies compared with bees from untreated control colonies. PMID:26938563

Square-wave voltammetry assays for glycoproteins on nanoporous gold

PubMed Central

Pandey, Binod; Bhattarai, Jay K.; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V.; Stine, Keith J.

2014-01-01

Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

Comparative performance of precommercial cellulases hydrolyzing pretreated corn stover

PubMed Central

2011-01-01

Background Cellulases and related hydrolytic enzymes represent a key cost factor for biochemical conversion of cellulosic biomass feedstocks to sugars for biofuels and chemicals production. The US Department of Energy (DOE) is cost sharing projects to decrease the cost of enzymes for biomass saccharification. The performance of benchmark cellulase preparations produced by Danisco, DSM, Novozymes and Verenium to convert pretreated corn stover (PCS) cellulose to glucose was evaluated under common experimental conditions and is reported here in a non-attributed manner. Results Two hydrolysis modes were examined, enzymatic hydrolysis (EH) of PCS whole slurry or washed PCS solids at pH 5 and 50°C, and simultaneous saccharification and fermentation (SSF) of washed PCS solids at pH 5 and 38°C. Enzymes were dosed on a total protein mass basis, with protein quantified using both the bicinchoninic acid (BCA) assay and the Bradford assay. Substantial differences were observed in absolute cellulose to glucose conversion performance levels under the conditions tested. Higher cellulose conversion yields were obtained using washed solids compared to whole slurry, and estimated enzyme protein dosages required to achieve a particular cellulose conversion to glucose yield were extremely dependent on the protein assay used. All four enzyme systems achieved glucose yields of 90% of theoretical or higher in SSF mode. Glucose yields were reduced in EH mode, with all enzymes achieving glucose yields of at least 85% of theoretical on washed PCS solids and 75% in PCS whole slurry. One of the enzyme systems ('enzyme B') exhibited the best overall performance. However in attaining high conversion yields at lower total enzyme protein loadings, the relative and rank ordered performance of the enzyme systems varied significantly depending upon which hydrolysis mode and protein assay were used as the basis for comparison. Conclusions This study provides extensive information about the performance of four precommercial cellulase preparations. Though test conditions were not necessarily optimal for some of the enzymes, all were able to effectively saccharify PCS cellulose. Large differences in the estimated enzyme dosage requirements depending on the assay used to measure protein concentration highlight the need for better consensus methods to quantify enzyme protein. PMID:21899748

Research Advances

ERIC Educational Resources Information Center

King, Angela G.

2004-01-01

Nanotechnology are employed by researchers at Northwestern University to develop a method of labeling disease markers present in blood with unique DNA tags they have dubbed "bio-bar-codes". The preparation of nanoparticle and magnetic microparticle probes and a nanoparticle-based PSR-less DNA amplification scheme are involved by the DNA-BCA assay.

Gold nanoparticles on titanium and interaction with prototype protein.

PubMed

Padmos, J Daniel; Duchesne, Paul; Dunbar, Michael; Zhang, Peng

2010-10-01

Modifying titanium (Ti) implant surfaces with functional proteins can strengthen the interface between prosthesis and bone. A prototype system was developed using gold nanoparticles (AuNPs) to immobilize proteins onto Ti. An electroless (galvanic displacement) deposition method was first used to form AuNPs of controlled size and coverage on commercial Ti foil (giving Ti-AuNPs). Parameters were then modified to create two groups of discs (n = 26) with different average AuNP diameters. Scanning electron microscopy and X-ray photoelectron spectroscopy were used to characterize the morphology and surface structure of Ti-AuNPs. To study the interaction of Ti-AuNPs with proteins, Ti discs (n = 8) modified with plain AuNPs and discs (n = 8) modified with thiol (HS--R--COOH)-functionalized AuNPs were treated with lysozyme solution. The amount and activity of the lysozyme on the discs were examined with Micro-BCA and enzymatic assays. Lysozyme was immobilized onto the discs, and the assays showed that the discs with thiol-functionalized AuNPs, discs with bare AuNPs, and Ti controls had average lysozyme adsorptions of 23 x 10(4), 2.3 x 10(4), and 5.7 x 10(4) microg/m2, respectively. The activity assays showed that 21.5, 18.4, and 12.5% of the adsorbed lysozyme was active on the discs with thiol-functionalized AuNPs, discs with bare AuNPs, and Ti controls, respectively. This technique holds promise for binding functional biomolecules to surgical implants, hence possibly creating implant surfaces that react to their local environment. Copyright 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010.

Surface conjugation of poly (dimethyl siloxane) with itaconic acid-based materials for antibacterial effects

NASA Astrophysics Data System (ADS)

Birajdar, Mallinath S.; Cho, Hyunjoo; Seo, Youngmin; Choi, Jonghoon; Park, Hansoo

2018-04-01

Poly (dimethyl siloxane) (PDMS) is widely used in various biomedical applications. However, the PDMS surface is known to cause bacterial adhesion and protein absorption issues due to its high hydrophobicity. Therefore, the development of antibacterial and anti-protein products is necessary to prevent these problems. In this study, to improve its antibacterial property and prevent protein adsorption, PDMS surfaces were conjugated with itaconic acid (IA) and poly (itaconic acid) (PIA) via a chemical method. Additionally, IA and PIA were physically blended with PDMS to compare the antibacterial properties of these materials with those of the chemically conjugated PDMS surfaces. The successful synthesis of the PIA polymer structure was confirmed by proton nuclear magnetic resonance (1H NMR) spectroscopy. The successful conjugation of IA and PIA on PDMS was confirmed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), water contact angle measurements, and microbicinchoninic acid (BCA) protein assay analyses. The PDMS surfaces functionalized with IA and PIA by the conjugation method better prevented protein adsorption than the bare PDMS. Therefore, these surface-conjugated PDMS can be used in various biomedical applications.

Carbonic anhydrase inhibitors: in vitro inhibition of α isoforms (hCA I, hCA II, bCA III, hCA IV) by flavonoids.

PubMed

Ekinci, Derya; Karagoz, Lutfi; Ekinci, Deniz; Senturk, Murat; Supuran, Claudiu T

2013-04-01

A series of flavonoids, such as quercetin, catechin, apigenin, luteolin, morin, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA). The compounds were tested against four α-CA isozymes purified from human and bovine (hCA I, hCA II, bCA III, hCA IV) tissues. The four isozymes showed quite diverse inhibition profiles with these compounds. The flavonoids inhibited hCA I with K(I)-s in the range of 2.2-12.8 μM, hCA II with K(I)-s in the range of 0.74-6.2 μM, bCA III with K(I)-s in the range of 2.2-21.3 μM, and hCA IV with inhibition constants in the range of 4.4-15.7, with an esterase assay using 4-nitrophenyl acetate as substrate. Some simple phenols/sulfonamides were also investigated as standard inhibitors. The flavonoids incorporate phenol moieties which inhibit these CAs through a diverse, not yet determined inhibition mechanism, compared to classic inhibitors such as the sulfonamide/sulfamate ones.

MSA Bladder Reference Set Application: Charles Rosser-Hawaii (2014) — EDRN Public Portal

Cancer.gov

The goal of this proposal is straightforward. We wish to assay in a discovery set, reference set from EDRN, both PAI-1 and ANG promoters and genes for mutations. Then the results will be confirmed in a test cohort comprised of DNA extracted from fresh frozen tissue (n = 80 BCa patients). DNA from matching buffy coat from these 80 patients will serve as control. Extracted RNA can be assessed for difference in transcription. Furthermore, matched voided urine samples from these 80 patients are available to assess protein levels of PAI-1 and ANG by ELISA in addition to assessing activity of PAI-1 and ANG. At the end, we will link any genetic alteration with changes in RNA, protein and protein activity level as well as clinical features (e.g., age, race, tobacco history, grade, stage and outcomes). This comprehensive study will allow us with certainty to state if there are mutations in the promoters and genes of PAI-1 and ANG that are functional and thus may lead to the growth advantage that we previously demonstrated in our experiments.

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

PubMed

Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

2018-01-24

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Estimation of antigenic tetanus toxoid extracted from biodegradable microspheres.

PubMed

Xing, D K; McLellan, K; Corbel, M J; Sesardic, D

1996-03-01

Microspheres made from poly (lactic/glycolic acid) polymers have been considered as a new delivery system for single-dose tetanus toxoid (TT) vaccines. One of the most critical properties of the proposed vaccines is the loading and distribution of TT as this will have a profound effect on immunogenicity. As the concentration of TT in microspheres is very low sensitive assay methods are required. An assay incorporating monoclonal antibody (MAb) recognizing a neutralizing epitope and cross-reacting with TT was developed (MAp capture ELISA) which provided a sensitivity of 0.001 Lf/ml. An extraction procedure was devised which did not destroy the antigenicity and gave a recovery of 90.6 +/- 3.39% when applied to different preparations. The extracted TT was then quantified by MAb capture ELISA which was estimated to be 250-fold more sensitive than single-site ELISA for toxoid. The loading of 20 microspheres preparations (12 filled and 8 placebo) was determined by both protein micro-BCA assay and the developed assay for TT. The TT content obtained for the 12 filled microspheres preparations from different sources varied up to 400-fold (range 0.01-4.0 Lf/mg microspheres). The utility of the MAb capture ELISA for detection of total antigenic content in microspheres was confirmed by the observation that the determine TT loading correlated with the theoretical loading predicted from the protein content for the best preparations. Preparations with high loading gave the greatest peak response. There was no relationship between dose and the in vivo immunogenic response, suggesting that encapsulated vaccines with differential loading, release properties and presence of excipients will have different response curves in vivo. Hence, the present assay, when combined with information on toxoid release rate and presence and effect of excipients may be of value in predicting in vivo response.

All0809/8/7 is a DevBCA-like ABC-type efflux pump required for diazotrophic growth in Anabaena sp. PCC 7120.

PubMed

Staron, Peter; Maldener, Iris

2012-10-01

Efflux pumps export a wide variety of proteinaceous and non-proteinaceous substrates across the Gram-negative cell wall. For the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the ATP-driven glycolipid efflux pump DevBCA-TolC has been shown to be crucial for the differentiation of N(2)-fixing heterocysts from photosynthetically active vegetative cells. In this study, a homologous system was described. All0809/8/7-TolC form a typical ATP-driven efflux pump as shown by surface plasmon resonance. This putative exporter is also involved in diazotrophic growth of Anabaena sp. PCC 7120. A mutant in all0809 encoding the periplasmic membrane fusion protein of the pump was not able to grow without combined nitrogen. Although heterocysts of this mutant were not distinguishable from those of the wild-type in light and electron micrographs, they were impaired in providing the microoxic environment necessary for N(2) fixation. RT-PCR of all0809 transcripts and localization studies on All0807-GFP revealed that All0809/8/7 was initially downregulated during heterocyst maturation and upregulated at later stages of heterocyst formation in all cells of the filament. A substrate of the efflux pump could not be identified in ATP hydrolysis assays. We discuss a role for All0809/8/7-TolC in maintaining the continuous periplasm and how this would be of special importance for heterocyst differentiation.

Effects of Early Altitude Exposure Following Traumatic Injury and Hemorrhagic Shock

DTIC Science & Technology

2017-06-27

chemokines by multiplex enzyme-linked immunosorbent assay ( ELISA ) (Quansys, Logan, UT), including the following: interleukin 1 alpha and beta (IL...Tissue Cytokine Profiles Fourteen cytokines and chemokines were analyzed from serum and intestinal tissues via multiplex ELISA . There were no...2017-3567, 25 Jul 2017. LIST OF ABBREVIATIONS AND ACRONYMS AE aeromedical evacuation BCA bicinchoninic acid ELISA enzyme-linked immunosorbent

Characterisation of Atherogenic Effects of Low Carbohydrate, High Protein Diet (LCHP) in ApoE/LDLR-/- Mice.

PubMed

Kostogrys, R B; Johann, C; Czyżyńska, I; Franczyk-Żarów, M; Drahun, A; Maślak, E; Jasztal, A; Gajda, M; Mateuszuk, Ł; Wrobel, T P; Baranska, M; Wybrańska, I; Jezkova, K; Nachtigal, P; Chlopicki, S

2015-08-01

Low Carbohydrate High Protein diet represents a popular strategy to achieve weight loss. The aim of this study was to characterize effects of low carbohydrate, high protein diet (LCHP) on atherosclerotic plaque development in brachiocephalic artery (BCA) in apoE/LDLR-/- mice and to elucidate mechanisms of proatherogenic effects of LCHP diet. Atherosclerosis plaques in brachiocephalic artery (BCA) as well as in aortic roots, lipoprotein profile, inflammation biomarkers, expression of SREBP-1 in the liver as well as mortality were analyzed in Control diet (AIN-93G) or LCHP (Low Carbohydrate High Protein) diet fed mice. Area of atherosclerotic plaques in aortic roots or BCA from LCHP diet fed mice was substantially increased as compared to mice fed control diet and was characterized by increased lipids and cholesterol contents (ORO staining, FT-IR analysis), increased macrophage infiltration (MOMA-2) and activity of MMPs (zymography). Pro-atherogenic phenotype of LCHP fed apoE/LDLR-/- mice was associated with increased plasma total cholesterol concentration, and in LDL and VLDL fractions, increased TG contents in VLDL, and a modest increase in plasma urea. LCHP diet increased SCD-1 index, activated SREBP-1 transcription factor in the liver and triggered acute phase response as evidence by an increased plasma concentration of haptoglobin, CRP or AGP. Finally, in long-term experiment survival of apoE/LDLR-/- mice fed LCHP diet was substantially reduced as compared to their counterparts fed control diet suggesting overall detrimental effects of LCHP diet on health. The pro-atherogenic effect of LCHP diet in apoE/LDLR-/- mice is associated with profound increase in LDL and VLDL cholesterol, VLDL triglicerides, liver SREBP-1 upregulation, and systemic inflammation.

Soot Aerosol In The Atmosphere: Pole-to-Pole Distribution And Contributions by Aircraft

NASA Technical Reports Server (NTRS)

Pueschel, R. F.; Verma, S.; Howard, S. D.; Ferry, G. V.; Goodman, J.; Allen, D. A.; Strawa, Anthony W. (Technical Monitor)

1995-01-01

Interest in the distribution of black carbon (soot) aerosol (BCA) in the atmosphere is warranted for the following reasons: (1) BCA has the highest absorption cross section of any compound known, thus it can absorb solar radiation to cause atmospheric warming; (2) BCA is a strong adsorber of gases, thus it can catalyze heterogeneous chemical reactions to modify the chemical composition of the atmosphere; (3) If aircraft emission is the major source of atmospheric BCA, it can serve as an atmospheric tracer of aircraft exhaust. We collect BCA particles greater than or equal to 0.02 micrometer diameter by wires mounted on both the DC-8 and ER-2 aircraft. After return to the laboratory, the wires are examined with a field emission scanning electron microscope to identify BCA particles by their characteristic morphology. Typically, BCA exists in the atmosphere as small particles of complex morphology. The particle sizes at the source are measured in tens of Angstrom units; after a short residence time in the atmosphere, individual particles coalesce to loosely packed agglomerates of typical dimensions 0.01 to 0.1 micrometer. We approximate the size of each BCA aggregate by that of a sphere of equivalent volume. This is done by computing the volume of a sphere whose diameter is the mean between averaged minimum and maximum dimensions of the BCA particle. While this procedure probably underestimates the actual surface area, it permits us to compare BCA size distributions among themselves and with other types of aerosols. When statistically justified, we fit lognormal distributions to the data points to determine number concentrations, geometric mean radii, standard deviations, BCA surface areas and volumes. Results to date permit the following conclusions: (1) BCA concentration in the northern stratosphere averages 0.6 ng per cubic meters. This amount is one part in 10(exp 4) after a volcanic eruption (e.g., Pinatubo) increasing to about one percent during volcanic quiescence. In the northern troposphere, BCA concentration averages 3.2 ng per cubic meters, or 0.3 percent of the background aerosol. (2) Applying an BCA emission index EI(BCA)=5 x 10(exp -5), measured in the exhaust wake of a Concorde supersonic jet aircraft, to realistic estimates of fuel burnt by the current and projected fleets permits us to conclude that: (i) Most BCA in the northern stratosphere results from aircraft emissions; (ii) Most BCA in the northern troposphere results from other sources than aircraft; (iii) A projected supersonic fleet will increase the northern stratospheric BCA concentration by one order of magnitude, unless the emission index is substantially reduced. (3) A strong gradient between the northern and southern hemispheres indicates that mixing across the equator is greatly inhibited in relation to atmospheric residence times of BCA. (4) The single scatter albedo of BCA/"background" aerosol mixtures suggests a cooling effect for most of the globe; an exemption is the Arctic because of the high surface albedo of the snow/ice covered earth's surface.

Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.

PubMed

Tran, Duc T; Banerjee, Sambuddha; Alayash, Abdu I; Crumbliss, Alvin L; Fitzgerald, Michael C

2012-02-07

Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application. © 2011 American Chemical Society

Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

PubMed

Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

2009-10-01

Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

Physical Properties and Cellular Responses to Crosslinkable Poly(Propylene Fumarate)/Hydroxyapatite Nanocomposites

PubMed Central

Lee, Kee-Won; Wang, Shanfeng; Yaszemski, Michael J.; Lu, Lichun

2008-01-01

A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity. PMID:18403013

Comparison of Plasma Exosomes by Differential Ultracentrifugation and Solvent Precipitation Methods.

PubMed

Peng, Qiao; Zhang, Jing; Zhou, Gang

2018-06-01

Emerging evidence has identified that exosomes play a pivotal role in intercellular signal transmission. However, the standardized purification techniques to isolate high quality exosomes are still deficient at present. This study was to evaluate reproducibility and efficiency of differential ultracentrifugation and solvent precipitation-based kits by isolating plasma-derived exosomes from oral lichen planus patients. Morphology, exosomal biomarkers, particle size distribution, proteomic components, and protein yield of isolated exosomes were evaluated by transmission electron microscope, western blot, laser diffraction instrument, Coomassie staining, and BCA protein assay kit, respectively. TEM displayed representative cup-shaped morphology of exosomes and western blot identified exosomal biomarkers CD9 and CD63. The size distribution showed that particles by differential ultracentrifugation were mainly from 26.15 nm to 166.5 nm, while some of the particles obtained by solvent precipitation kits were larger than 1,000 nm. In addition, exosomes isolated by solvent precipitation kits showed a significantly higher amount of protein yield due to plasma albumin contamination. Both differential ultracentrifugation and precipitation based kits could successfully isolate plasma exosomes, and exosomes by differential ultracentrifugation were purer and more appropriate for further proteomic analysis.

Silibinin suppresses bladder cancer cell malignancy and chemoresistance in an NF-κB signal-dependent and signal-independent manner.

PubMed

Sun, Yi; Guan, Zhenfeng; Zhao, Wencai; Jiang, Yazhuo; Li, Qing; Cheng, Yongyi; Xu, Yonggang

2017-10-01

Because bladder cancer (BCa) is the 9th most common malignant tumor and 13th leading cause of death due to cancer, therapeutic approaches have attracted a great deal of attention from both clinicians and BCa patients. Although the development of surgery and targeted drugs has brought new challenges for the traditional concept of BCa therapy, various types of chemotherapy remain the final treatment method for many BCa patients. However, chemoresistance inevitably appears, leading to the failure of chemotherapy. Silibinin, a polyphenolic flavonoid component isolated from the fruits or seeds of milk thistle, has been reported to play important roles in inhibiting tumor chemoresistance in breast cancer and head and neck squamous cell carcinomas. Our previous study indicated that silibinin inhibited BCa progression in some mechanisms but with no conclusion of chemoresistance inhibition. Therefore, in the present study, we dissected the role of silibinin in BCa progression and chemoresistance. Our results revealed that in BCa, chemodrug-induced chemoresistance was reversed in the presence of silibinin. Further mechanistic study indicated that silibinin suppressed chemoresistance and BCa malignancy in an NF-κB-dependent and -independent manner. In addition, all of the inhibitory effects were dose‑dependent. Thus, our results provide a potential use for silibinin in BCa therapeutics.

Soot Aerosols in the Atmosphere: Contributions by Aircraft

NASA Technical Reports Server (NTRS)

Pueschel, R. F.; Verma, S.; Howard, S. D.; Goodman, J.; Ferry, G. V.; Allen, D. A.; Gore, Warren J. Y. (Technical Monitor)

1995-01-01

Interest in the distribution of black carbon (soot) aerosol (BCA) in the atmosphere is based on the following: (1) Because BCA has the highest absorption cross section of any compound know, it can absorb solar radiation to cause atmospheric warming; (2) Because BCA is a strong adsorber of gases, it can catalyze heterogeneous chemical reactions to modify the chemical composition of the atmosphere; (3) If aircraft emission is the major source of BCA, it can serve as an atmospheric tracer of aircraft exhaust. We collect BCA particles as small as 0.02 micrometers by wires mounted on both the DC-8 and ER-2 aircraft. After return to the laboratory, the wires are examined with a field emission scanning electron microscope to identify BCA particles by their characteristics morphology, Typically, BCA exists in the atmosphere as small particles of complex morphology. The particle sizes at the source are measured in tens of Angstrom units; after a short residence time in the atmosphere, individual particles coalesce to loosely packed agglomerates of typical dimensions 0.01 to 0.1 micrometer. We approximate the size of each BCA aggregate by that of a sphere of equivalent volume. This is done by computing the volume of a sphere whose diameter is the mean between averaged minimum and maximum dimensions of the BCA particle. While this procedure probably underestimates the actual surface area, it permits us to compare BCA size distributions among themselves and with other types of aerosols.

Suppression of ERβ signaling via ERβ knockout or antagonist protects against bladder cancer development.

PubMed

Hsu, Iawen; Chuang, Kun-Lung; Slavin, Spencer; Da, Jun; Lim, Wei-Xun; Pang, See-Tong; O'Brien, Jeanne H; Yeh, Shuyuan

2014-03-01

Epidemiological studies showed that women have a lower bladder cancer (BCa) incidence, yet higher muscle-invasive rates than men, suggesting that estrogen and the estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), may play critical roles in BCa progression. Using in vitro cell lines and an in vivo carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced mouse BCa model, we found that ERβ plays a positive role in promoting BCa progression. Knockdown of ERβ with ERβ-shRNA in ERβ-positive human BCa J82, 647v and T24 cell lines led to suppressed cell growth and invasion. Mice lacking ERβ have less cancer incidence with reduced expression of the proliferation marker Ki67 in BBN-induced BCa. Consistently, our results show that non-malignant urothelial cells with ERβ knockdown are more resistant to carcinogen-induced malignant transformation. Mechanism dissection found that targeting ERβ suppressed the expression of minichromosome maintenance complex component 5 (MCM5), a DNA replication licensing factor that is involved in tumor cell growth. Restoring MCM5 expression can partially reverse ERβ knockdown-mediated growth reduction. Supportively, treating cells with the ERβ-specific antagonist, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), reduced BCa cell growth and invasion, as well as MCM5 expression. Furthermore, we provide the first evidence that BCa burden and mortality can be controlled by PHTPP treatment in the carcinogen-induced BCa model. Together, these results demonstrate that ERβ could play positive roles in promoting BCa progression via MCM5 regulation. Targeting ERβ through ERβ-shRNA, PHTPP or via downstream targets, such as MCM5, could serve as potential therapeutic approaches to battle BCa.

Barriers to Cervical Cancer and Breast Cancer Screening Uptake in Low-Income and Middle-Income Countries: A Systematic Review

PubMed Central

Islam, Rakibul M; Billah, Baki; Hossain, Md Nassif; Oldroyd, John

2017-01-01

Background: Cervical cancer (CCa) and breast cancer (BCa) are the two leading cancers in women worldwide. Early detection and education to promote early diagnosis and screening of CCa and BCa greatly increases the chances for successful treatment and survival. Screening uptake for CCa and BCa in low and middle - income countries (LMICs) is low, and is consequently failing to prevent these diseases. We conducted a systematic review to identify the key barriers to CCa and BCa screening in women in LMICs. Methods: We performed a systematic literature search using Ovid MEDLINE, EMBASE, PsycINFO, SCOPUS, CINHAL Plus, and Google scholar to retrieve all English language studies from inception to 2015. This review was done in accordance with the PRISMA-P guidelines. Results: 53 eligible studies, 31 CCa screening studies and 22 BCa screening studies, provided information on 81,210 participants. We found fewer studies in low-income and lower - middle - income countries than in upper - middle - income countries. Lack of knowledge about CCa and BCa, and understanding of the role of screening were the key barriers to CCa and BCa screening in LMICs. Factors that are opportunities for knowledge acquisition, such as level of education, urban living, employment outside the home, facilitated CCa and BCa screening uptake in women in LMICs. Conclusions: Improvements to CCa and BCa screening uptake in LMICs must be accompanied by educational interventions which aim to improve knowledge and understanding of CCa and BCa and screening to asymptomatic women. It is imperative for governments and health policy makers in LMICs to implement screening programmes, including educational interventions, to ensure the prevention and early detection of women with CCa and BCa. These programmes and policies will be an integral part of a comprehensive population-based CCa and BCa control framework in LMICs. PMID:28749101

Barriers to Cervical Cancer and Breast Cancer Screening Uptake in Low-Income and Middle-Income Countries: A Systematic Review

PubMed

Islam, Rakibul M; Billah, Baki; Hossain, Md Nassif; Oldroyd, John

2017-07-27

Background: Cervical cancer (CCa) and breast cancer (BCa) are the two leading cancers in women worldwide. Early detection and education to promote early diagnosis and screening of CCa and BCa greatly increases the chances for successful treatment and survival. Screening uptake for CCa and BCa in low and middle - income countries (LMICs) is low, and is consequently failing to prevent these diseases. We conducted a systematic review to identify the key barriers to CCa and BCa screening in women in LMICs. Methods: We performed a systematic literature search using Ovid MEDLINE, EMBASE, PsycINFO, SCOPUS, CINHAL Plus, and Google scholar to retrieve all English language studies from inception to 2015. This review was done in accordance with the PRISMA-P guidelines. Results: 53 eligible studies, 31 CCa screening studies and 22 BCa screening studies, provided information on 81,210 participants. We found fewer studies in low-income and lower - middle - income countries than in upper - middle - income countries. Lack of knowledge about CCa and BCa, and understanding of the role of screening were the key barriers to CCa and BCa screening in LMICs. Factors that are opportunities for knowledge acquisition, such as level of education, urban living, employment outside the home, facilitated CCa and BCa screening uptake in women in LMICs. Conclusions: Improvements to CCa and BCa screening uptake in LMICs must be accompanied by educational interventions which aim to improve knowledge and understanding of CCa and BCa and screening to asymptomatic women. It is imperative for governments and health policy makers in LMICs to implement screening programmes, including educational interventions, to ensure the prevention and early detection of women with CCa and BCa. These programmes and policies will be an integral part of a comprehensive population-based CCa and BCa control framework in LMICs. Creative Commons Attribution License

[Effect of Shexiang Baoxin Pills on isoprenaline-induced myocardial cell hypertrophy and Cx43 expression].

PubMed

Tang, Fen; Jiang, Zhentao; Tan, Wenting; Long, Junrong; Liu, Shengquan; Chu, Chun

2017-08-28

To observe the effects of Shexiang Baoxin Pill (SBP) on isoprenaline (Iso)-induced changes in myocardial cell volume, shape, and connexin 43 (Cx43) expression.
 Methods: H9C2 myocardial cells were randomly divided into a control group, a Iso group and a Iso+SBP group. After 72 h of culture, the average surface area of H9C2 cells was measured under phase contrast microscope. Bicinchoninic acid (BCA) protein assay was carried out to determine the concentration of proteins. The survival rate of myocardial cells was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and the Cx43 expression was detected by Western blot.
 Results: The mean surface area and Cx43 concentration in Iso-treated myocardial cells were increased under the phase contrast microscope (P<0.05). Compared with the Iso group, the mean surface area was decreased, and the Cx43 concentration was reduced in the Iso+SBP group (both P<0.05). Compared with the control group, the Cx43 expression was obviously down-regulated in the H9C2 cells of the Iso group (P<0.05); while compared with the Iso group, the Cx43 expression was obviously up-regulated in the Iso+SBP group (P<0.05).
 Conclusion: Shexiang Baoxin Pills can prevent Iso-induced myocardial hypertrophy and down-regulate Cx43 expression.

Composition of enamel pellicle from dental erosion patients.

PubMed

Carpenter, G; Cotroneo, E; Moazzez, R; Rojas-Serrano, M; Donaldson, N; Austin, R; Zaidel, L; Bartlett, D; Proctor, G

2014-01-01

Oral health is dependent upon a thin mobile film of saliva on soft and hard tissues. Salivary proteins adhere to teeth to form the acquired enamel pellicle which is believed to protect teeth from acid erosion. This study investigated whether patients suffering diet-induced dental erosion had altered enamel pellicles. Thirty patients suffering erosion were compared to healthy age-matched controls. Subjects wore a maxillary splint holding hydroxyapatite and human enamel blocks for 1 h. The acquired enamel pellicle was removed from the blocks and compared to the natural incisor pellicle. Basic Erosive Wear Examination scores confirmed that dental erosion was present in erosion patients and absent from healthy age-matched controls. Erosion patients had half the amount of proteins (BCA assay) within the acquired pellicle forming on splint blocks compared to normal controls (p < 0.05). In particular, statherin, a calcium-binding protein, was 35% less abundant (p < 0.05). Calcium concentration within the acquired pellicle was also reduced by 50% in erosion patients (p < 0.001). In contrast, the natural pellicle on the incisor had similar amounts of total protein in erosion patients and healthy controls. In summary, the formation of new acquired pellicles on surfaces was reduced in erosion patients, which may explain their greater susceptibility to acid erosion of teeth. © 2014 S. Karger AG, Basel.

Protein loss in human hair from combination straightening and coloring treatments.

PubMed

França-Stefoni, Simone Aparecida; Dario, Michelli Ferrera; Sá-Dias, Tânia Cristina; Bedin, Valcinir; de Almeida, Adriano José; Baby, André Rolim; Velasco, Maria Valéria R

2015-09-01

Hair chemical treatments, such as dyeing and straightening products, are known to cause damage that can be assessed by protein loss. The aim of this study was to evaluate the hair protein loss caused by combined chemical treatments (dye and relaxer) using the validated bicinchoninic acid (BCA) method. Three kinds of straighteners, based on ammonium thioglycolate, guanidine hydroxide and sodium hydroxide, were evaluated and the least harmful combination indicated. Caucasian virgin dark brown hair tresses were treated with developed natural brown color oxidative hair dyeing and/or straightening commercial products based on ammonium thioglycolate, sodium hydroxide, or guanidine hydroxide. Protein loss quantification was assessed by the validated BCA method which has several advantages for quantifying protein loss in chemically treated hair. When both treatments (straightening and dyeing) were combined, a higher negative effect was observed, particularly for dyed hair treated with sodium hydroxide. In this case, a 356% increase in protein loss relative to virgin hair was observed and 208% in relation to only dyed hair. The combination of dying and relaxers based on ammonium thioglycolate or guanidine hydroxide caused a small increase in protein loss, suggesting that these straightening products could be the best alternatives for individuals wishing to combine both treatments. These results indicated that when application of both types of products is desired, ammonium thioglycolate or guanidine hydroxide should be chosen for the straightening process. © 2015 Wiley Periodicals, Inc.

Effect of biochanin A on corn grain (Zea mays) fermentation by bovine rumen amylolytic bacteria.

PubMed

Harlow, B E; Flythe, M D; Aiken, G E

2017-04-01

The objective was to determine the effect of biochanin A (BCA), an isoflavone produced by red clover (Trifolium pratense L.), on corn fermentation by rumen micro-organisms. When bovine rumen bacterial cell suspensions (n = 3) were incubated (24 h, 39°C) with ground corn, amylolytic bacteria including group D Gram-positive cocci (GPC; Streptococcus bovis; enterococci) proliferated, cellulolytic bacteria were inhibited, lactate accumulated and pH declined. Addition of BCA (30 μg ml -1 ) inhibited lactate production, and pH decline. BCA had no effect on total amylolytics, but increased lactobacilli and decreased GPC. The initial rate and total starch disappearance was decreased by BCA addition. BCA with added Strep. bovis HC5 supernatant (containing bacteriocins) inhibited the amylolytic bacteria tested (Strep. bovis JB1; Strep. bovis HC5; Lactobacillus reuteri, Selenemonas ruminatium) to a greater extent than either addition alone. BCA increased cellulolytics and dry matter digestibility of hay with corn starch. These results indicate that BCA mitigates changes associated with corn fermentation by bovine rumen bacteria ex vivo. BCA could serve as an effective mitigation strategy for rumen acidosis. Future research is needed to evaluate the effect of BCA on mitigating rumen acidosis in vivo. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Targeting estrogen/estrogen receptor alpha enhances Bacillus Calmette-Guérin efficacy in bladder cancer

PubMed Central

Shang, Zhiqun; Li, Yanjun; Hsu, Iawen; Zhang, Minghao; Tian, Jing; Wen, Simeng; Han, Ruifa; Messing, Edward M.; Chang, Chawnshang; Niu, Yuanjie; Yeh, Shuyuan

2016-01-01

Recent studies showed the potential linkage of estrogen/estrogen receptor signaling with bladder tumorigenesis, yet detailed mechanisms remain elusive. Here we found a new potential therapy with the combination of Bacillus Calmette Guerin (BCG) and the anti-estrogen ICI 182,780 led to better suppression of bladder cancer (BCa) than BCG alone. Mechanism dissection found ICI 182,780 could promote BCG attachment/internalization to the BCa cells through increased integrin-α5β1 expression and IL-6 release, which may enhance BCG-induced suppression of BCa cell growth via recruiting more monocytes/macrophages to BCa cells and increased TNF-α release. Consistently, in vivo studies found ICI 182,780 could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models. Together, these in vitro and in vivo results suggest that combining BCG with anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence. PMID:27092883

Targeting estrogen/estrogen receptor alpha enhances Bacillus Calmette-Guérin efficacy in bladder cancer.

PubMed

Shang, Zhiqun; Li, Yanjun; Hsu, Iawen; Zhang, Minghao; Tian, Jing; Wen, Simeng; Han, Ruifa; Messing, Edward M; Chang, Chawnshang; Niu, Yuanjie; Yeh, Shuyuan

2016-05-10

Recent studies showed the potential linkage of estrogen/estrogen receptor signaling with bladder tumorigenesis, yet detailed mechanisms remain elusive. Here we found a new potential therapy with the combination of Bacillus Calmette-Guerin (BCG) and the anti-estrogen ICI 182,780 led to better suppression of bladder cancer (BCa) than BCG alone. Mechanism dissection found ICI 182,780 could promote BCG attachment/internalization to the BCa cells through increased integrin-α5β1 expression and IL-6 release, which may enhance BCG-induced suppression of BCa cell growth via recruiting more monocytes/macrophages to BCa cells and increased TNF-α release. Consistently, in vivo studies found ICI 182,780 could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models. Together, these in vitro and in vivo results suggest that combining BCG with anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence.

Stratospheric Sulfuric Acid and Black Carbon Aerosol Measured During POLARIS and its Role in Ozone Chemistry

NASA Technical Reports Server (NTRS)

Strawa, Anthony W.; Pueschel, R. F.; Drdla, K.; Verma, S.; Gore, Warren J. (Technical Monitor)

1998-01-01

Stratospheric aerosol can affect the environment in three ways. Sulfuric acid aerosol have been shown to act as sites for the reduction of reactive nitrogen and chlorine and as condensation sites to form Polar Stratospheric Clouds, under very cold conditions, which facilitate ozone depletion. Recently, modeling studies have suggested a link between BCA (Black Carbon Aerosol) and ozone chemistry. These studies suggest that HNO3, NO2, and O3 may be reduced heterogeneously on BCA particles. The ozone reaction converts ozone to oxygen molecules, while HNO3 and NO2 react to form NOx. Finally, a buildup of BCA could reduce the single-scatter albedo of aerosol below a value of 0.98, a critical value that has been postulated to change the effect of stratospheric aerosol from cooling to warming. Correlations between measured BCA amounts and aircraft usage have been reported. Attempts to link BCA to ozone chemistry and other stratospheric processes have been hindered by questions concerning the amount of BCA that exists in the stratosphere, the magnitude of reaction probabilities, and the scarcity of BCA measurements. The Ames Wire Impactors (AWI) participated in POLARIS as part of the complement of experiments on the NASA ER-2. One of our main objectives was to determine the amount of aerosol surface area, particularly BCA, available for reaction with stratospheric constituents and assess if possible, the importance of these reactions. The AWI collects aerosol and BCA particles on thin Palladium wires that are exposed to the ambient air in a controlled manner. The samples are returned to the laboratory for subsequent analysis. The product of the AWI analysis is the size, surface area, and volume distributions, morphology and elemental composition of aerosol and BCA. This paper presents results from our experiments during POLARIS and puts these measurements in the context of POLARIS and other missions in which we have participated. It describes modifications to the AWI data analysis procedures in which the collection of BCA is modeled as a fractal aggregate. The new analysis results in an increase in BCA surface area of approximately 24 and an increase in mass of 7-10 from the previous method. For the current study, BCA surface area is used in computer models that attempt to predict measured NOx/NOy ratios and O3 depletion rates. Inclusion of the HNO3 reaction with BCA in one model tends to improve the agreement of calculated to measured NOx/NOy ratio. However, it was found that these trends are viable only if the reactions are catalytic.

Core-top calibration of B/Ca in Pacific Ocean Neogloboquadrina incompta and Globigerina bulloides as a surface water carbonate system proxy

NASA Astrophysics Data System (ADS)

Quintana Krupinski, Nadine B.; Russell, Ann D.; Pak, Dorothy K.; Paytan, Adina

2017-05-01

Practical methods for reconstructing past ocean carbonate chemistry are needed to study past periods of ocean acidification and improve understanding of the marine carbonate system's role in the global climate cycles. Planktic foraminiferal B/Ca may fill this role, but requires better understanding and improved proxy calibrations. We used Pacific Ocean core-top sediments to generate new calibrations of the B/Ca proxy for past carbonate system parameters in two upwelling/subpolar species of asymbiotic planktic foraminifera (Globigerina bulloides and Neogloboquadrina incompta). Both species show significant positive correlation of B/Ca with calcite saturation (Ωcalcite) and carbonate ion concentration ([3 2-CO]) across a broad range of environmental conditions. This suggests a calcification rate control on B/Ca incorporation (as Ωcalcite regulates calcification rate), in agreement with recent inorganic calcite studies. This is also consistent with a surface entrapment model of trace element incorporation into CaCO3. In neither species is B/Ca significantly correlated with pH, suggesting that pH does not directly regulate boron incorporation, and that calculation of pH directly from foraminiferal B/Ca is not suitable. Correlations between B/Ca and [B(OH)4-], [B(OH)4-/HCO3-], and [B(OH)4-]/DIC) are weaker than with Ωcalcite. Boron partition coefficients (KD =[ B / Ca ] solid /[ B4 -(OH) /HCO-3 ] seawater) show little or no correlation with [CO32-] or temperature and vary widely, providing no support for application of KD to calculate carbonate system parameters from B/Ca. We also discuss potential effects of depth-related dissolution, temperature, and salinity on B/Ca. These empirical calibrations linking foraminiferal calcite B/Ca with Ωcalcite provide a strong tool for reconstructing the past ocean carbonate system and improve our understanding of the proxy's geochemical basis.

Biochanin-A ameliorates behavioural and neurochemical derangements in cognitive-deficit mice for the betterment of Alzheimer's disease.

PubMed

Biradar, S M; Joshi, H; Chheda, T K

2014-04-01

Biochanin-A (BCA), a potent phytoconstituent, has been previously used as an antitumour, a dopaminergic neuron protective agent, an antioxidant, an anticholinergic and on other pharmacological activities including neuroprotection. The present study was aimed to evaluate the behavioural and neurochemical evidence of BCA in cognitive-deficit mice in scopolamine challenged and natural aged-induced amnesia models in young and aged mice, respectively. BCA has exhibited decrease in the transfer latency and increase in step through latency significantly (p < 0.001) in scopolamine-treated and natural aged mice of exteroceptive behavioural models such as elevated plus maze and passive shock avoidance paradigm, respectively. A decrease in acetylcholinesterase activity of whole brain was seen in scopolamine and aged mice with standard piracetam (Pira; p < 0.001) and BCA in dose-dependent manner. The antioxidant property of BCA was proven by increase in GSH (p < 0.01) and decrease in thiobarbituric acid reactive substances level significantly in a scopolamine-challenged and aged mice. The scopolamine-treated mice exhibited significant (p < 0.01) increase in the content of noradrenalin and dopamine, which is a sign of dementia, and these excess increased neurotransmitters were reversed by BCA 40 mg kg(-1) (p < 0.05), BCA 20 mg kg(-1) (p > 0.05), BCA 10 mg kg(-1) (p < 0.05) and standard Pira (p < 0.05) when compared with scopolamine group. Furthermore, in histopathology of hippocampus, the Pira and BCA-treated mice were protected from the formation of pyknotic neurons, increases in the viable cells count and decreases in the number of degenerative cells compared with the scopolamine group. Hence, BCA could be potential enough for the betterment of Alzheimer's disease.

N-3 poly-unsaturated fatty acids shift estrogen signaling to inhibit human breast cancer cell growth.

PubMed

Cao, Wenqing; Ma, ZhiFan; Rasenick, Mark M; Yeh, ShuYan; Yu, JiangZhou

2012-01-01

Although evidence has shown the regulating effect of n-3 poly-unsaturated fatty acid (n-3 PUFA) on cell signaling transduction, it remains unknown whether n-3 PUFA treatment modulates estrogen signaling. The current study showed that docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) shifted the pro-survival and proliferative effect of estrogen to a pro-apoptotic effect in human breast cancer (BCa) MCF-7 and T47D cells. 17 β-estradiol (E2) enhanced the inhibitory effect of n-3 PUFAs on BCa cell growth. The IC50 of DHA or EPA in MCF-7 cells decreased when combined with E2 (10 nM) treatment (from 173 µM for DHA only to 113 µM for DHA+E2, and from 187 µm for EPA only to 130 µm for EPA+E2). E2 also augmented apoptosis in n-3 PUFA-treated BCa cells. In contrast, in cells treated with stearic acid (SA, C18:0) as well as cells not treated with fatty acid, E2 promoted breast cancer cell growth. Classical (nuclear) estrogen receptors may not be involved in the pro-apoptotic effects of E2 on the n-3 PUFA-treated BCa cells because ERα agonist failed to elicit, and ERα knockdown failed to block E2 pro-apoptotic effects. Subsequent studies reveal that G protein coupled estrogen receptor 1 (GPER1) may mediate the pro-apoptotic effect of estrogen. N-3 PUFA treatment initiated the pro-apoptotic signaling of estrogen by increasing GPER1-cAMP-PKA signaling response, and blunting EGFR, Erk 1/2, and AKT activity. These findings may not only provide the evidence to link n-3 PUFAs biologic effects and the pro-apoptotic signaling of estrogen in breast cancer cells, but also shed new insight into the potential application of n-3 PUFAs in BCa treatment.

miR-1271 inhibits ERα expression and confers letrozole resistance in breast cancer.

PubMed

Yu, Tao; Yu, Hai-Ru; Sun, Jia-Yi; Zhao, Zhao; Li, Shuang; Zhang, Xin-Feng; Liao, Zhi-Xuan; Cui, Ming-Ke; Li, Juan; Li, Chan; Zhang, Qiang

2017-12-05

Attenuation of estrogen receptor α (ERα) expression via unknown mechanism(s) is a hallmark of endocrine-resistant breast cancer (BCa) progression. Here, we report that miR-1271 was significantly down-regulated in letrozole-resistant BCa tissues and in letrozole-resistant BCa cells. miR-1271 directly targeted the chromatin of DNA damage-inducible transcript 3 (DDIT3) gene. miR-1271 expression level was inversely correlated to DDIT3 mRNA level in BCa biopsies. Form a mechanistic standpoint, reintroduction of exogenous miR-1271 could effectively restore ERα level via inhibiting DDIT3 expression, thereby potentiating letrozole sensitivity in BCa cells. Moreover, DDIT3 deregulation promoted letrozole-resistance by acting as a potent corepressor of ESR1 transcription. Taken together, we have identified that disruption of the miR-1271/DDIT3/ERα cascade plays a causative role in the pathogenesis of letrozole resistance in BCa.

Does stress increase risk of breast cancer? A 15-year prospective study.

PubMed

Butow, Phyllis; Price, Melanie; Coll, Joseph; Tucker, Katherine; Meiser, Bettina; Milne, Roger; Wilson, Judith; Heiniger, Louise; Baylock, Brandi; Bullen, Tracey; Weideman, Prue; Phillips, Kelly-Anne

2018-04-20

The possible impact of stress on cancer incidence remains controversial. We prospectively evaluated associations between life event stressors, social support, personality characteristics (optimism, anger control, antiemotionality), and risk of developing primary breast cancer (BCa), in women at increased familial risk of BCa. A prospective cohort, repeated measures design was used. Recruitment was through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer, which collects genetic, epidemiological, and clinical data from Australasian families with multiple BCa cases. Acute and chronic stressors for the prior 3 years and psychosocial, clinical, and epidemiological variables were measured at cohort entry and at 3-yearly intervals. Cox proportional hazard regression analysis controlling for BCa risk factors and familial clustering was undertaken. The primary outcome was histopathologically confirmed BCa (invasive or ductal carcinoma in situ, including occult cases diagnosed during risk-reducing mastectomy). Of 3595 consecutive women invited to participate, 3054 (85.0%) consented. Of these, 2739 (89.7%) from 990 families (range 1-16 per family) completed at least 1 assessment point. During the study, 103 women were diagnosed with BCa. No stressor or psychosocial variable or interaction between them was significantly associated with BCa in unadjusted or adjusted models (total acute stressors HR = 1.03 [0.99-1.08], P = .19; total chronic stressors HR = 1.0 [0.90-1.11], P = .98). This study did not demonstrate an association between acute and chronic stressors, social support, optimism, antiemotionality or anger control, and BCa risk. Women should focus on proven methods of BCa risk reduction. Copyright © 2018 John Wiley & Sons, Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandapaka, A; Ghebremedhin, A; Patyal, B

Purpose: Patients who undergo n-BCA glue embolization as part of treatment for AVMs are later referred for proton therapy. Knowing the relative stopping power of the glue accurately allows us to perform accurate dose calculations. In this study we experimentally determine the relative stopping power of an n-BCA mixture in a 126 MeV and 149.6 MeV proton beams. Methods: One unit of the TRUFILL™ n-BCA liquid embolic system consists of 1g unit of n-BCA, 1g unit of Tantalum powder and one 10mL vial of Ethiodized oil. The physician mixed 3:1 Ethiodized oil to n-BCA. Five units (20cc) of the n-BCAmore » liquid embolic glue were prepared and placed in a 6cm x 3cm x3cm Lucite container. The container was placed in front of a water tank in the proton beam path. A diamond detector (active volume 0.004mm3) was used to measure distal edge of depth dose of a modulated 126 MeV proton beam collimated using a 3cm brass aperture. The procedure was repeated with a container carrying the same amount of water placed in front of the water tank. The difference in the depth dose measured with glue and with water was used to determine the relative stopping power of the glue. The same determination was done earlier at 149.6 MeV using a different smaller sample (4cc) of n-BCA. Results: The relative stopping power of this particular n-BCA mixture was determined to be 1.06 at both 126 MeV and 149.6 MeV. We are working on obtaining the composition data of the n-BCA glue so we can perform Monte Carlo calculations. Conclusion: Accurate value of the stopping power of the n-BCA glue in the proton beam was determined to be 1.06. It will improve the accuracy of dose calculations in proton radiosurgery procedures on AVM patients with n-BCA embolization.« less

Induction of tumor cell death through targeting tubulin and evoking dysregulation of cell cycle regulatory proteins by multifunctional cinnamaldehydes.

PubMed

Nagle, Amrita A; Gan, Fei-Fei; Jones, Gavin; So, Choon-Leng; Wells, Geoffrey; Chew, Eng-Hui

2012-01-01

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G(2)/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G(2) phase. G(2) arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G(2) to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G(2) phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.

Cost-Plus-Percentage-of-Costs in Government Contracts

DTIC Science & Technology

1984-09-01

services to be performed under the contract. ASPR 3-405.6(a). cf. Martin-Marietta Corporation ASBCA No. 10062 , 65-2 BCA 14973. The granting of...371 F.2d 859 (1967); General Builders Supply Co. v. United States, 409 . F.2d 246 (1969); Martin-Marietta Corp., ASBCA 10062 , 65-2 BCA 1 4973 (1965...Inc., ASBCA 18730, 74-2 BCA 1 10,71.1 (1974). Kenimons-Wilson, Inc., ASBCA 16167, 72-2 BCA II 9689 (1972). Martin-Marietta Corp., ASBCA 10062 , 65-2

Health system costs for stage-specific breast cancer: a population-based approach

PubMed Central

Mittmann, N.; Porter, J.M.; Rangrej, J.; Seung, S.J.; Liu, N.; Saskin, R.; Cheung, M.C.; Leighl, N.B.; Hoch, J.S.; Trudeau, M.; Evans, W.K.; Dainty, K.N.; DeAngelis, C.; Earle, C.C.

2014-01-01

Objective The objective of the present analysis was to determine the publicly funded health care costs associated with the care of breast cancer (bca) patients by disease stage. Methods Incident cases of female invasive bca (2005–2009) were extracted from the Ontario Cancer Registry and linked to administrative datasets from the publicly funded system. The type and use of health care services were stratified by disease stage over the first 2 years after diagnosis. Mean costs and costs by type of clinical resource used in the care of bca patients were compared with costs for a matched control group. The attributable cost for the 2-year time horizon was determined in 2008 Canadian dollars. Results This cohort study involved 39,655 patients with bca and 190,520 control subjects. The average age in those groups was 61.1 and 60.9 years respectively. Most bca patients were classified as either stage i (34.4%) or stage ii (31.8%). Of the bca cohort, 8% died within the first 2 years after diagnosis. The overall mean cost per bca case from a public payer perspective in the first 2 years after diagnosis was $41,686. Over the 2-year time horizon, the mean cost increased by stage: i, $29,938; ii, $46,893; iii, $65,369; and iv, $66,627. The attributable cost of bca was $31,732. Cost drivers were cancer clinic visits, physician billings, and hospitalizations. Conclusions Costs of care increased by stage of bca. Cost drivers were cancer clinic visits, physician billings, and hospitalizations. These data will assist planning and decision-making for the use of limited health care resources. PMID:25489255

Awareness of breast cancer and barriers to breast screening uptake in Bangladesh: A population based survey.

PubMed

Islam, Rakibul M; Bell, Robin J; Billah, Baki; Hossain, Mohammad B; Davis, Susan R

2016-02-01

To investigate the awareness of breast cancer (BCa) and BCa screening amongst women at midlife in Bangladesh. A nationally representative cross-sectional survey of women aged 30-59 years was conducted in 7 districts of the 7 divisions in Bangladesh, using a multistage cluster sampling technique. The factors associated with the awareness of BCa and breast assessment of asymptomatic women were investigated separately, using multivariable logistic regression. Of the 1590 participants, mean age 42.3 (±8.0) years, 81.9% had ever heard of BCa and 64.2% of any methods of BCa screening, respectively. Awareness of BCa was associated with being aged 40-49 years (adjusted OR 2.04, 95% CI 1.46-2.84), aged 49-59 years (1.96, 1.32-2.91), being overweight (1.46, 1.07-2.01) and obesity (1.62, 1.01-2.62), while inversely associated with rural dwelling (0.37, 0.22-0.61), primary education (0.44, 0.27-0.70), having no education (0.23, 0.14-0.36) and parity (0.62, 0.44-0.87). Of the 750 women who were aware of clinical breast examination (CBE) or mammography, reasons provided for not undergoing screening included that they had no symptoms (92%) and that they did not know screening was needed (40%). 8% of women reported CBE. Women with no education were less likely to have undergone CBE (0.38, 0.141.04; p=0.059). Lack of understanding of the assessment of asymptomatic women is the key obstacle to BCa screening uptake in Bangladesh. Health education programs, especially BCa awareness programs, have the potential to increase BCa awareness and down-staging of the disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Oral health-related concerns, behavior, and communication with health care providers of patients with breast cancer: impact of different treatments.

PubMed

Taichman, L Susan; Van Poznak, Catherine H; Inglehart, Marita R

2018-01-01

The objectives are to compare responses of breast cancer (BCa) treatment groups (chemotherapy, tamoxifen, and aromatase inhibitors (AIs) to each other and a control regarding (a) subjective oral health, (b) oral health-related behaviors, (c) oral health-related concerns, and (d) communication with health care providers. Survey data were collected from 140 postmenopausal BCa patients and 41 healthy postmenopausal control respondents. BCa patients reported on average more frequent mouth sores/mucositis (5-point scale with 1 = never: 1.63 vs. 1.14; p < .01), glossadynia (1.60 vs. 1.07; p < .01), xerostomia (2.48 vs. 1.40; p < .01), and dysgeusia (2.10 vs. 1.46; p < .01) than the control respondents. Patients undergoing chemotherapy were more aware that cancer treatment can affect their oral health than patients on tamoxifen/AI (93% vs. 55%/56%; p < .001). BCa patients reported being more frequently informed by oncologists about oral health-related effects of cancer treatment than by dentists. Oncologists/nurses were more likely to communicate about oral health-related treatment effects with patients undergoing chemotherapy than patients on tamoxifen or AIs. Few BCa patients perceived dentists as knowledgeable about cancer treatment-related oral concerns and trusted them less than oncologists. BCa treatments impact oral health. Low percentages of BCa patients had received specific information about impacts of BCa treatments on oral health from their dentists. © 2018 Special Care Dentistry Association and Wiley Periodicals, Inc.

Metabolic Pathway Signatures Associated with Urinary Metabolite Biomarkers Differentiate Bladder Cancer Patients from Healthy Controls.

PubMed

Kim, Won Tae; Yun, Seok Joong; Yan, Chunri; Jeong, Pildu; Kim, Ye Hwan; Lee, Il Seok; Kang, Ho Won; Park, Sunghyouk; Moon, Sung Kwon; Choi, Yung Hyun; Choi, Young Deuk; Kim, Isaac Yi; Kim, Jayoung; Kim, Wun Jae

2016-07-01

Our previous high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA, based on our previously identified urinary metabolome. A total of 135 primary BCA samples and 26 control tissue samples from healthy volunteers were analyzed. The association between specific urinary metabolites and their related encoding genes was analyzed. Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n=135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to normal bladder tissues (p<0.05 all) of patients with non-muscle invasive BCA, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive BCA (p<0.05), with no changes in IDO1 expression. Alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, which were the most perturbed pathways in BCA, were determined.

Evaluation of miR-182/miR-100 Ratio for Diagnosis and Survival Prediction in Bladder Cancer.

PubMed

Chen, Zhanguo; Wu, Lili; Lin, Qi; Shi, Jing; Lin, Xiangyang; Shi, Liang

2016-09-01

Abnormal expression of microRNAs (miRNAs) plays an important role in development of several cancer types, including bladder cancer (BCa). However, the relationship between the ratio of miR-181/miR-100 and the prognosis of BCa has not been studied yet. The aim of this study was to evaluate the expression of miR-182, miR-100 and their clinical significance in BCa. Upregulation of miR-182 and down-regulation of miR-100 were validated in tissue specimens of 134 BCa cases compared with 148 normal bladder epithelia (NBE) specimens  using TaqMan-based real-time reverse transcription quantitative PCR (RT-qPCR). The diagnostic and prognostic evaluation of miR-182, miR-100, and miR-182/miR-100 ratio was also performed. miR-182 was upregulated in BCa and miR-100 was down-regulated in BCa compared with NBE (P < 0.001). The areas under receiver operating characteristic curves (AUCs-ROC) for miR-182 and miR-100 were 0.913 and 0.810, respectively. However, miR-182/miR-100 ratio increased the diagnostic performance, yielding an AUC of 0.981 (97.01% sensitivity and 90.54% specificity). Moreover, miR-182/miR-100 ratio was associated with pT-stage, histological grade, BCa recurrence and carcinoma in situ (P < 0.05 for all). Multivariate Cox regression analysis indicated that miR-182/miR-100 ratio was an independent prognostic factor for overall survival (Hazard ratio: 7.142; 95% CI: 2.106 - 9.891; P < 0.01). Furthermore, Kaplan-Meier curve analysis revealed that high-level of miR-182/miR-100 ratio was significantly correlated with shortened survival time for BCa patients (P < 0.01). The miR-182/miR-100 ratio may serve as a novel promising biomarker for diagnosis and survival prediction in BCa. Further studies are needed to elucidate the role of miR-182/miR-100 ratio as a non‑invasive diagnostic tool for BCa.

Calibration of the B/Ca proxy in symbiont-bearing planktonic foraminifera for application to the Paleocene-Eocene Thermal Maximum

NASA Astrophysics Data System (ADS)

Haynes, L.; Hoenisch, B.; Eggins, S.; Holland, K.; Rosenthal, Y.

2015-12-01

During the Paleocene-Eocene Thermal Maximum (PETM), rapid surface ocean acidification is indicated by a large decrease in the B/Ca ratios of planktic foraminiferal calcite, which is a proxy for the surface ocean carbonate system [1]. However, due to uncertainty in the effects of past seawater chemistry (e.g, different [Mg], [Ca], and [B]) on B/Ca, modern calibrations cannot be used to estimate the magnitude of acidification during this critical period. In addition, recent inorganic and sediment trap studies have respectively documented the controls of growth rate and light levels on B/Ca [2,3]. To extend the application of the B/Ca proxy to the PETM, we have conducted culturing experiments in O. universa, G. ruber, and G. sacculifer in which we simulated changes in pH and total DIC under Paleogene seawater conditions- high [Ca], low [Mg], and low [B]. We have further investigated the effects of variable light intensity (a control on symbiont activity), [Ca]seawater, and [B]seawater on the proxy. Results from O. universa confirm that B/Ca decreases with increasing DIC, decreasing pH, and decreasing [B]seawater, supporting a [B(OH)4-]/DIC control on the proxy [4]. In contrast, neither low light nor [Ca]seawater have a measurable effect on B/Ca, implying that influences of these parameters over the PETM were likely negligible. Critically, B/Ca appears to be more sensitive to pH at very low [B(OH)4-]/DIC in comparison to modern calibrations. Using estimates of surface ocean pH from boron isotopes, new calibrations can explain a larger proportion of the observed B/Ca excursion over the PETM. However, simulation of a large DIC pulse is necessary to explain the full excursion. New data will be presented from species that are more sensitive to pH, such as G. ruber and G. sacculifer, which will illuminate the range of responses of B/Ca to ocean acidification during the Paleogene. [1] Penman et al. 2014. Paleoceanography 29. [2] Uchikawa et al. 2015. GCA 150. [3] Babila et al. 2014. EPSL 404. [4] Allen et al. 2012. EPSL 351-352.

Anti-atherosclerotic effects of pravastatin in brachiocephalic artery in comparison with en face aorta and aortic roots in ApoE/LDLR-/- mice.

PubMed

Kostogrys, Renata B; Franczyk-Zarow, Magdalena; Gasior-Glogowska, Marlena; Kus, Edyta; Jasztal, Agnieszka; Wrobel, Tomasz P; Baranska, Malgorzata; Czyzynska-Cichon, Izabela; Drahun, Anna; Manterys, Angelika; Chlopicki, Stefan

2017-02-01

Cholesterol-dependent and independent mechanisms were proposed to explain anti-atherosclerotic action of statins in humans. However, their effects in murine models of atherosclerosis have not been consistently demonstrated. Here, we studied the effects of pravastatin on atherosclerosis in ApoE/LDLR -/- mice fed a control and atherogenic diet. ApoE/LDLR -/- mice were fed a control (CHOW) or an atherogenic (Low Carbohydrate High Protein, LCHP) diet. Two doses of pravastatin (40mg/kg and 100mg/kg) were used. The anti-atherosclerotic effects of pravastatin in en face aorta, cross-sections of aortic roots and brachiocephalic artery (BCA) were analysed. The lipid profile was determined. Fourier Transform Infrared Spectroscopy followed by Fuzzy C-Means (FCM) clustering was used for the quantitative assessment of plaque composition. Treatment with pravastatin (100mg/kg) decreased total and LDL cholesterol only in the LCHP group, but displayed a pronounced anti-atherosclerotic effect in BCA and abdominal aorta. The anti-atherosclerotic effect of pravastatin (100mg/kg) in BCA was associated with significant alterations of the chemical plaque composition, including a fall in cholesterol and cholesterol esters contents independently on total cholesterol and LDL concentration in plasma. Pravastatin at high (100mg/kg), but not low dose displayed a pronounced anti-atherosclerotic effect in ApoE/LDLR -/- mice fed a CHOW or LCHP diet that was remarkable in BCA, visible in en face aorta, whereas it was not observed in aortic roots, suggesting that previous inconsistencies might have been due to the various sites of atherosclerotic plaque analysis. Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase.

PubMed

Gao, Qun; Zhao, Xiangmin; Ahmad, Mansoor; Wolin, Michael S

2009-12-01

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

Group B streptococci causing neonatal infections in barcelona are a stable clonal population: 18-year surveillance.

PubMed

Martins, E R; Andreu, A; Correia, P; Juncosa, T; Bosch, J; Ramirez, M; Melo-Cristino, J

2011-08-01

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years.

Group B Streptococci Causing Neonatal Infections in Barcelona Are a Stable Clonal Population: 18-Year Surveillance▿

PubMed Central

Martins, E. R.; Andreu, A.; Correia, P.; Juncosa, T.; Bosch, J.; Ramirez, M.; Melo-Cristino, J.

2011-01-01

We analyzed 212 group B streptococci (GBS) from newborns with invasive infections in the area of Barcelona, Spain, between 1992 and 2009, with the aim of documenting changes in the prevalences of serotypes, antimicrobial resistance, and genetic lineages and evaluating their associations with either early-onset disease (EOD) or late-onset disease (LOD). Serotypes III (n = 118) and Ia (n = 47) together accounted for nearly 78% of the isolates. All isolates carried an alpha or alpha-like protein gene, and specific associations between genes and serotypes, such as serotype Ib and bca, serotype II and bca, serotype III and rib, and serotype V and alp3, reflected the presence of particular genetic lineages. Macrolide resistance (14.2%) was significantly associated with serotype V. Pulsed-field gel electrophoresis (PFGE) clustering was an excellent predictor of serotype and antibiotic resistance. The combination of PFGE and multilocus sequence typing revealed a large number of genetically distinct lineages. Still, specific lineages were dominant in our collection, particularly the serotype III/ST17/rib lineage, which had enhanced potential to cause LOD. Serotype Ia was concentrated in a single PFGE cluster composed of two genetic lineages: ST23/eps and ST24/bca. The ST24/bca sublineage of serotype Ia, which is found infrequently elsewhere, may be emerging as an important cause of neonatal invasive infections in the Mediterranean region. In spite of the introduction of prophylaxis, resulting in a pronounced decline in the frequency of EOD, the study revealed a remarkably stable clonal structure of GBS causing neonatal infections in Barcelona over a period of 18 years. PMID:21697333

Calibration of the B/Ca proxy in the planktic foraminifer Orbulina universa to Paleocene seawater conditions

NASA Astrophysics Data System (ADS)

Haynes, Laura L.; Hönisch, Bärbel; Dyez, Kelsey A.; Holland, Kate; Rosenthal, Yair; Fish, Carina R.; Subhas, Adam V.; Rae, James W. B.

2017-06-01

The B/Ca ratio of planktic foraminiferal calcite, a proxy for the surface ocean carbonate system, displays large negative excursions during the Paleocene-Eocene Thermal Maximum (PETM, 55.9 Ma), consistent with rapid ocean acidification at that time. However, the B/Ca excursion measured at the PETM exceeds a magnitude that modern pH calibrations can explain. Numerous other controls on the proxy have been suggested, including foraminiferal growth rate and the total concentration of dissolved inorganic carbon (DIC). Here we present new calibrations for B/Ca versus the combined effects of pH and DIC in the symbiont-bearing planktic foraminifer Orbulina universa, grown in culture solutions with simulated Paleocene seawater elemental composition (high [Ca], low [Mg], and low total boron concentration ([B]T). We also investigate the isolated effects of low seawater [B]T, high [Ca], reduced symbiont photosynthetic activity, and average shell growth rate on O. universa B/Ca in order to further understand the proxy systematics and to determine other possible influences on the PETM records. We find that average shell growth rate does not appear to determine B/Ca in high calcite saturation experiments. In addition, our "Paleocene" calibration shows higher sensitivity than the modern calibration at low [B(OH)4-]/DIC. Given a large DIC pulse at the PETM, this amplification of the B/Ca response can more fully explain the PETM B/Ca excursion. However, further calibrations with other foraminifer species are needed to determine the range of foraminifer species-specific proxy sensitivities under these conditions for quantitative reconstruction of large carbon cycle perturbations.

In silico study of breast cancer associated gene 3 using LION Target Engine and other tools.

PubMed

León, Darryl A; Cànaves, Jaume M

2003-12-01

Sequence analysis of individual targets is an important step in annotation and validation. As a test case, we investigated human breast cancer associated gene 3 (BCA3) with LION Target Engine and with other bioinformatics tools. LION Target Engine confirmed that the BCA3 gene is located on 11p15.4 and that the two most likely splice variants (lacking exon 3 and exons 3 and 5, respectively) exist. Based on our manual curation of sequence data, it is proposed that an additional variant (missing only exon 5) published in a public sequence repository, is a prediction artifact. A significant number of new orthologs were also identified, and these were the basis for a high-quality protein secondary structure prediction. Moreover, our research confirmed several distinct functional domains as described in earlier reports. Sequence conservation from multiple sequence alignments, splice variant identification, secondary structure predictions, and predicted phosphorylation sites suggest that the removal of interaction sites through alternative splicing might play a modulatory role in BCA3. This in silico approach shows the depth and relevance of an analysis that can be accomplished by including a variety of publicly available tools with an integrated and customizable life science informatics platform.

Immunoassays for Identification of Biological Agents in Sample Unknowns: NATO SlBCA Exercise VI

DTIC Science & Technology

2005-12-01

Yersiniapestis 103 cfu/mL Enzyme-linked immunosorbent assays Antibodies Unlabelled antibodies Antibody stocks developed under DRES contract by SciLab Consulting...goat anti-rabbit IgG, (whole molecule, lot no. 90H8990). Antibody purification Antibodies produced by Scilab Consulting Inc. were purified on a Bio...No. W7702-4-R430, Final Report. Scilab Consulting Inc. DRDC Suffield TM 2005-223 17 14. Fulton, R.E. and Thompson, H.G. Evaluation of the Rapid

FoxA and LIPG endothelial lipase control the uptake of extracellular lipids for breast cancer growth

PubMed Central

Slebe, Felipe; Rojo, Federico; Vinaixa, Maria; García-Rocha, Mar; Testoni, Giorgia; Guiu, Marc; Planet, Evarist; Samino, Sara; Arenas, Enrique J.; Beltran, Antoni; Rovira, Ana; Lluch, Ana; Salvatella, Xavier; Yanes, Oscar; Albanell, Joan; Guinovart, Joan J.; Gomis, Roger R.

2016-01-01

The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids. PMID:27045898

DELAYED PREPUTIAL SEPARATION (PPS) AND SP22 MEASUREMENT IN RATS ADMINISTERED BROMOCHLOROACETIC ACID (BCA) IN DRINKING WATER

EPA Science Inventory

Reproductive effects of BCA were determined in a dose range finding study (DRFS) and definitive two-generational study. Adult male and female CD� (SD) rats were administered BCA in drinking water for two weeks in the DRFS (10/sex/group) and ten weeks in the definitive study (25/s...

Breast Cancer Prognosis for Young Patients

PubMed Central

OWRANG, MEHDI; COPELAND, L. ROBERT JR; RICKS-SANTI, J. LUISEL; GASKINS, MELVIN; BEYENE, DESTA; DEWITTY, L. ROBERT JR; KANAAN, M. YASMINE

2017-01-01

Background/Aims: Breast cancer (BCa) prognostication is a vital element for providing effective treatment for patients with BCa. Studies suggest that ethnicity plays a greater role in the incidence and poor prognosis of BCa in younger women than in their older counterparts. Therefore, the goal of this study was to assess the association between age and ethnicity on the overall final prognosis. Materials and Methods: Nottingham Prognostic Index (NPI) was used to analyze BCa prognosis using Howard University Cancer Center Tumor Registry and the National Cancer Institute’s Surveillance, Epidemiology, and End Results BCa datasets. Patients were grouped according to their predicted prognosis based on NPI scheme. Results: There was no correlation between the younger patients compared to their older counterparts for any of the prognostic clusters. The significance of ethnicity in poorer prognosis for younger age is not conclusive either. Conclusion: An extended prognostic tool/system needs to be evaluated for its usefulness in a clinical practice environment. PMID:28652435

Influence of Past Changes in Atmospheric CO2 on Boron/Calcium of Planktic Fossil Foraminifera

NASA Astrophysics Data System (ADS)

Domeyko, R. A.; Allen, K. A.; deMenocal, P. B.

2014-12-01

Culture experiments have revealed that B/Ca of shells grown by the foraminiferal species Globigerinoides ruber increase with increasing seawater pH. Specifically, B/Ca responds to changes in the relative abundance of pH-sensitive dissolved carbon and boron species (Allen et al. 2011, 2012). Here, we present a high-resolution study on fossilized G. ruber from two sites in North Atlantic subtropical gyres (VM25-21 and ODP 1055B) through 20 ka BP to evaluate how B/Ca responds to past changes in atmospheric CO2. Forams were picked and crushed gently, then cleaned and dissolved using a variation of the Boyle and Keigwin (1985) and Barker et al. (2003) cleaning protocols prior to analysis. ODP 1055B (from Carolina Slope, West Atlantic) produced a high-resolution record with lower B/Ca values during the glacial period followed by a rapid shift to higher B/Ca values in the early deglaciation, with values remaining high through the Holocene. These results were not predicted by culture calibrations, but they are consistent with B/Ca records from the Caribbean (ODP 999, Foster et al. 2008), suggesting this pattern is characteristic of surface waters in the greater North Atlantic region.

Cyanogenesis - a general phenomenon in the lepidoptera

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witthohn, K.; Naumann, C.M.

1987-08-01

There are two different pathways known to be used for the detoxification of hydrocyanic acid in insects, viz., rhodanese and ..beta..-cyano-L-alanine synthase. The authors consider the latter to be indicative for cyanogenesis, while rhodanese might, in general, play a more important role in sulfur transfer for protein synthesis. This paper reports on the distribution of ..beta..-cyano-L-alanine (BCA) in the Lepidoptera. First reports of cyanogenesis are presented for the following families: Papilionidae, Pieridae, Lycaenidae, Hesperiidae, Lymantriidae, Arctiidae, Notodontidae, Megalopygidae, Limacodidae, Cymatophoridae, Noctuidae, Geometridae, and Yponomeutidae. New and old records for three other families, the Nymphalidae, Zygaenidae, and Heterogynidae, are included tomore » complete the present state of knowledge. Special emphasis has been laid on the Nymphalidae, where BCA has been detected in eight subfamilies. Taxonomic, geographic, and seasonal variation has been found in a number of cases. In all cases observed so far, the source of cyanogenesis in the Lepidoptera is most probably the cyanoglucosides linamarin and lotaustralin, although cyanogenesis based on mustard oil glucosides and cyclopentenoid glucosides might occur as well. BCA has been found in both cryptic and aposematic species, including taxa such as the Pieridae, Danainae, Ithomiinae, and Arctiidae, where the defensive biology is believed to be linked with other compounds, like mustard oil glucosides, cardenolides, or pyrrolizidinie alkaloids. The ecological interaction and significance of such secondary compounds is not yet understood.« less

Cytoplasmatic and Nuclear YAP1 and pYAP1 Staining in Urothelial Bladder Cancer.

PubMed

Latz, Stefan; Umbach, Tine; Goltz, Diane; Kristiansen, Glen; Müller, Stephan C; Ellinger, Jörg

2016-01-01

Yes-associated protein 1 (YAP1), the nuclear effector of the Hippo pathway, plays an important role in many tumor entities. We evaluated staining and clinical significance of YAP1 and phosphorylated YAP1 (pYAP1) in urothelial bladder cancer (BCA). We used a tissue micorarray with samples of patients with muscle-invasive bladder cancer (MIBC, n = 192), non-muscle-invasive bladder cancer (NMIBC, n = 192) and normal urothelial bladder tissue (CTRL, n = 38) to determine the immunhistochemical staining of YAP1 and pYAP1. Cytoplasmatic and nuclear levels were evaluated. The t test was used for comparative analysis. Overall survival and progression-free survival were evaluated by Kaplan-Meier estimates and the Cox proportional hazard regression model. Nuclear YAP1 as well as cytoplasmatic pYAP1 levels were higher in CTRL than in BCA, whereby both--NMIBC and MIBC--had lower levels than CTRL. Among patients with MIBC, cytoplasmatic YAP1 and pYAP1 staining decreased with advanced stage. YAP1 and pYAP1 staining did not correlate with the recurrence rate, progression-free, cancer-specific or overall survival. Immunhistochemical staining and subcellular localization of YAP1 and pYAP1 are different for BCA, NMIBC, MIBC and CTRL, indicating that the Hippo pathway is involved in urothelial carcinogenesis. © 2015 S. Karger AG, Basel.

Portable Microplate Analyzer with a Thermostatic Chamber Based on a Smartphone for On-site Rapid Detection.

PubMed

Wan, Zijian; Zhong, Longjie; Pan, Yuxiang; Li, Hongbo; Zou, Quchao; Su, Kaiqi; Wang, Ping

2017-01-01

A microplate method provides an efficient way to use modern detection technology. However, there are some difficulties concerning on-site detection, such as being non-portable and time-consuming. In this work, a novel portable microplate analyzer with a thermostatic chamber based on a smartphone was designed for rapid on-site detection. An analyzer with a wide-angle lens and an optical filter provides a proper environment for the microplate. A smartphone app-iPlate Monitor was used for RGB analyze of image. After a consistency experiment with a microtiter plate reader (MTPR), the normalized calibration curves were y = 0.7276x + 0.0243 (R 2 = 0.9906) and y = 0.3207x + 0.0094 (R 2 = 0.9917) with a BCA protein kit as well as y = 0.182x + 0.0134 (R 2 = 0.994) and y = 0.0674x + 0.0003 (R 2 = 0.9988) with a glucose kit. The times for obtaining the detection requirement were 15 and 10 min for the BCA protein kit and the glucose kit at 37°C; in contrast, it required more than 30 and 20 min at ambient temperature. Meanwhile, it also showed good repeatability for detections.

Testosterone and breast cancer prevention.

PubMed

Glaser, R; Dimitrakakis, C

2015-11-01

Testosterone (T) is the most abundant biologically active hormone in women. Androgen receptors (AR) are located throughout the body including the breast where T decreases tissue proliferation. However, T can be aromatized to estradiol (E2), which increases proliferation and hence, breast cancer (BCA) risk. Increased aromatase expression and an imbalance in the ratio of stimulatory estrogens to protective androgens impacts breast homeostasis. Recent clinical data supports a role for T in BCA prevention. Women with symptoms of hormone deficiency treated with pharmacological doses of T alone or in combination with anastrozole (A), delivered by subcutaneous implants, had a reduced incidence of BCA. In addition, T combined with A effectively treated symptoms of hormone deficiency in BCA survivors and was not associated with recurrent disease. Most notably, T+A implants placed in breast tissue surrounding malignant tumors significantly reduced BCA tumor size, further supporting T direct antiproliferative, protective and therapeutic effect. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Breast Cancer Prognosis for Young Patients.

PubMed

Owrang, Mehdi; Copeland, Robert L; Ricks-Santi, Luisel J; Gaskins, Melvin; Beyene, Desta; Dewitty, Robert L; Kanaan, Yasmine M

2017-01-01

Breast cancer (BCa) prognostication is a vital element for providing effective treatment for patients with BCa. Studies suggest that ethnicity plays a greater role in the incidence and poor prognosis of BCa in younger women than in their older counterparts. Therefore, the goal of this study was to assess the association between age and ethnicity on the overall final prognosis. Nottingham Prognostic Index (NPI) was used to analyze BCa prognosis using Howard University Cancer Center Tumor Registry and the National Cancer Institute's Surveillance, Epidemiology, and End Results BCa datasets. Patients were grouped according to their predicted prognosis based on NPI scheme. There was no correlation between the younger patients compared to their older counterparts for any of the prognostic clusters. The significance of ethnicity in poorer prognosis for younger age is not conclusive either. An extended prognostic tool/system needs to be evaluated for its usefulness in a clinical practice environment. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The New York State Bird Conservation Area (BCA) Program: A Model for the United States

Treesearch

M. F. Burger; D. J. Adams; T. Post; L. Sommers; B. Swift

2005-01-01

The New York State Bird Conservation Area (BCA) Program, modeled after the National Audubon Society?s Important Bird Areas Program, is based on legislation signed by Governor Pataki in 1997. New York is the first state in the nation to enact such a program. The BCA Program seeks to provide a comprehensive, ecosystem approach to conserving birds and their habitats on...

The Contrasting Role of the Mediator Subunit MED30 in the Progression of Bladder Cancer.

PubMed

Syring, Isabella; Weiten, Richard; Müller, Tim; Schmidt, Doris; Steiner, Susanne; Kristiansen, Glen; Müller, Stefan C; Ellinger, Jörg

2017-12-01

The Mediator complex is a key regulator of gene transcription, and several studies have demonstrated altered expression of particular subunits in diverse human diseases, especially cancer. To date, nothing is known about the role of MED30 in bladder cancer. We, therefore, performed an RNA expression and survival analysis of the subunit MED30 in 537 samples of bladder cancer by using the database cBioPortal. To validate these data on the protein level, we practiced immunohistochemical staining against MED30 on a tissue microarray containing 210 samples of all tumour stages and performed survival analyses. For functional analysis, the siRNA-mediated knockdown of MED30 was performed in the cell lines T24 and TCCSUP followed by proliferation, migration, and invasion assays. On the mRNA and protein levels, higher expression of MED30 is associated with better patient survival. In accordance with this, advanced T- and N-stages showed lower expression of MED30. In contrast, knockdown of MED30 led to reduction of the tumour parameters proliferation, migration, and invasion in the BCa cell lines. MED30 appears to be integrated in the progression of the urothelial tumour in the bladder. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Distinct breast cancer subtypes in women with early-onset disease across races

PubMed Central

Singh, Mandeep; Ding, Yi; Zhang, Li-Ying; Song, Dong; Gong, Yun; Adams, Sylvia; Ross, Dara S; Wang, Jin-Hua; Grover, Shruti; Doval, Dinesh Chandra; Shao, Charles; He, Zi-Li; Chang, Victor; Chin, Warren W; Deng, Fang-Ming; Singh, Baljit; Zhang, David; Xu, Ru-Liang; Lee, Peng

2014-01-01

Background: Racial disparities among breast cancer (BCa) patients are known but not well studied in early-onset BCa. We analyzed molecular subtypes in early-onset BCa across five major races. Methods: A total of 2120 cases were included from non-Hispanic White (NHW), African American (AA) and Hispanic, Chinese and Indian. Based on ER, PR and HER-2 status, BCa was classified into 4 intrinsic subtypes as Luminal A, Luminal B, HER2/neu overexpression and Triple negative BCa (TNBC) subtypes. Data was stratified according to race and age as younger/early-onset group (40-years and younger) and older group (50-years and older). Results: In early-onset BCa, incidence of TNBC was significantly higher (p = 0.0369) in Indian women followed by AA, Hispanic, NHW and Chinese women. Incidence of Her2 over-expression subtype also was highest in Indian women, followed by Hispanic, Chinese, AA and NHW women. In contrast, Luminal B subtype was most significantly higher in AA women (p = 0.0000) followed by NHW (p = 0.0002), Chinese (p = 0.0003), Hispanic (0.0128) and Indian (p = 0.0468) women. Luminal A subtype was most significantly reduced in Indian women (p = 0.0113) followed by Hispanic, AA, NHW and Chinese women. These results were based on statistical analysis with the mean of older group populations. Conclusions: These results show significant disparities in receptor subtypes across races. This study will contribute in developing optimal clinical trial protocols and personalized management strategies for early-onset BCa patients. PMID:25057437

MD and BCA simulations of He and H bombardment of fuzz in bcc elements

NASA Astrophysics Data System (ADS)

Klaver, T. P. C.; Zhang, S.; Nordlund, K.

2017-08-01

We present results of MD simulations of low energy He ion bombardment of low density fuzz in bcc elements. He ions can penetrate several micrometers into sparse fuzz, which allows for a sufficient He flux through it to grow the fuzz further. He kinetic energy falls off exponentially with penetration depth. A BCA code was used to carry out the same ion bombardment on the same fuzz structures as in MD simulations, but with simpler, 10 million times faster calculations. Despite the poor theoretical basis of the BCA at low ion energies, and the use of somewhat different potentials in MD and BCA calculations, the ion penetration depths predicted by BCA are only ∼12% less than those predicted by MD. The MD-BCA differences are highly systematic and trends in the results of the two methods are very similar. We have carried out more than 200 BCA calculation runs of ion bombardment of fuzz, in which parameters in the ion bombardment process were varied. For most parameters, the results show that the ion bombardment process is quite generic. The ion species (He or H), ion mass, fuzz element (W, Ta, Mo, Fe) and fuzz element lattice parameter turned out to have a modest influence on ion penetration depths at most. An off-normal angle of incidence strongly reduces the ion penetration depth. Increasing the ion energy increases the ion penetration, but the rate by which ion energy drops off at high ion energies follows the same exponential pattern as at lower energies.

Prognostic and Prediction Tools in Bladder Cancer: A Comprehensive Review of the Literature.

PubMed

Kluth, Luis A; Black, Peter C; Bochner, Bernard H; Catto, James; Lerner, Seth P; Stenzl, Arnulf; Sylvester, Richard; Vickers, Andrew J; Xylinas, Evanguelos; Shariat, Shahrokh F

2015-08-01

This review focuses on risk assessment and prediction tools for bladder cancer (BCa). To review the current knowledge on risk assessment and prediction tools to enhance clinical decision making and counseling of patients with BCa. A literature search in English was performed using PubMed in July 2013. Relevant risk assessment and prediction tools for BCa were selected. More than 1600 publications were retrieved. Special attention was given to studies that investigated the clinical benefit of a prediction tool. Most prediction tools for BCa focus on the prediction of disease recurrence and progression in non-muscle-invasive bladder cancer or disease recurrence and survival after radical cystectomy. Although these tools are helpful, recent prediction tools aim to address a specific clinical problem, such as the prediction of organ-confined disease and lymph node metastasis to help identify patients who might benefit from neoadjuvant chemotherapy. Although a large number of prediction tools have been reported in recent years, many of them lack external validation. Few studies have investigated the clinical utility of any given model as measured by its ability to improve clinical decision making. There is a need for novel biomarkers to improve the accuracy and utility of prediction tools for BCa. Decision tools hold the promise of facilitating the shared decision process, potentially improving clinical outcomes for BCa patients. Prediction models need external validation and assessment of clinical utility before they can be incorporated into routine clinical care. We looked at models that aim to predict outcomes for patients with bladder cancer (BCa). We found a large number of prediction models that hold the promise of facilitating treatment decisions for patients with BCa. However, many models are missing confirmation in a different patient cohort, and only a few studies have tested the clinical utility of any given model as measured by its ability to improve clinical decision making. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

[Cytologic diagnosis of adenoid cystic carcinoma of salivary glands and distinction from basal cell adenoma].

PubMed

Bai, Y P; Zhang, Y; Tian, C; Xing, L; Liu, H G

2018-04-08

Objective: To describe the cytologic features of adenoid cystic carcinoma (ADCC) of salivary glands, and to identify distinguishing cytologic features of ADCC and basal cell adenoma (BCA). Methods: A retrospective review of cytology smears of 30 cases of ADCC and 12 cases of BCA of salivary glands were performed. All cases were collected from Beijing Tongren Hospital, Capital Medical University from January 2010 to January 2017. Except for 2 aspirate smears of ADCC, all were touch imprint smears. All cases had further histological confirmation. Results: Neoplastic ductal cells of ADCC were arranged in three-dimensional clusters, sheets and singles. Hyaline globules were found in most cases (20/30, 66.7%). The nuclei were round to oval, showing varying degrees of nuclear atypia. These included (1) the nuclei were hyperchromatic, demonstrating coarse or slightly coarse, irregularly distributed chromatin; (2) the nuclei were slightly large and vary in size; (3) appearance of the nuclei had a different degree of irregularity (often mild). Nucleoli were common seen (21/30, 70.0%), and were prominent in some cases. Mitosis and necrosis were rare. Cytologically, BCA showed cell arrangements and nuclear features overlapped with those of ADCC. The cytologic difference between these two tumors included: (1) the tumor cells presented rarely in singles; (2) hyaline globules were very uncommon (1/12) in BCA; (3) nuclei of BCA were hypochromatic or slightly hyperchromatic, homogeneous and uniform in appearance and size, overall without nuclear atypia and they were smaller and slender then those of ADCC and (4) individual cells of BCA showed relatively abundant cytoplasm. Conclusions: The cytologic features of ADCC and BCA both overlap and different from each other. Most cases can be diagnosed by cytologic examination. The presence of hyaline globules is an important diagnostic clue of ADCC, although not pathognomonic. Nuclear atypia of neoplastic ductal cells is an essential cytological feature in the diagnosis of ADCC, and is the most reliable point for differential diagnosis of ADCC and BCA.

A numerical study of combined use of two biocontrol agents with different biocontrol mechanisms in controlling foliar pathogens.

PubMed

Xu, X-M; Jeffries, P; Pautasso, M; Jeger, M J

2011-09-01

Effective use of biocontrol agents is an important component of sustainable agriculture. A previous numerical study of a generic model showed that biocontrol efficacy was greatest for a single biocontrol agent (BCA) combining competition with mycoparasitism or antibiosis. This study uses the same mathematical model to investigate whether the biocontrol efficacy of combined use of two BCAs with different biocontrol mechanisms is greater than that of a single BCA with either or both of the two mechanisms, assuming that two BCAs occupy the same host tissue as the pathogen. Within the parameter values considered, a BCA with two biocontrol mechanisms always outperformed the combined use of two BCAs with a single but different biocontrol mechanism. Similarly, combined use of two BCAs with a single but different biocontrol mechanism is shown to be far less effective than that of a single BCA with both mechanisms. Disease suppression from combined use of two BCAs was very similar to that achieved by the more efficacious one. As expected, a higher BCA introduction rate led to increased disease suppression. Incorporation of interactions between two BCAs did not greatly affect the disease dynamics except when a mycoparasitic and, to a lesser extent, an antibiotic-producing BCA was involved. Increasing the competitiveness of a mycoparasitic BCA over a BCA whose biocontrol mechanism is either competition or antibiosis may lead to improved biocontrol initially and reduced fluctuations in disease dynamics. The present study suggests that, under the model assumptions, combined use of two BCAs with different biocontrol mechanisms in most cases only results in control efficacies similar to using the more efficacious one alone. These predictions are consistent with published experimental results, suggesting that combined use of BCAs should not be recommended without clear understanding of their main biocontrol mechanisms and relative competitiveness, and experimental evaluation.

Influence of solution chemistry on the boron content in inorganic calcite grown in artificial seawater

NASA Astrophysics Data System (ADS)

Uchikawa, Joji; Harper, Dustin T.; Penman, Donald E.; Zachos, James C.; Zeebe, Richard E.

2017-12-01

The ratio of boron to calcium (B/Ca) in marine biogenic carbonates has been proposed as a proxy for properties of seawater carbonate chemistry. Applying this proxy to planktic foraminifera residing in the surface seawater largely in equilibrium with the atmosphere may provide a valuable handle on past atmospheric CO2 concentrations. However, precise controls on B/Ca in planktic foraminifera remain enigmatic because it has been shown to depend on multiple physicochemical seawater properties. To help establish a firm inorganic basis for interpreting the B/Ca records, we examined the effect of a suite of chemical parameters ([Ca2+], pH, [DIC], salinity and [PO43-]) on B/Ca in inorganic calcite precipitated in artificial seawater. These parameters were primarily varied individually while keeping all others constant, but we also tested the influence of pH and [DIC] at a constant calcite precipitation rate (R) by concurrent [Ca2+] adjustments. In the simple [Ca2+], pH and [DIC] experiments, both R and B/Ca increased with these parameters. In the pH-[Ca2+] and [DIC]-[Ca2+] experiments at constant R, on the other hand, B/Ca was invariant at different pH and decreased with [DIC], respectively. These patterns agree with the behavior of solution [BTotal/DIC] ratio such that, at a fixed [BTotal], it is independent of pH but decreases with [DIC]. Based on these results, R and [BTotal/DIC] ratio appear to be the primary controls on B/Ca in inorganic calcite, suggesting that both B(OH)4- and B(OH)3 are possibly involved in B incorporation. Moreover, B/Ca modestly increased with salinity and [PO43-]. Inorganic calcite precipitated at higher R and in the presence of oxyanions such as SO42- and PO43- in growth solutions often undergoes surface roughening due to formation of crystallographic defects, vacancies and, occasionally, amorphous/hydrous CaCO3. These non-lattice sites may provide additional space for B, particularly B(OH)3. Consequently, besides the macroscopic influence of R and bulk solution chemistry, molecular-scale processes associated with calcite nucleation can be an important consideration for B incorporation, especially in complex ionic solutions. Lastly, the covariance of B/Ca with [DIC] and salinity observed here qualitatively agrees with those in planktic foraminifers. It follows that their impact on foraminiferal B/Ca is partly inorganically driven, which may explain why the effect is evident across different species.

Effects of Low Carbohydrate High Protein (LCHP) diet on atherosclerotic plaque phenotype in ApoE/LDLR-/- mice: FT-IR and Raman imaging.

PubMed

Wrobel, T P; Marzec, K M; Chlopicki, S; Maślak, E; Jasztal, A; Franczyk-Żarów, M; Czyżyńska-Cichoń, I; Moszkowski, T; Kostogrys, R B; Baranska, M

2015-09-22

Low Carbohydrate High Protein (LCHP) diet displays pro-atherogenic effects, however, the exact mechanisms involved are still unclear. Here, with the use of vibrational imaging, such as Fourier transform infrared (FT-IR) and Raman (RS) spectroscopies, we characterize biochemical content of plaques in Brachiocephalic Arteries (BCA) from ApoE/LDLR(-/-) mice fed LCHP diet as compared to control, recomended by American Institute of Nutrition, AIN diet. FT-IR images were taken from 6-10 sections of BCA from each mice and were complemented with RS measurements with higher spatial resolution of chosen areas of plaque sections. In aortic plaques from LCHP fed ApoE/LDLR(-/-) mice, the content of cholesterol and cholesterol esters was increased, while that of proteins was decreased as evidenced by global FT-IR analysis. High resolution imaging by RS identified necrotic core/foam cells, lipids (including cholesterol crystals), calcium mineralization and fibrous cap. The decreased relative thickness of the outer fibrous cap and the presence of buried caps were prominent features of the plaques in ApoE/LDLR(-/-) mice fed LCHP diet. In conclusion, FT-IR and Raman-based imaging provided a complementary insight into the biochemical composition of the plaque suggesting that LCHP diet increased plaque cholesterol and cholesterol esters contents of atherosclerotic plaque, supporting the cholesterol-driven pathogenesis of LCHP-induced atherogenesis.

Cryptic Species and Vital Effects of Planktonic Foraminifera and the Implications for the B/Ca Carbonate System Proxy

NASA Astrophysics Data System (ADS)

Osborne, E.; Thunell, R.; Bizimis, M.; Buckley, W. P., Jr.; Sadekov, A.

2017-12-01

The concentration of boron in marine carbonates (B/Ca ratio) has been proposed to covary with seawater carbonate chemistry. However, a number of studies have evaluated the B/Ca proxy in planktonic foraminifera and found that boron incorporation may not be a straightforward process. The majority of previous research has been dedicated to tropical species that host large photosynthetic symbionts, which interfere with ambient carbonate chemistry and the incorporation of boron into the calcite lattice. This study focuses on evaluating B/Ca of subtropical and subpolar species that are asymbiontic or host small intra-shell non-dinoflagellate symbionts (Globigerina bulloides, Neogloboquadrina incompta and Neogloboquadrina dutertrei). Size-fraction replicate B/Ca measurements spanning a year-long sediment trap time-series generally indicate no size-dependent uptake of boron for these species, further supporting that symbionts influence/complicate boron incorporation/concentration. Both Neogloboquadrina species indicate strong correlation with the calcite saturation of seawater, carbonate ion concentration and pH that agree well with a recently published Pacific core-top calibration. However the relationship between G. bulloides and these variables is more complex. The presence of two cryptic species of G. bulloides (encrusted and normal) in our study region with documented offsets in stable isotopic composition also causes the unexpected differences between pooled trace element sample values and carbonate chemistry. Individual shell geochemistries determined by laser ablation ICP-MS indicate that the encrusted morphospecies have higher boron concentrations relative to the normal morphospecies. Based on these results we conclude that B/Ca ratios of non-dinoflagellate/symbiont-barren foraminifera faithfully record seawater carbonate chemistry. However inter-species and morphospecies differences may be significant and careful application of the B/Ca proxy to a single morphospecies is essential to creating meaningful data.

Onyx versus nBCA and coils in the treatment of intracranial dural arteriovenous fistulas

PubMed Central

Choo, Douglas M

2016-01-01

Background and purpose Intracranial dural arteriovenous fistulas (DAVFs) with cortical venous drainage have significant morbidity and mortality. Complete closure of these lesions is necessary to reduce these risks. The purpose of our study was to compare the outcome of DAVFs treated with Onyx versus those treated with n-Butyl Cyanoacrylate (nBCA) and coil embolization in a case-control study. Compared with nBCA and coil embolization, we hypothesized that Onyx embolization for DAVF is safer and has a higher chance of complete obliteration, with no need for post-embolization surgery for the DAVF. Materials and methods From 1998 to 2015, 29 patients who had DAVFs were treated with endovascular embolization. Of these, 24 patients had imaging available for analysis. Successful closure rates, complications, and procedure time were compared between the embolization techniques. Results The chance of not requiring post-embolization surgery with Onyx (81.8%) was significantly higher (p = 0.005) than with nBCA (22.22%). The complication rate with Onyx (9.1%) tended to be lower compared with that of nBCA (22.22%; p = 0.37). Procedural time was not significantly different between Onyx (mean 267 minutes) and nBCA (mean 288 minutes) (p = 0.59). The odds ratio of a DAVF being treated with Onyx and then requiring no follow-up surgery was 17.5 (95% CI 1.97–155.4). Conclusion Our case-control study suggests that Onyx embolization is superior to nBCA and coil embolization in completely obliterating DAVFs, with higher odds of no post-embolization surgery. We also found that Onyx is safe for embolization of DAVFs, with no associated neurological mortality and morbidity. PMID:26748082

Onyx versus nBCA and coils in the treatment of intracranial dural arteriovenous fistulas.

PubMed

Choo, Douglas M; Shankar, Jai Jai Shiva

2016-04-01

Intracranial dural arteriovenous fistulas (DAVFs) with cortical venous drainage have significant morbidity and mortality. Complete closure of these lesions is necessary to reduce these risks. The purpose of our study was to compare the outcome of DAVFs treated with Onyx versus those treated with n-Butyl Cyanoacrylate (nBCA) and coil embolization in a case-control study. Compared with nBCA and coil embolization, we hypothesized that Onyx embolization for DAVF is safer and has a higher chance of complete obliteration, with no need for post-embolization surgery for the DAVF. From 1998 to 2015, 29 patients who had DAVFs were treated with endovascular embolization. Of these, 24 patients had imaging available for analysis. Successful closure rates, complications, and procedure time were compared between the embolization techniques. The chance of not requiring post-embolization surgery with Onyx (81.8%) was significantly higher (p = 0.005) than with nBCA (22.22%). The complication rate with Onyx (9.1%) tended to be lower compared with that of nBCA (22.22%; p = 0.37). Procedural time was not significantly different between Onyx (mean 267 minutes) and nBCA (mean 288 minutes) (p = 0.59). The odds ratio of a DAVF being treated with Onyx and then requiring no follow-up surgery was 17.5 (95% CI 1.97-155.4). Our case-control study suggests that Onyx embolization is superior to nBCA and coil embolization in completely obliterating DAVFs, with higher odds of no post-embolization surgery. We also found that Onyx is safe for embolization of DAVFs, with no associated neurological mortality and morbidity. © The Author(s) 2016.

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

PubMed

Chen, Dong-Bao; Feng, Lin; Hodges, Jennifer K; Lechuga, Thomas J; Zhang, Honghai

2017-09-01

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

BCA-1 is highly expressed in Helicobacter pylori–induced mucosa-associated lymphoid tissue and gastric lymphoma

PubMed Central

Mazzucchelli, Luca; Blaser, Andrea; Kappeler, Andreas; Schärli, Patrik; Laissue, Jean A.; Baggiolini, Marco; Uguccioni, Mariagrazia

1999-01-01

Infection with Helicobacter pylori (Hp) induces the formation of lymphoid tissue in the stomach and the occasional development of primary gastric B-cell lymphomas. We have studied the expression of 2 chemokines that attract B lymphocytes, BCA-1 and SLC, in gastric tissue samples obtained from patients with chronic gastritis induced by Hp infection or nonsteroidal anti-inflammatory drugs, as well as from patients with Hp-associated low-grade and high-grade gastric lymphomas. High-level expression of BCA-1 and its receptor, CXCR5, was observed in all mucosal lymphoid aggregates and in the mantle zone of all secondary lymphoid follicles in Hp-induced gastric mucosa-associated lymphoid tissue (MALT). Follicular dendritic cells and B lymphocytes are possible sources of BCA-1, which is not expressed by T lymphocytes, macrophages, or CD1a+ dendritic cells. Strong expression of BCA-1 and CXCR5 was also detected in the transformed B cells of gastric MALT lymphomas. By contrast, SLC was confined almost exclusively to endothelial cells in and outside the lymphoid tissue. Only scant, occasional SLC expression was observed in the marginal zone of MALT follicles. Our findings indicate that BCA-1, which functions as a homing chemokine in normal lymphoid tissue, is induced in chronic Hp gastritis and is involved in the formation of lymphoid follicles and gastric lymphomas of the MALT type. J. Clin. Invest. 104:R49–R54 (1999). PMID:10562310

Occupation and Bladder Cancer Phenotype: Identification of Workplace Patterns That Increase the Risk of Advanced Disease Beyond Overall Incidence.

PubMed

Noon, Aidan P; Martinsen, Jan Ivar; Catto, James W F; Pukkala, Eero

2016-07-20

We examined a national data set to determine if workers employed in specific occupations develop distinct bladder cancer (BCa) phenotypes. To compare the incidence and disease-specific mortality (DSM) of localized and advanced BCa in workers with different job titles. BCa incidence, stage at diagnosis, and DSM in 1.7 million Finnish men (13 717 with BCa) and 1.7 million women (4282 with BCa) with annotated occupational descriptions. Follow-up was 37 and 43 million person-years, respectively. The gender-specific incidence and BCa DSM within each occupational category was compared with the expected number of cases based on the entire Finnish population to generate standardized incidence ratios (SIRs) and standard mortality ratios (SMRs). Occupations were found that had significant differences in the incidence of localized (SIR loc ) and advanced (SIR adv , SMR adv ) BCa and DSM. Male chemical process workers (SIR loc /SIR adv : 5.19; 95% confidence interval [CI], 1.73-25.7), male military personnel (SIR loc /SIR adv : 6.4; 95% CI, 1.09-259.0), and male public safety workers (SIR loc /SIR adv : 1.77; 95% CI, 1.04-3.23) had significantly more localized than advanced tumors. In contrast, miscellaneous construction workers had more advanced than localized cancers for both genders (male SIR loc /SIR adv : 0.67; 95% CI, 0.53-0.86; female SIR loc /SIR adv : 0.12; 95% CI, 0.09-0.54). Male chemical process workers had fewer deaths from BCa than expected from advanced tumors (SMR adv : 0.32; 95% CI, 0.07-0.94), and miscellaneous constructions workers had more deaths from advanced tumors than expected (male SMR adv : 1.44; 95% CI, 1.10-1.85; female SMR adv : 3.35; 95% CI, 1.23-7.30). Limitations of this study are failure to control accurately for the effects of smoking and a lack of specific treatment information. Occupations exist that may differ in their risks for localized and advanced BCa and for DSM. Occupations have been identified that may have different patterns of bladder cancer than expected. These findings may be explained by confounding factors such as exposure to tobacco smoke; however, it could be that workers with these job titles are exposed to specific bladder carcinogens. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samuelson, Shaun D.; Louie, John D.; Sze, Daniel Y., E-mail: dansze@stanford.edu

Purpose. Avoidance of nontarget microsphere deposition via hepatoenteric anastomoses is essential to the safety of yttrium-90 radioembolization (RE). The hepatic hilar arterial network may remain partially patent after coil embolization of major arteries, resulting in persistent risk. We retrospectively reviewed cases where n-butyl cyanoacrylate (n-BCA) glue embolization was used to facilitate endovascular hepatic arterial skeletonization before RE. Methods. A total of 543 RE procedures performed between June 2004 and March 2012 were reviewed, and 10 were identified where n-BCA was used to embolize hepatoenteric anastomoses. Arterial anatomy, prior coil embolization, and technical details were recorded. Outcomes were reviewed to identifymore » subsequent complications of n-BCA embolization or nontarget RE. Results. The rate of complete technical success was 80 % and partial success 20 %, with one nontarget embolization complication resulting in a minor change in treatment plan. No evidence of gastrointestinal or biliary ischemia or infarction was identified, and no microsphere-related gastroduodenal ulcerations or other evidence of nontarget RE were seen. Median volume of n-BCA used was <0.1 ml. Conclusion. n-BCA glue embolization is useful to eliminate hepatoenteric networks that may result in nontarget RE, especially in those that persist after coil embolization of major vessels such as the gastroduodenal and right gastric arteries.« less

Repression of ESR1 transcription by MYOD potentiates letrozole-resistance in ERα-positive breast cancer cells.

PubMed

Zhang, Qiang; Liu, Xiao-Yan; Li, Shuang; Zhao, Zhao; Li, Juan; Cui, Ming-Ke; Wang, En-Hua

2017-10-21

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Rapid determination of trace copper in animal feed based on micro-plate colorimetric reaction and statistical partitioning correction.

PubMed

Niu, Yiming; Wang, Jiayi; Zhang, Chi; Chen, Yiqiang

2017-04-15

The objective of this study was to develop a micro-plate based colorimetric assay for rapid and high-throughput detection of copper in animal feed. Copper ion in animal feed was extracted by trichloroacetic acid solution and reduced to cuprous ion by hydroxylamine. The cuprous ion can chelate with 2,2'-bicinchoninic acid to form a Cu-BCA complex which was detected with high sensitivity by micro-plate reader at 354nm. The whole assay procedure can be completed within 20min. To eliminate matrix interference, a statistical partitioning correction approach was proposed, which makes the detection of copper in complex samples possible. The limit of detection was 0.035μg/mL and the detection range was 0.1-10μg/mL of copper in buffer solution. Actual sample analysis indicated that this colorimetric assay produced results consistent with atomic absorption spectrometry analysis. These results demonstrated that the developed assay can be used for rapid determination of copper in animal feed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Levels in neurotransmitter precursor amino acids correlate with mental health in patients with breast cancer.

PubMed

Hüfner, K; Oberguggenberger, A; Kohl, C; Geisler, S; Gamper, E; Meraner, V; Egeter, J; Hubalek, M; Beer, B; Fuchs, D; Sperner-Unterweger, B

2015-10-01

Breast cancer is the most common cancer among females. Approximately 30% of cancer patients develop depression or depressive adaptation disorder within 5 years post diagnosis. Low grade inflammation and subsequent changes in neurotransmitter levels could be the pathophysiological link. In the current study we investigated the association of neurotransmitter precursor amino acids with a diagnosis of depression or state anxiety in 154 subjects suffering from breast cancer (BCA(+)), depression (DPR(+)), both or neither. Sociodemographic parameters, severity of depressive symptoms, and state anxiety (ANX) were recorded. Neopterin, kynurenine/tryptophan and phenylalanine/tyrosine were analysed by HPLC or ELISA. Significantly higher serum neopterin values were found in DPR(+) patients (p = 0.034) and in ANX(+) subjects (p = 0.008), as a marker of Th1-related inflammation. The phenylalanine/tyrosine ratio (index of the catecholamine pathway) was associated with the factors "breast cancer" and "depression" and their interaction (all p < 0.001); it was highest in the DPR(+)BCA(+) group. The kynurenine/tryptophan ratio (index of the serotonin pathway) was significantly associated with the factors "breast cancer" and "state anxiety" and their interaction (p < 0.001, p = 0.026, p = 0.02, respectively); it was highest in the ANX(+)BCA(+) group. In BCA(+) patients kynurenine/tryptophan ratios correlated with severity of state anxiety (r = 0.226, p = 0.048, uncorrected) and phenylalanine/tyrosine ratios with severity of depressive symptoms (r = 0.376, p < 0.05, corrected). In conclusion, levels of neurotransmitter precursor amino acids correlate with mental health, an effect which was much more pronounced in BCA(+) patients than in BCA(-) subjects. Aside from identifying underlying pathophysiological mechanisms, these results could be the basis for future treatment studies: in BCA(+) patients with depression the use of serotonin-noradrenaline reuptake inhibitors might be recommended while in those with predominant anxiety selective serotonin reuptake inhibitors might be the treatment of choice. Copyright © 2015 Elsevier Ltd. All rights reserved.

Photodynamic therapy of hamster Greene melanoma in vitro and in vivo using bacteriochlorin-a as photosensitizer

NASA Astrophysics Data System (ADS)

Schuitmaker, J. J.; Van Best, Jaap A.; van Delft, J. L.; Jannink, J. E.; Oosterhuis, J. A.; Vrensen, Gijs F.; Ms Wolff-Rouendaal, Didi; Dubbelman, T. M.

1996-01-01

Efficient photodynamic therapy (PDT) of malignant melanoma may be possible with photosensitizers having absorption maxima in the far-red region e.g., above 700 nm. Bacteriochlorin a (BCA), a non toxic derivative of bacteriochlorphyllin a, has a high molecular absorption coefficient (32.000 M-1.cm-1) at 760 nm. At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low. In several series of experiments BCA was proven to be a very effective photosensitizer, in vitro and in vivo. It is preferentially retained in experimental hamster Greene melanoma, rhabdomyosarcoma, RIF- and mamma tumors. Its fluorescence can be detected in vivo, thus enabling early tumor detection and it is rapidly cleared from the tissues which promises no, or minor skin photosensitivity. The effects of BCA-PDT were studied in vitro and in vivo using the heavily pigmented Hamster Greene Melanoma (HGM) cell line as a model. In vitro it was found that the uptake of BCA was time, concentration and temperature dependant. Upon illumination (10 Mw/cm2, 756 nm) after incubation with 2.5 (mu) g/ml BCA for 1 h, almost complete cell kill was obtained within seconds. Hamster Greene Melanoma implanted in the anterior eye chamber of rabbits is an accepted in vivo model for ocular melanoma. The effects of BCA-PDT using this model were studied by light- and electron microscopy. Immediately after PDT intracellular spaces were enlarged and blood vessels were clotted with swollen erythrocytes. Electron microscopy showed fused inner and outer membranes and affected cristae mitchondriales of some mitochondria. With time, the severity of tissue and cell damage increased. One day after irradiation tumor growth had stopped; fluorescein angiography showed non perfusion of the tumor. Histopathology showed almost complete tumor necrosis with occasionally viable cells at the tumor periphery. It is concluded that the direct mitochondrial damage and the vascular damage both contribute to BCA-PDT induced tumor necrosis.

Validation of multi-frequency bioelectrical impedance analysis versus dual-energy X-ray absorptiometry to measure body fat percentage in overweight/obese Colombian adults.

PubMed

Ramírez-Vélez, Robinson; Tordecilla-Sanders, Alejandra; Correa-Bautista, Jorge Enrique; González-Ruíz, Katherine; González-Jiménez, Emilio; Triana-Reina, Hector Reynaldo; García-Hermoso, Antonio; Schmidt-RioValle, Jacqueline

2018-01-01

To verify the validity of multi-frequency bioelectrical impedance analysis (mBCA) for predicting body fat percentage (BF%) in overweight/obese adults using dual-energy X-ray absorptiometry (DXA) as the reference method. Forty-eight adults participated (54% women, mean age = 41.0 ± 7.3 years old). The Pearson's correlation coefficient was used to evaluate the correlation between BIA and BF% assessed by DXA. The concordance between BF% measured by both methods was obtained with Lin's concordance correlation coefficient and Bland-Altman difference plots. Measures of BF% were estimated as 39.0 (SD = 6.1) and 38.3 (SD = 6.5) using DXA and mBCA, respectively. The Pearson's correlation coefficient reflected a strong correlation (r =.921, P = .001). The paired t-test showed a significant mean difference between these methods for obese men BF% of -0.6 [(SD 1.95; 95% CI = -4.0 to 3.0), P =.037]. Overall, the bias of the mBCA was -0.6 [(SD 2.2; 95% CI = -5.0 to 3.7), P =.041], which indicated that the mBCA method significantly underestimated BF% in comparison to the reference method. Finally, in both genders, Lin's concordance correlation coefficient showed a strong agreement. More specifically the DXA value was ρc = 0.943 (95% CI = 0.775 to 0.950) and the mBCA value was ρc = 0.948 (95% CI = 0.778 to 0.978). Our analysis showed a strong agreement between the two methods as reflected in the range of BF%. These results show that mBCA and DXA are comparable methods for measuring body composition with higher body fat percentages. However, due to broad limits of agreement, we can only recommend mBCA for groups of populations. © 2017 Wiley Periodicals, Inc.

Chronic urinary tract infection and bladder carcinoma risk: a meta-analysis of case-control and cohort studies.

PubMed

Akhtar, Saeed; Al-Shammari, Ahmad; Al-Abkal, Jarrah

2018-02-05

This meta-analysis of published case-control and cohort studies sought to quantify the magnitude and direction of association between chronic UTI (defined as the infection of the urinary tract that either does not respond to treatment or keeps recurring) and risk of bladder carcinoma (BCa) (i.e., including mainly urothelial carcinoma, squamous cell carcinoma or adenocarcinoma). A literature search was conducted using Medline, Embase, Ovid, Web of Science, Science Direct and Cochrane Library, which was supplemented with manual search of reference lists of the identified articles. Case-control and cohort studies examining UTI as a predictor of BCa risk published through June 2016 were eligible. Using random-effects models, odds ratios (OR) or relative risks (RR) from eligible studies were combined to synthesize summary effect estimates. The included studies were assessed for methodological quality and potential publication bias. Heterogeneity by study characteristics was examined by sub-group and meta-regression analyses. Eighteen case-control and three cohort studies published between 1963 and 2016 were eligible. Random-effects models showed that UTI was significantly associated with an increased BCa risk both in case-control studies (summary OR RE  = 2.33; 95% CI 1.86, 2.92) and cohort studies (summary RR RE  = 2.88; 95% CI 1.20, 6.89). The observed relationship of UTI with an increased BCa risk was independent of the study characteristics considered. No significant publication bias was detected. Chronic UTI was significantly and independently associated with an increased BCa risk. However, due to the presence of high between-study heterogeneity and inconsistent patterns of adjusted confounding effects, more data are needed to clarify the role of chronic UTI in causation of BCa and if established, prompt and effective treatment of UTI may minimize a substantial proportion of BCa risk.

78 FR 61388 - TA-W-82,705, the Boeing Company Boeing Commercial Aircraft, (BCA) Including On-Site Leased...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-03

..., (BCA) Including On-Site Leased Workers From Adecco, USA, Inc., Cascade Engineering, CDI Corporation...., Cascade Engineering, CDI Corporation, Chipton Ross, Inc., Comforce Corporation, Computer Staffing Services...

ONYX versus n-BCA for embolization of cranial dural arteriovenous fistulas.

PubMed

Rabinov, James David; Yoo, Albert J; Ogilvy, Christopher S; Carter, Bob S; Hirsch, Joshua A

2013-07-01

To evaluate the efficacy of n-butyl-2-cyanoacrylate (Trufill n-BCA) versus ethylene vinyl alcohol copolymer (ONYX) for the embolization of cranial dural arteriovenous fistulas (DAVF). Fifty-three consecutive patients with cranial dural AVF were treated with liquid embolic agents from November, 2003 to November, 2008. These 53 patients had 56 lesions treated with arterial embolization. Patients embolized to completion underwent follow-up angiography at 3 months to assess for durable occlusion. Twenty-one lesions were treated with n-BCA. Seven patients treated with n-BCA had initial angiographic occlusion of their DAVF, which were durable at 3 months. Six patients had adjunctive treatment with coils and/or polyvinyl alcohol particles, but none of these were occluded by endovascular treatment alone. Eleven patients underwent post-embolization surgery for closure of their DAVF. There was one death related to intractable status epilepticus at presentation. One patient developed a major stroke from venous sinus thrombosis after embolization. Thirty-five lesions were treated with ONYX in 34 patients. Twenty-nine patients treated with ONYX had initial angiographic occlusion of their DAVF by embolization alone. One patient had recurrence at 3 months and was re-treated out of 27 total follow-ups. Four patients underwent post-embolization surgical obliteration of their lesions. No deaths or major strokes occurred in this cohort. Initial angiographic occlusion (p=0.0004) and durable angiographic occlusion (p=0.0018) rates for embolization of cranial DAVF show a statistically significant higher efficacy with ONYX compared with n-BCA. Patients embolized with ONYX underwent surgery less frequently compared with those treated with n-BCA (p=0.0015).

Biopolymer-modified graphite oxide nanocomposite films based on benzalkonium chloride-heparin intercalated in graphite oxide

NASA Astrophysics Data System (ADS)

Meng, Na; Zhang, Shuang-Quan; Zhou, Ning-Lin; Shen, Jian

2010-05-01

Heparin is a potent anticoagulant agent that interacts strongly with antithrombin III to prevent the formation of fibrin clots. In the present work, poly(dimethylsiloxane)(PDMS)/graphite oxide-benzalkonium chloride-heparin (PDMS/modified graphite oxide) nanocomposite films were obtained by the solution intercalation technique as a possible drug delivery system. The heparin-benzalkonium chloride (BAC-HEP) was intercalated into graphite oxide (GO) layers to form GO-BAC-HEP (modified graphite oxide). Nanocomposite films were characterized by XRD, SEM, TEM, ATR-FTIR and TGA. The modified graphite oxide was observed to be homogeneously dispersed throughout the PDMS matrix. The effect of modified graphite oxide on the mechanical properties of the nanocomposite film was investigated. When the modified graphite oxide content was lower than 0.2 wt%, the nanocomposites showed excellent mechanical properties. Furthermore, nanocomposite films become delivery systems that release heparin slowly to make the nanocomposite films blood compatible. The in vitro studies included hemocompatibility testing for effects on platelet adhesion, platelet activation, plasma recalcification profiles, and hemolysis. Results from these studies showed that the anticoagulation properties of PDMS/GO-BCA-HEP nanocomposite films were greatly superior to those for no treated PDMS. Cell culture assay indicated that PDMS/GO-BCA-HEP nanocomposite films showed enhanced cell adhesion.

Preclinical optimization of a broad-spectrum anti-bladder cancer tri-drug regimen via the Feedback System Control (FSC) platform

NASA Astrophysics Data System (ADS)

Liu, Qi; Zhang, Cheng; Ding, Xianting; Deng, Hui; Zhang, Daming; Cui, Wei; Xu, Hongwei; Wang, Yingwei; Xu, Wanhai; Lv, Lei; Zhang, Hongyu; He, Yinghua; Wu, Qiong; Szyf, Moshe; Ho, Chih-Ming; Zhu, Jingde

2015-06-01

Therapeutic outcomes of combination chemotherapy have not significantly advanced during the past decades. This has been attributed to the formidable challenges of optimizing drug combinations. Testing a matrix of all possible combinations of doses and agents in a single cell line is unfeasible due to the virtually infinite number of possibilities. We utilized the Feedback System Control (FSC) platform, a phenotype oriented approach to test 100 options among 15,625 possible combinations in four rounds of assaying to identify an optimal tri-drug combination in eight distinct chemoresistant bladder cancer cell lines. This combination killed between 82.86% and 99.52% of BCa cells, but only 47.47% of the immortalized benign bladder epithelial cells. Preclinical in vivo verification revealed its markedly enhanced anti-tumor efficacy as compared to its bi- or mono-drug components in cell line-derived tumor xenografts. The collective response of these pathways to component drugs was both cell type- and drug type specific. However, the entire spectrum of pathways triggered by the tri-drug regimen was similar in all four cancer cell lines, explaining its broad spectrum killing of BCa lines, which did not occur with its component drugs. Our findings here suggest that the FSC platform holdspromise for optimization of anti-cancer combination chemotherapy.

75 FR 78798 - Airport Improvement Program: Proposed Changes to Benefit Cost Analysis (BCA) Threshold

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-16

...The Federal Aviation Administration (FAA) is issuing this Notice to advise that FAA has developed draft guidance modifying its policy requiring benefit cost analyses (BCA) for capacity projects when applying for Airport Improvement Program (AIP) grants for capacity projects at the discretion of the Secretary of Transportation. This modification proposes to raise the threshold at which BCAs are required, from $5 million to $10 million in AIP Discretionary funds. FAA invites airport sponsors and other interested parties to comment on the draft guidance. FAA will consider these comments in promulgating final BCA guidance for airport sponsors.

Automated tubule nuclei quantification and correlation with oncotype DX risk categories in ER+ breast cancer whole slide images

NASA Astrophysics Data System (ADS)

Romo-Bucheli, David; Janowczyk, Andrew; Romero, Eduardo; Gilmore, Hannah; Madabhushi, Anant

2016-03-01

Early stage estrogen receptor positive (ER+) breast cancer (BCa) treatment is based on the presumed aggressiveness and likelihood of cancer recurrence. The primary conundrum in treatment and management of early stage ER+ BCa is identifying which of these cancers are candidates for adjuvant chemotherapy and which patients will respond to hormonal therapy alone. This decision could spare some patients the inherent toxicity associated with adjuvant chemotherapy. Oncotype DX (ODX) and other gene expression tests have allowed for distinguishing the more aggressive ER+ BCa requiring adjuvant chemotherapy from the less aggressive cancers benefiting from hormonal therapy alone. However these gene expression tests tend to be expensive, tissue destructive and require physical shipping of tissue blocks for the test to be done. Interestingly breast cancer grade in these tumors has been shown to be highly correlated with the ODX risk score. Unfortunately studies have shown that Bloom-Richardson (BR) grade determined by pathologists can be highly variable. One of the constituent categories in BR grading is the quantification of tubules. The goal of this study was to develop a deep learning neural network classifier to automatically identify tubule nuclei from whole slide images (WSI) of ER+ BCa, the hypothesis being that the ratio of tubule nuclei to overall number of nuclei would correlate with the corresponding ODX risk categories. The performance of the tubule nuclei deep learning strategy was evaluated with a set of 61 high power fields. Under a 5-fold cross-validation, the average precision and recall measures were 0:72 and 0:56 respectively. In addition, the correlation with the ODX risk score was assessed in a set of 7513 high power fields extracted from 174 WSI, each from a different patient (At most 50 high power fields per patient study were used). The ratio between the number of tubule and non-tubule nuclei was computed for each WSI. The results suggests that for BCa cases with both low ODX score and low BR grade, the mean tubule nuclei ratio was significantly higher than that obtained for the BCa cases with both high ODX score and high BR grade (p < 0:01). The low ODX and low BR grade cases also presented a significantly higher average tubule nuclei ratio when compared with the rest of the BCa cases (p < 0:05). Finally, the BCa cases that presented both a high ODX and high BR grade show a mean tubule nuclei to total number of nuclei ratio which was significantly smaller than that obtained for the rest of BCa cases (p < 0:01).

1 alpha, 25-dihydroxylvitamin D3 promotes Bacillus Calmette-Guérin immunotherapy of bladder cancer

PubMed Central

Hsu, Jong-Wei; Yin, Peng-Nien; Wood, Ronald; Messing, James; Messing, Edward; Lee, Yi-Fen

2013-01-01

Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD. PMID:24353168

An evaluation of the behaviour-change techniques used on Canadian cancer centre Web sites to support physical activity behaviour for breast cancer survivors

PubMed Central

Sylvester, B.D.; Zammit, K.; Fong, A.J.; Sabiston, C.M.

2017-01-01

Background Cancer centre Web sites can be a useful tool for distributing information about the benefits of physical activity for breast cancer (bca) survivors, and they hold potential for supporting health behaviour change. However, the extent to which cancer centre Web sites use evidence-based behaviour change techniques to foster physical activity behaviour among bca survivors is currently unknown. The aim of our study was to evaluate the presentation of behaviour-change techniques on Canadian cancer centre Web sites to promote physical activity behaviour for bca survivors. Methods All Canadian cancer centre Web sites (n = 39) were evaluated by two raters using the Coventry, Aberdeen, and London–Refined (calo-re) taxonomy of behaviour change techniques and the eEurope 2002 Quality Criteria for Health Related Websites. Descriptive statistics were calculated. Results The most common behaviour change techniques used on Web sites were providing information about consequences in general (80%), suggesting goal-setting behaviour (56%), and planning social support or social change (46%). Overall, Canadian cancer centre Web sites presented an average of M = 6.31 behaviour change techniques (of 40 that were coded) to help bca survivors increase their physical activity behaviour. Evidence of quality factors ranged from 90% (sites that provided evidence of readability) to 0% (sites that provided an editorial policy). Conclusions Our results provide preliminary evidence that, of 40 behaviour-change techniques that were coded, fewer than 20% were used to promote physical activity behaviour to bca survivors on cancer centre Web sites, and that the most effective techniques were inconsistently used. On cancer centre Web sites, health promotion specialists could focus on emphasizing knowledge mobilization efforts using available research into behaviour-change techniques to help bca survivors increase their physical activity. PMID:29270056

Mid- and long-term outcomes of carotid-cavernous fistula endovascular management with Onyx and n-BCA: experience of a single tertiary center.

PubMed

Barber, Sean M; Rangel-Castilla, Leonardo; Zhang, Y Jonathan; Klucznik, Richard; Diaz, Orlando

2015-10-01

Endovascular therapy is the preferred treatment for most carotid-cavernous fistulas (CCFs). Early reports have documented excellent initial clinical and radiographic outcomes after embolization of CCFs with Onyx or n-butyl cyanoacrylate (n-BCA), but little evidence is available about the long-term durability of this technique. To characterize the long-term durability of CCF liquid embolization. The authors retrospectively reviewed a database of 24 CCFs in 21 consecutive patients who underwent Onyx or n-BCA embolization of a CCF from 2006 to 2013 at our institution. A total of 25 Onyx or n-BCA embolization procedures were attempted and 24 successfully completed during the study, resulting in complete or near-complete occlusion by the end of the study in all 24 CCFs (obliteration success, 100%). Attempted embolization in a single CCF failed initially, but was performed successfully at a later date by a different approach. None of the 24 CCFs recanalized, regrew, or required any further treatment subsequent to Onyx or n-BCA embolization throughout a mean 12.4 months of angiographic follow-up (range 1-36 months). Clinically significant complications were seen in three embolization procedures, including cranial nerve palsies (n=1), embolic infarct (n=1), and intraperitoneal hemorrhage (n=1). Early evidence has indicated that endovascular embolization with Onyx is relatively safe and effective at achieving an initial angiographic cure for CCFs. Results of our series suggest that angiographic and clinical outcomes of Onyx and n-BCA embolization remain stable at mid- and long-term follow-up. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Awareness of breast cancer and breast self-examination among female undergraduate students in a higher teachers training college in Cameroon.

PubMed

Sama, Carlson-Babila; Dzekem, Bonaventure; Kehbila, Jules; Ekabe, Cyril Jabea; Vofo, Brice; Abua, Naomi Liteba; Dingana, Therence Nwana; Angwafo, Fru

2017-01-01

The incidence of breast cancer (BCa) in Cameroon is on the rise and accounts for a leading cause of mortality. An understanding of the knowledge and practices on breast cancer and breast self-examination (BSE) among teachers are important first steps which will guide the designing of interventions aimed at raising awareness across the general population. We conducted a cross-sectional study in April 2016 involving 345 consenting female undergraduate students in the Higher Teachers Training College, Bambili, Cameroon. Data was collected using a pretested self-administered questionnaire and analysed using descriptive methods. The mean age of the respondents was 22.5±3.2years and a vast majority (n = 304, 88.1%) had heard about BCa primarily from the television/radio (n=196, 64.5%). Overall, less than a quarter (n=65, 21.4%) of respondents who had heard about BCa had sufficient knowledge on its risk factors and signs/symptoms. A plurality (53.3%) thought BCa can be prevented via vaccination while over a third (38.7%) opined that BCa can be treated spiritually. Less than half (47%) of respondents who had heard about BCa had heard about BSE amongst which only 55 (38.5%) had ever practiced it. Though most students are aware of the existence of breast cancer, their overall knowledge on its risk factors and clinical presentation is insufficient with a concomitant low practice of BSE. These highlighted gaps warrants intensification of sensitization campaigns and educational programmes in order to raise knowledge levels and enhance prevention strategies that would aid in reducing the burden of breast cancer in Cameroon.

An evaluation of the behaviour-change techniques used on Canadian cancer centre Web sites to support physical activity behaviour for breast cancer survivors.

PubMed

Sylvester, B D; Zammit, K; Fong, A J; Sabiston, C M

2017-12-01

Cancer centre Web sites can be a useful tool for distributing information about the benefits of physical activity for breast cancer (bca) survivors, and they hold potential for supporting health behaviour change. However, the extent to which cancer centre Web sites use evidence-based behaviour change techniques to foster physical activity behaviour among bca survivors is currently unknown. The aim of our study was to evaluate the presentation of behaviour-change techniques on Canadian cancer centre Web sites to promote physical activity behaviour for bca survivors. All Canadian cancer centre Web sites ( n = 39) were evaluated by two raters using the Coventry, Aberdeen, and London-Refined (calo-re) taxonomy of behaviour change techniques and the eEurope 2002 Quality Criteria for Health Related Websites. Descriptive statistics were calculated. The most common behaviour change techniques used on Web sites were providing information about consequences in general (80%), suggesting goal-setting behaviour (56%), and planning social support or social change (46%). Overall, Canadian cancer centre Web sites presented an average of M = 6.31 behaviour change techniques (of 40 that were coded) to help bca survivors increase their physical activity behaviour. Evidence of quality factors ranged from 90% (sites that provided evidence of readability) to 0% (sites that provided an editorial policy). Our results provide preliminary evidence that, of 40 behaviour-change techniques that were coded, fewer than 20% were used to promote physical activity behaviour to bca survivors on cancer centre Web sites, and that the most effective techniques were inconsistently used. On cancer centre Web sites, health promotion specialists could focus on emphasizing knowledge mobilization efforts using available research into behaviour-change techniques to help bca survivors increase their physical activity.

Probing the internal calcification chemistry of O. universa using B/Ca

NASA Astrophysics Data System (ADS)

Holland, K.; Eggins, S.; Hoenisch, B.; Haynes, L.; Russell, A. D.

2014-12-01

The B/Ca, U/Ca ratio values of calcitic foraminifer shells are all influenced by seawater carbonate chemistry (seawater [B(OH)4-/HCO3-], [CO32-], and pH respectively), and as such are increasingly being used as proxies to reconstruct past changes in ocean inorganic carbon content, saturation state and pH. However, the behavior of these proxies is subject to modification by foraminifers' efforts to regulate the conditions under which they grow their shells. We have undertaken experiments on Orbulina universa that manipulate synthetic culture water DIC, pH and [Ca] in an effort to disentangle the biological versus environmental influences of seawater carbonate system and saturation state on B/Ca, U/Ca and Mg/Ca ratio into foraminiferal calcite. Experiments were designed to be able determine the extent to which foraminifers are able to modify the chemical composition of their (vacuolized?) internal calcification fluid, in particular by using B/Ca and U/Ca as sensors for calcification chemistry (i.e. internal [B(OH)4-/HCO3-] and [CO32-]) . We have used a high resolution LA-ICPMS depth profiling techniques to characterize the amplitude of B/Ca, U/Ca, Mg/Ca, and Sr/Ca ratio values across and the thickness (calcification rate) of diurnal bands that are developed in individual shells grown under different synthetic seawater compositions. Results indicate Orbulina universa modify the chemistry of their calcification fluid far from that of external seawater, but are not able to mitigate changes in external seawater. This most likely achieved through the interactive effects of internal pH manipulation and a carbon concentration mechanism. Our results are likely to have important implications for the interpretation of Mg/Ca, B/Ca and U/Ca as proxies seawater temperatures and carbonate system parameters.

Fluorescence and photodynamic effects of bacteriochlorin a observed in vivo in 'sandwich' observation chambers.

PubMed Central

van Leengoed, H. L.; Schuitmaker, J. J.; van der Veen, N.; Dubbelman, T. M.; Star, W. M.

1993-01-01

Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in vitro and in vivo. BCA has a major absorption peak at 760 nm where tissue penetration is optimal. This property, together with rapid tissue clearance promises minor skin photosensitivity. The tissue localising and photodynamic properties of BCA were studied using isogeneic RMA mammary tumours, transplanted into subcutaneous tissue in transparent 'sandwich' observation chambers on the back of WAG/Rij rats. The fluorescence kinetics following an i.v. administration of 20 mg kg-1 BCA was assessed in blood vessels, tumour and normal tissue. Subsequently, the development of vascular- and tissue damage after a therapeutic light dose (760 nm, 600 J cm-2) was observed. Fifteen minutes post injection (p.i.), the fluorescence of BCA in the tumour reached a plateau value of 2.5 times the fluorescence in the normal tissue. From 1 h post injection the tumour fluorescence diminished gradually; after 24 h, the tumour fluorescence signal did not exceed that of the normal tissue. Following photodynamic therapy (PDT), 24 h p.i., complete vascular stasis was observed 2 h post treatment in the tumour only, with subsequent recovery. The presence of viable tumour cells following PDT was assessed by histology and re-transplantation of treated tumour tissue from the chamber into the flank immediately or 7 days after treatment. In both cases tumour regrowth was observed. BCA-PDT (20 mg kg-1, 760 nm, 100 J cm-2) 1 h after BCA administration, an interval which gives the optimal differential between tumour and normal tissue, was sufficient to prevent tumour regrowth. However, this only occurred when re-transplantation was performed 7 days after PDT. During PDT, 1 h p.i., vascular damage in tumour and normal tissue was considerable. Complete vascular shut-down was observed in the tumour 2 h after therapy and in the surrounding tissues at 24 h. Circulation damage was associated with vascular spasm and occlusion probably due to thrombi formation. Oedema was notable, especially following PDT with 600 J cm-2 at 24 h p.i. Images Figure 1 PMID:8494722

Effects of varying doses of β-nerve growth factor on the timing of ovulation, plasma progesterone concentration and corpus luteum size in female alpacas (Vicugna pacos).

PubMed

Stuart, C C; Vaughan, J L; Kershaw-Young, C M; Wilkinson, J; Bathgate, R; de Graaf, S P

2015-11-01

Ovulation in camelids is induced by the seminal plasma protein ovulation-inducing factor (OIF), recently identified as β-nerve growth factor (β-NGF). The present study measured the total protein concentration in alpaca seminal plasma using a bicinchoninic acid (BCA) protein quantification assay and found it to be 22.2±2.0mgmL(-1). To measure the effects of varying doses of β-NGF on the incidence and timing of ovulation, corpus luteum (CL) size and plasma progesterone concentration, 24 female alpacas were synchronised and treated with either: (1) 1mL 0.9% saline (n=5); (2) 4µg buserelin (n=5); (3) 1mg β-NGF protein (n=5); (4) 0.1mg β-NGF (n=5); or (5) 0.01mg β-NGF (n=4). Females were examined by transrectal ultrasonography at 1-2-h intervals between 20 and 45h after treatment or until ovulation occurred, as well as on Day 8 to observe the size of the CL, at which time blood was collected to measure plasma progesterone concentrations. Ovulation was detected in 0/5, 5/5, 5/5, 3/5 and 0/4 female alpacas treated with saline, buserelin, 1, 0.1 and 0.01mg β-NGF, respectively. Mean ovulation interval (P=0.76), CL diameter (P=0.96) and plasma progesterone concentration (P=0.96) did not differ between treatments. Mean ovulation interval overall was 26.2±1.0h. In conclusion, buserelin and 1mg β-NGF are equally effective at inducing ovulation in female alpacas, but at doses ≤0.1mg, β-NGF is not a reliable method for the induction of ovulation.

Interaction of fibrinogen and albumin with titanium dioxide nanoparticles of different crystalline phases

NASA Astrophysics Data System (ADS)

Marucco, Arianna; Fenoglio, Ivana; Turci, Francesco; Fubini, Bice

2013-04-01

TiO2 nanoparticles (NPs) are contained in different kinds of industrial products including paints, self-cleaning glasses, sunscreens. TiO2 is also employed in photocatalysis and it has been proposed for waste water treatment. Micrometric TiO2 is generally considered a safe material, while there is concern on the possible health effects of nanometric titania. Due to their small size NPs may migrate within the human body possibly entering in the blood stream. Therefore studies on the interaction of NPs with plasma proteins are needed. In fact, the interaction with proteins is believed to ultimately influences the NPs biological fate. Fibrinogen and albumin are two of the most abundant plasma proteins. They are involved in several important physiological functions. Furthermore, fibrinogen is known to trigger platelet adhesion and inflammation. For these reasons the study of the interaction between these protein and nanoparticles is an important step toward the understanding of the behavior of NPs in the body. In this study we investigated the interaction of albumin and fibrinogen with TiO2 nanoparticles of different crystal phases (rutile and anatase) using an integrated set of techniques. The amount of adsorbed fibrinogen and albumin for each TiO2 surface was investigated by using the bicinchoninic acid assay (BCA). The variation of the surface charge of the NP-protein conjugates respect to the naked NPs was used to indirectly estimate both surface coverage and reversibility of the adsorption upon dilution. Surface charge was monitored by measuring the ζ potential with a conventional electrophoretic light scattering (ELS) system. The extent of protein deformation was evaluated by Raman Spectroscopy. We found that both proteins adsorb irreversibly against electrostatic repulsion, likely undergoing conformational changes or selective orientation upon adsorption. The size of primary particles and the particles aggregation rather than the crystal phase modulate the affinity of fibrinogen for the TiO2 surfaces.

Physical and Optical/Radiative Properties of Arctic Aerosols: Potential Effects on Arctic Climate

NASA Technical Reports Server (NTRS)

Pueschel, R. F.; Kinne, S. A.; Gore, Warren J. (Technical Monitor)

1994-01-01

We have determined the abundance of light-scattering sulfuric acid (H2SO4/H2O) and light-absorbing black carbon aerosol (BCA) in Spring 1992 in the Arctic atmosphere by airborne in situ sampling with impactors, and measured particle sizes and morphologies by scanning electron microscopy. The mass of BCA in the Arctic troposphere is one percent of the total aerosol, reduced to one part in 104 in the stratosphere. A Mie algorithm permits the calculation of the optical properties of the various aerosol components, and an algorithm developed by Ackerman and Toon and modified to serve our needs lets us calculate the optical effects of the black carbon aerosol that is mixed internally with the sulfuric acid aerosol. It follows that the effect of internally-mixed BCA on the aerosol scattering and absorption properties depends on its location within the droplet. BCA concentrated near the droplet surface has a greater effect on absorption of solar radiation than does the same amount of BCA located near its center. Single scatter albedos of the combined system are omega(sub 0)=1.0 in the post-Pinatubo Arctic stratosphere, and as low as 0.94 in the troposphere. The aerosol has the potential to regionally warm the Arctic earth-atmosphere system, because of the high surface albedo of the snow-covered Arctic.

Prostaglandin receptors EP1-4 as a potential marker for clinical outcome in urothelial bladder cancer.

PubMed

von der Emde, Laura; Goltz, Diane; Latz, Stefan; Müller, Stefan C; Kristiansen, Glen; Ellinger, Jörg; Syring, Isabella

2014-01-01

Prostaglandins, especially prostaglandin E2 (PGE2), and COX-2 play an important role in carcinogenesis of many tumors including bladder cancer (BCA). The PGE2 receptors EP1-4 regulate tumor cell growth, invasion and migration in different tumor entities but EP expression in BCA remains to be determined. In the present study we examined the expression of EP1-4 in non-muscle invasive bladder cancer (NMIBC), muscle invasive bladder cancer (MIBC) and normal urothelial tissue (NU) using immunohistochemistry. Nuclear and cytoplasmic EP1-4 expression was correlated with clinicopathological parameters and survival of BCA patients. EP1, EP2 and EP3 were significantly less expressed in the cytoplasm und nucleus of NMIBC and MIBC than in NU; EP4 cytoplasmic staining in MIBC was significantly higher compared to NU. The cytoplasmic staining was significantly more abundant in MIBC than in NMIBC in all investigated receptors except EP2. The level of EP staining in NMIBC was correlated with staging and grading, especially cytoplasmic EP1. Nuclear staining of EP1 was an independent predictor of BCA recurrence-free survival in NMIBC patients. EP receptors are dysregulated in BCA. The increase of EP1 may be used as prognostic parameter in NMIBC patients and its dysregulation could be targeted by specific EP1 inhibitors.

Prostaglandin receptors EP1-4 as a potential marker for clinical outcome in urothelial bladder cancer

PubMed Central

von der Emde, Laura; Goltz, Diane; Latz, Stefan; Müller, Stefan C; Kristiansen, Glen; Ellinger, Jörg; Syring, Isabella

2014-01-01

Prostaglandins, especially prostaglandin E2 (PGE2), and COX-2 play an important role in carcinogenesis of many tumors including bladder cancer (BCA). The PGE2 receptors EP1-4 regulate tumor cell growth, invasion and migration in different tumor entities but EP expression in BCA remains to be determined. In the present study we examined the expression of EP1-4 in non-muscle invasive bladder cancer (NMIBC), muscle invasive bladder cancer (MIBC) and normal urothelial tissue (NU) using immunohistochemistry. Nuclear and cytoplasmic EP1-4 expression was correlated with clinicopathological parameters and survival of BCA patients. EP1, EP2 and EP3 were significantly less expressed in the cytoplasm und nucleus of NMIBC and MIBC than in NU; EP4 cytoplasmic staining in MIBC was significantly higher compared to NU. The cytoplasmic staining was significantly more abundant in MIBC than in NMIBC in all investigated receptors except EP2. The level of EP staining in NMIBC was correlated with staging and grading, especially cytoplasmic EP1. Nuclear staining of EP1 was an independent predictor of BCA recurrence-free survival in NMIBC patients. EP receptors are dysregulated in BCA. The increase of EP1 may be used as prognostic parameter in NMIBC patients and its dysregulation could be targeted by specific EP1 inhibitors. PMID:25520883

HALOACID INDUCED ALTERATIONS IN FERTILITY AND THE SPERM BIOMARKER SP22 IN THE RAT ARE ADDITIVE: VALIDATION OF AN ELISA

EPA Science Inventory

Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of cauda epididymal rat sperm an...

The relationship of biofilm production to biocontrol activity of Burkholderia pyrrocinia FP62

USDA-ARS?s Scientific Manuscript database

Foliar biocontrol agent (BCA) efficacy is often inconsistent due to poor colonization and survival on plant surfaces. Burkholderia pyrrocinia FP62, a superior leaf colonist and BCA of Botrytis cinerea, forms unsaturated biofilms on plant surfaces. To determine the relationship between biocontrol act...

DOD Product Support Business Case Analysis Guidebook

DTIC Science & Technology

2011-04-01

Mitigation Plans ................................................................................................ 30 4.8 Sensitivity Analysis...Product Support BCA concludes with a recommendation and associated specific actions and an implementation plan to achieve stated organizational...primary executer of the actions and recommendations derived out of the BCA. Within the program office, the PSM has the responsibility to plan

Endoglucanase (EG) Activity Assays.

PubMed

Rahman, M Shafiqur; Fernando, Sheran; Ross, Brian; Wu, Jiangning; Qin, Wensheng

2018-01-01

Cellulosic biomass, the most common organic compound of primary energy source on earth, is a network of interwoven biopolymers of plant cell walls. Degradation of cellulose is important for global carbon recycling. Moreover, biofuel, a renewable fuel whose energy can be derived from cellulosic biomass by enzymatic hydrolysis of cellulases. Among cellulases are endoglucases that act synergistically for subsequent hydrolytic reactions to break down the polymeric cellulose. However, in cellulolytic enzyme activity endoglucanase plays a prominent role in initiating and sustaining the hydrolytic process. Endoglucanase randomly cleaves the cellulose polymer into smaller sugar and oligomeric polysaccharides. Characterization and quantification of endoglucanase activity is important for industry and in the overall study of cellulose degradation. All assays including those for endoglucanase fall into two broad categories either qualitative or quantitative. Quantitative assays can tell if the enzyme is present, how much and its activity. Measurement can be done indirectly using a secondary colorimetric product like (1) molybdenum blue, (2) 3-amino-5-nitrosalicylic acid, (3) bicinchoninic acid (BCA), and (4) 2-cyanoacetamide or directly using an antibody in an ELISA. In this chapter, we discuss several common protocols for the measurement of endoglucanase activity.

Norms and Standards for Computer Education (MCA, BCA) through Distance Mode.

ERIC Educational Resources Information Center

Rausaria, R.R., Ed.; Lele, Nalini A., Ed.; Bhushan, Bharat, Ed.

This document presents the norms and standards for computer education in India through distance mode, including the Masters in Computer Applications (MCA) and Bachelor in Computer Applications (BCA) programs. These norms and standards were considered and approved by the Distance Education Council, Indira Gandhi National Open University (India), at…

Raise the Bar

ERIC Educational Resources Information Center

Williams, Dana

2004-01-01

Detroit's Benjamin Carson Academy (BCA) is believed to be the nation's first charter school for juvenile offenders. Opened in 1999, BCA is housed in the newly built Wayne County Juvenile Detention Facility, a state of the art, 89,300-square-foot building in downtown Detroit with half a dozen gymnasiums, two computer labs, a media center, mental…

Defense AT and L. Volume 41, Number 2, March-April 2012

DTIC Science & Technology

2012-03-01

a philosophical perspective, but let’s put some “shoe leather ” on this discussion. What will it take to prepare and groom these current and future...is up to the BCA team, working in tan - dem with strategic level senior thinkers, to explore and assess BCA alternatives’ effects on other

Herbivore-induced shifts in carbon and nitrogen allocation in red oak seedlings

Treesearch

Christopher J. Frost; Mark D. Hunter

2008-01-01

A dual-isotope, microcosm experiment was conducted with Quercus rubra (red oak) seedlings to test the hypothesis that foliar herbivory would increase belowground carbon allocation (BCA), carbon (C) rhizodeposition and nitrogen (N) uptake. Plant BCA links soil ecosystems to aboveground processes and can be affected by insect herbivores, though the...

Relationship between mycoparasites lifestyles and biocontrol behaviors against Fusarium spp. and mycotoxins production.

PubMed

Kim, Seon Hwa; Vujanovic, Vladimir

2016-06-01

Global food security research is seeking eco-friendly solutions to control mycotoxins in grain infected by fungi (molds). In particular, mycotoxigenic Fusarium spp. outbreak is a chronic threat for cereal grain production, human, and animal health. In this review paper, we discuss up-to-date biological control strategies in applying mycoparasites as biological control agents (BCA) to prevent plant diseases in crops and mycotoxins in grain, food, and feed. The aim is to increase food safety and to minimize economic losses due to the reduced grain yield and quality. However, recent papers indicate that the study of the BCA specialists with biotrophic lifestyle lags behind our understanding of the BCA generalists with necrotrophic lifestyle. We examine critical behavioral traits of the two BCA groups of mycoparasites. The goal is to highlight their major characteristics in the context of future research towards an efficient biocontrol strategy against mycotoxin-producing Fusarium species. The emphasis is put on biocontrol of Fusarium graminearum, F. avenaceum, and F. culmorum causing Fusarium head blight (FHB) in cereals and their mycotoxins.

Cryptophane Nanoscale Assemblies Expand 129Xe NMR Biosensing.

PubMed

Zemerov, Serge D; Roose, Benjamin W; Greenberg, Mara L; Wang, Yanfei; Dmochowski, Ivan J

2018-06-19

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129 Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129 Xe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129 Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.

Black Carbon (Soot) Aerosol in the Lower Stratosphere and Upper Troposphere. Revised

NASA Technical Reports Server (NTRS)

Pueschel, R. F.; Black, D. F.; Snetsinger, K. G.; Hansen, A. D. A.; Verna, S.; Kato, K.

1992-01-01

We have used two approaches to measure BCA in the stratosphere. The first method consists of collecting aerosols by impaction on quartz rods, upon which are mounted strips of polystyrene filter material. Each 25 mm by 0.5 mm filter strip is alternately masked and exposed in 5 mm long sections. After sampling, the optical density of the exposed sections is measured and compared with the optical density of the masked (unexposed) sections. The filter strip is a Lambertian collector, the scattering property of which is not affected by the deposition of liquid sulfuric acid droplets which dominate the stratospheric aerosol. Hence, all attenuation greater in the exposed than in the non-exposed sections is due to absorption by BCA or rare opaque materials such as meteoritic iron. In a second approach we expose carbon-coated gold or palladium wires to air outside the aircraft boundary layer [Farlow et al., 1979; Pueschel et al., 1989]. With a field emission scanning electron microscope we are able to characterize BCA particles in addition to the dominant sulfuric acid droplets. Typically, soot is comprised of 20 nm diameter spheres linked together as branching chains or loosely packed aggregates (Figure 1a). The microscope has a lateral image resolution of 1.5 nm and can clearly resolve individual spheres making up BCA aggregates. Their morphology is uniquely different from that of the liquid sulfuric acid-water aerosol (Figure 1b). We approximate the size of each BCA particle by that of a sphere of equivalent volume and fit a lognormal size distribution to both the BCA and H2SO4/H2O aerosols. Both sampler types have been mounted on NASA's ER-2 high-altitude research aircraft (altitude ceiling of 70,000 feet), and on NASA's DC-8 (40,000 feet ceiling) aircraft to sample aerosols from a significant portion of the atmosphere.

The Influence of Race on Overall Survival in Patients with Newly Diagnosed Bladder Cancer.

PubMed

DeDeugd, Casey; Miyake, Makito; Palacios, Diego Aguilar; Rosser, Charles J

2015-03-01

Previous studies have reported significant lower incidence yet greater risk of death from bladder cancer (BCa) in African-Americans compared with Caucasians. In this study, the overall survival amongst African-Americans and Caucasians with BCa within the state of Florida is evaluated. The Florida Cancer Data System and the Florida Agency for Health Care Administration data sets were linked on the basis of unique identifiers, which identified 28,786 patients (27,811 Caucasian and 975 African-Americans) with newly diagnosed BCa from January 1994-December 2009. Data in the database included race/ethnicity, age, smoking history, insurance status, treatment, tumor grade, tumor stage, and overall survival. Chi-square and Mann-Whitney U tests were used to compare variables between African-Americans and Caucasians. Survival rates were calculated by the Kaplan-Meier method while univariate effects were tested by the log-rank test, and multivariate effects were tested by Cox proportional-hazard regression model. P values less than 0.05 were considered statistically significant. Higher clinical stage bladder tumors including T3/4 disease (14.5 % vs. 8.0 %, p < 0.001), lymph node involvement (7.3 % vs. 3.4 %, p < 0.001), and metastatic disease (5.3 % vs. 1.7 %, p < 0.001), as well as higher grade disease (60.2 % vs. 48 %, p < 0.001) were more commonly reported in African-Americans than in Caucasians with newly diagnosed BCa. African-Americans tended to be treated with more aggressive therapies (e.g., radical cystectomy). After adjusting for all covariates, African-Americans actually had more favorable outcomes as related to overall survival (HR = 0.35, 95 % CI, 0.12-0.98, p = 0.045). Though African-Americans initially present with more aggressive BCa, African-Americans actually have an improved overall survival compared with Caucasians. Though contrary to previous reports, our results may signify a more complex relationship between race and BCa outcomes and thus warrants further attention.

Heterodimer formation by Oct4 and Smad3 differentially regulates epithelial-to-mesenchymal transition-associated factors in breast cancer progression.

PubMed

Mandal, Gunjan; Biswas, Subir; Roy Chowdhury, Sougata; Chatterjee, Annesha; Purohit, Suman; Khamaru, Poulomi; Chakraborty, Sayan; Mandal, Palash Kumar; Gupta, Arnab; de la Mare, Jo-Anne; Edkins, Adrienne Lesley; Bhattacharyya, Arindam

2018-06-01

The multifunctional cytokine TGF-β crucially participates in breast cancer (BCa) metastasis and works differently in the disease stages, thus contributing in BCa progression. We address connections between TGF-β and the stem cell-related transcription factor (TF) Oct4 in BCa. In 147 BCa patients with infiltrating duct carcinoma, we identified a significantly higher number of cases with both moderate/high Oct4 expression and high TGF-β in late stages compared to early stages of the disease. In vitro studies showed that TGF-β elevated Oct4 expression, which in turn, regulated Epithelial-to-Mesenchymal transition (EMT)-regulatory gene (Snail and Slug) expression, migratory ability, chemotactic invasiveness and extracellular matrix (ECM) degradation potential of BCa cells. Putative binding sites for Oct4 on the snail, slug and cxcl13 promoters and for Smad3 on the snail and slug promoters were identified. Promoter activities of snail and slug were greater in dual-treated cells than only TGF-β-treated or Oct4-overexpressing cells. CXCL13 mRNA fold changes, however, were low in cells induced with TGF-β, compared to dual-treated or Oct4-overexpressing cells. Our co-IP studies confirmed that Oct4 and Smad3 form heterodimers that recognize specific promoter sequences to promote Snail and Slug expression, but which in turn, indirectly inhibits Smad3-mediated repression of CXCL13 expression, allowing Oct4 to act as a positive TF for CXCL13. Taken together, these data suggest that TGF-β signaling and Oct4 cooperate to induce expression of EMT-related genes Snail, Slug and CXCL13, which accelerates disease progression, particularly in the late stages, and may indicate a poor prognosis for BCa patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

PubMed

Hess, A; Wang-Lauenstein, L; Braun, A; Kolle, S N; Landsiedel, R; Liebsch, M; Ma-Hock, L; Pirow, R; Schneider, X; Steinfath, M; Vogel, S; Martin, C; Sewald, K

2016-04-01

In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23 h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (μM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

REPRODUCTIVE AND GENOMIC EFFECTS IN TESTES FROM MICE EXPOSED TO THE WATER DISINFECTANT BYPRODUCT BROMOCHLOROACETIC ACID

EPA Science Inventory

ABSTRACT

A byproduct of drinking water disinfection, bromochloroacetic acid (BCA), acts as a reproductive toxicant in rats. To determine if BCA produces similar reproductive toxicity in mice, juvenile and adult C57BL/6 males were exposed to 0, 8, 24, 72 or 216 mg/kg of BC...

Benefit-Cost Analysis of Drug Abuse Prevention Programs: A Macroscopic Approach.

ERIC Educational Resources Information Center

Kim, Sehwan; And Others

1995-01-01

Determines the overall strategy for initiating benefit-cost analysis (BCA) in relation to drug abuse prevention programs, followed by definitions of BCA and cost-effectiveness analysis. Determines the most likely population benefit-cost efficiency ratio of 15:1, indicating that there is a $15 savings on every dollar spent on drug abuse education.…

REPRODUCTIVE EFFECTS OF THE WATER DISINFECTANT BYPRODUCT BROMOCHLOROACETIC ACID (BCA) IN ADULT AND JUVENILE MALE C57BL/6 MICE

EPA Science Inventory

REPRODUCTIVE EFFECTS OF THE WATER DISINFECTANT BYPRODUCT BROMOCHLOROACETIC ACID (BCA) IN ADULT AND JUVENILE MALE C57BL/6 MICE.
JC Rockett, JC Luft, JB Garges and DJ Dix. Reproductive Toxicology Division, USEPA, RTP, NC, USA.
Sponsor: G Klinefelter
The development of wate...

Boron incorporation in the foraminifer Amphistegina lessonii under a decoupled carbonate chemistry

NASA Astrophysics Data System (ADS)

Kaczmarek, K.; Langer, G.; Nehrke, G.; Horn, I.; Misra, S.; Janse, M.; Bijma, J.

2014-12-01

A number of studies have shown that the boron isotopic composition (δ11B) and the B/Ca ratio of biogenic carbonates (mostly foraminifers) can serve as proxies for two parameters of the ocean's carbonate chemistry, rendering it possible to calculate the entire carbonate system. However, the B incorporation mechanism into marine carbonates is still not fully understood and analyses of field samples show species specific and hydrographic effects on the B proxies complicating their application. Identifying the carbonate system parameter influencing boron incorporation is difficult due to the co-variation of pH, CO32-, and B(OH)4-. To shed light on the question which parameter of the carbonate system is related to the boron incorporation, we performed culture experiments with the benthic symbiont-bearing foraminifer Amphistegina lessonii using a decoupled pH-CO32- chemistry. The determination of the boron isotopic composition and B/Ca ratios was performed simultaneously by means of a new in situ technique combining optical emission spectroscopy and laser ablation MC-ICP-MS. The boron isotopic composition in the tests gets heavier with increasing pH and B/Ca increases with increasing BOH4-/HCO3- of the culture media. The latter indicates that boron uptake of A. lessonii features a competition between B(OH)4- and HCO3-. Furthermore, the simultaneous determination of B/Ca and δ11B on single specimens allows for assessing the relative variability of these parameters. Among different treatments the B/Ca shows an increasing variability with increasing boron concentration in the test whereas the variability in the isotope distribution is constant.

Clinical practice guidelines in breast cancer

PubMed Central

Tyagi, N. Kumar; Dhesy-Thind, S.

2018-01-01

Background A number of clinical practice guidelines (cpgs) concerning breast cancer (bca) screening and management are available. Here, we review the strengths and weaknesses of cpgs from various professional organizations and consensus groups with respect to their methodologic quality, recommendations, and implementability. Methods Guidelines from four groups were reviewed with respect to two clinical scenarios: adjuvant ovarian function suppression (ofs) in premenopausal women with early-stage estrogen receptor–positive bca, and use of sentinel lymph node biopsy (slnb) after neoadjuvant chemotherapy (nac) for locally advanced bca. Guidelines from the American Society of Clinical Oncology (asco); Cancer Care Ontario’s Program in Evidence Based Care (cco’s pebc); the U.S. National Comprehensive Cancer Network (nccn); and the St. Gallen International Breast Cancer Consensus Conference were reviewed by two independent assessors. Guideline methodology and applicability were evaluated using the agree ii tool. Results The quality of the cpgs was greatest for the guidelines developed by asco and cco’s pebc. The nccn and St. Gallen guidelines were found to have lower scores for methodologic rigour. All guidelines scored poorly for applicability. The recommendations for ofs were similar in three guidelines. Recommendations by the various organizations for the use of slnb after nac were contradictory. Conclusions Our review demonstrated that cpgs can be heterogeneous in methodologic quality. Low-quality cpg implementation strategies contribute to low uptake of, and adherence to, bca cpgs. Further research examining the barriers to recommendations—such as intrinsic guideline characteristics and the needs of end users—is required. The use of bca cpgs can improve the knowledge-to-practice gap and patient outcomes.

In vivo prediction of CYP-mediated metabolic interaction potential of formononetin and biochanin A using in vitro human and rat CYP450 inhibition data.

PubMed

Arora, Sumit; Taneja, Isha; Challagundla, Muralikrishna; Raju, Kanumuri Siva Rama; Singh, Sheelendra Pratap; Wahajuddin, Muhammad

2015-11-19

Formononetin (FMN) and Biochanin A (BCA) are the principal isoflavones present in commercially available extracts of red clover that are widely been consumed for various health benefits. We investigated the in vitro effects of FMN and BCA on catalytic activity of human/rat cytochrome P450 enzymes to assess the drug interaction potential of red clover. IC50 and Ki values of FMN and BCA for CYPs were determined in human/rat liver microsomes. FMN and BCA showed concentration-dependent inhibition of CYP1A2 activity with IC50 values of 13.42 and 24.98μM in human liver microsomes and 38.57 and 11.86μM in rat liver microsomes, respectively. The mode of inhibition of human CYP1A2 by FMN was found to be competitive with apparent Ki value of 10.13±1.96μM. FMN also inhibited human CYP2D6. BCA exerted moderately inhibitory effects on human CYP2C9. The predicted in vivo inhibition for CYP1A2 was insignificant (R value <1.1) at hepatic level while at intestinal level, it was significant (R value >11). The inhibitory effects on other CYPs were found to be minimal. Red clover may be considered safe to be consumed along with co-prescribed medications; however, precaution must be taken while co-administering it with CYP1A2 substrates. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A comparison of basal and eye-flush tears for the analysis of cat tear proteins.

PubMed

Petznick, Andrea; Evans, Margaret D M; Madigan, Michele C; Markoulli, Maria; Garrett, Qian; Sweeney, Deborah F

2011-02-01

To identify a rapid and effective tear collection method providing sufficient tear volume and total protein content (TPC) for analysis of individual proteins in cats. Domestic adult short-haired cats (12-37 months; 2.7-6.6 kg) were used in the study. Basal tears without stimulation and eye-flush tears after instillation of saline (10 μl) were collected using microcapillary tubes from animal eyes either unwounded control or wounded with 9-mm central epithelial debridement giving four groups with n = 3. Tear comparisons were based on total time and rate for tear collection, TPC using micro bicinchoninic acid (BCA), tear immunoglobulin A (IgA), total matrix-metalloproteinase (MMP)-9 concentration using sandwich enzyme-linked immunosorbent assay (ELISA) and MMP-9 activity. Eye-flush tears were collected significantly faster than basal tears in wounded eyes with higher rates for tear collection in unwounded control and wounded eyes. TPC was significantly lower in eye-flush tears compared to basal tears. The relative proportion of tear IgA normalized to TPC (% IgA of TPC) was not significantly different between basal and eye-flush tears. In unwounded control eyes, MMP-9 was slightly higher in eye-flush than in basal tears; activity of MMP-9 in both tear types was similar. In wounded eyes, eye-flush tears showed highest MMP-9 levels and activity on Day 1, which subsequently decreased to Day 7. MMP-9 activity in basal tears from wounded eyes did not display changes in expression. Eye-flush tears can be collected rapidly providing sufficient tear volume and TPC. This study also indicates that eye-flush tears may be more suitable than basal tears for the analysis of MMPs following corneal wounding. © 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.

d-limonene ameliorates diabetes and its complications in streptozotocin-induced diabetic rats.

PubMed

Bacanlı, Merve; Anlar, Hatice Gül; Aydın, Sevtap; Çal, Tuğbagül; Arı, Nuray; Ündeğer Bucurgat, Ülkü; Başaran, A Ahmet; Başaran, Nurşen

2017-12-01

It is known that diabetes causes some complications including alterations in lipid profile, hepatic enzyme levels but also it causes oxidative stress. Limonene, a major component of Citrus oils, has important health beneficial effects in lowering the level of oxidative stress due to its antioxidant activity. The aim of this study was to investigate the effects of D-limonene on streptozotocin (STZ)-induced diabetes in Wistar albino rats. For this purpose, DNA damage was evaluated by alkaline comet assay. Changes in the activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GSHPx) and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), total glutathione (GSH), malondialdehyde (MDA), insulin, total bilirubin and BCA protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol and triglyceride were also evaluated. D-limonene treatment was found to significantly decrease DNA damage, GR enzyme activities and MDA levels and significantly increase GSH levels and CAT, SOD and GSH-Px enzyme activities and altered lipid and liver enzyme parameters in diabetic rats. According to our results, it seems that D-limonene might have a role in the prevention of the complication of diabetes in rats. Copyright © 2017 Elsevier Ltd. All rights reserved.

Grassland Bird Conservation Efforts in Missouri and Iowa: How Will We Measure Success?

Treesearch

Brad Jacobs; Rolf R. Koford; Frank R. Thompson III; Hope Woodward; Mike Hubbard; Jane A. Fitzgerald; James R. Herkert

2005-01-01

Missouri and Iowa have adopted the Bird Conservation Area (BCA) model of Partners in Flight and applied the BCA model to ten areas managed to benefit grassland birds. These ten BCAs have large core areas of continuous grassland centered on active lek sites of Greater Prairie-Chickens (Tympanuchus cupido). Management is focused on expanding the number...

Comparison of virulence factors and capsular types of Streptococcus agalactiae isolated from human and bovine infections.

PubMed

Emaneini, Mohammad; Khoramian, Babak; Jabalameli, Fereshteh; Abani, Samira; Dabiri, Hossein; Beigverdi, Reza

2016-02-01

Streptococcus agalactiae is a leading cause of human and bovine infections. A total of 194 S. agalactiae isolates, 55 isolates from bovines and 139 from humans, were analyzed for capsular types, virulence genes (scpB, hly, rib, bca and bac) and mobile genetic elements (IS1548 and GBSi1) using polymerase chain reaction (PCR) and multiplex PCR. Capsular type III was predominant (61%), followed by types V, II, Ib, and IV. The scpB, hly, bca and bac virulence genes were only found among human isolates. Twelve and 2 distinct virulence gene profiles were identified among human and bovine isolates respectively. The virulence gene profiles scpB- hly- IS1548- rib-bca (51%) and scpB- hly- IS1548- bca (19%) were only predominant among human isolates. The rib gene was the most common virulence gene in both human and bovine isolates. The study showed a high prevalence of virulence genes in S. agalactiae strains isolated from human infections, these result can support the idea that S. agalactiae isolated from humans and bovines are generally unrelated and probably belonged to separate populations. Copyright © 2015 Elsevier Ltd. All rights reserved.

76 FR 65769 - Airport Improvement Program: Modifications to Benefit Cost Analysis (BCA) Threshold

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-24

...This document announces the publication of the final policy changes to the Federal Aviation Administration's policy requiring a benefit cost analysis (BCA) for capacity projects funded by Airport Improvement Program (AIP) discretionary funds. On December 16, 2010, the FAA issued a Notice of Availability of Draft Guidance and Request for Comments with regard to the modification of its policy requiring benefit cost analyses (BCA) for capacity projects, which was published in the Federal Register. (78 FR 78798-02, December 16, 2010). The FAA now is (1) Issuing the final policy modifying the threshold at which BCAs are required from $5 million to $10 million in Airport Improvement Program (AIP) Discretionary funds, and (2) responding to comments requested in the Notice on December 16, 2010.

Cibicidodes Pachyderma B/Ca as a Shalow Water Carbonate Saturation State Proxy

NASA Astrophysics Data System (ADS)

Wojcieszek, D. E.; Flower, B. P.; Moyer, R. P.; Byrne, R. H.

2012-12-01

Since the industrial revolution, the oceans have absorbed about 25% of anthropogenic CO2 emissions to the atmosphere, leading to a decrease in seawater pH (termed ocean acidification: OA) as well as many associated effects, including decreased saturation states. Assessment of the effects of OA on marine ecosystems is presently based on <50 years of observations. Reconstructions of past seawater chemistry and its impact on biota over much longer time scales can provide essential context for likely future consequences of OA. Reliable oceanic paleo-proxies for influential chemical variables such as pH and carbonate saturation state are crucial components for examining ancient environments affected by OA. Addition of CO2 to seawater leads to not only decreases in seawater pH and saturation state, but also the extent to which boron (B) is incorporated into CaCO3 during biotic calcification. Consequently, the abundance of B in calcite could reflect pH and/or saturation state of the water in which calcification occurred. Recent studies indicate a linear relationship between the ratio of boron to calcium (B/Ca) in benthic foraminifera shells ( Cibicidoides wuellerstorfi, C. mundulus) and the degree of carbonate saturation (Δ[CO32-]), defined as a difference between [CO32-]in situ and [CO32-]saturation. However, the observed relationship between B/Ca and Δ[CO32-] was only established for depths >1000m. Thus, since OA most immediately affects the upper 1000 m of the water column, a reliable shallow water (<1000 m) carbonate chemistry proxy is desirable. We are testing the utility of B/Ca in Cibicidoides pachyderma as a shallow water Δ[CO32-] proxy. C. pachyderma is an epibenthic species and therefore records the composition of bottom, rather than interstitial, waters. It usually inhabits depths between 200 and 1000 m, and is a common species in the Gulf of Mexico. The gently sloping West Florida Shelf (WFS) is an excellent setting for this kind of study as it provides a full range of depths habitable by C.pachyderma. Nine surface sediment samples were collected along a 150-1400 m depth transect across the WFS. Bottom water pH along the transect ranged between 7.82 and 8.12, corresponding to Δ[CO32-] values between 35 and 200 μmol/kg. Preliminary B/Ca data range between 109 and 174 μmol/mol and display high variability. Our results suggest that WFS bottom water chemistry may be intermittently affected by phenomena such as eutrophication-induced hypoxia or boundary layer differences between the lower water column and the sediment-water interface. Additional B/Ca and bottom water measurements are planned to characterize the relationship between C.pachyderma B/Ca and Δ[CO32-], and extend global benthic foraminifera B/Ca calibrations to depths shallower than 1000 m.

Nanoparticle based bio-bar code technology for trace analysis of aflatoxin B1 in Chinese herbs.

PubMed

Yu, Yu-Yan; Chen, Yuan-Yuan; Gao, Xuan; Liu, Yuan-Yuan; Zhang, Hong-Yan; Wang, Tong-Ying

2018-04-01

A novel and sensitive assay for aflatoxin B1 (AFB1) detection has been developed by using bio-bar code assay (BCA). The method that relies on polyclonal antibodies encoded with DNA modified gold nanoparticle (NP) and monoclonal antibodies modified magnetic microparticle (MMP), and subsequent detection of amplified target in the form of bio-bar code using a fluorescent quantitative polymerase chain reaction (FQ-PCR) detection method. First, NP probes encoded with DNA that was unique to AFB1, MMP probes with monoclonal antibodies that bind AFB1 specifically were prepared. Then, the MMP-AFB1-NP sandwich compounds were acquired, dehybridization of the oligonucleotides on the nanoparticle surface allows the determination of the presence of AFB1 by identifying the oligonucleotide sequence released from the NP through FQ-PCR detection. The bio-bar code techniques system for detecting AFB1 was established, and the sensitivity limit was about 10 -8  ng/mL, comparable ELISA assays for detecting the same target, it showed that we can detect AFB1 at low attomolar levels with the bio-bar-code amplification approach. This is also the first demonstration of a bio-bar code type assay for the detection of AFB1 in Chinese herbs. Copyright © 2017. Published by Elsevier B.V.

δ11B as monitor of calcification site pH in divergent marine calcifying organisms

NASA Astrophysics Data System (ADS)

Sutton, Jill N.; Liu, Yi-Wei; Ries, Justin B.; Guillermic, Maxence; Ponzevera, Emmanuel; Eagle, Robert A.

2018-03-01

The boron isotope composition (δ11B) of marine biogenic carbonates has been predominantly studied as a proxy for monitoring past changes in seawater pH and carbonate chemistry. However, a number of assumptions regarding chemical kinetics and thermodynamic isotope exchange reactions are required to derive seawater pH from δ11B biogenic carbonates. It is also probable that δ11B of biogenic carbonate reflects seawater pH at the organism's site of calcification, which may or may not reflect seawater pH. Here, we report the development of methodology for measuring the δ11B of biogenic carbonate samples at the multi-collector inductively coupled mass spectrometry facility at Ifremer (Plouzané, France) and the evaluation of δ11BCaCO3 in a diverse range of marine calcifying organisms reared for 60 days in isothermal seawater (25 °C) equilibrated with an atmospheric pCO2 of ca. 409 µatm. Average δ11BCaCO3 composition for all species evaluated in this study range from 16.27 to 35.09 ‰, including, in decreasing order, coralline red alga Neogoniolithion sp. (35.89 ± 3.71 ‰), temperate coral Oculina arbuscula (24.12 ± 0.19 ‰), serpulid worm Hydroides crucigera (19.26 ± 0.16 ‰), tropical urchin Eucidaris tribuloides (18.71 ± 0.26 ‰), temperate urchin Arbacia punctulata (16.28 ± 0.86 ‰), and temperate oyster Crassostrea virginica (16.03 ‰). These results are discussed in the context of each species' proposed mechanism of biocalcification and other factors that could influence skeletal and shell δ11B, including calcifying site pH, the proposed direct incorporation of isotopically enriched boric acid (instead of borate) into biogenic calcium carbonate, and differences in shell/skeleton polymorph mineralogy. We conclude that the large inter-species variability in δ11BCaCO3 (ca. 20 ‰) and significant discrepancies between measured δ11BCaCO3 and δ11BCaCO3 expected from established relationships between abiogenic δ11BCaCO3 and seawater pH arise primarily from fundamental differences in calcifying site pH amongst the different species. These results highlight the potential utility of δ11B as a proxy of calcifying site pH for a wide range of calcifying taxa and underscore the importance of using species-specific seawater-pH-δ11BCaCO3 calibrations when reconstructing seawater pH from δ11B of biogenic carbonates.

EFFECT OF GESTATIONAL EXPOSURE TO ETHANE DIMETHANESULFONATE (EDS), BROMOCHLOROACETIC ACID (BCA) AND MOLINATE ON REPRODUCTIVE FUNCTION IN CD-1 MALE MICE

EPA Science Inventory

EFFECT OF GESTATIONAL EXPOSURE TO ETHANE DIMETHANESULFONATE (EDS), BROMOCHLOROACETIC ACID (BCA) AND MOLINATE ON REPRODUCTIVE FUNCTION IN CD-1 MALE MICE. D.K. Tarka1,2 , G.R. Klinefelter2, J.C. Rockett2, J.D. Suarez2, N.L. Roberts2 and J.M. Rogers1,2. 1 University of North Carol...

Regenerative Stem Cell Therapy for Breast Cancer Bone Metastasis

DTIC Science & Technology

2014-09-01

13. SUPPLEMENTARY NOTES 14. ABSTRACT Bone is the most common site of metastasis for human breast cancer (BCa), which results in significant...to all major bones as in human patients. 15. SUBJECT TERMS Bone metastasis; osteolysis; osteoprotegerin 16. SECURITY CLASSIFICATION OF: 17...metastasis for human breast cancer (BCa), which results in significant morbidity and mortality in patients with advanced disease. A vicious cycle

--No Title--

Science.gov Websites

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Non-toxic approach for treatment of breast cancer and its cutaneous metastasis: Capecitabine (Xeloda) enhanced photodynamic therapy in a murine tumor model

NASA Astrophysics Data System (ADS)

Anand, Sanjay; Denisyuk, Anton; Bullock, Taylor; Govande, Mukul; Maytin, Edward V.

2018-02-01

Breast cancer (BCA) is the most frequently diagnosed cancer in women, with distant metastases to lung, liver, bone and skin occurring in approximately 40% of cases. Radiation therapy (RT) has been successfully employed for the treatment of BCA; however, multiple rounds of RT are associated with undesirable cutaneous side effects. This study explores PDT as a therapeutic alternative, to be given alone or in combination with RT and chemotherapy. Earlier, we had developed differentiation-enhanced combination photodynamic therapy (cPDT) using a neoadjuvant (5-fluorouracil; 5FU) prior to PDT. The neoadjuvant increases the levels of PpIX, leading to better efficacy following aminolevulinate (ALA)- based PDT. Here, to avoid the toxicity of systemic 5FU, we used a nontoxic 5FU precursor (Capecitabine; CPBN) in a new cPDT regimen. CBPN, a standard chemotherapeutic for BCA, is metabolized to 5FU specifically within tumor tissue. Murine (4T1) BCA cells were injected into breast fat pads of nude mice. CPBN was administered by oral gavage followed by intraperitoneal ALA and red light for PDT. CPBN pretreatment of 4T1 tumors led to increased tumor cell differentiation (3.5 fold), homogenous elevation of intratumoral PpIX levels (4.5 fold), and enhanced tumor cell death post-PDT (5 fold), relative to vehicle control. Using an in vivo imaging system (IVIS), a decline in tumor growth following CPBN-PDT was observed. Results showing the effect of CPBN-PDT on distant metastases of BCA to lung, lymph nodes and skin will be presented. In summary, CPBN-PDT, a novel combination approach, has a significant potential for translation into the clinic.

Metastasis Risk Reduction Related with Beta-Blocker Treatment in Mexican Women with Breast Cancer.

PubMed

Parada-Huerta, E; Alvarez-Dominguez, Tp; Uribe-Escamilla, R; Rodriguez-Joya, Jf; Ponce-Medrano, Ja Diaz; Padron-Lucio, S; Alfaro-Rodriguez, A; Bandala, C

2016-01-01

Breast Cancer (BCa) is the most common malignant tumour in Mexican women. In BCa, several studies have linked β2-adrenergic receptor activation with increased tumour growth and progression as related with Epinephrine-NorEpinephrine (E-NE) stimulation. The aim of this study was to describe Beta-Blocker (BB) treatment related with reduction of the risk of metastasis in Mexican patients with BCa. We collected data of 120 patients seen at the High-Specialty Naval General Hospital in Mexico City (HOSGENAES), all of these with a histopathological diagnosis of BCa. Four groups of patients were divided as follows: without Systemic Arterial Hypertension (SAH); with SAH treatment with non-selective BB; with SAH treatment with selective BB, and with SAH treatment with other antihypertensive drugs. Chi-square, Mantel- Haenszel, Student t, and ANOVA tests were performed for data analysis. On average, patients were 54.8±11.8 years of age. Risk factors such as smoking and consuming alcohol exhibited a frequency of 33 and 36.5% respectively. Clinical stages III- IV were found in 50% of patients, while, 30% of patients had arterial hypertension (n=29 and N=96, respectively) and 17.5% used BB. One hundred percent of patients with arterial hypertension treated with BB for β1 - and β2 -adrenergic-receptors did not present metastasis globally, but patients treated with β1 BB presented 30% of metastasis while patients treated with no BB or without SAH had around 70% of metastasis. In Mexican patients with BCa and SAH treated with non-selective (β1- and β2-adrenergic receptors) BB, a decrease in the risk for metastasis was observed at the time of diagnosis.

Minimally toxic approach for treatment of cutaneous breast cancer metastases: capecitabine-enhanced photodynamic therapy

NASA Astrophysics Data System (ADS)

Anand, Sanjay; Bullock, Taylor; Maytin, Edward V.

2017-02-01

Cutaneous metastasis (CM) occurs in 20% of patients with breast carcinoma (BCA), and is extremely difficult to treat. These CM are relatively resistant to chemotherapy, generally responding only to ionizing radiation (IR). Multiple rounds of IR, however, lead to debilitating fibrosis and radiation dermatitis. An alternative to IR is needed for better management of BCA/CM. In our laboratory, we have developed differentiation-enhanced combination PDT (cPDT), a concept in which a pro-differentiating agent (methotrexate; vitamin D; or 5-fluorouracil, 5FU) is used as a neoadjuvant prior to PDT. After using these neoadjuvants, levels of protoporphyrin IX (PpIX) were elevated in animal tumor models of skin, prostate, and BCA, leading to better PDT efficacy. However, all the agents have toxicity issues. Here, we use a nontoxic 5FU precursor called Capecitabine (CPBN) for cPDT. CBPN is a standard chemotherapeutic for metastatic BCA, and is metabolized to 5FU specifically within tumor tissue. Murine (4T1) and human (MCF-7) BCA cell lines were injected into breast fat pads of nude mice. After tumor nodules appeared, CPBN (400-600 mg/kg/day) was administered by oral gavage for five days followed by intraperitoneal ALA administration on day 6. Mice were sacrificed and tumors harvested. CPBN pretreatment led to a 4-fold elevation of PpIX levels in tumors, relative to vehicle control. Not only did PpIX levels increase, but also PpIX distribution became more homogeneous after CPBN pretreatment. In summary, the use of non-toxic CPBN as a neoadjuvant prior to PDT is a combination approach with significant potential for translation into the clinic.

Modeling Security Bridge Certificate Authority Architecture

NASA Astrophysics Data System (ADS)

Ren, Yizhi; Li, Mingchu; Sakurai, Kouichi

Current Public Key Infrastructures suffer from a scaling problem, and some may have security problems, even given the topological simplification of bridge certification authorities. This paper analyzes the security problems in Bridge Certificate Authorities (BCA) model by using the concept of “impersonation risk, ” and proposes a new modified BCA model, which enhances its security, but is a bit more complex incertification path building and implementation than the existing one.

Carcinoma ex basal cell adenoma of the parotid gland: A report of an extremely rare case.

PubMed

Kusafuka, Kimihide; Kawasaki, Takuya; Nakajima, Takashi; Sugino, Takashi

2017-07-01

Malignant non-basaloid tumors that arise from basal cell adenoma (BCA) are extremely rare. The patient was a 72-year-old Japanese male, who had noticed swelling of the left parotid region 21 years ago. A superficial lobectomy was performed. About 60% of the tumor was made up of cribriform and trabecular tissue composed of basaloid cells, which exhibited mild atypia and nuclear expression of β-catenin. This portion of the tumor was considered to be a BCA. In the other part of the tumor, the proliferation of large eosinophilic atypical cells, most of which formed intraductal structures, was observed. These tumor cells displayed cellular atypia, and some of them formed Roman bridge structures or contributed to intracapsular invasion. Immunohistochemically, these cells were positive for cytokeratin 7, gross cystic disease fluid proten-15 (GCDFP-15), androgen receptor (AR), and mammaglobin (MMG) and exhibited a high Ki-67 labeling index. So, this portion of the tumor was considered to be a salivary duct carcinoma (SDC). The tumor's final diagnosis was SDC ex BCA (intracapsular type), which is extremely rare. GCDFP-15, AR, MMG, and Ki-67 are useful immunohistochemical markers for diagnosing SDC ex BCA. © 2017 The Authors. Pathology International Published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Si amorphization by focused ion beam milling: Point defect model with dynamic BCA simulation and experimental validation.

PubMed

Huang, J; Loeffler, M; Muehle, U; Moeller, W; Mulders, J J L; Kwakman, L F Tz; Van Dorp, W F; Zschech, E

2018-01-01

A Ga focused ion beam (FIB) is often used in transmission electron microscopy (TEM) analysis sample preparation. In case of a crystalline Si sample, an amorphous near-surface layer is formed by the FIB process. In order to optimize the FIB recipe by minimizing the amorphization, it is important to predict the amorphous layer thickness from simulation. Molecular Dynamics (MD) simulation has been used to describe the amorphization, however, it is limited by computational power for a realistic FIB process simulation. On the other hand, Binary Collision Approximation (BCA) simulation is able and has been used to simulate ion-solid interaction process at a realistic scale. In this study, a Point Defect Density approach is introduced to a dynamic BCA simulation, considering dynamic ion-solid interactions. We used this method to predict the c-Si amorphization caused by FIB milling on Si. To validate the method, dedicated TEM studies are performed. It shows that the amorphous layer thickness predicted by the numerical simulation is consistent with the experimental data. In summary, the thickness of the near-surface Si amorphization layer caused by FIB milling can be well predicted using the Point Defect Density approach within the dynamic BCA model. Copyright © 2017 Elsevier B.V. All rights reserved.

Shrinkage of spray-freeze-dried microparticles of pure protein for ballistic injection by manipulation of freeze-drying cycle.

PubMed

Straller, Georg; Lee, Geoffrey

2017-10-30

Spray-freeze-drying was used to produce shrivelled, partially-collapsed microparticles of pure proteins that may be suitable for use in a ballistic injector. Various modifications of the freeze drying cycle were examined for their effects on collapse of the pure protein microparticles. The use of annealing at a shelf temperature of up to +10°C resulted in no visible particle shrinkage. This was because of the high T g ' of the pure protein. Inclusion of trehalose or sucrose led to particle shrinkage because of the plasticizing effects of the disaccharides on the protein. Only by extending the duration of primary drying from 240 to 2745min at shelf temperatures in the range -12 to -8°C were shrivelled, wrinkled particles of bSA and bCA of reduced porosity obtained. Manipulation of the freeze-drying cycle used for SFD can therefore be used to modify particle morphology and increase particle density. Copyright © 2017 Elsevier B.V. All rights reserved.

Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

PubMed Central

Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2013-01-01

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

PubMed

Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2013-05-24

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

The Right to Terminate for Default

DTIC Science & Technology

1992-01-01

increment had been 85-90% completed; however, believing that the specification resulted in an inoperative holster strap on the vest, Switlik suspended...submit conforming goods four days after originally rejected for noncompliance). 1L3 ooden Pump , Ltd., ASBCA No. 27084, 83-2 BCA 1 16,895 at 84,067 (10...7Gooden Pump , Ltd., ASBCA No.27084, 83-2 BCA 1 16,895 (10 days reasonable time to allow contractor to cmplete performance of contract); Boston

Analysis prediction of Indonesian banks (BCA, BNI, MANDIRI) using adaptive neuro-fuzzy inference system (ANFIS) and investment strategies

NASA Astrophysics Data System (ADS)

Trianto, Andriantama Budi; Hadi, I. M.; Liong, The Houw; Purqon, Acep

2015-09-01

Indonesian economical development is growing well. It has effect for their invesment in Banks and the stock market. In this study, we perform prediction for the three blue chips of Indonesian bank i.e. BCA, BNI, and MANDIRI by using the method of Adaptive Neuro-Fuzzy Inference System (ANFIS) with Takagi-Sugeno rules and Generalized bell (Gbell) as the membership function. Our results show that ANFIS perform good prediction with RMSE for BCA of 27, BNI of 5.29, and MANDIRI of 13.41, respectively. Furthermore, we develop an active strategy to gain more benefit. We compare between passive strategy versus active strategy. Our results shows that for the passive strategy gains 13 million rupiah, while for the active strategy gains 47 million rupiah in one year. The active investment strategy significantly shows gaining multiple benefit than the passive one.

Dosimetric measurements of an n-butyl cyanoacrylate embolization material for arteriovenous malformations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Labby, Zacariah E., E-mail: zelabby@humonc.wisc.edu; Chaudhary, Neeraj; Gemmete, Joseph J.

2015-04-15

Purpose: The therapeutic regimen for cranial arteriovenous malformations often involves both stereotactic radiosurgery and endovascular embolization. Embolization agents may contain tantalum or other contrast agents to assist the neurointerventionalists, leading to concerns regarding the dosimetric effects of these agents. This study investigated dosimetric properties of n-butyl cyanoacrylate (n-BCA) plus lipiodol with and without tantalum powder. Methods: The embolization agents were provided cured from the manufacturer with and without added tantalum. Attenuation measurements were made for the samples and compared to the attenuation of a solid water substitute using a 6 MV photon beam. Effective linear attenuation coefficients (ELAC) were derivedmore » from attenuation measurements made using a portal imager and derived sample thickness maps projected in an identical geometry. Probable dosimetric errors for calculations in which the embolized regions are overridden with the properties of water were calculated using the ELAC values. Interface effects were investigated using a parallel plate ion chamber placed at set distances below fixed samples. Finally, Hounsfield units (HU) were measured using a stereotactic radiosurgery CT protocol, and more appropriate HU values were derived from the ELAC results and the CT scanner’s HU calibration curve. Results: The ELAC was 0.0516 ± 0.0063 cm{sup −1} and 0.0580 ± 0.0091 cm{sup −1} for n-BCA without and with tantalum, respectively, compared to 0.0487 ± 0.0009 cm{sup −1} for the water substitute. Dose calculations with the embolized region set to be water equivalent in the treatment planning system would result in errors of −0.29% and −0.93% per cm thickness of n-BCA without and with tantalum, respectively. Interface effects compared to water were small in magnitude and limited in distance for both embolization materials. CT values at 120 kVp were 2082 and 2358 HU for n-BCA without and with tantalum, respectively; dosimetrically appropriate HU values were estimated to be 79 and 199 HU, respectively. Conclusions: The dosimetric properties of the embolization agents are very close to those of water for a 6 MV beam. Therefore, treating the entire intracranial space as uniform in composition will result in less than 1% dosimetric error for n-BCA emboli smaller than 3.4 cm without added tantalum and n-BCA emboli smaller than 1.1 cm with added tantalum. Furthermore, when effective embolization can be achieved by the neurointerventionalist using n-BCA without tantalum, the dosimetric impact of overriding material properties will be lessened. However, due to the high attenuation of embolization agents with and without added tantalum for diagnostic energies, artifacts may occur that necessitate additional imaging to accurately identify the spatial extent of the region to be treated.« less

The Rab GTPase RabG3b Positively Regulates Autophagy and Immunity-Associated Hypersensitive Cell Death in Arabidopsis1[W

PubMed Central

Kwon, Soon Il; Cho, Hong Joo; Kim, Sung Ryul; Park, Ohkmae K.

2013-01-01

A central component of the plant defense response to pathogens is the hypersensitive response (HR), a form of programmed cell death (PCD). Rapid and localized induction of HR PCD ensures that pathogen invasion is prevented. Autophagy has been implicated in the regulation of HR cell death, but the functional relationship between autophagy and HR PCD and the regulation of these processes during the plant immune response remain controversial. Here, we show that a small GTP-binding protein, RabG3b, plays a positive role in autophagy and promotes HR cell death in response to avirulent bacterial pathogens in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing a constitutively active RabG3b (RabG3bCA) displayed accelerated, unrestricted HR PCD within 1 d of infection, in contrast to the autophagy-defective atg5-1 mutant, which gradually developed chlorotic cell death through uninfected sites over several days. Microscopic analyses showed the accumulation of autophagic structures during HR cell death in RabG3bCA cells. Our results suggest that RabG3b contributes to HR cell death via the activation of autophagy, which plays a positive role in plant immunity-triggered HR PCD. PMID:23404918

The Sovereign Acts Doctrine in the Law of Government Contracts

DTIC Science & Technology

1992-09-30

Franchi Construction Co., ASBCA No. 16735, 74-2 BCA para. 10,654 (1974) (local ban on burning, to assuage residents, not a public and general act...act. See, Franchi Construction Co., ASBCA No. 16735, 74-2 BCA para. 10,654 (1974). 37 sovereign capacity" will be afforded the protection available...qualify for protection under the sovereign acts doctrine. In Franchi Construction Co.,12 the Board was confronted with a local act that affected a

A new chromogenic medium for isolation of Bacteroides fragilis suitable for screening for strains with antimicrobial resistance.

PubMed

Tierney, Daniel; Copsey, Sarah D; Morris, Trefor; Perry, John D

2016-06-01

There have been an increasing number of reports describing the acquisition of antimicrobial resistance by Bacteroides fragilis including the occurrence of strains with resistance to multiple antimicrobials that are relied upon for treatment of infections. The aim of this study was to design a chromogenic selective medium for isolation of B. fragilis that could be adapted for specific isolation of antimicrobial-resistant strains. Bacteroides chromogenic agar (BCA) was the result of this endeavour and allowed growth of Bacteroides spp. as black colonies and the efficient inhibition of almost all other genera tested. The medium also allowed some differentiation of B. fragilis from other members of the B. fragilis group. When compared with an adaptation of Bacteroides bile-esculin agar (BBE) for the isolation of B. fragilis from 100 stool samples, 30 isolates of B. fragilis were recovered on BCA compared with 19 isolates recovered on BBE (P = 0.022). When supplemented with meropenem (4 μg/ml) or metronidazole (2 μg/ml), BCA could be used to select for the growth of B. fragilis isolates with resistance to these agents. We conclude that BCA is a useful research tool for surveillance studies to assess the prevalence of B. fragilis and, in particular, the occurrence of antimicrobial-resistant strains. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of polygenic risk scores for predicting breast and prostate cancer risk.

PubMed

Machiela, Mitchell J; Chen, Chia-Yen; Chen, Constance; Chanock, Stephen J; Hunter, David J; Kraft, Peter

2011-09-01

Recently, polygenic risk scores (PRS) have been shown to be associated with certain complex diseases. The approach has been based on the contribution of counting multiple alleles associated with disease across independent loci, without requiring compelling evidence that every locus had already achieved definitive genome-wide statistical significance. Whether PRS assist in the prediction of risk of common cancers is unknown. We built PRS from lists of genetic markers prioritized by their association with breast cancer (BCa) or prostate cancer (PCa) in a training data set and evaluated whether these scores could improve current genetic prediction of these specific cancers in independent test samples. We used genome-wide association data on 1,145 BCa cases and 1,142 controls from the Nurses' Health Study and 1,164 PCa cases and 1,113 controls from the Prostate Lung Colorectal and Ovarian Cancer Screening Trial. Ten-fold cross validation was used to build and evaluate PRS with 10 to 60,000 independent single nucleotide polymorphisms (SNPs). For both BCa and PCa, the models that included only published risk alleles maximized the cross-validation estimate of the area under the ROC curve (0.53 for breast and 0.57 for prostate). We found no significant evidence that PRS using common variants improved risk prediction for BCa and PCa over replicated SNP scores. © 2011 Wiley-Liss, Inc.

Online Educational Tool to Promote Bone Health in Cancer Survivors.

PubMed

des Bordes, Jude K A; Suarez-Almazor, Maria E; Volk, Robert J; Lu, Huifang; Edwards, Beatrice; Lopez-Olivo, Maria A

2017-10-01

Osteoporosis burden is significant in cancer survivors. Websites providing health information abound, but their development, quality, and source of information remain unclear. Our aim was to use a systematic and transparent approach to create an educational website on bone health, and to evaluate its potential to improve knowledge, self-management, and awareness in prostate cancer (PCa) and breast cancer (BCa) survivors. Guided by the Health Belief Model, we created a website using international standards and evaluated it in 10 PCa and 10 BCa survivors with self-administered questionnaire before, after, and 1 month after navigating the website. The mean scores on the knowledge questionnaire at baseline, postintervention and 1 month were, respectively, 5.1 (±2.0), 6.9 (±2.5), and 6.7 (±2.4), p < .008, in PCa and 3.4 (±2.7), 7.6 (±3.0), and 6.5 (±3.8), p  = .016, in BCa survivors. Acceptability ratings ranged from 60% to 100%. Participants found the website useful, helpful, and able to raise bone health awareness. Our website improved bone health knowledge in both PCa and BCa survivors. A systematic and transparent approach to the development of online educational websites could result in a tool capable of meeting the educational needs of targeted consumers. Cancer survivors could benefit from proven online educational tools.

The neonatal splice variant of Nav1.5 potentiates in vitro invasive behaviour of MDA-MB-231 human breast cancer cells

PubMed Central

Brackenbury, William J.; Chioni, Athina-Myrto; Diss, James K. J.; Djamgoz, Mustafa B. A.

2014-01-01

Upregulation of functional voltage-gated Na+ channels (VGSCs) occurs in metastatic human breast cancer (BCa) in vitro and in vivo. The present study aimed to ascertain the specific involvement of the ‘neonatal’ splice variant of Nav1.5 (nNav1.5), thought to be predominant, in the VGSC-dependent invasive behaviour of MDA-MB-231 cells. Functional activity of nNav1.5 was suppressed by two different methods targeting nNav1.5: (i) small interfering RNA (siRNA), and (ii) a polyclonal antibody (NESO-pAb); effects upon migration and invasion were determined. nNav1.5 mRNA, protein and signalling were measured using real-time PCR, Western blotting, and patch clamp recording, respectively. Treatment with the siRNA rapidly reduced (by ~90 %) the level of nNav1.5 (but not adult Nav1.5) mRNA, but the protein reduction was much smaller (~30 %), even after 13 days. Nevertheless, the siRNA reduced peak VGSC current density by 33 %, and significantly increased the cells’ sensitivity to nanomolar tetrodotoxin (TTX). Importantly, the siRNA suppressed in vitro migration by 43 %, and eliminated the normally inhibitory effect of TTX. Migrated MDA-MB-231 cells expressed more nNav1.5 protein at the plasma membrane than non-migrated cells. Furthermore, NESO-pAb reduced migration by up to 42 %, in a dose-dependent manner. NESO-pAb also reduced Matrigel invasion without affecting proliferation. TTX had no effect on cells already treated with NESO-pAb. It was concluded that nNav1.5 is primarily responsible for the VGSC-dependent enhancement of invasive behaviour in MDA-MB-231 cells. Accordingly, targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa. PMID:16838113

Evaluation of gas and carbon transport in a methanogenic bioelectrochemical system (BES).

PubMed

Dykstra, Christy M; Pavlostathis, Spyros G

2017-05-01

Bioelectrochemical systems (BESs) may be used to upgrade anaerobic digester biogas by directly converting CO 2 to CH 4 . The objective of this study was to evaluate gas (N 2 , CO 2 , CH 4 , and H 2 ) and carbon transport within a methanogenic BES. Four BES configurations were evaluated: abiotic anode with abiotic cathode (AAn-ACa), bioanode with abiotic cathode (BAn-ACa), abiotic anode with biocathode (AAn-BCa), and bioanode with biocathode (BAn-BCa). Transport of N 2 , a gas commonly used for flushing anoxic systems, out of the anode headspace ranged from 3.7 to 6.2 L/d-atm-m 2 , normalized to the proton exchange membrane (PEM) surface area and net driving pressure (NDP). CO 2 was transported from the cathode to the anode headspace at rates from 3.7 to 5.4 L/d-atm-m 2 . The flux of H 2 from cathode to anode headspace was 48% greater when the system had a biocathode (AAn-BCa) than when H 2 was produced at an abiotic cathode (BAn-ACa), even though the abiotic cathode headspace had 75% more H 2 than the AAn-BCa biocathode at the end of 1 day. A 7-day carbon balance of a batch-fed BAn-BCa BES showed transient microbial carbon storage and a net transport of carbon from anode to cathode. After a 7-day batch incubation, the CH 4 production in the biocathode was 27% greater on a molar basis than the initial CO 2 supplied to the biocathode headspace, indicating conversion of CO 2 produced in the anode. This research expands the current understanding of methanogenic BES operation, which may be used in improving the assessment of BES performance and/or in the development of alternative BES designs and mathematical models. Biotechnol. Bioeng. 2017;114: 961-969. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Functional social support, psychological capital, and depressive and anxiety symptoms among people living with HIV/AIDS employed full-time.

PubMed

Liu, Li; Pang, Ran; Sun, Wei; Wu, Ming; Qu, Peng; Lu, Chunming; Wang, Lie

2013-12-01

Psychological distress (e.g., depression and anxiety) has been regarded as the main cause of leaving work for people living with HIV/AIDS (PLWHA) in workplaces. This study aims to explore the associations of functional social support (FSS) and psychological capital (PC) with depressive and anxiety symptoms among PLWHA employed full-time. This cross-sectional study was performed in Liaoning, China, during the period of December 2010-April 2011. The Center for Epidemiologic Studies Depression Scale, the Zung Self-Rating Anxiety Scale, the Duke-UNC Functional Social Support Questionnaire, and the Psychological Capital Questionnaire were completed by PLWHA employed full-time. Structural equation modeling was used to test the proposed relationships between variables. Asymptotic and resampling strategies were performed to explore the mediating roles of PC and its components (self-efficacy, hope, optimism, resilience). Of 320 participants surveyed, 66.3% had depressive symptoms, and 45.6% had anxiety symptoms. Significant negative associations of FSS and PC with depressive and anxiety symptoms were revealed. PC (a*b = -0.209, BCa 95% CI: -0.293, -0.137, p < 0.05), hope (a*b = -0.103, BCa 95% CI: -0.192, -0.034, p < 0.05), and optimism (a*b = -0.047, BCa 95% CI: -0.106, -0.008, p < 0.05) significantly mediated the association between FSS and depressive symptoms. PC (a*b = -0.151, BCa 95% CI: -0.224, -0.095, p < 0.05) and self-efficacy (a*b = -0.080, BCa 95% CI: -0.158, -0.012, p < 0.05) significantly mediated the FSS-anxiety symptoms association. FSS and PC could help reduce depressive and anxiety symptoms among PLWHA employed full-time. PC fully mediates the associations of FSS with depressive and anxiety symptoms. In addition to enhancing FSS, PC development could be included in the prevention and treatment strategies for depressive and anxiety symptoms targeted at PLWHA employed full-time.

Characterization and in vitro biological evaluation of mineral/osteogenic growth peptide nanocomposites synthesized biomimetically on titanium

NASA Astrophysics Data System (ADS)

Chen, Cen; Kong, Xiangdong; Zhang, Sheng-Min; Lee, In-Seop

2015-04-01

Nanocomposite layers of mineral/osteogenic growth peptide (OGP) were synthesized on calcium phosphate coated titanium substrates by immersing in calcium-phosphate buffer solution containing OGP. Peptide incorporated mineral was characterized by determining quantity loaded, effects on mineral morphology and structure. Also, the biological activity was investigated by cell adhesion, proliferation assay, and measurement of alkaline phosphatase (ALP) activity. X-ray photoelectron spectroscopy (XPS) and micro-bicinchoninic acid (BCA) assay revealed that OGP was successfully incorporated with mineral and the amount was increased with immersion time. Incorporated OGP changed the mineral morphology from sharp plate-like shape to more rounded one, and the octacalcium phosphate structure of the mineral was gradually transformed into apatite. With confocal microscopy to examine the incorporation of fluorescently labeled peptide, OGP was evenly distributed throughout mineral layers. Mineral/OGP nanocomposites promoted cell adhesion and proliferation, and also increased ALP activity of mesenchymal stem cells (MSCs). Results presented here indicated that the mineral/OGP nanocomposites formed on titanium substrates had the potential for applications in dental implants.

A Methodological Approach for Conducting a Business Case Analysis (BCA) of Zephyr Joint Capability Technology Demonstration (JCTD)

DTIC Science & Technology

2008-12-01

Approved OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the...Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188) Washington DC 20503. 1 . AGENCY USE ONLY...on Investment (ROI) of the Zephyr system. This is achieved by ( 1 ) Developing a model to carry out Business Case Analysis (BCA) of JCTDs, including

[New methods of patient selection for improved anticholinergic therapy].

PubMed

Neuhaus, J; Schwalenberg, T; Schlichting, N; Schulze, M; Horn, L-C; Stolzenburg, J-U

2007-09-01

M3-specific inhibitors are currently preferred for anticholinergic therapy of OAB. However, not all of the patients profit from this regimen. This might reflect a heterogeneity of the patient group. The aim of this work is to define subgroups of patients with specific alterations of receptor expression and to profile the receptor expression individually. These receptor profiles might be used for the development of evidence-based "tailored" therapies. Detrusor probes from bladder carcinoma patients (BCa, n=9 F, n=7 male) and interstitial cystitis patients (IC, n=9 female) were examined using confocal immunofluorescence and PCR. M2, M3, P2X1-3, and H1-3 mRNAs were demonstrated in detrusor tissue. As revealed by immunofluorescence, the M2 receptor expression was significantly higher in female compared to male BCa tissues. In addition, the M2 receptor was further upregulated in IC vs BCa in female detrusor. IC patients showed specific alterations of their receptor profile. Individual receptor profiles might be used to optimize medicinal therapies.

Crystal structure of a β-aminopeptidase from an Australian Burkholderia sp.

PubMed

John-White, Marietta; Dumsday, Geoff J; Johanesen, Priscilla; Lyras, Dena; Drinkwater, Nyssa; McGowan, Sheena

2017-07-01

β-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal β-amino acids from synthetic β-peptides. β-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of β-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a β-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0 Å and showed a tetrameric assembly typical of the β-aminopeptidases. Each monomer consists of an α-subunit (residues 1-238) and a β-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known β-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.

BCA-kMC Hybrid Simulation for Hydrogen and Helium Implantation in Material under Plasma Irradiation

NASA Astrophysics Data System (ADS)

Kato, Shuichi; Ito, Atsushi; Sasao, Mamiko; Nakamura, Hiroaki; Wada, Motoi

2015-09-01

Ion implantation by plasma irradiation into materials achieves the very high concentration of impurity. The high concentration of impurity causes the deformation and the destruction of the material. This is the peculiar phenomena in the plasma-material interaction (PMI). The injection process of plasma particles are generally simulated by using the binary collision approximation (BCA) and the molecular dynamics (MD), while the diffusion of implanted atoms have been traditionally solved by the diffusion equation, in which the implanted atoms is replaced by the continuous concentration field. However, the diffusion equation has insufficient accuracy in the case of low concentration, and in the case of local high concentration such as the hydrogen blistering and the helium bubble. The above problem is overcome by kinetic Monte Carlo (kMC) which represents the diffusion of the implanted atoms as jumps on interstitial sites in a material. In this paper, we propose the new approach ``BCA-kMC hybrid simulation'' for the hydrogen and helium implantation under the plasma irradiation.

Nanoporous Aluminum Oxide Membranes Coated with Atomic Layer Deposition-Grown Titanium Dioxide for Biomedical Applications: An In Vitro Evaluation.

PubMed

Petrochenko, Peter E; Kumar, Girish; Fu, Wujun; Zhang, Qin; Zheng, Jiwen; Liang, Chengdu; Goering, Peter L; Narayan, Roger J

2015-12-01

The surface topographies of nanoporous anodic aluminum oxide (AAO) and titanium dioxide (TiO2) membranes have been shown to modulate cell response in orthopedic and skin wound repair applications. In this study, we: (1) demonstrate an improved atomic layer deposition (ALD) method for coating the porous structures of 20, 100, and 200 nm pore diameter AAO with nanometer-thick layers of TiO2 and (2) evaluate the effects of uncoated AAO and TiO2-coated AAO on cellular responses. The TiO2 coatings were deposited on the AAO membranes without compromising the openings of the nanoscale pores. The 20 nm TiO2-coated membranes showed the highest amount of initial protein adsorption via the micro bicinchoninic acid (micro-BCA) assay; all of the TiO2-coated membranes showed slightly higher protein adsorption than the uncoated control materials. Cell viability, proliferation, and inflammatory responses on the TiO2-coated AAO membranes showed no adverse outcomes. For all of the tested surfaces, normal increases in proliferation (DNA content) of L929 fibroblasts were observed over from 4 hours to 72 hours. No increases in TNF-alpha production were seen in RAW 264.7 macrophages grown on TiO2-coated AAO membranes compared to uncoated AAO membranes and tissue culture polystyrene (TCPS) surfaces. Both uncoated AAO membranes and TiO2-coated AAO membranes showed no significant effects on cell growth and inflammatory responses. The results suggest that TiO2-coated AAO may serve as a reasonable prototype material for the development of nanostructured wound repair devices and orthopedic implants.

The Effectiveness of Voriconazole in Therapy of Candida glabrata's Biofilms Oral Infections and Its Influence on the Matrix Composition and Gene Expression.

PubMed

Rodrigues, Célia F; Gonçalves, Bruna; Rodrigues, Maria Elisa; Silva, Sónia; Azeredo, Joana; Henriques, Mariana

2017-08-01

Candida glabrata is one of most prevalent yeast in fungal infections, especially in immunocompromised patients. Its azole resistance results in a low therapeutic response, particularly when associated with biofilms. The main goal of this work was to study the effectiveness of voriconazole (Vcz) against C. glabrata biofilms oral pathologies, as esophageal or oropharyngeal candidiasis. Antifungal susceptibilities were determined in pre-formed 24-h-biofilms and ERG genes expression was determined by qRT-PCR. Protein quantification was performed using BCA ® Kit, carbohydrate was estimated according to the Dubois assay and β-1,3 glucans concentration were determined using Glucatell ® kit. Finally, ergosterol, Vcz, and fluconazole (Flu) concentrations within the biofilm matrices were determined by RP-HPLC. Results showed that C. glabrata biofilms were more susceptible to Vcz than to Flu and that ERG genes expression evidenced an overexpression of the three ERG genes in the presence of both azoles. The matrix content presented a remarked decrease in proteins and an increase in carbohydrates, namely β-1,3 glucans. Ergosterol was successfully detected and quantified in the biofilm matrices, with no differences in all the considered conditions. Vcz demonstrated better diffusion through the biofilms and better cell penetration capacities, than Flu, indicating that the structure of the drug molecule fully influences its dissemination through the biofilm matrices. This work showed that Vcz is notably more effective than Flu for the treatment of resistant C. glabrata oral biofilms, which demonstrates a clinical relevance in its future use for the treatment of oropharyngeal/esophageal candidiasis caused by this species.

Promoter hypermethylation of HS3ST2, SEPTIN9 and SLIT2 combined with FGFR3 mutations as a sensitive/specific urinary assay for diagnosis and surveillance in patients with low or high-risk non-muscle-invasive bladder cancer.

PubMed

Roperch, Jean-Pierre; Grandchamp, Bernard; Desgrandchamps, François; Mongiat-Artus, Pierre; Ravery, Vincent; Ouzaid, Idir; Roupret, Morgan; Phe, Véronique; Ciofu, Calin; Tubach, Florence; Cussenot, Olivier; Incitti, Roberto

2016-09-01

Non-muscle-invasive bladder cancer (NMIBC) is a high incidence form of bladder cancer (BCa), where genetic and epigenetic alterations occur frequently. We assessed the performance of associating a FGFR3 mutation assay and a DNA methylation analysis to improve bladder cancer detection and to predict disease recurrence of NMIBC patients. We used allele specific PCR to determine the FGFR3 mutation status for R248C, S249C, G372C, and Y375C. We preselected 18 candidate genes reported in the literature as being hypermethylated in cancer and measured their methylation levels by quantitative multiplex-methylation specific PCR. We selected HS3ST2, SLIT2 and SEPTIN9 as the most discriminative between control and NMIBC patients and we assayed these markers on urine DNA from a diagnostic study consisting of 167 NMIBC and 105 controls and a follow-up study consisting of 158 NMIBC at diagnosis time's and 425 at follow-up time. ROC analysis was performed to evaluate the diagnostic accuracy of each assay alone and in combination. For Diagnosis: Using a logistic regression analysis with a model consisting of the 3 markers' methylation values, FGFR3 status, age and known smoker status at the diagnosis time we obtained sensitivity/specificity of 97.6 %/84.8 % and an optimism-corrected AUC of 0.96. With an estimated BCa prevalence of 12.1 % in a hematuria cohort, this corresponds to a negative predictive value (NPV) of 99.6 %. For Follow-up: Using a logistic regression with FGFR3 mutation and the CMI at two time points (beginning of the follow-up and current time point), we got sensitivity/specificity/NPV of 90.3 %/65.1 %/97.0 % and a corrected AUC of 0.84. We also tested a thresholding algorithm with FGFR3 mutation and the two time points as described above, obtaining sensitivity/specificity/NPV values of, respectively, 94.5 %/75.9 %/98.5 % and an AUC of 0.82. We showed that combined analysis of FGFR3 mutation and DNA methylation markers on urine can be a useful strategy in diagnosis, surveillance and for risk stratification of patients with NMIBC. These results provide the basis for a highly accurate noninvasive test for population screening and allowing to decrease the frequency of cystoscopy, an important feature for both patient quality of life improvement and care cost reduction.

The effects of some tumor markers on human erythrocyte (HCA-I and HCA-II), bovine erythrocyte (BCA) and bovine lung (CA-IV) carbonic anhydrase enzyme activities in vitro.

PubMed

Demir, N; Nadaroglu, H; Gungor, A A; Demir, Y

2015-01-01

The influence of prostatic acid phosphatase (PAP) and human chorionic gonadotropin (HCG), tumor markers have been investigated on human erythrocyte carbonic anhydrase (HCA-I and HCA-II) and bovine erythrocyte (BCA) and bovine lung carbonic anhydrase (CA-IV) in vitro. Tumor markers are substances that can often be detected in higher-than-normal amounts in the blood, urine, or body tissues of some patients with certain types of cancer. Tumor markers are produced either by the tumor itself or by the body in response to the presence of cancer or certain benign (noncancerous) conditions. In addition to their role in cancer diagnosis, some tumor marker levels are measured before treatment to help doctors plan appropriate therapy. All of the tumor markers were determined to have inhibition effect, on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes. The effect of each tumor marker on CA was investigated by Wilbur-Andersen method modified by Rickly et al Inhibition effects of two different tumor markers on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes were determined by using the CO2-Hydratase method by plotting activity % vs (tumor markers). I50 values of tumor markers exhibiting inhibition effects were found by means of these graphs (Tab.1, Fig. 2, Ref. 20).

Recovery of Indirect Costs in the Pricing of Equitable Adjustments and Terminations for Convenience

DTIC Science & Technology

1988-05-01

per unit of production decreases.2 0 5 For example, a contractor buys a machine for $10,000 that will be obsolete after one year. If the contractor...convenience may reduce the quantity of material needed for performance and the volume of purchases made by the contractor. As a result of not buying in...Ocean Technology, ASBCA 21363, 78-1 BCA 13204 at 64,586; Franchi Constr., ASBCA 16735, 74-2 BCA 10654 at 50,598; Warren Painting, ASBCA 18456, 74-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandapaka, A; Ghebremedhin, A; Patyal, B

Purpose: To validate the treatment planning system predicted proton dose beyond a heterogeneity (n-BCA glue) by making a measurement in a custom acrylic phantom. Methods: A custom cubic acrylic phantom was designed for this experiment. A container was designed to fit in the phantom. This container was filled with TRUFILL™ n-Butyl Cyanoacrylate(n-BCA) glue. When the container was placed in the phantom, its center was at a distance of 7.4cm from the entrance. This depth allows us to make measurements around the center of modulation of a 126 MeV proton beam with a 3cm spread-out-Bragg peak. To make measurements at othermore » beam energies, additional acrylic can be added in front of the phantom, to adjust the depth of the heterogeneity. A diamond detector was cross calibrated against a standard cylindrical ion chamber in a 126MeV beam. The diamond detector was then used to make dose measurements beyond the inhomogeneity. The measurement was repeated with the container filled with water. Several measurements were made at each setup, to check reproducibility of measurements. Results: For the same number of Tic3R1 counts, the dose measured with the diamond detector beyond n-BCA glue was 1.053 times the dose measured beyond the water filled container. This result is in agreement with the measured stopping power of glue (1.06). These measurements were in agreement with the dose predicted by the treatment planning system when the electron density of the heterogeneity was replaced with 1.06 before the dose calculation. Conclusion: Our initial measurements validate the dose predicted by our treatment plan in the presence of heterogeneity in a phantom. The material tested (n-BCA glue) is commonly used in the treatment of AVM’s prior to an SRS treatment. An error in dose predicted by the treatment plan in the presence of the glue can be detrimental in a single fraction high dose SRS treatment I received the n-BCA liquid embolic system samples from Codman and Shurtleff, Inc.« less

Streptomyces globosus UAE1, a Potential Effective Biocontrol Agent for Black Scorch Disease in Date Palm Plantations.

PubMed

Saeed, Esam E; Sham, Arjun; Salmin, Zeinab; Abdelmowla, Yasmeen; Iratni, Rabah; El-Tarabily, Khaled; AbuQamar, Synan

2017-01-01

Many fungal diseases affect date palm causing considerable losses in date production worldwide. We found that the fungicide Cidely ® Top inhibited the mycelial growth of the soil-borne pathogenic fungus Thielaviopsis punctulata , the causal agent of black scorch disease of date palm, both in vitro and in vivo . Because the use of biocontrol agents (BCAs) can minimize the impact of pathogen control on economic and environmental concerns related to chemical control, we aimed at testing local actinomycete strains isolated from the rhizosphere soil of healthy date palm cultivated in the United Arab Emirates (UAE) against T. punctulata . The selected isolate can thus be used as a potential agent for integrated disease management programs. In general, the BCA showed antagonism in vitro and in greenhouse experiments against this pathogen. The most promising actinomycete isolate screened showed the highest efficacy against the black scorch disease when applied before or at the same time of inoculation with T. punctulata , compared with BCA or fungicide application after inoculation. The nucleotide sequence and phylogenetic analyses using the 16 S ribosomal RNA gene with other Streptomyces spp. in addition to morphological and cultural characteristics revealed that the isolated UAE strain belongs to Streptomyces globosus UAE1. The antagonistic activity of S. globosus against T. punctulata , was associated with the production by this strain of diffusible antifungal metabolites i.e., metabolites that can inhibit mycelial growth of the pathogen. This was evident in the responses of the vegetative growth of pure cultures of the pathogen when exposed to the culture filtrates of the BCA. Altogether, the pathogenicity tests, disease severity indices and mode of action tests confirmed that the BCA was not only capable of suppressing black scorch disease symptoms, but also could prevent the spread of the pathogen, as a potential practical method to improve disease management in the palm plantations. This is the first report of an actinomycete, naturally occurring in the UAE with the potential for use as a BCA in the management of the black scorch disease of date palms in the region.

Reduction in Surface Ocean Carbon Storage across the Middle Miocene

NASA Astrophysics Data System (ADS)

Babila, T. L.; Sosdian, S. M.; Foster, G. L.; Lear, C. H.

2017-12-01

During the Middle Miocene, Earth underwent a profound climate shift from the warmth of the Miocene Climatic Optimum (MCO; 14-17 Ma) to the stable icehouse of today during the Middle Miocene Climate transition (MMCT). Elevated atmospheric carbon dioxide concentrations (pCO2) revealed by boron isotope records (δ11B) link massive volcanic outputs of Columbia River Flood Basalts to the general warmth of MCO. Superimposed on the long-term cooling trend (MMCT) is a gradual pCO2 decline and numerous positive carbon isotope (δ13C) excursions that indicate dynamic variations in the global carbon cycle. Enhanced organic carbon burial via marine productivity, increased silicate weathering and volcanic emission cessation are each invoked to explain the drawdown of pCO2. To better constrain the oceanic role in carbon sequestration over the Middle Miocene detailed records of carbonate chemistry are needed. We present high resolution Boron/Calcium (B/Ca) and δ13C records in planktonic foraminifer T.trilobus spanning 12-17 Ma at ODP 761 (tropical eastern Indian Ocean) to document changes in surface ocean carbonate chemistry. An overall 30% increase in B/Ca ratios is expressed as two stepwise phases occurring at 14.7 and 13 Ma. Cyclic B/Ca variations are coherent with complimentary δ13C records suggesting a tight coupling between ocean carbonate chemistry parameters. Lower resolution B/Ca data at DSDP 588 (Pacific) and ODP 926 (Atlantic) corroborate the trends observed at ODP 761. We employ a paired approach that combines B/Ca (this study) to δ11B (Foster et al., 2012) and an ad hoc calibration to estimate changes in surface ocean dissolved inorganic carbon (DIC). We estimate a substantial decrease in surface ocean DIC spanning the Middle Miocene that culminates with modern day like values. This gradual decline in surface ocean DIC is coeval with existing deep-ocean records which together suggests a whole ocean reduction in carbon storage. We speculate that enhanced weathering rather than short term processes are responsible for mediating global carbon reservoirs over the Middle Miocene.

Benefit-cost analysis of lane departure warning and roll stability control in commercial vehicles.

PubMed

Medina-Flintsch, Alejandra; Hickman, Jeffrey S; Guo, Feng; Camden, Matthew C; Hanowski, Richard J; Kwan, Quon

2017-09-01

This paper presents the cost benefits of two different onboard safety systems (OSS) installed on trucks as they operated during normal revenue deliveries. Using a formal economic analysis approach, the study quantified the costs and benefits associated with lane departure warning (LDW) systems and roll stability control (RSC) systems. The study used data collected from participating carriers (many of these crashes were not reported to state or Federal agencies), and the research team also reviewed each crash file to determine if the specific OSS would have mitigated or prevented the crash. The deployment of each OSS was anticipated to increase the safety of all road users, but impact different sectors of society in different ways. Benefits that were inherent in each group (e.g., industry, society) were considered, and different benefit-cost analyses (BCAs) were performed. This paper presents two BCAs: a BCA focused on the costs and benefits in the carrier industry by implementing each OSS, and a BCA that measured the societal benefits of each OSS. In addition, a BCA for a theoretical mandatory deployment option for each OSS is presented. BCA results for LDW and RSC clearly showed their benefits outweighed their costs for the carrier and society. Practical applications: Cost information is a crucial factor in purchasing decisions in carriers; similarly, regulators must consider the cost burden prior to mandating technologies. The results in this study provide carrier decision makers and regulators with information necessary to make an informed decision regarding RSC and LDW. Copyright © 2017 National Safety Council and Elsevier Ltd. All rights reserved.

Websites on Bladder Cancer: an Appropriate Source of Patient Information?

PubMed

Salem, Johannes; Paffenholz, Pia; Bolenz, Christian; von Brandenstein, Melanie; Cebulla, Angelika; Haferkamp, Axel; Kuru, Timur; Lee, Cheryl T; Pfister, David; Tsaur, Igor; Borgmann, Hendrik; Heidenreich, Axel

2018-01-08

A growing number of patients search for health information online. An early investigation of websites about bladder cancer (BCa) revealed mostly incomplete and particularly inaccurate information. We analyzed the quality, readability, and popularity of the most frequented websites on BCa. An Internet search on www.google.com was performed for the term "bladder cancer." After selecting the most frequented websites for patient information, HONcode quality certification, Alexa popularity rank, and readability scores (according to US grade levels) were investigated. A 36-point checklist was used to assess the content according to the EAU guidelines on BCa, which was categorized into seven topics. The popularity of the 49 websites analyzed was average, with a median Alexa popularity rank of 41,698 (interquartile range [IQR] 7-4,671,246). The readability was rated difficult with 11 years of school education needed to understand the information. Thirteen (27%) websites were HONcode certified. Out of 343 topics (seven EAU guideline topics each on 49 websites), 79% were mentioned on the websites. Of these, 10% contained incorrect information, mostly outdated or biased, and 34% contained incomplete information. Publically provided websites mentioned more topics per website (median [IQR] 7 [5.5-7] vs. 5.5 [3.3-7]; p = 0.022) and showed less incorrect information (median [IQR] 0 [0-1] vs. 1 [0-1]; p = 0.039) than physician-provided websites. Our study revealed mostly correct but partially incomplete information on BCa websites for patients. Physicians and public organizations should strive to keep their website information up-to-date and unbiased to optimize patients' health literacy.

YouTube™ as a Source of Instructional Videos on Bowel Preparation: a Content Analysis.

PubMed

Ajumobi, Adewale B; Malakouti, Mazyar; Bullen, Alexander; Ahaneku, Hycienth; Lunsford, Tisha N

2016-12-01

Instructional videos on bowel preparation have been shown to improve bowel preparation scores during colonoscopy. YouTube™ is one of the most frequently visited website on the internet and contains videos on bowel preparation. In an era where patients are increasingly turning to social media for guidance on their health, the content of these videos merits further investigation. We assessed the content of bowel preparation videos available on YouTube™ to determine the proportion of YouTube™ videos on bowel preparation that are high-content videos and the characteristics of these videos. YouTube™ videos were assessed for the following content: (1) definition of bowel preparation, (2) importance of bowel preparation, (3) instructions on home medications, (4) name of bowel cleansing agent (BCA), (5) instructions on when to start taking BCA, (6) instructions on volume and frequency of BCA intake, (7) diet instructions, (8) instructions on fluid intake, (9) adverse events associated with BCA, and (10) rectal effluent. Each content parameter was given 1 point for a total of 10 points. Videos with ≥5 points were considered by our group to be high-content videos. Videos with ≤4 points were considered low-content videos. Forty-nine (59 %) videos were low-content videos while 34 (41 %) were high-content videos. There was no association between number of views, number of comments, thumbs up, thumbs down or engagement score, and videos deemed high-content. Multiple regression analysis revealed bowel preparation videos on YouTube™ with length >4 minutes and non-patient authorship to be associated with high-content videos.

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yongbaek; Thai-Vu Ton; De Angelo, Anthony B.

2006-07-15

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCRmore » on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/{beta}-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.« less

A 20 million year record of planktic foraminiferal B/Ca ratios: Systematics and uncertainties in pCO 2 reconstructions

NASA Astrophysics Data System (ADS)

Tripati, Aradhna K.; Roberts, Christopher D.; Eagle, Robert A.; Li, Gaojun

2011-05-01

We use new and published data representing a 20 million long record to discuss the systematics of interpreting planktic foraminiferal B/Ca ratios. B/Ca-based reconstructions of seawater carbonate chemistry and atmospheric pCO 2 assume that the incorporation of boron into foraminiferal tests can be empirically described by an apparent partition coefficient, KD={B/Ca}/{B(OH4-/HCO)} ( Hemming and Hanson, 1992). It has also been proposed that there is a species-specific relationship between K D and temperature ( Yu et al., 2007). As we discuss, although these relationships may be robust, there remain significant uncertainties over the controls on boron incorporation into foraminifera. It is difficult to be certain that the empirically defined correlation between temperature and K D is not simply a result of covariance of temperature and other hydrographic variables in the ocean, including carbonate system parameters. There is also some evidence that K D may be affected by solution [HCO3-]/[CO32-] ratios (i.e., pH), or by [CO32-]. In addition, the theoretical basis for the definition of K D and for a temperature control on K D is of debate. We also discuss the sensitivity of pCO 2 reconstructions to different K D-temperature calibrations and seawater B/Ca. If a K D-temperature calibration is estimated using ice core pCO 2 values between 0 and 200 ka, B/Ca ratios can be used to reasonably approximate atmospheric pCO 2 between 200 and 800 ka; however, the absolute values of pCO 2 calculated are sensitive to the choice of K D-temperature relationship. For older time periods, the absolute values of pCO 2 are also dependent on the evolution of seawater B concentrations. However, we find that over the last 20 Ma, reconstructed changes in declining pCO 2 across the Mid-Pleistocene Transition, Pliocene glacial intensification, and the Middle Miocene Climate Transition are supported by the B/Ca record even if a constant coretop K D is used, or different K D-temperature calibrations and models of seawater B evolution are applied to the data. The inferred influence of temperature on K D from coretop data therefore cannot itself explain the structure of a published pCO 2 reconstruction ( Tripati et al., 2009). We conclude the raw B/Ca data supports a coupling between pCO 2 and climate over the past 20 Ma. Finally, we explore possible implications of B/Ca-based pCO 2 estimates for the interpretation of other marine pCO 2 proxies.

Tripeptide GGH as the Inhibitor of Copper-Amyloid-β-Mediated Redox Reaction and Toxicity.

PubMed

Hu, Xiaoyu; Zhang, Qian; Wang, Wei; Yuan, Zhi; Zhu, Xushan; Chen, Bing; Chen, Xingyu

2016-09-21

The Aβ complexes of some redox-active species, such as Cu, cause oxidative stress and induce severe toxicity by generating reactive oxygen species (ROS). Thus, Cu chelation therapy should be considered as a valuable strategy for the treatment of Alzheimer's disease (AD). However, more attention should be paid to the specific chelating ability of these chelating agents. Herein, a tripeptide GGH was used to selectively chelate the Cu(2+) in Aβ-Cu complex in the presence of other metal ions (e.g., K(+), Ca(2+), Ni(2+), Mg(2+), and Zn(2+)) as shown by isothermal titration calorimetry results. GGH decreased the level of HO(•) radicals by preventing the formation of intermediate Cu(I) ion. Thus, the Cu species completely lost its catalytic activity at a superequimolar GGH/Cu(II) ratio (4:1) as observed by UV-visible spectroscopy, coumarin-3-carboxylic acid fluorescence, and BCA assay. Moreover, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay indicates that GGH increased PC-12 cell viability from 36% to 63%, and neurotoxicity partly triggered by ROS decreased. These results indicate potential development of peptide chelation therapy for AD treatment.

Antiquorum sensing, antibiofilm formation and cytotoxicity activity of commonly used medicinal plants by inhabitants of Borabu sub-county, Nyamira County, Kenya

PubMed Central

Omwenga, Eric Omori; Hensel, Andreas; Pereira, Susana; Shitandi, Alfred Anakalo

2017-01-01

Productions of various bacterial traits like production of virulence factors (e.g. toxins, enzymes), biofilm formation, luminescence among others, have been known to be controlled by quorum sensing (QS), a process that is dependent on chemical signals or autoinducers (AIs). Bacteria known to rely on such AIs are known to be virulent and tend to be resistant against various antimicrobial agents. Therefore, strategies aimed at the inhibition of QS pathways, are regarded as potential novel therapies in managing bacterial virulence hence reducing their ability to induce infections in humans. In the present study, a portfolio of 25 medicinal plant extracts (ethanol 50% v/v) used in southwestern Kenya were assayed against a transformed E. coli Top 10 reporter QS strain. This biosensor responds to the exogenous addition of 3-oxo-N-hexanoyl homoserine lactone (3OC6HSL) expressing green fluorescent protein (GFP). The large majority of the screened medicinal plants seemed to exhibit toxic effects and almost none of them induced antiquorum sensing (AQS) activity. This could be the consequence of the presence of mixed compounds in the extracts. Elaeodendron buchananii Loes and Acacia gerrardii Benth extracts that seemed to show AQS activity were further proved found to possess mild AQS but with defined antimicrobial activities, and no antibiofilm formation inhibition. As a control, an E. coli pBCA9145_jtk2828::sfGFP strain that produces constitutively GFP was used and confirmed that none of the two extracts quenched the fluorescence of sfGFP. Cytotoxicity assays with mammalian MDCK cells also did indicate that the selected extracts with putative AQS activity, also reduced the cell viability. Therefore, further studies will be needed to separate and re-test the individual compounds especially from the selected two promising plants. PMID:29091715

A Lanthanide MOF Thin-Film Fixed with Co3 O4 Nano-Anchors as a Highly Efficient Luminescent Sensor for Nitrofuran Antibiotics.

PubMed

Zhang, Feng; Yao, Hua; Chu, Tianshu; Zhang, Gaowei; Wang, Yi; Yang, Yangyi

2017-08-01

Nitrofurans are a group of widely used veterinary antibiotics, which have been banned due to antibiotics pollution. Development of a rapid and effective method for the detection of nitrofuran antibiotics (NFAs) is an important challenge. Herein, we designed a chemical sensor based on a thin-film composed of the lanthanide metal-organic framework (Ln-MOF) {[Eu 2 (BCA) 3 (H 2 O)(DMF) 3 ]⋅0.5DMF⋅H 2 O} n (Eu-BCA, in which BCA is 2,2'-biquinoline-4,4'-dicarboxylate) coated on a cost-effective stainless steel wire mesh (SSWM) by Co 3 O 4 nano-anchor fixation method. The MOF coatings were well adhered to the SSWM, resulting in a three-dimensional porous, flexible, and processable sensor. The structure of the as-prepared MOF thin-film was confirmed by powder X-ray diffraction (PXRD), and the surface morphology was examined by scanning electron microscopy (SEM). Significantly, the Eu-BCA thin-film was highly selective and sensitive to NFAs, and yet remained unaffected by other common antibiotics that may be present. The limits of detection for nitrofurantoin (NFT) and nitrofurazone (NFZ) are 0.21 and 0.16 μm, respectively. NFAs were also successfully detected in water from the Pearl River in Guangzhou, and from bovine serum samples. Hence, the reported Ln-MOF thin-film is a promising sensor for the detection of NFAs, thereby helping to protect human beings from all manner of hazards that arise from the abuse of antibiotics in livestock breeding. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Gadolinium compounds signaling through TLR4 and TLR7 in normal human macrophages: establishment of a proinflammatory phenotype and implications for the pathogenesis of nephrogenic systemic fibrosis.

PubMed

Wermuth, Peter J; Jimenez, Sergio A

2012-07-01

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity pathways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-κB, and increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-κB-inducible reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-1R-associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression. Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-6, TGF-β, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis.

Shoulder impairment before breast cancer surgery.

PubMed

Flores, Ann Marie; Dwyer, Kathleen

2014-09-01

To compare pre- and post-operative shoulder active range of motion (AROM) values from female breast cancer survivors to population norm values for shoulder AROM; and to compare shoulder AROM differences pre- and post-surgery between female African American and White breast cancer survivors (BCA). This pilot study used a convenience sample and longitudinal design measuring participants 2 times (T0 = baseline, after biopsy but within 2 weeks before BCA surgery; T1 = 2 nd postoperative week). The U.S. has the largest BCA survivor population in history and yet the mortality burden remains highest among AA BCA survivors. AAs may also have greater burden of physical and functional side effects compared to whites and the general population. The data were collected from a convenience sample (n = 33; n AA = 9, n W = 24) and included data on shoulder AROM, medical chart review for pre- and co-morbid conditions, and self-reported demographics and medical history. We used t-tests to compare sample AROM means to population norms. We then compared our sample across 2 timepoints (T0 = pre-surgery; T1 = 2 weeks post-surgery) using independent samples t-tests and repeated measures analysis of variance (p < .05) to compare AA to White sub-samples AROM means. African Americans had significantly less shoulder abduction (at T0) and flexion (at T1) than whites. However, 100% had significantly reduced AROM for all movements at T0 (prior to surgery but after biopsy) when compared to population norms. The significant reduction in shoulder AROM after biopsy but before surgery points to a possible unmet need for early physical therapy intervention. Further research using randomized controlled trial design is recommended.

Disparities in the Use of Radiation Therapy in Patients With Local-Regionally Advanced Breast Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez, Steve R., E-mail: steve.martinez@ucdmc.ucdavis.ed; Beal, Shannon H.; Chen, Steven L.

2010-11-01

Background: Radiation therapy (RT) is indicated for the treatment of local-regionally advanced breast cancer (BCa). Hypothesis: We hypothesized that black and Hispanic patients with local-regionally advanced BCa would receive lower rates of RT than their white counterparts. Methods: The Surveillance Epidemiology and End Results database was used to identify white, black, Hispanic, and Asian patients with invasive BCa and {>=}10 metastatic lymph nodes diagnosed between 1988 and 2005. Univariate and multivariate logistic regression evaluated the relationship of race/ethnicity with use of RT. Multivariate models stratified for those undergoing mastectomy or lumpectomy. Results: Entry criteria were met by 12,653 patients. Approximatelymore » half of the patients did not receive RT. Most patients were white (72%); the remainder were Hispanic (10.4%), black (10.3%), and Asian (7.3%). On univariate analysis, Hispanics (odd ratio [OR] 0.89; 95% confidence interval [CI], 0.79-1.00) and blacks (OR 0.79; 95% CI, 0.70-0.89) were less likely to receive RT than whites. On multivariate analysis, blacks (OR 0.76; 95% CI, 0.67-0.86) and Hispanics (OR 0.80; 95% CI, 0.70-0.90) were less likely than whites to receive RT. Disparities persisted for blacks (OR 0.74; 95% CI, 0.64-0.85) and Hispanics (OR 0.77; 95% CI, 0.67-0.89) who received mastectomy, but not for those who received lumpectomy. Conclusions: Many patients with local-regionally advanced BCa do not receive RT. Blacks and Hispanics were less likely than whites to receive RT. This disparity was noted predominately in patients who received mastectomy. Future efforts at improving rates of RT are warranted. Efforts at eliminating racial/ethnic disparities should focus on black and Hispanic candidates for postmastectomy RT.« less

Endogenous Hydrogen Sulfide Enhances Cell Proliferation of Human Gastric Cancer AGS Cells.

PubMed

Sekiguchi, Fumiko; Sekimoto, Teruki; Ogura, Ayaka; Kawabata, Atsufumi

2016-01-01

Hydrogen sulfide (H2S), the third gasotransmitter, is endogenously generated by certain H2S synthesizing enzymes, including cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) from L-cysteine in the mammalian body. Several studies have shown that endogenous and exogenous H2S affects the proliferation of cancer cells, although the effects of H2S appear to vary with cell type, being either promotive or suppressive. In the present study, we determined whether endogenously formed H2S regulates proliferation in human gastric cancer AGS cells. CSE, but not CBS, was expressed in AGS cells. CSE inhibitors, DL-propargylglycine (PPG) and β-cyano-L-alanine (BCA), significantly suppressed the proliferation of AGS cells in a concentration-dependent manner. CSE inhibitors did not increase lactate dehydrogenase (LDH) release in the same concentration range. The inhibitory effects of PPG and BCA on cell proliferation were reversed by repetitive application of NaHS, a donor of H2S. Interestingly, nuclear condensation and fragmentation were detected in AGS cells treated with PPG or BCA. These results suggest that endogenous H2S produced by CSE may contribute to the proliferation of gastric cancer AGS cells, most probably through anti-apoptotic actions.

[Peculiarities of the course of arterial hypertension in patients with atherosclerotic stenoses of brachiocephalic arteries before and after surgery on carotid arteries].

PubMed

Kosacheva, N B; Tuev, A V

2013-01-01

Study aim was to investigate relationship between atherosclerosis of carotid arteries (CA) and the presence of arterial hypertension (AH) and possibilities of correction of AH after surgery. We examined 70 patients with episodes of elevation of arterial pressure (AP) scheduled for surgical interventions on brachiocephalic arteries (BCA) because of CA stenoses (n=50, group 1) or anomalies of BCA development (n=20, group 2). Before operation patients of group 1 had higher mean AP than patients of group 2 (103.6+/-11.3 and 91.7+/-6.6 mm Hg, respectively, p=0.00007). This difference disappeared after surgery. In postoperative period in patients with CA stenoses levels of both systolic and diastolic AP decreased (from 145.1+/-14.7 to 135.6+/-12.3, p=0.02; and from 83.3+/-10.2 78.1+/-9.7 mm Hg, =0.02, respectively). In group 2 no significant AP reduction was observed. Thus AP elevation in patients with involvement of BCA to a considerable degree is caused by CA stenosis. Surgical treatment of these patients facilitates lowering of AP.

The mediational role of helplessness in psychological outcomes in systemic lupus erythematosus.

PubMed

Mills, S D; Azizoddin, D; Gholizadeh, S; Racaza, G Z; Nicassio, P M

2018-06-01

Objective Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can result in disability and psychological distress. Although pain has been associated with depressive symptomatology and stress in SLE, a paucity of theoretical models have been used to explain the relationship between pain and psychological distress in this population. Thus, the present study examined helplessness as a mediator of the relationship between pain and psychological distress among patients with SLE. Methods Multiple mediation analysis was used to examine the hypothesis that learned helplessness mediates the relationship between pain and symptoms of anxiety, depression, and stress in a sample of patients with SLE ( N = 136) receiving medical care at Cedars Sinai Medical Center. Results The mean score on the Helplessness subscale was 14.5 ( SD = 5.4). Helplessness fully mediated the relationship between pain vitality and symptoms of anxiety (BCa 95% CI (-0.073, -0.015)), depression (BCa 95% CI (-0.502, -0.212)), and stress (BCa 95% CI (-0.063, -0.027)). Conclusion Participants reported a high level of perceived inability to control one's disease. Helplessness fully mediated the relationship between pain and measures of anxiety, depression, and perceived stress among patients with SLE.

Detection of gear cracks in a complex gearbox of wind turbines using supervised bounded component analysis of vibration signals collected from multi-channel sensors

NASA Astrophysics Data System (ADS)

Li, Zhixiong; Yan, Xinping; Wang, Xuping; Peng, Zhongxiao

2016-06-01

In the complex gear transmission systems, in wind turbines a crack is one of the most common failure modes and can be fatal to the wind turbine power systems. A single sensor may suffer with issues relating to its installation position and direction, resulting in the collection of weak dynamic responses of the cracked gear. A multi-channel sensor system is hence applied in the signal acquisition and the blind source separation (BSS) technologies are employed to optimally process the information collected from multiple sensors. However, literature review finds that most of the BSS based fault detectors did not address the dependence/correlation between different moving components in the gear systems; particularly, the popular used independent component analysis (ICA) assumes mutual independence of different vibration sources. The fault detection performance may be significantly influenced by the dependence/correlation between vibration sources. In order to address this issue, this paper presents a new method based on the supervised order tracking bounded component analysis (SOTBCA) for gear crack detection in wind turbines. The bounded component analysis (BCA) is a state of art technology for dependent source separation and is applied limitedly to communication signals. To make it applicable for vibration analysis, in this work, the order tracking has been appropriately incorporated into the BCA framework to eliminate the noise and disturbance signal components. Then an autoregressive (AR) model built with prior knowledge about the crack fault is employed to supervise the reconstruction of the crack vibration source signature. The SOTBCA only outputs one source signal that has the closest distance with the AR model. Owing to the dependence tolerance ability of the BCA framework, interfering vibration sources that are dependent/correlated with the crack vibration source could be recognized by the SOTBCA, and hence, only useful fault information could be preserved in the reconstructed signal. The crack failure thus could be precisely identified by the cyclic spectral correlation analysis. A series of numerical simulations and experimental tests have been conducted to illustrate the advantages of the proposed SOTBCA method for fatigue crack detection. Comparisons to three representative techniques, i.e. Erdogan's BCA (E-BCA), joint approximate diagonalization of eigen-matrices (JADE), and FastICA, have demonstrated the effectiveness of the SOTBCA. Hence the proposed approach is suitable for accurate gear crack detection in practical applications.

Positive resources for combating depressive symptoms among Chinese male correctional officers: perceived organizational support and psychological capital.

PubMed

Liu, Li; Hu, Shu; Wang, Lie; Sui, Guoyuan; Ma, Lei

2013-03-19

Although correctional officers (COs) clearly suffer from depression, positive resources for combating depression have been rarely studied in this population. The purpose of the study was to examine the associations of perceived organizational support (POS) and psychological capital (PsyCap) with depressive symptoms among Chinese COs. A cross-sectional survey was conducted in a province of northeast China during March-April 2011. A self-administered questionnaire was distributed to 1900 male COs from four male prisons. Depressive symptoms, POS, and PsyCap (self efficacy, hope, resilience, and optimism) were measured anonymously. A total of 1428 effective respondents with 953 frontline COs (FL-COs) and 475 non-frontline COs (NFL-COs) became our final sample. Hierarchical linear regression was performed to explore the factors associated with depressive symptoms. Asymptotic and resampling strategies were used to examine the mediating roles of PsyCap and its four components. The level of depressive symptoms of FL-COs was significantly higher than that of NFL-COs (t = 2.28, p = 0.023). There were significant negative associations of POS, PsyCap, hope, resilience, and optimism with depressive symptoms among FL-COs. In NFL-COs, POS, PsyCap, and optimism were negatively associated with depressive symptoms. POS was positively associated with PsyCap and its four components among both FL-COs and NFL-COs. For FL-COs, PsyCap (a*b = -0.143, BCa 95% CI: -0.186, -0.103, p < 0.05), resilience (a*b = -0.052, BCa 95% CI: -0.090, -0.017, p < 0.05), and optimism (a*b = -0.053, BCa 95% CI: -0.090, -0.016, p < 0.05) significantly mediated the association between POS and depressive symptoms. For NFL-COs, PsyCap (a*b = -0.126, BCa 95% CI: -0.186, -0.065, p < 0.05) and optimism (a*b = -0.066, BCa 95% CI: -0.116, -0.008, p < 0.05) significantly mediated the association. Perceived organizational support and psychological capital could be positive resources for combating depressive symptoms in Chinese male COs. Psychological capital and its components (resilience and optimism) partially mediate the association between perceived organizational support and depressive symptoms. Therefore, organizational support and psychological capital investment (especially resilience and optimism) should be included in depression preventions and treatments targeting Chinese male COs.

Comparative differences in the behavior of TiO2 and SiO2 food additives in food ingredient solutions

NASA Astrophysics Data System (ADS)

Yusoff, Ridhwan; Nguyen, Luong T. H.; Chiew, Paul; Wang, Zheng Ming; Ng, Kee Woei

2018-03-01

Nanotechnology is widely used in the food industry to improve the color, taste, and texture of food products. However, concerns regarding potential undesirable health effects remain. It is expected that interaction of engineered nanomaterials (ENMs) with food ingredients will influence their behavior and the resulting corona. Nonetheless, there are limited systematic studies conducted to clarify this understanding to date. Herein, we investigated the behavior and corona formation of food grade titanium dioxide (TiO2) and silicon dioxide (SiO2) in solutions of model food ingredients including bovine serum albumin (BSA) and sucrose. Measurements using dynamic light scattering (DLS) showed that both TiO2 and SiO2 nanoparticles displayed a decrease in agglomerate sizes in the presence of both food ingredients. Both particles were negatively charged in all the conditions tested. Corona adsorption studies were carried out using multiple complementary methods including Fourier transform infrared (FTIR) spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS), transmission electron microscopy (TEM), micro bicinchoninic acid (BCA) protein assay, and thermogravimetric analysis (TGA). Comparative investigation showed that sucrose could disperse both particles more effectively than BSA and that SiO2 displayed greater adsorption capacity for both BSA and sucrose, compared to TiO2. Taken collectively, this study demonstrated the importance of considering food ingredient effects when mapping the behavior of ENMs in food products. Such understanding could be significant in the evaluation of biological effects, such as toxicity, of ENMs used in food products.

[Biological characteristics of exosomes secreted by human bone marrow mesenchymal stem cells].

PubMed

Feng, Ying; Lu, Shi-Hong; Wang, Xin; Cui, Jun-Jie; Li, Xue; DU, Wen-Jing; Wang, Ying; Li, Juan-Juan; Song, Bao-Quan; Chen, Fang; Ma, Feng-Xia; Chi, Ying; Yang, Shao-Guang; Han, Zhong-Chao

2014-06-01

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.

Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia

PubMed Central

Paranjape, Geeta S.; Terrill, Shana E.; Gouwens, Lisa K.; Ruck, Benjamin M.; Nichols, Michael R.

2012-01-01

Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1–42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia. PMID:23242692

[Screening methods for mild cognitive impairment in primary care].

PubMed

Freire Pérez, Alberto

2017-06-01

Diagnosis of mild cognitive impairment (MCI) is always clinical and screening methods only indicate that the patient has a higher risk of this condition. In MCI, there is a slight decline in some cognitive abilities that does not affect activities of daily living and therefore does not produce social or occupational disability. The definitive diagnosis of MCI requires a considerable time investment that is very rarely possible to provide in primary care (PC) consultations. Hence the need for PC physicians to employ rapid and simple screening methods (brief cognitive assessment -BCA-) that allow objective identification of patients likely to have MCI in a few minutes. This article reviews the BCA tools that can truly be applied in less than 10 minutes. The phototest is a brief screening tool that is easy to use and interpret by physicians and is well accepted by patients. Consequently, it is one of the most useful tests in PC for screening of both MCI and dementia. In addition to BCA, instrumental activities of daily living scales should also be applied to differentiate MCI from dementia. Copyright © 2017 Sociedad Española de Geriatría y Gerontología. Publicado por Elsevier España, S.L.U. All rights reserved.

Groundwater vulnerability assessment for the Banyas Catchment of the Syrian coastal area using GIS and the RISKE method.

PubMed

Kattaa, Bassam; Al-Fares, Walid; Al Charideh, Abdul Rahman

2010-05-01

Vulnerability assessment to delineate areas that are more susceptible to contamination from anthropogenic sources has become an important element for sensible resource management and landuse planning. This contribution aims at estimating aquifer vulnerability by applying the RISKE model in Banyas Catchment Area (BCA), Tartous Prefecture, west Syria. An additional objective is to demonstrate the combined use of the RISKE model and a geographical information system (GIS) as an effective method for groundwater pollution risk assessment. The RISKE model uses five environmental parameters (Rock of aquifer media, Infiltration, Soil media, Karst, and Epikarst) to characterize the hydro-geological setting and evaluate aquifer vulnerability. The elevated eastern and low western part of the study area was dominated by high vulnerability classes, while the middle part was characterized by moderate vulnerability classes. Based on the vulnerability analysis, it was found that 2% and 39% of BCA is under low and high vulnerability to groundwater contamination, respectively, while more than 52% and 5% of the area of BCA can be designated as an area of moderate and very high vulnerability to groundwater contamination, respectively. The GIS technique has provided an efficient environment for analyses and high capabilities of handling a large amount of spatial data. Copyright 2009 Elsevier Ltd. All rights reserved.

Single-stage excision of localized head and neck venous malformations using preoperative glue embolization.

PubMed

Tieu, David D; Ghodke, Basavaraj V; Vo, Nghia J; Perkins, Jonathan A

2013-04-01

Describe single-stage removal of head and neck venous malformations using percutaneous embolization with n-butyl cyanoacrylate (n-BCA) glue prior to surgical resection. Case series with chart review. Tertiary-care pediatric hospital. A total of 169 venous malformations were identified between 2000 and 2012, and 102 (60.1%) were in the head and neck. Thirty-five of 102 (34.3%) were observed, 56 of 102 (54.9%) had invasive therapy, and 11 of 102 (10.8%) underwent n-BCA embolization and surgery ("GES procedure"). The median age of the glue embolization and surgery cohort was 14 years (range, 6-19), and 7 of 11 (63.6%) were female. Treated venous malformations involved the oral cavity/tongue (4/11; 36.4%) and parotid/face (7/11; 63.6%). During facial lesion excision, intraoperative facial nerve monitoring was used. All surgical sites (11/11) were closed primarily. No patient in this cohort had any posttreatment nerve deficits, dysarthria, and dysphagia or lesion persistence. Localized venous malformations can be treated with preoperative percutaneous embolization with n-BCA glue followed by surgical excision. This technique, with selective motor nerve monitoring, appears safe and allows for complete venous malformation removal with limited nerve dissection, to allow maximal tissue and functional preservation.

Lung cancer, pulmonary emphysema and pleural effusion: An autopsy study.

PubMed

Marel, Miloslav; Koubkova, Leona; Kovarikova, Zuzana; Grandcourtova, Alzbeta; Petrik, Frantisek; Hroudova, Hana; Capkova, Linda; Kodet, Roman; Fila, Libor

2015-12-01

To determine the exact incidence of lung cancer, pulmonary emphysema and pleural effusion we decided to carry out an autopsy study. In this autopsy study carried out over two years, we compared the results of autopsy findings with the clinical data in accompanying records of the deceased. Among the 708 deceased subjects, there were 398 males and 310 females with a median age of 71 years. At autopsy, 55 cases of lung carcinoma (BCA) were found, of which 24 have not been identified during life (44%). Among the deceased with BCA, emphysema was also observed at autopsy in 40% of the cases. Pulmonary emphysema was described macroscopically in 28% of the full set of 708 deceased, whereas the accompanying records of the deceased described this condition in only 12% of the cases. Microscopic changes compatible with emphysema were identified in 54% of the examined lungs. Pleural effusions were described in the accompanying records of 13% of the deceased, while the autopsies showed this condition in 33% of the deceased. BCA was accompanied by effusion in 25% of the cases. The obtained results show that the studied conditions are present in more cases than are reported by clinicians. The study confirms the commonly accepted association between lung cancer and emphysema.

Biological effects of functionalizing copolymer scaffolds with nanodiamond particles.

PubMed

Xing, Zhe; Pedersen, Torbjorn O; Wu, Xujun; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Kloss, Frank R; Waag, Thilo; Krueger, Anke; Steinmüller-Nethl, Doris; Mustafa, Kamal

2013-08-01

Significant evidence has indicated that poly(L-lactide)-co-(ɛ-caprolactone) [(poly(LLA-co-CL)] scaffolds could be one of the suitable candidates for bone tissue engineering. Oxygen-terminated nanodiamond particles (n-DP) were combined with poly(LLA-co-CL) and revealed to be positive for cell growth. In this study, we evaluated the influence of poly(LLA-co-CL) scaffolds modified by n-DP on attachment, proliferation, differentiation of bone marrow stromal cells (BMSCs) in vitro, and on bone formation using a sheep calvarial defect model. BMSCs were seeded on either poly(LLA-co-CL)- or n-DP-coated scaffolds and incubated for 1 h. Scanning electron microscopy (SEM) and fluorescence microscopy were used in addition to protein and DNA measurements to evaluate cellular attachment on the scaffolds. To determine the effect of n-DP on proliferation of BMSCs, cell/scaffold constructs were harvested after 3 days and evaluated by Bicinchoninic Acid (BCA) protein assay and SEM. In addition, the osteogenic differentiation of cells grown for 2 weeks on the various scaffolds and in a dynamic culture condition was evaluated by real-time RT-PCR. Unmodified and modified scaffolds were implanted into the calvaria of six-year-old sheep. The expression of collagen type I (COL I) and bone morphogenetic protein-2 (BMP-2) after 4 weeks as well as the formation of new bone after 12 and 24 weeks were analyzed by immunohistochemistry and histology. Scaffolds modified with n-DP supported increased cell attachment and the mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), and BMP-2 were significantly increased after 2 weeks of culture. The BMSCs had spread well on the various scaffolds investigated after 3 days in the study with no significant difference in cell proliferation. Furthermore, the in vivo data revealed more positive staining of COL I and BMP-2 in relation to the n-DP-coated scaffolds after 4 weeks and presented more bone formation after 12 and 24 weeks. n-DP modification significantly increased cell attachment and differentiation of BMSCs on poly(LLA-co-CL) scaffolds in vitro and enhanced bone formation in vivo.

Efficacy of fungal and bacterial antagonists for controlling growth, FUM1 gene expression and fumonisin B1 production by Fusarium verticillioides on maize cobs of different ripening stages.

PubMed

Samsudin, Nik Iskandar Putra; Rodriguez, Alicia; Medina, Angel; Magan, Naresh

2017-04-04

This study was carried out to examine the efficacy of two biocontrol agents (Clonostachys rosea 016, BCA1; Gram-negative bacterium, BCA5) for control of FUM1 gene expression and fumonisin B 1 (FB 1 ) production by F. verticillioides FV1 on maize cobs of different ripening stages: R 3 , Milk (0.985 a w ); R 4 , Dough (0.976 a w ); R 5 , Dent (0.958 a w ). Initially, temporal studies on FUM1 gene expression and FB 1 production were performed on maize kernels for up to 14days. This revealed that day 10 was optimum for both parameters, and was used in the biocontrol studies. Maize cobs were inoculated with 50:50 mixtures of the pathogen:antagonist inoculum and incubated in environmental chambers to maintain the natural a w conditions for ten days at 25 and 30°C. The growth rates of F. verticillioides FV1, the relative expression of the FUM1 gene and FB 1 production were quantified. It was found that, a w ×temp had significant impacts on growth, FUM1 gene expression and FB 1 production by F. verticillioides FV1 on maize cobs of different maturities. The fungal antagonist (BCA1) significantly reduced FB 1 contamination on maize cobs by >70% at 25°C, and almost 60% at 30°C regardless of maize ripening stage. For the bacterial antagonist (BCA5) however, FB 1 levels on maize cobs were significantly decreased only in some treatments. These results suggest that efficacy of antagonists to control mycotoxin production in ripening maize cobs needs to take account of the ecophysiology of the pathogen and the antagonists, as well as the physiological status of the maize during silking to ensure effective control. Copyright © 2017 Elsevier B.V. All rights reserved.

Post-Surgical Depressive Symptoms and Pro-Inflammatory Cytokine Elevations in Women Undergoing Primary Treatment for Breast Cancer

PubMed Central

Bouchard, Laura C.; Antoni, Michael H.; Blomberg, Bonnie B.; Stagl, Jamie M.; Gudenkauf, Lisa M.; Jutagir, Devika R.; Diaz, Alain; Lechner, Suzanne; Glück, Stefan; Derhagopian, Robert P.; Carver, Charles S.

2015-01-01

Objective Depression and inflammation may independently promote breast cancer (BCa) disease progression and poorer clinical outcomes. Depression has been associated with increased levels of inflammatory markers in medically healthy individuals and cancer patients. However, inconsistencies in study time frames complicate interpretation of results within specific cancer types. This study examined relationships between depressive symptoms and inflammation in women with early stage BCa before beginning adjuvant treatment. Method Women with stage 0–III BCa were recruited approximately 4–8 weeks post-surgery. Depressive symptoms were assessed using the Hamilton Rating Scale for Depression and blood samples were collected to quantify circulating levels of IL-1β, IL-6, and TNF-α by ELISA. ANCOVAs were used to test for group differences (elevated vs. low depressive symptoms) in levels of cytokines. Multiple regression analyses were used to examine relationships between continuous severity of depressive symptoms and levels of cytokines adjusting for relevant biobehavioral covariates. Results Thirty-six of 89 (40%) patients showed elevated levels of depressive symptoms, and in adjusted models had marginally higher levels of IL-1β (M=14.49, 95% CI [6.11, 32.65] vs. M=4.68, 95% CI [1.96, 9.86]) and significantly higher levels of TNF-α (M=17.07, 95% CI [8.27, 34.32] vs. M=6.94, 95% CI [3.58, 12.80]) than women with low depressive symptoms. Across the spectrum of depressive symptoms, greater magnitude of depressive symptoms was related to greater levels of IL-1β (β=0.06, p=0.006, R2=0.25) and TNF-α (β=0.06, p=0.003, R2=0.27). Conclusions Post-surgery and pre-adjuvant treatment for early stage BCa, depressive symptoms covary with elevated levels of multiple pro-inflammatory cytokines. Findings have implications for psychosocial and biological interventions concurrently focusing on depression and inflammation. PMID:26569533

Melon chlorotic leaf curl virus: characterization and differential reassortment with closest relatives reveal adaptive virulence in the squash leaf curl virus clade and host shifting by the host-restricted bean calico mosaic virus.

PubMed

Idris, A M; Mills-Lujan, K; Martin, K; Brown, J K

2008-02-01

The genome components of the Melon chlorotic leaf curl virus (MCLCuV) were cloned from symptomatic cantaloupe leaves collected in Guatemala during 2002. The MCLCuV DNA-A and DNA-B components shared their closest nucleotide identities among begomoviruses, at approximately 90 and 81%, respectively, with a papaya isolate of MCLCuV from Costa Rica. The closest relatives at the species level were other members of the Squash leaf curl virus (SLCV) clade, which is endemic in the southwestern United States and Mexico. Biolistic inoculation of cantaloupe seedlings with the MCLCuV DNA-A and -B components resulted in the development of characteristic disease symptoms, providing definitive evidence of causality. MCLCuV experimentally infected species within the Cucurbitaceae, Fabaceae, and Solanaceae. The potential for interspecific reassortment was examined for MCLCuV and its closest relatives, including the bean-restricted Bean calico mosaic virus (BCaMV), and three other cucurbit-infecting species, Cucurbit leaf crumple virus (CuLCrV), SLCV, and SMLCV. The cucurbit viruses have distinct but overlapping host ranges. All possible reassortants were established using heterologous combinations of the DNA-A or DNA-B components. Surprisingly, only certain reassortants arising from MCLCuV and BCaMV, or MCLCuV and CuLCrV, were viable in bean, even though it is a host of all of the "wild-type" (parent) viruses. The bean-restricted BCaMV was differentially assisted in systemically infecting the cucurbit test species by the components of the four cucurbit-adapted begomoviruses. In certain heterologous combinations, the BCaMV DNA-A or -B component was able to infect one or more cucurbit species. Generally, the reassortants were less virulent in the test hosts than the respective wild-type (parent) viruses, strongly implicating adaptive modulation of virulence. This is the first illustration of reassortment resulting in the host range expansion of a host-restricted begomovirus.

Strongly anisotropic thermal conductivity and adequate breathability of bilayered films for heat management of on-skin electronics

NASA Astrophysics Data System (ADS)

Zhou, Tianle; Wei, Hao; Tan, Huaping; Wang, Xin; Zeng, Haibo; Liu, Xiaoheng; Nagao, Shijo; Koga, Hirotaka; Nogi, Masaya; Sugahara, Tohru; Suganuma, Katsuaki

2018-07-01

Thin-film wearable electronics are required to be directly laminated on to human skin for reliable, sensitive bio-sensing but with minimal irritation to the user after long-time use. Excellent heat management films with strongly anisotropic thermal conductivity (K) and adequate breathability are increasingly desirable for shielding the skin from heating while allowing the skin to breathe properly. Here, interfacial self-assembly of a graphene oxide (GO) film covering an ambient-dried bacterial cellulose aerogel (AD-BCA) film followed by laser reduction was proposed to prepare laser-reduced GO (L-rGO)/AD-BCA bilayered films. The AD-BCA substrate provides low cross-plane K (K ⊥  ≈  0.052 W mK‑1), high breathability, and high compressive and tensile resistance by ‘partially’ inheriting the pore structure from bacterial cellulose (BC) gel. The introduction of an upper L-rGO film, which is only 0.31 wt% content, dramatically increases the in-plane K (K // ) from 0.3 W mK‑1 in AD-BCA to 10.72 W mK‑1 owing to the highly in-plane oriented, continuous, uniform assembling geometry of the GO film; while K ⊥ decreases to a lower value of 0.033 W mK‑1, mainly owing to the air pockets between L-rGO multilayers caused by the laser reduction. The bilayered films achieve a K // /K ⊥ of 325, which is substantially larger even than that of graphite and similar polymer composites. They permit high transmission rates for water vapor (416.78 g/m2/day, >204 g/m2/day of normal skin) and O2 (449.35 cm3/m2/day). The combination of strongly anisotropic thermal conductivity and adequate breathability facilitates applications in heat management in on-skin electronics.

Trace element evidence for abrupt changes in deep South Atlantic Ocean nutrient and carbonate chemistry across the Mid-Pleistocene Transition

NASA Astrophysics Data System (ADS)

Farmer, J. R.; Hoenisch, B.; Haynes, L.; Kroon, D.; Bell, D. B.; Jung, S.; Seguí, M. J.; Raymo, M. E.; Goldstein, S. L.; Pena, L. D.

2016-12-01

Pleistocene glaciations underwent a profound transition from lower amplitude 40 kyr cycles to high amplitude 100 kyr cycles between 1.2 and 0.8 Ma, an interval termed the Mid-Pleistocene Transition (MPT). While the underlying causes of the MPT are uncertain, previous studies show quasi-contemporaneous reductions in North Atlantic Deep Water (NADW) export1 and glacial atmospheric pCO22 around 0.9 Ma. Although this suggests a possible role for enhanced deep-ocean carbon storage in amplifying climate change across the MPT, few direct records of deep ocean carbonate chemistry exist for this interval to test this hypothesis. Here we present South Atlantic benthic foraminiferal B/Ca and Cd/Ca records from International Ocean Discovery Program Sites 1088, 1264 and 1267 (2.1 to 4.3 km water depth) as part of a larger study of Atlantic-wide changes in deep ocean chemistry and circulation spanning the MPT. Results show an abrupt 15-20% decrease in benthic B/Ca and 40-50% increase in Cd/Ca at 4.3 km depth (Site 1267) between 1.0 and 0.9 Ma. Site 1088, which at 2.1 km depth is sensitive to input of southern-sourced Upper Circumpolar Deep Water, shows a prolonged 25% decrease in B/Ca and 50% increase in Cd/Ca from 1.0 to 0.6 Ma. In contrast, at Site 1264 ( 2.5 km depth within the core of modern NADW) B/Ca and Cd/Ca changes across the MPT are more modest (-5% and +10%, respectively). These observations reflect on the accumulation of regenerated carbon and nutrients in the deep South Atlantic, and varying contributions of northern- and southern-sourced watermasses to each core site. Implications for deep-ocean carbon storage and forcing of the MPT will be discussed. 1Pena, L. and Goldstein, S. (2014), Science 345, 318 2Hönisch, B. et al. (2009), Science 324, 1551

Effectiveness of urine fibronectin as a non-invasive diagnostic biomarker in bladder cancer patients: a systematic review and meta-analysis.

PubMed

Dong, Fan; Shen, Yifan; Xu, Tianyuan; Wang, Xianjin; Gao, Fengbin; Zhong, Shan; Chen, Shanwen; Shen, Zhoujun

2018-03-21

Previous researches pointed out that the measurement of urine fibronectin (Fn) could be a potential diagnostic test for bladder cancer (BCa). We conducted this meta-analysis to fully assess the diagnostic value of urine Fn for BCa detection. A systematic literature search in PubMed, ISI Web of Science, EMBASE, Cochrane library, and CBM was carried out to identify eligible studies evaluating the urine Fn in diagnosing BCa. Pooled sensitivity, specificity, and diagnostic odds ratio (DOR) with their 95% confidence intervals (CIs) were calculated, and summary receiver operating characteristic (SROC) curves were established. We applied the STATA 13.0, Meta-Disc 1.4, and RevMan 5.3 software to the meta-analysis. Eight separate studies with 744 bladder cancer patients were enrolled in this meta-analysis. The pooled sensitivity, specificity, and DOR were 0.80 (95%CI = 0.77-0.83), 0.79 (95%CI = 0.73-0.84), and 15.18 (95%CI = 10.07-22.87), respectively, and the area under the curve (AUC) of SROC was 0.83 (95%CI = 0.79-0.86). The diagnostic power of a combined method (urine Fn combined with urine cytology) was also evaluated, and its sensitivity and AUC were significantly higher (0.86 (95%CI = 0.82-0.90) and 0.89 (95%CI = 0.86-0.92), respectively). Meta-regression along with subgroup analysis based on various covariates revealed the potential sources of the heterogeneity and the detailed diagnostic value of each subgroup. Sensitivity analysis supported that the result was robust. No threshold effect and publication bias were found in this meta-analysis. Urine Fn may become a promising non-invasive biomarker for bladder cancer with a relatively satisfactory diagnostic power. And the combination of urine Fn with cytology could be an alternative option for detecting BCa in clinical practice. The potential value of urine Fn still needs to be validated in large, multi-center, and prospective studies.

Deciphering the Boron Proxy Records of the Paleocene-Eocene Thermal Maximum

NASA Astrophysics Data System (ADS)

Hoenisch, B.; Haynes, L.; Harper, D. T.; Penman, D. E.; Holland, K.; Rosenthal, Y.; Zachos, J. C.

2016-12-01

Rapid surface ocean acidification at the PETM has been documented by pronounced decreases in the boron isotope and B/Ca proxies measured in surface dwelling planktic foraminifera (Babila et al., 2016; Penman et al., 2014). However, translating these geochemical signatures to past seawater carbonate chemistry is challenging due to the different-from-modern elemental and isotopic composition of seawater, in addition to the lack of constraints on vital effects in foraminifer species that are now extinct. While the pH decrease can be reasonably quantified from boron isotopes, the application of modern laboratory calibrations to translate the B/Ca signal yields unfeasible estimates, thus raising questions about how well we understand fundamental proxy systematics. Here we present a possible solution to this conundrum from laboratory culture experiments performed under simulated Paleocene seawater conditions, with lower [B] and [Mg], higher [Ca] and across a range of dissolved inorganic carbon and pH. These experiments suggest that raising DIC in addition to acidification amplifies the B/Ca decrease recorded in planktic foraminifera shells, thus providing an opportunity to deconvolve the B/Ca record into pH and DIC signals. Using the boron proxy records in ODP 1209 from Shatsky Rise in the Pacific Ocean as a case study, we will perform a series of sensitivity studies to better constrain the carbon perturbation at the PETM, and the long-term evolution of surface ocean chemistry from the Paleocene into the Eocene. Our results will be compared to LOSCAR model estimates of different carbon input scenarios at the PETM. Babila, T.L., Rosenthal, Y., Wright, J.D. and Miller, K.G. (2016) A continental shelf perspective of ocean acidification and temperature evolution during the Paleocene-Eocene Thermal Maximum. Geology 44, 275-278. Penman, D.E., Hönisch, B., Zeebe, R.E., Thomas, E. and Zachos, J.C. (2014) Rapid and sustained surface ocean acidification during the Paleocene-Eocene Thermal Maximum. Paleoceanography 29, 2014PA002621.

Influence of fertilisation with sewage sludge-derived preparation on selected soil properties and prairie cordgrass yield.

PubMed

Ociepa, Ewa; Mrowiec, Maciej; Lach, Joanna

2017-07-01

The aim of the study was to evaluate the effect of using a fertilizer obtained from waste substances on selected physical and chemical properties of soil and biomass yield Spartina pectinate. The fertilizer used for soil (C) fertilisation contained sewage sludge (SS), waste soil fractions of brown coal (BC), brown coal ash (BCA) enriched with mineral potassium (K) fertilizer (C+SS+BC+BCA+K). The composition of the preparation was developed by the authors and adjusted to the quality of the fertilised soil and the individual characteristics of the plant. It was assumed that the preparation should replace expensive conventional fertilisation methods, allow for management of waste substances and improve soil properties, leading to a high yield of Spartina pectinata used as energy crop. The plants were grown on the soil from the Huta Częstochowa steelworks effect zone. The soil was light, with acid reaction (pH KCl =5.5), with small contents of such contaminants as Cd and Zn and elevated Pb content. Based on a three-year pot experiment, the paper presents the results of the examinations concerning the effect of fertilisation on soil pH, hydrolytic acidity, sorptive properties, content of humic acids, organic coal and total nitrogen in soil and crop yielding. The effect of the use of the fertilizer (C+SS+BC+BCA+K) was compared with the use of the sludge (C+SS), sludge with mineral fertilizers (C+SS+NPK), mixture of brown coal and mineral fertilizers (C+BC+NPK) and effect of only mineral fertilizers (C+NPK). Fertilisation with (C+SS+BC+BCA+K) led to the increase in soil pH from 5.5 to 6.0, which is considered sufficient for light soils. The fertilised soil was characterized by sorption capacity of ca. 5.8 cmol(+)/kg, and, after fertilisation with O+W+P, reached the value of ca. 8.0 cmol(+)kg -1 . Consequently the soil can be regarded as of good quality in terms of its capability to store nutrients. The C:N ratio was also extended from 11:1 (control soil) to 14:1 (C+SS+BC+BCA+K). The yield of Spartina pectinata in the first year was 1.6 and in the third year 2.7 times higher in the case of fertilisation with the investigated mixture as compared to the control. Copyright © 2017 Elsevier Inc. All rights reserved.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

Preoperative Embolization of Venous Malformations Using n-Butyl Cyanoacrylate.

PubMed

Uller, Wibke; El-Sobky, Sherif; Alomari, Ahmad I; Fishman, Steven J; Spencer, Samantha A; Taghinia, Amir H; Chaudry, Gulraiz

2018-05-01

The purpose of this study was to evaluate the safety and efficacy of preoperative percutaneous n-butyl cyanoacrylate (nBCA) embolization of venous malformations in children. Clinical data were retrospectively reviewed in children who underwent embolization using nBCA followed by resection of venous malformations. A total of 17 embolizations were performed in 14 patients (9 females, mean age: 5.5 years; median age: 3 years; range 0.1-16 years). The venous malformations involved the lower extremity and the knee joint (n = 7), the trunk (n = 4), head and neck (n = 2), and hand (n = 1). n-Butyl cyanoacrylate was diluted with iodized oil at a ratio of 1:3 to 1:5. The mean and median volume of nBCA per procedure were 2.1 and 2 mL, respectively (range: 0.5-8 mL). There were no complications associated with the procedures. The mean and median time between final embolization and resection were 3.6 and 2 days, respectively. All children underwent successful resection of the symptomatic lesions. The estimated mean and median blood loss were 75 and 50 mL, respectively (range: 5-350 mL). The postprocedure course was uneventful, the days to discharge ranged between 1 and 6 days (mean 3 days). Initial results suggest that preoperative percutaneous n-butyl cyanoacrylate embolization of venous malformations is safe and effective in children, with the potential for minimizing blood loss and inpatient stay.

Validity of Binary Collision Theory in Ion-Surface Interactions at 50-500 eV

NASA Astrophysics Data System (ADS)

Gordon, Michael; Giapis, Kostas

2003-10-01

Ion-surface interactions in the 50-500 eV regime have become increasingly important in plasma processing. Concerns exist in literature about the validity of the binary collision approximation (BCA) at low impact energies because peculiarities are frequently seen in the scattered ion energy distribution. Sub-surface processes, multiple bouncing, and super-elastic phenomena have all been hypothesized. This talk will explore the usefulness of BCA theory in predicting energy transfer during ion-surface collisions in the 50-500 eV energy range. Well-defined beams of rare gas ions (Ne, Ar, Kr) were scattered off semiconductor (Si, Ge) and metal surfaces (Ag, Au, Ni, Nb) to measure energy loss upon impact. The ion beams were produced from a floating ICP reactor coupled to a small accelerator beamline for transport and mass filtering. Exit channel energies were measured using a 90 gegree electrostatic sector coupled to a quadrupole mass filter with single ion detection capability. Although the BCA presents an over-simplified picture of the collision process, our results demonstrate that it is remarkably accurate in the low energy range for a variety of projectile-target combinations. In addition, reactive ion scattering of O2+ and O+ on inert and reactive surfaces (Au vs. Ag, Pt) suggests there may be rather high energy threshold processes which determine exit channel selectivity.

Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

PubMed

Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

2015-01-01

Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

Capturing the global signature of surface ocean acidification during the PETM

NASA Astrophysics Data System (ADS)

Babila, T. L.; Penman, D. E.; Hoenisch, B.; Kelly, D. C.; Bralower, T. J.; Rosenthal, Y.; Zachos, J. C.

2016-12-01

Anthropogenic greenhouse gas emissions over the last century have elevated atmospheric carbon dioxide concentrations while concomitantly acidifying the oceans. Instrumental records are sparse and limited in duration, making it difficult to separate regional from global trends of ocean acidification. Geologically rapid carbon perturbations such as the Paleocene-Eocene Thermal Maximum (PETM, 56 Ma) are arguably the closest paleo analogue to present climate change. Marine ecosystems experienced dynamic changes during the event, and parallel environmental changes, including acidification and warming. Here we present a synthesis of new and published geochemical reconstructions from various oceanographic settings to determine the magnitude and spatial extent of surface ocean acidification. In the deep ocean, acidification is inferred from widespread dissolution of seafloor carbonates, whereas evidence for surface ocean acidification has emerged from planktonic foraminifera boron proxy records (B/Ca and δ11B) (Penman et al. 2014; Babila et al. 2016). B/Ca and δ11B in surface and thermocline planktonic foraminifera suggest a simultaneous decrease at the PETM onset in all pelagic and shelf sites. Salinity, diagenesis and foraminiferal symbiont loss can complicate the interpretation of boron proxy records. Local salinity changes (based on paired Mg/Ca and δ18O) account for a relatively small component of total B/Ca change. The large range in environmental conditions between sites could explain the subtle differences in absolute values exhibited by the records. Shelf sites (ODP 174AX Bass River and Ancora, NJ) reveal similar absolute values and trends compared to pelagic sites (ODP 1209, N. Pacific), precluding a significant preservation bias on the geochemical records. Southern Ocean sites (ODP 689 and 690) are located in colder surface waters and exhibit a similar decrease in B/Ca, suggesting that temperature and symbiont loss are likely not major factors. We conclude that while the mass of released carbon is comparable to anthropogenic emissions, the rate is much slower, resulting in a less severe degree of undersaturation. Furthermore, the consistent latitudinal pattern of acidification suggests that thermal stress rather than acidification contributed to the observed biotic responses.

Effects of coexisting BDE-47 on the migration and biodegradation of BDE-99 in river-based aquifer media recharged with reclaimed water.

PubMed

Yan, Y; Li, Y; Ma, M; Ma, W; Cheng, X; Xu, K

2018-02-01

Two prominent polybrominated diphenyl ether (PBDE) congeners have been included in the persistent organic pollutant list, 2,2',4,4',5-tetrabromodiphenyl ether (BDE-99) and 2,2,4,4'-tetrabromodiphenyl ether (BDE-47), which have been detected in treated municipal wastewater, river water, and sediments in China. A lab-scale column experiment was established to investigate the effects of the competitive sorption of BDE-47 on BDE-99 biodegradation and migration in two types of river-based aquifer soils during groundwater recharge with reclaimed water. Two types of recharge columns were used, filled with either silty clay (SC) or black carbon-amended silty clay (BCA). The decay rate constants of BDE-99 in the BCA and SC systems were 0.186 and 0.13 m -1 in the single-solute system and 0.128 and 0.071 m -1 in the binary-solute system, respectively, showing that the decay of BDE-99 was inhibited by the coexistence of BDE-47. This was particularly evident in the SC system because the higher hydrophobicity of BDE-99 determined the higher affinity and competition for sorption sites onto black carbon. The biodegradation of BDE-99 was suppressed by the coexistence of BDE-47, especially in the SC system. Lesser-brominated congeners (BDE-47 and BDE-28) and higher-brominated congeners (BDE-100, BDE-153, BDE-154, and BDE-183) were generated in the four recharge systems, albeit at different ratios. Bacterial biodiversity was influenced by the presence of BDE-47 in the SC system, while it had no significant effect on the BCA system, because the high sorption capacity of black carbon on the hydrophobic PBDEs effectively reduced their toxicity. The ranking order of the most abundant classes changed markedly due to the coexistence of BDE-47 in both the SC and BCA systems. The ranking order of the most abundant genera changed from Azospira, Methylotenera, Desulfovibrio, Methylibium, and Bradyrhizobium to Halomonas, Hyphomicrobium, Pseudomonas, Methylophaga, and Shewanella, which could be involved in PBDE degradation.

Benefit-Cost Analysis of Undergraduate Education Programs: An Example Analysis of the Freshman Research Initiative.

PubMed

Walcott, Rebecca L; Corso, Phaedra S; Rodenbusch, Stacia E; Dolan, Erin L

2018-01-01

Institutions and administrators regularly have to make difficult choices about how best to invest resources to serve students. Yet economic evaluation, or the systematic analysis of the relationship between costs and outcomes of a program or policy, is relatively uncommon in higher education. This type of evaluation can be an important tool for decision makers considering questions of resource allocation. Our purpose with this essay is to describe methods for conducting one type of economic evaluation, a benefit-cost analysis (BCA), using an example of an existing undergraduate education program, the Freshman Research Initiative (FRI) at the University of Texas Austin. Our aim is twofold: to demonstrate how to apply BCA methodologies to evaluate an education program and to conduct an economic evaluation of FRI in particular. We explain the steps of BCA, including assessment of costs and benefits, estimation of the benefit-cost ratio, and analysis of uncertainty. We conclude that the university's investment in FRI generates a positive return for students in the form of increased future earning potential. © 2018 R. L. Walcott et al. CBE—Life Sciences Education © 2018 The American Society for Cell Biology. This article is distributed by The American Society for Cell Biology under license from the author(s). It is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Family history of breast cancer increases the risk of prostate cancer: results from the EPICAP study.

PubMed

Lamy, Pierre-Jean; Trétarre, Brigitte; Rebillard, Xavier; Sanchez, Marie; Cénée, Sylvie; Ménégaux, Florence

2018-05-04

Familial aggregation is now well established with an increased risk of prostate cancer in patients with a family history of prostate cancer in first degree relatives. The aim of this paper was to investigate the role of family history of cancer in first degree relatives in prostate cancer risk. As expected, a family history of prostate cancer in first-degree relatives was more frequent in cases than in controls (OR 3.10, 95% CI 2.32-4.15). A family history of early BCa (before age 50) in first-degree relatives was more frequent in cases than in controls (OR 1.79, 95% CI 1.09-2.94) with higher risk of aggressive PCa. The association was more pronounced for BCa in daughters (OR 15.26 95% CI 1.95-120). In summary, a family history of BCa in first degree relatives before age 50 may increases the risk of PCa with higher Gleason score. This finding could suggest a specific prostate surveillance and/or genetic counselling for men who present such familial history. EPIdemiological study of Prostate CAncer (EPICAP) is a population-based case-control study specifically designed to investigate the role of environmental and genetic factors in prostate cancer. Detailed information on family history of cancer in first degree relatives (parents, brothers and sisters, children) was collected as well as the age of occurrence and the localization of each cancer. Overall, 819 cases and 879 controls have been included.

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Cost benefit analysis: applications and future opportunities.

DOT National Transportation Integrated Search

2016-06-01

Cost Benefit Analysis (CBA or Benefit Cost Analysis BCA) is an evaluation tool that state transportation agencies can : use to compare infrastructure project options across transportation modes and gauge if the discounted value of benefits : exce...

Quantitation of proteins using a dye-metal-based colorimetric protein assay.

PubMed

Antharavally, Babu S; Mallia, Krishna A; Rangaraj, Priya; Haney, Paul; Bell, Peter A

2009-02-15

We describe a dye-metal (polyhydroxybenzenesulfonephthalein-type dye and a transition metal) complex-based total protein determination method. The binding of the complex to protein causes a shift in the absorption maximum of the dye-metal complex from 450 to 660 nm. The dye-metal complex has a reddish brown color that changes to green on binding to protein. The color produced from this reaction is stable and increases in a proportional manner over a broad range of protein concentrations. The new Pierce 660 nm Protein Assay is very reproducible, rapid, and more linear compared with the Coomassie dye-based Bradford assay. The assay reagent is room temperature stable, and the assay is a simple and convenient mix-and-read format. The assay has a moderate protein-to-protein variation and is compatible with most detergents, reducing agents, and other commonly used reagents. This is an added advantage for researchers needing to determine protein concentrations in samples containing both detergents and reducing agents.

Novel protein-repellent dental adhesive containing 2-methacryloyloxyethyl phosphorylcholine

PubMed Central

Zhang, Ning; Melo, Mary Anne S.; Bai, Yuxing; Xu, Hockin H. K.

2015-01-01

Objectives Biofilms at tooth-restoration margins can produce acids and cause secondary caries. A protein-repellent adhesive resin can potentially inhibition bacteria attachment and biofilm growth. However, there has been no report on protein-repellent dental resins. The objectives of this study were to develop a protein-repellent bonding agent incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC), and to investigate its resistance to protein adsorption and biofilm growth for the first time. Methods MPC was incorporated into Scotchbond Multi-Purpose (SBMP) at 0%, 3.75%, 7.5%, 11.25%, and 15% by mass. Extracted human teeth were used to measure dentin shear bond strengths. Protein adsorption onto resins was determined by a micro bicinchoninic acid (BCA) method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to measure biofilm metabolic activity and colony-forming unit (CFU) counts. Results Adding 7.5% MPC into primer and adhesive did not decrease the dentin bond strength, compared to control (p > 0.1). Incorporation of 7.5% of MPC achieved the lowest protein adsorption, which was 20-fold less than that of control. Incorporation of 7.5% of MPC greatly reduced bacterial adhesion, yielding biofilm total microorganism, total streptococci, and mutans streptococci CFU that were an order of magnitude less than control. Conclusions A protein-repellent dental adhesive resin was developed for the first time. Incorporation of MPC into primer and adhesive at 7.5% by mass greatly reduced the protein adsorption and bacterial adhesion, without compromising the dentin bond strength. The novel protein-repellent primer and adhesive are promising to inhibit biofilm formation and acid production, to protect the tooth-restoration margins and prevent secondary caries. PMID:25234652

A MODIFIED PROTEIN ASSAY FROM MICROGRAM TO LOW NANOGRAM LEVELS IN DILUTE SAMPLES

PubMed Central

Heda, Ghanshyam D.; Kunwar, Upasana; Heda, Rajiv P.

2013-01-01

In this paper we present a modified and improved protein assay that was previously described as ‘amidoschwarz assay’ by Schaffner and Weissmann (Anal. Biochem. 56, 1973, 502–514). Our improved protein assay is user-friendly and 30 to 40 times more sensitive than the earlier method. The assay was developed into 3 formats (maco, micro, and nanoassay) with TCA as protein precipitating agent; measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots, bound to nitrocellulose membrane with lowest protein measurements to 1 μg and 0.1 μg respectively. The nanoassay on the other hand with combination staining of amido black followed by colloidal gold can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 μg/ml, prior to their biochemical analysis such as in comparative proteomics. PMID:24135655

Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Detection of patient's bed statuses in 3D using a Microsoft Kinect.

PubMed

Li, Yun; Berkowitz, Lyle; Noskin, Gary; Mehrotra, Sanjay

2014-01-01

Patients spend the vast majority of their hospital stay in an unmonitored bed where various mobility factors can impact patient safety and quality. Specifically, bed positioning and a patient's related mobility in that bed can have a profound impact on risks such as pneumonias, blood clots, bed ulcers and falls. This issue has been exacerbated as the nurse-per-bed (NPB) ratio has decreased in recent years. To help assess these risks, it is critical to monitor a hospital bed's positional status (BPS). Two bed positional statuses, bed height (BH) and bed chair angle (BCA), are of critical interests for bed monitoring. In this paper, we develop a bed positional status detection system using a single Microsoft Kinect. Experimental results show that we are able to achieve 94.5% and 93.0% overall accuracy of the estimated BCA and BH in a simulated patient's room environment.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

NASA Astrophysics Data System (ADS)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

Amyloid-like aggregates formation by bovine apo-carbonic anhydrase in various alcohols: A comparative study.

PubMed

Es-Haghi, Ali; Ebrahim-Habibi, Azadeh; Sabbaghian, Marjan; Nemat-Gorgani, Mohsen

2016-11-01

Peptides and proteins convert from their native states to amyloid fibrillar aggregates in a number of pathological conditions. Characterizing these species could provide useful information on their pathogenicity and the key factors involved in their generation. In this study, we have observed the ability of the model protein apo-bovine carbonic anhydrase (apo-BCA) to form amyloid-like aggregates in the presence of halogenated and non-halogenated alcohols. Far-UV circular dichroism, ThT fluorescence, atomic force microscopy and dynamic light scattering were used to characterize these structures. The concentration required for effective protein aggregation varied between the solvents, with non-halogenated alcohols acting in a wider range. These aggregates show amyloid-like structures as determined by specific techniques used for characterizing amyloid structures. Oligomers were obtained with various size distributions, but fibrillar structures were not observed. Use of halogenated alcohols resulted into smaller hydrodynamic radii, and most stable oligomers were formed in hexafluoropropan-2-ol (HFIP). At optimal concentrations used to generate these structures, the non-halogenated alcohols showed higher hydrophobicity, which may be related to the lower stability of the generated oligomers. These oligomers have the potential to be used as models in the search for effective treatments in proteinopathies. Copyright © 2016 Elsevier B.V. All rights reserved.

Probing the crystal structure landscape by doping: 4-bromo, 4-chloro and 4-methylcinnamic acids.

PubMed

Desiraju, Gautam R; Chakraborty, Shaunak; Joseph, Sumy

2018-06-11

Accessing the data points in the crystal structure landscape of a molecule is a challenging task, either experimentally or computationally. We have charted the crystal structure landscape of 4-bromocinnamic acid (4BCA) experimentally and computationally: experimental doping is achieved with 4-methylcinnamic acid (4MCA) to obtain new crystal structures; computational doping is performed with 4-chlorocinnamic acid (4CCA) as a model system, because of the difficulties associated in parameterizing the Br-atom. The landscape of 4CCA is explored experimentally in turn, also by doping it with 4MCA, and is found to bear a close resemblance to the landscape of 4BCA, justifying the ready miscibility of these two halogenated cinnamic acids to form solid solutions without any change in crystal structure. In effect, 4MCA, 4CCA and 4BCA form a commutable group of crystal structures, which may be realized experimentally or computationally, and constitute the landscape. Unlike the results obtained by Kitaigorodskii and others, all but two of the multiple solid solutions obtained in the methyl-doping experiments take structures that are different from the hitherto observed crystal forms of the parent compounds. Even granted that the latter might be inherently polymorphic, this unusual observation provokes the suggestion that solid solution formation may be used to probe the crystal structure landscape. The influence of pi...pi interactions, weak hydrogen bonds and halogen bonds in directing the formation of these new structures is also seen. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Periodontal Health in Women With Early-Stage Postmenopausal Breast Cancer Newly on Aromatase Inhibitors: A Pilot Study.

PubMed

Taichman, L Susan; Inglehart, Marita R; Giannobile, William V; Braun, Thomas; Kolenic, Giselle; Van Poznak, Catherine

2015-07-01

Aromatase inhibitor (AI) use results in low estrogen levels, which in turn affect bone mineral density (BMD). Periodontitis, alveolar bone loss, and tooth loss are associated with low BMD. The goal of this study is to assess the prevalence of periodontitis and perceived oral health and evaluate salivary biomarkers in postmenopausal women who are survivors of early-stage (I to IIIA) breast cancer (BCa) and receive adjuvant AI therapy. Participants included 58 postmenopausal women: 29 with BCa on AIs and 29 controls without BCa diagnoses. Baseline periodontal status was assessed with: 1) periodontal probing depth (PD); 2) bleeding on probing (BOP); and 3) attachment loss (AL). Demographic and dental utilization information was gathered by questionnaire. Linear regression modeling was used to analyze the outcomes. No differences were found in mean PD or number of teeth. The AI group had significantly more sites with BOP (27.8 versus 16.7; P = 0.02), higher worst-site AL (5.2 versus 4.0 mm; P <0.01), and more sites with dental calculus (18.2 versus 6.4; P <0.001) than controls. Linear regression adjusted for income, tobacco use, dental insurance, and previous radiation and chemotherapy exposure demonstrated that AI use increased AL by >2 mm (95% confidence interval, 0.46 to 3.92). Median salivary osteocalcin and tumor necrosis factor-α levels were significantly higher in the AI group than the control group. This first investigation of the periodontal status of women initiating adjuvant AI therapy identifies this population as having an increased risk for periodontitis.

Single-Particle Measurements of Midlatitude Black Carbon and Light-Scattering Aerosols from the Boundary Layer to the Lower Stratosphere

NASA Technical Reports Server (NTRS)

Schwartz, J. P.; Gao, R. S.; Fahey, D. W.; Thomson, D. S.; Watts, L. A.; Wilson, J. C.; Reeves, J. M.; Darbeheshti, M.; Baumgardner, D. G.; Kok, G. L.;

Endovascular Embolization of Intracranial Infectious Aneurysms in Patients Undergoing Open Heart Surgery Using n-Butyl Cyanoacrylate.

PubMed

Cheng-Ching, Esteban; John, Seby; Bain, Mark; Toth, Gabor; Masaryk, Thomas; Hui, Ferdinand; Hussain, Muhammad Shazam

2017-03-01

Mycotic aneurysms are a serious complication of infective endocarditis with increased risk of intracranial hemorrhage. Patients undergoing open heart surgery for valve repair or replacement are exposed to anticoagulants, increasing the risk of aneurysm bleeding. These patients may require endovascular or surgical aneurysm treatment prior to heart surgery, but data on this approach are scarce. Retrospective review of consecutive patients with infectious endocarditis and mycotic aneurysms treated endovascularly with Trufill n-butyl cyanoacrylate (n-BCA) at the Cleveland Clinic between January 2013 and December 2015. Nine patients underwent endovascular treatment of mycotic aneurysms with n-BCA (mean age of 39 years). On imaging, 4 patients had intracerebral hemorrhage, 2 had multiple embolic infarcts, and the rest had no imaging findings. Twelve mycotic aneurysms were detected (3 patients with 2 aneurysms). Seven aneurysms were in the M4 middle cerebral artery segment, 4 in the posterior cerebral artery distribution, and 1 in the callosomarginal branch. n-BCA was diluted in ethiodized oil (1:1 to 1:2). Embolization was achieved in a single rapid injection with immediate microcatheter removal. Complete aneurysm exclusion was achieved in all cases without complications. All patients underwent open heart surgery and endovascular embolization within a short interval, 2 with both procedures on the same day. There were no new hemorrhages after aneurysm embolization. Endovascular embolization of infectious intracranial aneurysms with liquid embolics can be performed successfully in critically ill patients requiring immediate open heart surgery and anticoagulation. Early embolization prior to and within a short interval from open heart surgery is feasible.

Smart roadside initiative macro benefit analysis project report.

DOT National Transportation Integrated Search

2015-03-31

Through the Smart Roadside Initiative (SRI), a Benefit-Cost Analysis (BCA) tool was developed for the evaluation of various new transportation technologies at a State level and to provide results that could support technology adoption by a State Depa...

Benefit-cost analysis framework for evaluating inter-city transit investment.

DOT National Transportation Integrated Search

2008-10-01

This report describes the development and application of a benefit/cost analysis (BCA) model to support the evaluation of investment decisions for intercity bus services. The model recognizes two principle types of intercity bus benefits: benefits th...

Development of the North American cargo securement standard

DOT National Transportation Integrated Search

2012-08-01

This report presents the test plan for conducting the Benefit-Cost Analysis (BCA) for the United States Department of Transportation (U.S. DOT) evaluation of the Dallas U.S. 75 Integrated Corridor Management (ICM) Initiative Demonstration. The ICM pr...

Optical improvements in the diagnosis of bladder cancer: implications for clinical practice.

PubMed

Schubert, Tina; Rausch, Steffen; Fahmy, Omar; Gakis, Georgios; Stenzl, Arnulf

2017-11-01

For over 100 years white-light cystoscopy has remained the gold-standard technique for the detection of bladder cancer (BCa). Some limitations in the detection of flat lesions (CIS), the differentiation between inflammation and malignancy, the inaccurate determination of the tumor margin status as well as the tumor depth, have led to a variety of technological improvements. The aim of this review is to evaluate the impact of these improvements in the diagnosis of BCa and their effectiveness in clinical practice. A systematic literature search was conducted according to the PRISMA statement to identify studies reporting on imaging modalities in the diagnosis of NMIBC between 2000 and 2017. A two-stage selection process was utilized to determine eligible studies. A total of 74 studies were considered for final analysis. Optical imaging technologies have emerged as an adjunct to white-light cystoscopy and can be classified according to their scope as macroscopic, microscopic and molecular. Macroscopic techniques including photodynamic diagnosis (PDD), narrow-band imaging (NBI) and the Storz Professional Image Enhancement System (IMAGE1 S, formerly known as SPIES) are similar to white-light cystoscopy, but are superior in the detection of bladder tumors by means of contrast enhancement. Especially the detection rate of very mute lesions in the bladder mucosa (CIS) could be significantly increased by the use of these methods. Microscopic imaging techniques like confocal laser endomicroscopy and optical coherence tomography permit a real-time high-resolution assessment of the bladder mucosa at a cellular and sub-cellular level with spatial resolutions similar to histology, enabling the surgeon to perform an 'optical biopsy'. Molecular techniques are based on the combination of optical imaging technologies with fluorescence labeling of cancer-specific molecular agents like antibodies. This labeling is intended to favor an optical distinction between benign and malignant tissue. Optical improvements of the standard white-light cystoscopy have proven their benefit in the detection of BCa and have found their way into clinical practice. Especially the combination of macroscopic and microscopic techniques may improve diagnostic accuracy. Nevertheless, HAL-PDD guided cystoscopy is the only approach approved for routine use in the diagnosis of BCa by most urological associations in the EU and USA to date.

Investigation of Seal-to-Floor Effects on Semi-Span Transonic Models

NASA Technical Reports Server (NTRS)

Sleppy, Mark A.; Engel, Eric A.; Watson, Kevin T.; Atler, Douglas M.

2009-01-01

In an effort to achieve the maximum possible Reynolds number (Re) when conducting production testing for flight loads aerodynamic databases, it has been the preferred practice of The Boeing Company / Commercial Airplanes (BCA) -- Loads and Dynamics Group since the early 1990's to test large scale semi-span models in the 11- By 11-Foot Transonic Wind Tunnel (TWT) leg of the Unitary Plan Wind Tunnel (UPWT) at the NASA Ames Research Center (ARC). There are many problems related to testing large scale semi-span models of high aspect ratio flexible transport wings, such as; floor boundary layer effects, wing spanwise wall effects, solid blockage buoyancy effects, floor mechanical interference effects, airflow under the model effects, or tunnel flow gradient effects. For most of these issues, BCA has developed and implemented either standard testing methods or numerical correction schemes and these will not be discussed in this document. Other researchers have reported on semi-span transonic testing correction issues, however most of the reported research has been for low Mach testing. Some of the reports for low Mach testing address the difficult problem of preventing undesirable airflow under a semi-span model while ensuring unrestricted main balance functionality, however, for transonic models this issue has gone unresolved. BCA has been cognizant for sometime that there are marked differences in wing pressure distributions from semi-span transonic model testing than from full model or flight testing. It has been suspected that these differences are at least in part due to airflow under the model. Previous efforts by BCA to address this issue have proven to be ineffective or inconclusive and in one situation resulted in broken hardware. This paper reports on a Boeing-NASA collaborative investigation based on a series of small tests conducted between June 2006 and November 2007 in the 11 by 11 foot Transonic Wind Tunnel at NASA Ames on three large commercial jet transport configurations to assess the effects of sealing a semi-span model to the floor and to investigate efficient sealing and testing techniques. This document will show how sealing the model to the floor has a small but remarkably far reaching spanwise effect on wing pressures, wing local section forces and wing force summations.

Performance Evaluation of Protein Chip Assay for Rapid Diagnosis of Hepatitis C Virus Infection in Injection Drug Abusers.

PubMed

Dai, Zhiyan; Shu, Xin; Li, Gang; Xi, Weidong

2018-04-01

We evaluated the performance of a protein chip assay for the detection of antibodies to hepatitis C virus (HCV) peptides among injection drug abusers (IDAs) by comparing the assay with existing methods, including enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and recombinant immunoblot assay (RIBA). Seventy serum samples collected from IDAs were analyzed by protein chip assay. ELISA, RT-PCR, and RIBA assay were used to validate the results. The protein chips could detect different peptides' antibodies against HCV-C, HCV-NS3, HCV-NS4, HCV-NS5, and HCV-mix antigen; no cross reactivity between antigens was observed. Results of the protein-chip assay were compared with those of ELISA. Any inconsistency in results was validated by both RT-PCR and RIBA. Concordance between the results of the protein-chip assay and ELISA was 96.3% for positive samples and 100% for negative samples. The protein chip had a higher specificity than ELISA, a higher sensitivity than RTPCR, and similar specificity and sensitivity as compared to RIBA. The limit of detection of HCV antibodies in the protein chip assay was examined and calculated by incubation of model array with different dilutions. The protein chip assay required smaller amounts of both samples and reagents; it detected serum antibody in sample quantity as low as 3 ng/mL and at antibody dilution as low as 1:1000; its cost was low as well. The positive rates in the antiC, anti-NS3, anti-NS4, and anti-NS5 groups were significantly associated with levels of HCV RNA and the viral load. The HCV RNA and protein chip positive rate in the injection equipment-sharing group was higher than that in the non-injection equipment-sharing group. The protein chip assay is a faster and simpler approach to simultaneously screen for all HCV peptide antibodies accurately and to provide a rapid diagnosis of HCV infection in IDAs. The dominant positive HCV peptide antibodies were significantly associated with HCV RNA load, especially in the injection equipment-sharing group.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

PubMed

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

PubMed

Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

2016-02-01

Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

2016-02-12

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative tomore » other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.« less

Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

PubMed

Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

2016-01-01

The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

PubMed Central

Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

2016-01-01

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

U-2012: An improved Lowry protein assay, insensitive to sample color, offering reagent stability and enhanced sensitivity.

PubMed

Upreti, Girish C; Wang, Yanming; Finn, Alona; Sharrock, Abigail; Feisst, Nicholas; Davy, Marcus; Jordan, Robert B

2012-03-01

Traditional colorimetric protein assays such as Biuret, Lowry, and modified Lowry (U-1988) are unsuitable for colored biological samples. Here we describe an improved Lowry protein assay (U-2012), which utilizes stable reagents and offers enhanced sensitivity over the U-1988 assay. U-2012 circumvents interference from colored pigments and other substances (for example sugars) bound to perchloric acid (PCA) precipitated proteins by hydrogen peroxide (H2O2) induced oxidation at 50°C. Unused hydrogen peroxide is neutralized with sodium pyruvate before protein estimation for a stable end color. The U-2012 assay is carried out on the PCA precipitated protein pellet after neutralization (with Na2CO3 plus NaOH), solubilization (in Triton-NaCl), decolorization (by H2O2) and pyruvate treatment. Protein contents in red wine and homogenates of beetroot and blueberry are calculated from standard curves established for various proteins and generated using a rectangular hyperbola with parameters estimated with Microsoft Excel's Solver add-in. The U-2012 protein assay represents an improvement over U-1988 and gives a more accurate estimation of protein content.

Comparative biochemical studies of fresh frozen plasma and pooled solvent/detergent-treated plasma (octaplasLG® ) with focus on protein S and its impact in different thrombin generation assay set-ups.

PubMed

Heger, A; Janisch, S; Pock, K; Römisch, J

2016-10-01

The solvent/detergent treatment enables effective and robust inactivation of all lipid-enveloped viruses, but also inactivates partly sensitive plasma proteins such as protein S. The aim of this study was to investigate the thrombin generation capacity of octaplasLG ® , in particular focusing on the function of protein S in thrombin generation assay and the impact of assay settings. Sixteen octaplasLG ® batches and 32 units of single donor fresh frozen plasma (FFP) were investigated. For protein S, both functional activity and free antigen levels were measured. Thrombin generation assay was performed using two fluorogenic tests with different triggers. Finally, rotational thromboelastometry was performed. Mean protein S levels were lower in octaplasLG ® , but a wider range of values was found for FFP. Clotting parameters and thrombin generation capacities overlapped between the two plasma groups as demonstrated using both thrombin generation assays and different triggers. Spiking studies with protein S-depleted plasma, human purified protein S or antibodies against protein S confirmed a correlation between protein S and thrombin generation capacity under specific assay conditions, especially in an assay with low tissue factor concentration. Correlation between protein S and thrombin generation capacity was demonstrated in the TGA. Due to higher variability in protein S content in the FFP group, overlapping haemostatic potentials of the two plasma groups were found. © 2016 International Society of Blood Transfusion.

An Analysis of Internal Controls for DoD Contract Management

DTIC Science & Technology

2015-12-01

61 xiv THIS PAGE INTENTIONALLY LEFT BLANK xv LIST OF ACRONYMS AND ABBREVIATIONS BCA Business Case Analysis BPA ...indefinite quantity (IDIQ) and blanket purchase agreements ( BPA ), which can provide the government flexibility on ordering recurring requirements. Figure 5

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

PubMed

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine.

PubMed

Akimoto, Masaru; Hokazono, Eisaku; Ota, Eri; Tateishi, Takiko; Kayamori, Yuzo

2016-01-01

Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results. We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components. Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations. The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein. © The Author(s) 2015.

A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

DOE PAGES

Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

2015-07-14

Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Westernmore » blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.« less

Nucleolar molecular signature of pluripotent stem cells.

PubMed

Pliss, Artem; Kuzmin, Andrey N; Kachynski, Aliaksandr V; Jiang, Houbo; Hu, Zhixing; Ren, Yong; Feng, Jian; Prasad, Paras N

2013-04-02

Induced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells to the pluripotent state. Identification and quantitative characterization of changes in the molecular organization of the cell during the process of cellular reprogramming is valuable for stem cell research and advancement of its therapeutic applications. Here we employ quantitative Raman microspectroscopy and biomolecular component analysis (BCA) for a comparative analysis of the molecular composition of nucleoli in skin fibroblasts and iPSC derived from them. We report that the cultured fibroblasts obtained from different human subjects, share comparable concentrations of proteins, RNA, DNA, and lipids in the molecular composition of nucleoli. The nucleolar molecular environment is drastically changed in the corresponding iPSC. We measured that the transition from skin fibroblasts to iPSC is accompanied by a statistically significant increase in protein concentrations ~1.3-fold, RNA concentrations ~1.3-fold, and DNA concentrations ~1.4-fold, while no statistically significant difference was found for the lipid concentrations. The analysis of molecular vibrations associated with diverse aminoacids and protein conformations indicates that nucleoli of skin fibroblasts contain similar subsets of proteins, with prevalence of tyrosine. In iPSC, we observed a higher signal from tryptophan with an increase in the random coil and α helix protein conformations, indicating changes in the subset of nucleolar proteins during cell reprogramming. At the same time, the concentrations of major types of macromolecules and protein conformations in the nucleoli of iPSC and human embryonic stem cells (hESC) were found to be similar. We discuss these results in the context of nucleolar function and conclude that the nucleolar molecular content is correlated with the cellular differentiation status. The approach described here shows the potential for spectroscopically monitoring changes in macromolecular organization of the cell at different stages of reprogramming.

A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

PubMed Central

Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

2017-01-01

Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

De novo self-assembling collagen heterotrimers using explicit positive and negative design.

PubMed

Xu, Fei; Zhang, Lei; Koder, Ronald L; Nanda, Vikas

2010-03-23

We sought to computationally design model collagen peptides that specifically associate as heterotrimers. Computational design has been successfully applied to the creation of new protein folds and functions. Despite the high abundance of collagen and its key role in numerous biological processes, fibrous proteins have received little attention as computational design targets. Collagens are composed of three polypeptide chains that wind into triple helices. We developed a discrete computational model to design heterotrimer-forming collagen-like peptides. Stability and specificity of oligomerization were concurrently targeted using a combined positive and negative design approach. The sequences of three 30-residue peptides, A, B, and C, were optimized to favor charge-pair interactions in an ABC heterotrimer, while disfavoring the 26 competing oligomers (i.e., AAA, ABB, BCA). Peptides were synthesized and characterized for thermal stability and triple-helical structure by circular dichroism and NMR. A unique A:B:C-type species was not achieved. Negative design was partially successful, with only A + B and B + C competing mixtures formed. Analysis of computed versus experimental stabilities helps to clarify the role of electrostatics and secondary-structure propensities determining collagen stability and to provide important insight into how subsequent designs can be improved.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

PubMed

Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan

2005-05-31

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination.

PubMed

Field, Anjalie; Field, Jeffrey

2010-08-01

In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives.

Management of congenital neck lesions in children.

PubMed

Erikci, Volkan; Hoşgör, Münevver

2014-09-01

A retrospective clinical trial was conducted to evaluate the clinical features and treatment outcomes and to determine the incidence of complications in children with congenital neck lesions (CNLs) treated at our institution with a special emphasis on thyroglossal duct remnant (TGDR), branchial cleft anomaly (BCA), and dermoid cyst (DC). This series had 72 patients with CNL. The diagnosis of CNL was made by physical examination, ultrasound (US) in most, and for a potential extension of the mass computed tomography (CT) or magnetic resonance imaging (MRI) in a few patients and confirmed by histopathological examination in all of the children. Of the patients in this series, 39 (54.2%) children had thyroglossal duct remnant (TGDR). The most common surgical procedure (n = 36) in these children was Sistrunk's procedure. Four children (10.3%) with TGDR had associated anomalies including Turner syndrome and Morgagni hernia. During the study period, 25 (34.7%) children with branchial cleft anomaly (BCA) were treated and most of these were second branchial anomalies. There were eight children (11.1%) with dermoid cyst (DC). TGDR is the most common CNL and is presented clinically rather late with regard to BCA and DC in this series. Surgical resection is optimal choice of therapy in CNLs not only for aesthetic reasons but also for the recurrent infections and the potential danger of malignancy. Definitive surgery may be associated with high morbidity, especially recurrence. Associated anomalies may be observed, especially in children with TGDR. Although the Sistrunk's procedure is a safe and successful technique, life-threatening complications should also be kept in mind during the management of these lesions and early and adequate surgical treatment is suggested. Copyright © 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Prognostic Factors, Treatment, and Outcomes in Early Stage, Invasive Papillary Breast Cancer: A SEER Investigation of Less Aggressive Treatment in a Favorable Histology.

PubMed

Fakhreddine, Mohamad H; Haque, Waqar; Ahmed, Awad; Schwartz, Mary R; Farach, Andrew M; Paulino, Arnold C; Bonefas, Elizabeth; Miltenburg, Darlene; Niravath, Polly; Butler, E Brian; Teh, Bin S

2018-06-01

Invasive papillary breast cancer (IPBCA) represents 0.5% of invasive BCA, and is thought to carry a favorable prognosis. This population-based study reports on prognostic factors, treatment, and outcomes of early-stage IPBCA to explore whether there is any evidence to support less aggressive treatment. IPBCA cases from 1990 to 2009 of the recent Surveillance, Epidemiology, and End Results were analyzed. Inclusion criteria included patients with stage T1-2, N0 IPBCA. Univariate and multivariate analyses were performed using the variables of treatment, stage, race, hormone receptor status, grade (G1-3), and age. Treatment modalities included lumpectomy alone (LA), lumpectomy with radiation treatment (LRT), and mastectomy alone (MA). Among 10,485 patients, median follow-up was 56 months. Five and 10-year overall survival (OS) were 93.1% and 76.8%, respectively. Patients treated with LRT had superior mean OS 16.8 versus 14.9 years for MA (P=0.0004) and 14.2 years for LA (P=0.0003). Improved OS also correlated with lower histologic grade (P<0.0001), lower T-stage (P<0.0001), and younger age (P<0.0001). Black patients had the worst OS (12.7 y, P<0.0001). LRT is associated with superior OS for early-stage invasive papillary BCA patients, when compared with LA or MA in this population-based study. The findings support the standard of care breast conservation approach for patients with invasive papillary BCA. Other prognostic factors associated with worse OS include increased age, higher T-stage, higher histologic grade, and black race.

Associations of perceived social support and positive psychological resources with fatigue symptom in patients with rheumatoid arthritis

PubMed Central

Xu, NeiLi; Zhao, Shuai; Xue, HongXia; Fu, WenYi; Liu, Li; Zhang, TianQi; Huang, Rui; Zhang, Ning

2017-01-01

Objective This study aimed to assess the association between perceived social support (PSS) and fatigue and the roles of hope, optimism, general self-efficacy and resilience as mediators or moderators on PSS-fatigue association among Rheumatoid Arthritis (RA) patients in China. Methods A multi-center, cross-sectional study was conducted withinpatients diagnosed with RA in northeast China, in which 305 eligible inpatients were enrolled. The Multidimensional Fatigue Inventory, Multidimensional Scale of Perceived Social Support, Herth Hope Index, Life Orientation Test Revised, General Self-Efficacy Scale and Ego-Resiliency Scale were completed. The associations of PSS, hope, optimism, general self-efficacy and resilience with fatigue and the moderating roles of these positive psychological constructs were tested by hierarchical linear regression. Asymptotic and resampling strategies were utilized to assess the mediating roles of hope, optimism, general self-efficacy and resilience. Results The mean score of the MFI was 57.88 (SD = 9.50). PSS, hope, optimism and resilience were negatively associated with RA-related fatigue, whereas DAS28-CRP was positively associated. Only resilience positively moderated the PSS-fatigue association (B = 0.03, β = 0.13, P<0.01). Hope, optimism and resilience may act as partial mediators in the association between PSS and fatigue symptoms (hope: a*b = -0.16, BCa 95%CI: -0.27, -0.03; optimism: a*b = -0.20, BCa 95%CI: -0.30, -0.10; resilience: a*b = -0.12, BCa 95%CI: -0.21–0.04). Conclusions Fatigue is a severe symptom among RA patients. Resilience may positively moderate the PSS-fatigue association. Hope, optimism and resilience may act as partial mediators in the association. PSS, hope, optimism and resilience may contribute as effective recourses to alleviate fatigue, upon which PSS probably has the greatest effect. PMID:28291837

Associations of perceived social support and positive psychological resources with fatigue symptom in patients with rheumatoid arthritis.

PubMed

Xu, NeiLi; Zhao, Shuai; Xue, HongXia; Fu, WenYi; Liu, Li; Zhang, TianQi; Huang, Rui; Zhang, Ning

2017-01-01

This study aimed to assess the association between perceived social support (PSS) and fatigue and the roles of hope, optimism, general self-efficacy and resilience as mediators or moderators on PSS-fatigue association among Rheumatoid Arthritis (RA) patients in China. A multi-center, cross-sectional study was conducted withinpatients diagnosed with RA in northeast China, in which 305 eligible inpatients were enrolled. The Multidimensional Fatigue Inventory, Multidimensional Scale of Perceived Social Support, Herth Hope Index, Life Orientation Test Revised, General Self-Efficacy Scale and Ego-Resiliency Scale were completed. The associations of PSS, hope, optimism, general self-efficacy and resilience with fatigue and the moderating roles of these positive psychological constructs were tested by hierarchical linear regression. Asymptotic and resampling strategies were utilized to assess the mediating roles of hope, optimism, general self-efficacy and resilience. The mean score of the MFI was 57.88 (SD = 9.50). PSS, hope, optimism and resilience were negatively associated with RA-related fatigue, whereas DAS28-CRP was positively associated. Only resilience positively moderated the PSS-fatigue association (B = 0.03, β = 0.13, P<0.01). Hope, optimism and resilience may act as partial mediators in the association between PSS and fatigue symptoms (hope: a*b = -0.16, BCa 95%CI: -0.27, -0.03; optimism: a*b = -0.20, BCa 95%CI: -0.30, -0.10; resilience: a*b = -0.12, BCa 95%CI: -0.21-0.04). Fatigue is a severe symptom among RA patients. Resilience may positively moderate the PSS-fatigue association. Hope, optimism and resilience may act as partial mediators in the association. PSS, hope, optimism and resilience may contribute as effective recourses to alleviate fatigue, upon which PSS probably has the greatest effect.

Protein subcellular localization assays using split fluorescent proteins

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

Expression profiles and clinical value of plasma exosomal Tim-3 and Galectin-9 in non-small cell lung cancer.

PubMed

Gao, Jianwei; Qiu, Xiangyu; Li, Xinying; Fan, Hang; Zhang, Fang; Lv, Tangfeng; Song, Yong

2018-04-06

Exosomes are membrane-bound, virus-sized vesicles present in circulating blood. Tumor cells are avid producers of exosomes, which are thought to mimic molecular features of parent tumor cells. T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) is a the next-generation immune checkpoint that can be activated by its ligand Galectin-9 to negatively regulate the anti-tumor immune response. However, the characteristics of plasma exosomal Tim-3 and Galectin-9 (Exo-T/G) in cancer remained unknown. This study was conducted to investigate the expression patterns and clinical value of plasma exosomal total protein (Exo-pro) and Exo-T/G in non-small cell lung cancer (NSCLC). Plasma was collected from 103 NSCLC patients including 60 early stages and 43 advanced stages disease samples as well as 56 healthy subjects. Exosomes were isolated from plasma by commercial exosome precipitation solution and identified by western blotting of CD63 and transmission electron microscopy. Exo-pro concentration was measured by the BCA assay. Enzyme-linked immunosorbent assay was used to quantify Exo-T/G. Additionally, 34 NSCLC samples were applied to directly detect plasma TIM-3 (Plas-T) and Galectin-9 (Plas-G). Our results showed that Exo-pro, Exo-T, and Exo-G were significantly increased in NSCLC plasma compared to that in the healthy samples. High levels of Exo-T and Exo-G were all positively correlated with several malignant parameters, including larger tumor size, advanced stages, and more distant metastasis. High levels of Exo-pro and Exo-T were also correlated with more lymph node metastasis. Additionally, plasma from lung squamous cell carcinoma showed higher Exo-T and Exo-G compared with that from lung adenocarcinoma. ALK-positive patients showed to have decreased Exo-T and Exo-G levels. Pearson's correlation analysis revealed a significant correlation between Exo-pro and Exo-T/G, Exo-T and Exo-G, Exo-T and Plas-T, Exo-G and Plas-G, and Plas-T and Plas-G. Together, our data revealed that Exo-pro, especially Exo-T and Exo-G could be potential biomarkers for NSCLC. Further studies focusing on pure tumor-derived exosomes isolated from plasma were needed. Copyright © 2018 Elsevier Inc. All rights reserved.

Advantages and application of label-free detection assays in drug screening.

PubMed

Cunningham, Brian T; Laing, Lance G

2008-08-01

Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

Protein and cell wall polysaccharide carbonyl determination by a neutral pH 2,4-dinitrophenylhydrazine-based photometric assay.

PubMed

Georgiou, Christos D; Zisimopoulos, Dimitrios; Argyropoulou, Vasiliki; Kalaitzopoulou, Electra; Salachas, George; Grune, Tilman

2018-04-10

A new 2,4-dinitrophenylhydrazine (DNPH)-based photometric assay is developed for the quantification of carbonyls in protein samples from any biological source by protein carbonyl-DNPH hydrazone formation at acidic pH in the presence of denaturing urea, and subsequent hydrazone solubilization in the presence of SDS and stabilization from acid hydrolysis at pH 7.0. At this neutral (ntr) pH, interfering unreacted DNPH is uncharged and its thus increased hydrophobicity permits its 100% effective removal from the solubilizate with ethyl acetate/hexane wash. The ntrDNPH assay is more reliable and sensitive than the standard (std) DNPH photometric assay because it eliminates its main limitations: (i) interfering unreacted DNPH (pKa 1.55) that is nonspecifically bound to the TCA (pKa 0.7)-protein pellet is not effectively removed after wash with EtOH: ethyl acetate because it is positively charged, (ii) acid (TCA-induced) hydrolysis of the protein carbonyl-DNPH hydrazone, (iii) sample protein concentration re-determination, (iv) loss of sample acid (TCA)-soluble proteins, (v) DNA interference, and (vi) requires high protein quantity samples (≥ 1 mg). Considering ntrDNPH assay's very low protein limit (1 µg), its cumulative and functional sensitivities are 2600- and 2000-fold higher than those of the stdDNPH assay, respectively. The present study elucidates the DNA interference mechanism on the stdDNPH assay, and also develops a standardized protocol for sample protein treatment and fractionation (into cytoplasmic/aqueous, membrane/lipid-bound, and histone/DNA-bound proteins; see Supplement section V) in order to ensure reproducible carbonyl determination on defined cell protein fractions, and to eliminate assay interference from protein samples containing (i) Cys sulfenic acid groups (via their neutralization with dithiothreitol), and (ii) DNA (via its removal by streptomycin sulfate precipitation). Lastly, the ntrDNPH assay determines carbonyl groups on cell wall polysaccharides, thus paving the way on studies to investigate cell walls acting as antioxidant defense in plants, fungi, bacteria and lichens. Copyright © 2018. Published by Elsevier B.V.

[Effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice].

PubMed

Jin, Xin; Zhang, Hui-xin; Zhang, Yan-fen; Cui, Wen-wen; Bi, Yao; He, Qi-long; Zhou, Sheng-shan

2015-03-01

To study the effect of Jinlida on changes in expression of skeletal muscle lipid transport enzymes in fat-induced insulin resistance ApoE -/- mice. Eight male C57BL/6J mice were selected in the normal group (NF), 40 male ApoE -/- mice were fed for 16 weeks, divided into the model group (HF), the rosiglitazone group ( LGLT), the Jinlida low-dose group (JLDL), the Jinlida medium-dose group (JLDM), the Jinlida high-dose group (JLDH) and then orally given drugs for 8 weeks. The organization free fatty acids, BCA protein concentration determination methods were used to determine the skeletal muscle FFA content. The Real-time fluorescent quantitative reverse transcription PCR ( RT-PCR) and Western blot method were adopted to determine mRNA and protein expressions of mice fatty acids transposition enzyme (FAT/CD36), carnitine palm acyltransferase 1 (CPT1), peroxide proliferators-activated receptor α( PPAR α). Jinlida could decrease fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), free fatty acid (FFA) and fasting insulin (FIns) and raise insulin sensitive index (ISI) in mice to varying degrees. It could also up-regulate mRNA and protein expressions of CPT1 and PPARα, and down-regulate mRNA and protein levels of FAT/CD36. Jinlida can improve fat-induced insulin resistance ApoE -/- in mice by adjusting the changes in expression of skeletal muscle lipid transport enzymes.

Integrated corridor management initiative : demonstration phase evaluation – San Diego benefit-cost analysis test plan.

DOT National Transportation Integrated Search

2012-08-01

This report presents the test plan for conducting the Benefit-Cost Analysis (BCA) for the United States Department of Transportation (U.S. DOT) evaluation of the San Diego Integrated Corridor Management (ICM) Initiative Demonstration. The ICM project...

Biochanin A improves fiber fermentation by cellulolytic bacteria

USDA-ARS?s Scientific Manuscript database

The objective was to determine the effect of the isoflavone biochanin A (BCA) on rumen cellulolytic bacteria and consequent fermentative activity. When bovine microbial rumen cell suspensions (n = 3) were incubated (24 h, 39 °C) with ground hay, cellulolytic bacteria proliferated, short chain fatty...

How Can Bee Colony Algorithm Serve Medicine?

PubMed Central

Salehahmadi, Zeinab; Manafi, Amir

2014-01-01

Healthcare professionals usually should make complex decisions with far reaching consequences and associated risks in health care fields. As it was demonstrated in other industries, the ability to drill down into pertinent data to explore knowledge behind the data can greatly facilitate superior, informed decisions to ensue the facts. Nature has always inspired researchers to develop models of solving the problems. Bee colony algorithm (BCA), based on the self-organized behavior of social insects is one of the most popular member of the family of population oriented, nature inspired meta-heuristic swarm intelligence method which has been proved its superiority over some other nature inspired algorithms. The objective of this model was to identify valid novel, potentially useful, and understandable correlations and patterns in existing data. This review employs a thematic analysis of online series of academic papers to outline BCA in medical hive, reducing the response and computational time and optimizing the problems. To illustrate the benefits of this model, the cases of disease diagnose system are presented. PMID:25489530

How can bee colony algorithm serve medicine?

PubMed

Salehahmadi, Zeinab; Manafi, Amir

2014-07-01

Healthcare professionals usually should make complex decisions with far reaching consequences and associated risks in health care fields. As it was demonstrated in other industries, the ability to drill down into pertinent data to explore knowledge behind the data can greatly facilitate superior, informed decisions to ensue the facts. Nature has always inspired researchers to develop models of solving the problems. Bee colony algorithm (BCA), based on the self-organized behavior of social insects is one of the most popular member of the family of population oriented, nature inspired meta-heuristic swarm intelligence method which has been proved its superiority over some other nature inspired algorithms. The objective of this model was to identify valid novel, potentially useful, and understandable correlations and patterns in existing data. This review employs a thematic analysis of online series of academic papers to outline BCA in medical hive, reducing the response and computational time and optimizing the problems. To illustrate the benefits of this model, the cases of disease diagnose system are presented.

Low publication rate of 2005 conference presentations: implications for practitioners serving individuals with autism and intellectual disabilities.

PubMed

Richling, Sarah M; Rapp, John T; Funk, Janie A; D'Agostini, Jaimie; Garrido, Natalia; Moreno, Vicki

2014-11-01

This study determined the percentage of presentations at the annual conference of the Association for Behavior Analysis in 2005 with the autism (AUT) and developmental disabilities (DDA) codes (N=880) that (a) provided continuing education credits (CEs) for Board Certified Behavior Analysts (BCBAs) and Board Certified Assistant Behavior Analysts (BCaBAs) and (b) included content that was published in a peer-reviewed outlet. Results indicate that only 77 (8.8%) presentations were ultimately published. Although posters were not eligible for CEs, posters accounted for 57.1% of the published presentations. Specifically, posters presented by a university-affiliated presenter accounted for 44.2% of presentations with published content. As a whole, only 10.4% of AUT and DDA presentations offering CEs contained data sets that were published. Considered together, these results suggest that the content provided to BCBAs and BCaBAs for CEs may not be adequately measured or sufficiently rigorous to guide clinical practices. Copyright © 2014 Elsevier Ltd. All rights reserved.

RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

PubMed

Roussis, Ioannis M; Guille, Matthew; Myers, Fiona A; Scarlett, Garry P

2016-01-01

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation-Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.

Protein immobilization techniques for microfluidic assays

PubMed Central

Kim, Dohyun; Herr, Amy E.

2013-01-01

Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers

PubMed Central

2018-01-01

Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described. PMID:29641569

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

PubMed

Ni, Yan G; Yuan, Xiling; Newitt, John A; Peterson, Jon E; Gleason, Carol R; Haulenbeek, Jonathan; Santockyte, Rasa; Lafont, Virginie; Marsilio, Frank; Neely, Robert J; DeSilva, Binodh; Piccoli, Steven P

2015-07-01

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Smart roadside initiative macro benefit analysis : user’s guide for the benefit-cost analysis tool.

DOT National Transportation Integrated Search

2015-03-01

Through the Smart Roadside Initiative (SRI), a Benefit-Cost Analysis (BCA) tool was developed for the evaluation of various new transportation technologies at a State level and to provide results that could support technology adoption by a State Depa...

Confidence Interval Coverage for Cohen's Effect Size Statistic

ERIC Educational Resources Information Center

Algina, James; Keselman, H. J.; Penfield, Randall D.

2006-01-01

Kelley compared three methods for setting a confidence interval (CI) around Cohen's standardized mean difference statistic: the noncentral-"t"-based, percentile (PERC) bootstrap, and biased-corrected and accelerated (BCA) bootstrap methods under three conditions of nonnormality, eight cases of sample size, and six cases of population…

Cupric Oxide (CuO) Oxidation Detects Pyrogenic Carbon in Burnt Organic Matter and Soils.

PubMed

Hatten, Jeff; Goñi, Miguel

2016-01-01

Wildfire greatly impacts the composition and quantity of organic carbon stocks within watersheds. Most methods used to measure the contributions of fire altered organic carbon-i.e. pyrogenic organic carbon (Py-OC) in natural samples are designed to quantify specific fractions such as black carbon or polyaromatic hydrocarbons. In contrast, the CuO oxidation procedure yields a variety of products derived from a variety of precursors, including both unaltered and thermally altered sources. Here, we test whether or not the benzene carboxylic acid and hydroxy benzoic acid (BCA) products obtained by CuO oxidation provide a robust indicator of Py-OC and compare them to non-Py-OC biomarkers of lignin. O and A horizons from microcosms were burned in the laboratory at varying levels of fire severity and subsequently incubated for 6 months. All soils were analyzed for total OC and N and were analyzed by CuO oxidation. All BCAs appeared to be preserved or created to some degree during burning while lignin phenols appeared to be altered or destroyed to varying extents dependent on fire severity. We found two specific CuO oxidation products, o-hydroxybenzoic acid (oBd) and 1,2,4-benzenetricarboxylic acid (BTC2) that responded strongly to burn severity and withstood degradation during post-burning microbial incubations. Interestingly, we found that benzene di- and tricarboxylic acids (BDC and BTC, respectively) were much more reactive than vanillyl phenols during the incubation as a possible result of physical protection of vanillyl phenols in the interior of char particles or CuO oxidation derived BCAs originating from biologically available classes of Py-OC. We found that the ability of these compounds to predict relative Py-OC content in burned samples improved when normalized by their respective BCA class (i.e. benzene monocarboxylic acids (BA) and BTC, respectively) and when BTC was normalized to total lignin yields (BTC:Lig). The major trends in BCAs imparted by burning persisted through a 6 month incubation suggesting that fire severity had first order control on BCA and lignin composition. Using original and published BCA data from soils, sediments, char, and interfering compounds we found that BTC:Lig and BTC2:BTC were able to distinguish Py-OC from compounds such as humic materials, tannins, etc. The BCAs released by the CuO oxidation procedure increase the functionality of this method in order to examine the relative contribution of Py-OC in geochemical samples.

Life satisfaction among elderly patients in Nepal: associations with nutritional and mental well-being.

PubMed

Ghimire, Saruna; Baral, Binaya Kumar; Karmacharya, Isha; Callahan, Karen; Mishra, Shiva Raj

2018-06-07

Nepal's demography is aging rapidly, yet few studies to date have examined how this has affected the health and well-being of the elderly, defined as those above 60 years in Nepal's Senior Citizen Act (2006). Our study, abbreviated NepEldQOLII, aims to assess perceived life satisfaction, and evaluate its relationship with nutritional health and mental well-being among the burgeoning Nepalese elderly population. A cross-sectional survey among 289 Nepalese elderly, aged ≥60 years, attending an outpatient clinic of a hospital in Kathmandu, Nepal was conducted. Nutritional status, depression, and life satisfaction were assessed by a mini-nutritional assessment scale (range: 0-14), a geriatric depression scale (range: 0-15), and a satisfaction with life scale (range: 5-35), respectively. Mediation analyses, adjusted for age, sex, marital status, and family type, were used to assess mediating relationships between nutritional and mental wellbeing with life satisfaction as the outcome. Approximately 21% of the participants were dissatisfied with their life. Life satisfaction was positively associated with being married, high family income, involvement in active earning, and a high nutritional score. Conversely, life satisfaction was inversely associated with living in a nuclear (as opposed to joint) family, the perception of having worse health than peers, the perception of being ignored/hated due to old age, and a higher depression score. In mediation analyses, both nutrition (β = 0.48, bias-corrected and accelerated (BCa) 95% CI: 0.27, 0.69) and depression (β = - 0.87, BCa 95% CI: -1.01, - 0.74) had significant direct associations with life satisfaction. Furthermore, both nutrition (β = 0.30, BCa 95% CI: 0.13, 0.49) and depression (β = - 0.07, BCa 95% CI: -0.14, - 0.03) mediate each other's association with life satisfaction. Nutritional score mediated 7% of the total association between depression and life satisfaction; depression mediated 38% of the total association between nutrition and life satisfaction. Life satisfaction shows a pattern of decline as nutritional and mental health status decrease. Both depression and under-nutrition had a significant association with life satisfaction. The pathway by which nutrition affects life satisfaction is influenced by depression as a mediator. Moreover, nutritional status explained a small portion of the relationship between depression and life satisfaction. These observed preliminary findings should be confirmed in future longitudinal studies.

Cupric Oxide (CuO) Oxidation Detects Pyrogenic Carbon in Burnt Organic Matter and Soils

PubMed Central

Hatten, Jeff; Goñi, Miguel

2016-01-01

Wildfire greatly impacts the composition and quantity of organic carbon stocks within watersheds. Most methods used to measure the contributions of fire altered organic carbon–i.e. pyrogenic organic carbon (Py-OC) in natural samples are designed to quantify specific fractions such as black carbon or polyaromatic hydrocarbons. In contrast, the CuO oxidation procedure yields a variety of products derived from a variety of precursors, including both unaltered and thermally altered sources. Here, we test whether or not the benzene carboxylic acid and hydroxy benzoic acid (BCA) products obtained by CuO oxidation provide a robust indicator of Py-OC and compare them to non-Py-OC biomarkers of lignin. O and A horizons from microcosms were burned in the laboratory at varying levels of fire severity and subsequently incubated for 6 months. All soils were analyzed for total OC and N and were analyzed by CuO oxidation. All BCAs appeared to be preserved or created to some degree during burning while lignin phenols appeared to be altered or destroyed to varying extents dependent on fire severity. We found two specific CuO oxidation products, o-hydroxybenzoic acid (oBd) and 1,2,4-benzenetricarboxylic acid (BTC2) that responded strongly to burn severity and withstood degradation during post-burning microbial incubations. Interestingly, we found that benzene di- and tricarboxylic acids (BDC and BTC, respectively) were much more reactive than vanillyl phenols during the incubation as a possible result of physical protection of vanillyl phenols in the interior of char particles or CuO oxidation derived BCAs originating from biologically available classes of Py-OC. We found that the ability of these compounds to predict relative Py-OC content in burned samples improved when normalized by their respective BCA class (i.e. benzene monocarboxylic acids (BA) and BTC, respectively) and when BTC was normalized to total lignin yields (BTC:Lig). The major trends in BCAs imparted by burning persisted through a 6 month incubation suggesting that fire severity had first order control on BCA and lignin composition. Using original and published BCA data from soils, sediments, char, and interfering compounds we found that BTC:Lig and BTC2:BTC were able to distinguish Py-OC from compounds such as humic materials, tannins, etc. The BCAs released by the CuO oxidation procedure increase the functionality of this method in order to examine the relative contribution of Py-OC in geochemical samples. PMID:27011012

Differences in MYB expression and gene abnormalities further confirm that salivary cribriform basal cell tumors and adenoid cystic carcinoma are two distinct tumor entities.

PubMed

Tian, Zhen; Li, Lei; Zhang, Chun-Ye; Gu, Ting; Li, Jiang

2016-10-01

In practices, some cases of salivary basal cell tumors that consist mainly of cribriform growth pattern are difficult to differentiate from adenoid cystic carcinoma (AdCC). Identification of reliable molecular biomarkers for the differential diagnosis between them is required. Twenty-two cases of cribriform salivary basal cell tumors (at least 10% cribriform pattern present in each tumor) comprising 18 cases of basal cell adenoma (BCA) and four cases of basal cell adenocarcinoma (BcAC) were collected between 1985 and 2008. Twenty cases of cribriform AdCC were retrieved from our archives. MYB protein expression and gene abnormalities were detected in all cases by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) analyses, respectively. Neither MYB protein nor split genes were detected in any of the cases of cribriform basal cell tumors, while 55% (11/20) of cases of cribriform AdCC had MYB protein expression. High MYB expression was detected in 81.8% (9/11) cases, while low expression was found in the remaining cases. FISH analysis indicated that nine AdCC tumors with high MYB protein expression were split gene-positive, while MYB gene splitting was not detected in the 11 cases with low or absent MYB protein expression. The molecular changes in AdCC differ from those associated with cribriform basal cell tumors, which further confirms that cribriform basal cell tumors and AdCC are two distinct tumor entities. Simultaneous detection of MYB protein expression and the associated molecular changes could be beneficial in differentiating salivary cribriform basal cell tumors from AdCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics.

PubMed

Sun, Kaiwen; Zheng, Yuyu; Zhu, Ziqiang

2017-11-20

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

PubMed Central

Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei

2012-01-01

A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation. PMID:23346268

Cysteine residue is not essential for CPM protein thermal-stability assay.

PubMed

Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

2015-05-01

A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

PubMed

Tsuganezawa, Keiko; Shinohara, Yoshiyasu; Ogawa, Naoko; Tsuboi, Shun; Okada, Norihisa; Mori, Masumi; Yokoyama, Shigeyuki; Noda, Nobuo N; Inagaki, Fuyuhiko; Ohsumi, Yoshinori; Tanaka, Akiko

2013-10-01

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

Evaluation of Multiplexed Foot-and-Mouth Disease Nonstructural Protein Antibody Assay Against Standardized Bovine Serum Panel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perkins, J; Parida, S; Clavijo, A

2007-05-14

Liquid array technology has previously been used to show proof-of-principle of a multiplexed non structural protein serological assay to differentiate foot-and-mouth infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B and 3D and recombinant protein signature 3ABC in combination with four controls. To determine diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright,more » UK. This sera panel has been used to assess the performance of other singleplex ELISA-based non-structural protein antibody assays. The 3ABC signature in the multiplexed assay showed comparative performance to a commercially available non-structural protein 3ABC ELISA (Cedi test{reg_sign}) and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex is acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promotes further assay development and optimization to generate an assay for routine use in foot-and-mouth disease surveillance.« less

Parallel Force Assay for Protein-Protein Interactions

PubMed Central

Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

Parallel force assay for protein-protein interactions.

PubMed

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Comparison of total protein concentration in skeletal muscle as measured by the Bradford and Lowry assays.

PubMed

Seevaratnam, Rajini; Patel, Barkha P; Hamadeh, Mazen J

2009-06-01

The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland-Altman analysis revealed considerable bias (15.8 +/- 29.7%). Both Lowry reagents and its protein-reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05-0.50 mg/ml) than the Bradford (0-2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein-reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats

EPA Science Inventory

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo a...

Coefficient Omega Bootstrap Confidence Intervals: Nonnormal Distributions

ERIC Educational Resources Information Center

Padilla, Miguel A.; Divers, Jasmin

2013-01-01

The performance of the normal theory bootstrap (NTB), the percentile bootstrap (PB), and the bias-corrected and accelerated (BCa) bootstrap confidence intervals (CIs) for coefficient omega was assessed through a Monte Carlo simulation under conditions not previously investigated. Of particular interests were nonnormal Likert-type and binary items.…

Bootstrapping Confidence Intervals for Robust Measures of Association.

ERIC Educational Resources Information Center

King, Jason E.

A Monte Carlo simulation study was conducted to determine the bootstrap correction formula yielding the most accurate confidence intervals for robust measures of association. Confidence intervals were generated via the percentile, adjusted, BC, and BC(a) bootstrap procedures and applied to the Winsorized, percentage bend, and Pearson correlation…

Atmospheric black carbon in the Russian Arctic: anthropogenic inputs in comparison with average or extremal wood fires' ones

NASA Astrophysics Data System (ADS)

Vinogradova, Anna A.; Smirnov, Nikolay S.; Korotkov, Vladimir N.

2016-04-01

Model estimates of atmospheric black carbon concentrations were made for different points of the Russian Arctic. Anthropogenic BC emissions and wood fires' ones were calculated from Russian official statistics for the 2000s. We used the data of Ministry of Natural Resources and Environment of RF on anthropogenic air emissions of pollution in Russian cities and regions [1], as well as the data of Federal Forestry Agency of RF (Rosleshoz) [2] on wood fires. We considered the area within (50-72)N and (20-180)E, which covers about 94% of the Russian territory, where both anthropogenic and fire BC emissions have been arranged through grid cells (1×1) deg. Anthropogenic BC emissions are estimated as annual values based on the data for 54 regions and more than 100 cities. Total emission is estimated as (220 ± 30) Gg BC in 2010 [3], including emissions from open flares associated with gas/oil extractive industry which are about (25 ± 8) Gg/yr. We analyzed the data on wood fires (detailing crown, ground and underground fires in forests and fires on non-forest lands) with their spatial and seasonal variations during 15 years (2000-2014). Different combustion factors [4] and BC emission coefficients [5] were used in calculations for different types of burning. Russian total average annual BC emission from fires, occurring mainly in summertime, was estimated as 30 Gg with large variations (4-100 Gg/yr) from year to year. Asian territory emits about 90% of this value. We estimated anthropogenic (BC_A) and fires' (BC_F) contributions to BC air concentrations at different Russian Arctic points using the approach [6] - decadal back-trajectory analysis combined with spatial distribution of sensitivity pollution emission function (SPEF). Extraordinary atmospheric circulation causing, to a great extent, abnormally intensive fires in the middle latitudes often leads to a decrease in SPEF values for these territories. As a result, fires are not so dangerous for the whole Arctic, as it is believed. But there are distinctions at various points: Kola Peninsula - annually BC concentrations in air are not sizable and BCA prevails, but BCF prevails in summer. SE of Arkhangelsk region - annually BCA prevails, but in summer BCA is equal to BC_F, and in summer 2010 BCF was 2 times higher. Nenetsky Nature Reserve - BCF always prevails. Gydansky Nature Reserve - BCA prevails through a year, previously from oil/gas flares. Ust'-Lensky Nature Reserve - annually BC concentrations in air are not sizable, but in summer 2012 BCF prevails and is near the same as at Nenetsky Nature Reserve. The work was supported by RFBR, grants: 14-05-00059, 14-05-93089. _____________________ 1. Yearbook 2010 on Atmospheric Emissions of Pollution in Towns and Regions of Russian Federation. St.Petersburg, SRI Atmosphere, 2011. - 560 pp. [in Russian]. 2. http://www.rosleshoz.gov.ru/; http://www.aviales.ru/ 3. Vinogradova A.A. Anthropogenic Black Carbon Emissions to the Atmosphere: Surface Distribution through Russian Territory // DOI: 10.1134/S1024856015020141 4. http://www.ipcc-nggip.iges.or.jp/public/2006gl/vol4.html 5. Akagi S.K. et al. Emission factors for open and domestic biomass burning for use in atmospheric models // DOI: 10.5194/acp-11-4039-2011 6. Vinogradova A.A. Distant evaluation of atmospheric pollution influence on the remote territories // DOI:10.1134/S0001433815070099

Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe.

PubMed

Ma, Changbei; Lv, Xiaoyuan; Wang, Kemin; Jin, Shunxin; Liu, Haisheng; Wu, Kefeng; Zeng, Weimin

2017-11-02

Protein kinase A was detected by quantifying the amount of ATP used after a protein kinase reaction. The ATP assay was performed using the T4 DNA ligase and a molecular beacon (MB). In the presence of ATP, DNA ligase catalyzed the ligation of short DNA. The ligation product then hybridized to MB, resulting in a fluorescence enhancement of the MB. This assay was capable of determining protein kinase A in the range of 12.5∼150 nM, with a detection limit of 1.25 nM. Furthermore, this assay could also be used to investigate the effect of genistein on protein kinase A. It was a universal, non-radioisotopic, and homogeneous method for assaying protein kinase A.

A novel assay to identify the trafficking proteins that bind to specific vesicle populations

PubMed Central

Bentley, Marvin; Banker, Gary

2016-01-01

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin–binding domain (FRB)–tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled. PMID:26621371

A scalable double-barcode sequencing platform for characterization of dynamic protein-protein interactions.

PubMed

Schlecht, Ulrich; Liu, Zhimin; Blundell, Jamie R; St Onge, Robert P; Levy, Sasha F

2017-05-25

Several large-scale efforts have systematically catalogued protein-protein interactions (PPIs) of a cell in a single environment. However, little is known about how the protein interactome changes across environmental perturbations. Current technologies, which assay one PPI at a time, are too low throughput to make it practical to study protein interactome dynamics. Here, we develop a highly parallel protein-protein interaction sequencing (PPiSeq) platform that uses a novel double barcoding system in conjunction with the dihydrofolate reductase protein-fragment complementation assay in Saccharomyces cerevisiae. PPiSeq detects PPIs at a rate that is on par with current assays and, in contrast with current methods, quantitatively scores PPIs with enough accuracy and sensitivity to detect changes across environments. Both PPI scoring and the bulk of strain construction can be performed with cell pools, making the assay scalable and easily reproduced across environments. PPiSeq is therefore a powerful new tool for large-scale investigations of dynamic PPIs.

Linearization of the bradford protein assay.

PubMed

Ernst, Orna; Zor, Tsaffrir

2010-04-12

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

PubMed

Redmile-Gordon, M A; Armenise, E; White, R P; Hirsch, P R; Goulding, K W T

2013-12-01

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

Multifocal canalicular adenoma of the minor labial salivary glands

PubMed Central

Samar, María Elena; Avila, Rodolfo Esteban; Fonseca, Ismael Bernardo; Anderson, William; Fonseca, Gabriel M; Cantín, Mario

2014-01-01

Canalicular adenoma (CA) is an uncommon benign neoplasia of salivary glands which is clinically difficult to recognise. Despite having an excellent prognosis, the histological diagnosis and clinical management of this entity can be troublesome. While the main differential diagnosis to consider is basal cell adenoma (BCA), similar histological patterns and multifocality have been observed in adenoid cystic carcinoma (ACC) and polymorphous low-grade adenocarcinoma (PLGA), both locally-aggressive malignancies which require radically different treatment to CA. An emphasis has been placed on the value of immunohistochemistry in avoiding diagnostic and surgical errors. CA is positive for AE1/AE3, CD117 and S-100 protein, and negative for p63, α-SMA, Ki 67 and vimentin. Here we discuss the case of a 61-year-old female with CA in her right upper lip, showing multifocal growth histologically. The differential diagnosis with other adenomas is discussed in addition to the role of immunohistochemical studies that can confirm the clinical and surgical findings. PMID:25550873

A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays.

PubMed

Hampf, Mathias; Gossen, Manfred

2006-09-01

We established a quantitative reporter gene protocol, the P/Rluc assay system, allowing the sequential measurement of Photinus and Renilla luciferase activities from the same extract. Other than comparable commercial reporter assay systems and their noncommercial counterparts, the P/Rluc assay system was formulated under the aspect of full compatibility with standard methods for protein assays. This feature greatly expands the range of applications for assay systems quantifying the expression of multiple luciferase reporters.

Employment of colorimetric enzyme assay for monitoring expression and solubility of GST fusion proteins targeted to inclusion bodies.

PubMed

Mačinković, Igor S; Abughren, Mohamed; Mrkic, Ivan; Grozdanović, Milica M; Prodanović, Radivoje; Gavrović-Jankulović, Marija

2013-12-01

High levels of recombinant protein expression can lead to the formation of insoluble inclusion bodies. These complex aggregates are commonly solubilized in strong denaturants, such as 6-8M urea, although, if possible, solubilization under milder conditions could facilitate subsequent refolding and purification of bioactive proteins. Commercially available GST-tag assays are designed for quantitative measurement of GST activity under native conditions. GST fusion proteins accumulated in inclusion bodies are considered to be undetectable by such assays. In this work, solubilization of recombinantly produced proteins was performed in 4M urea. The activity of rGST was assayed in 2M urea and it was shown that rGST preserves 85% of its activity under such denaturing conditions. A colorimetric GST activity assay with 1-chloro-2, 4-dinitrobenzene (CDNB) was examined for use in rapid detection of expression targeted to inclusion bodies and for the identification of inclusion body proteins which can be solubilized in low concentrations of chaotropic agents. Applicability of the assay was evaluated by tracking protein expression of two GST-fused allergens of biopharmaceutical value in E. coli, GST-Der p 2 and GST-Mus a 5, both targeted to inclusion bodies. Copyright © 2013 Elsevier B.V. All rights reserved.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

PubMed

Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

2014-01-01

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

A simple method to generate stable cell lines for the analysis of transient protein-protein interactions.

PubMed

Savage, Emilia Elizabeth; Wootten, Denise; Christopoulos, Arthur; Sexton, Patrick Michael; Furness, Sebastian George Barton

2013-04-01

Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energy transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins.

Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

PubMed

Michnick, Stephen W; Landry, Christian R; Levy, Emmanuel D; Diss, Guillaume; Ear, Po Hien; Kowarzyk, Jacqueline; Malleshaiah, Mohan K; Messier, Vincent; Tchekanda, Emmanuelle

2016-11-01

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics. © 2016 Cold Spring Harbor Laboratory Press.

The Use of AlphaScreen Technology in HTS: Current Status

PubMed Central

Eglen, Richard M; Reisine, Terry; Roby, Philippe; Rouleau, Nathalie; Illy, Chantal; Bossé, Roger; Bielefeld, Martina

2008-01-01

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal. In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions. Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems. Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular. PMID:20161822

Development of novel dental adhesive with double benefits of protein-repellent and antibacterial capabilities.

PubMed

Zhang, Ning; Weir, Michael D; Romberg, Elaine; Bai, Yuxing; Xu, Hockin H K

2015-07-01

Secondary caries at the tooth-restoration margins remains a main reason for restoration failure. The objectives of this study were to: (1) combine protein-repellent 2-methacryloyloxyethyl phosphorylcholine (MPC) with quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM) to develop a new dental adhesive with double benefits of protein-repellent and antibacterial capabilities for the first time; and (2) investigate the effects on protein adsorption, anti-biofilm activity, and dentin bond strength. MPC and DMAHDM were incorporated into Scotchbond Multi-Purpose (SBMP) primer and adhesive. Dentin shear bond strengths were measured using extracted human molars. Protein adsorption onto the adhesive resin surfaces was determined by the micro bicinchoninic acid (BCA) method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to investigate biofilm metabolic activity, colony-forming unit (CFU) counts, lactic acid production and live/dead staining of biofilms on resins. Incorporation of 7.5% MPC and 5% DMAHDM into primer and adhesive did not adversely affect the dentin shear bond strength (p>0.1). The resin with 7.5% MPC+5% DMAHDM had protein adsorption that was nearly 20-fold less than SBMP control (p<0.05). The resin with 7.5% MPC+5% DMAHDM had much stronger antibacterial effects than using MPC or DMAHDM alone (p<0.05). Biofilm CFU counts on the resin with 7.5% MPC+5% DMAHDM were reduced by more than 4 orders of magnitude, compared to SBMP control. The use of double agents (protein-repellent MPC+antibacterial DMAHDM) in dental adhesive achieved much stronger inhibition of biofilms than using each agent alone. The novel protein-repellent and antibacterial bonding agent is promising to reduce biofilm/plaque buildup and reduce recurrent caries at the tooth-restoration margins. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ANCA-associated vasculitis

PubMed Central

Monach, Paul A; Warner, Roscoe L; Tomasson, Gunnar; Specks, Ulrich; Stone, John H; Ding, Linna; Fervenza, Fernando C; Fessler, Barri J; Hoffman, Gary S; Iklé, David; Kallenberg, Cees GM; Krischer, Jeffrey; Langford, Carol A; Mueller, Mark; Seo, Philip; St. Clair, E William; Spiera, Robert; Tchao, Nadia; Ytterberg, Steven R; Johnson, Kent J; Merkel, Peter A

2016-01-01

Objective To identify circulating proteins that distinguish between active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission in a manner complementary to markers of systemic inflammation. Methods Twenty-eight serum proteins representing diverse aspects of the biology of AAV were measured before and 6 months after treatment in a large clinical trial of AAV. Subjects (n=186) enrolled in the Rituximab in ANCA-Associated Vasculitis (RAVE) trial were studied. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels were available for comparison. The primary outcome was the ability of markers to distinguish severe AAV (Birmingham Vasculitis Activity Score for Wegener’s granulomatosis (BVAS/WG)≥3 at screening) from remission (BVAS/WG=0 at month 6), using areas under receiver operating characteristic (ROC) curve (AUC). Results All subjects had severe active vasculitis (median BVAS/WG=8) at screening. In the 137 subjects in remission at month 6, 24 of the 28 markers showed significant declines. ROC analysis indicated that levels of CXCL13 (BCA-1), matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) best discriminated active AAV from remission (AUC>0.8) and from healthy controls (AUC>0.9). Correlations among these markers and with ESR or CRP were low. Conclusions Many markers are elevated in severe active AAV and decline with treatment, but CXCL13, MMP-3 and TIMP-1 distinguish active AAV from remission better than the other markers studied, including ESR and CRP. These proteins are particularly promising candidates for future studies to address unmet needs in the assessment of patients with AAV. PMID:22975753

Effect of biochanin A on corn grain (Zea Mays) fermentation by bovine rumen amylolytic bacteria

USDA-ARS?s Scientific Manuscript database

The objective of this research was to determine the effect of biochanin A (BCA), an isoflavone produced by red clover (Trifolium pratense L.), on corn fermentation by rumen microorganisms. When bovine rumen cell suspensions (n = 3) were incubated (24 h, 39 °C) with corn, amylolytic bacteria includi...

Developmental toxicity of mixtures: the water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture

EPA Science Inventory

The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA) and bromochloro (BCA) acetic...

THE FERTILITY BIOMARKER (SP22) IS COMPROMISED IN AN ADDITIVE FASHION BY HALOACIDS: COMPARATIVE QUANTITATION BY IMMUNOASSAY AND 2D-GEL ANALYSIS

EPA Science Inventory

Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water known to produce defects in spermatogenesis and fertility in adult rats. Previous work in our laboratory demonstated a high correlation between fertility of sperm fr...

Book Review: Caves and Karst of the Yorkshire Dales

NASA Astrophysics Data System (ADS)

Westaway, Rob

2015-10-01

The British Cave Research Association (BCRA) is the research division of the British Caving Association (BCA), itself the principal society in Britain for those interested in caving, with activities including provision of training and safety certification for cavers and managing access to caves. Although some UK cave-related research is carried out by academics, this tends to be restricted to archaeological investigations of caves that have served as human habitations, and to be focused more on the occupants than the caves themselves. In contrast, most cave exploration is undertaken as a leisure activity, under the auspices of clubs affiliated to the BCA/BCRA, this being indeed virtually the only field of Earth science where amateur investigators can continue to make significant discoveries. Many cave explorers are also affiliated with academic researchers, such as managers of dating laboratories; the synergy between these two groups is highly productive, having resulted for instance in the discovery and exploration in recent years of the vast Ogof Draenen cave system in South Wales, which probably dates back to the Early Pleistocene (e.g., Farrant et al., 2014).

Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

DOE PAGES

Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

2017-07-19

Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling.

PubMed

Buntin, Nirunya; Hongpattarakere, Tipparat; Ritari, Jarmo; Douillard, François P; Paulin, Lars; Boeren, Sjef; Shetty, Sudarshan A; de Vos, Willem M

2017-01-15

The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development. Copyright © 2016 American Society for Microbiology.

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling

PubMed Central

Buntin, Nirunya; Hongpattarakere, Tipparat; Ritari, Jarmo; Douillard, François P.; Paulin, Lars; Boeren, Sjef; Shetty, Sudarshan A.

2016-01-01

ABSTRACT The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. β-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes β-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum. The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored β-fructosidase were induced to a high abundance when inulin was present as a carbon source. IMPORTANCE Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development. PMID:27815279

Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands

PubMed Central

Crowther, Gregory J.; Napuli, Alberto J.; Thomas, Andrew P.; Chung, Diana J.; Kovzun, Kuzma V.; Leibly, David J.; Castaneda, Lisa J.; Bhandari, Janhavi; Damman, Christopher J.; Hui, Raymond; Hol, Wim G. J.; Buckner, Frederick S.; Verlinde, Christophe L. M. J.; Zhang, Zhongsheng; Fan, Erkang; Van Voorhis, Wesley C.

2010-01-01

In the last decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. Here we present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. We conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal. PMID:19470714

A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

PubMed

Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

2015-02-01

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

Enzyme-linked immunosorbent assay detection and bioactivity of Cry1Ab protein fragments

USDA-ARS?s Scientific Manuscript database

Enzyme-linked immunosorbent assay (ELISA) has emerged as the preferred detection method for Cry proteins in environmental matrices. Concerns exist that ELISAs are capable of detecting fragments of Cry proteins, which may lead to an over-estimation of the concentration of these proteins in the enviro...

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

Screening Carbohydrate Libraries for Protein Interactions Using the Direct ESI-MS Assay. Applications to Libraries of Unknown Concentration

NASA Astrophysics Data System (ADS)

Kitova, Elena N.; El-Hawiet, Amr; Klassen, John S.

2014-08-01

A semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤103 M-1) ligands from moderate and high affinity (>105 M-1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3.

Emory University: Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

Evaluation of five commercially available assays and measurement of serum total protein concentration via refractometry for the diagnosis of failure of passive transfer of immunity in foals.

PubMed

Davis, Rachel; Giguère, Steeve

2005-11-15

To determine and compare sensitivity, specificity, accuracy, and predictive values of measurement of serum total protein concentration by refractometry as well as 5 commercially available kits for the diagnosis of failure of passive transfer (FPT) of immunity in foals. Prospective study. 65 foals with various medical problems and 35 clinically normal foals. IgG concentration in serum was assessed by use of zinc sulfate turbidity (assay C), glutaraldehyde coagulation (assay D), 2 semiquantitative immunoassays (assays F and G), and a quantitative immunoassay (assay H). Serum total protein concentration was assessed by refractometry. Radial immunodiffusion (assays A and B) was used as the reference method. For detection of IgG < 400 mg/dL, sensitivity of assay H (100%) was not significantly different from that of assays C, E, and G (88.9%). Specificity of assays H (96.0%) and G (95.8%) was significantly higher than that of assays C (79.4%) and E (78.1 %). For detection of IgG < 800 mg/dL, sensitivities of assays H (976%), D (92.9%), C (81.0%), and G (81.0%) were significantly higher than that of assay F (52.4%). Specificity of assays F (100%), G (94.7%), and H (82.8%) was significantly higher than that of assays C (56.9%) and D (58.6%). Serum total protein concentration < or = 4.5 g/dL was suggestive of FPT, whereas values > or = 6.0 g/dL indicated adequate IgG concentrations. Most assays were adequate as initial screening tests. However, their use as a definitive test would result in unnecessary treatment of foals with adequate IgG concentrations.

Conditions of Mytilus edulis extracellular body fluids and shell composition in a pH-treatment experiment: Acid-base status, trace elements and δ11B

NASA Astrophysics Data System (ADS)

Heinemann, Agnes; Fietzke, Jan; Melzner, Frank; BöHm, Florian; Thomsen, JöRn; Garbe-SchöNberg, Dieter; Eisenhauer, Anton

2012-01-01

Mytilus edulis were cultured for 3 months under six different seawater pCO2 levels ranging from 380 to 4000 μatm. Specimen were taken from Kiel Fjord (Western Baltic Sea, Germany) which is a habitat with high and variable seawater pCO2 and related shifts in carbonate system speciation (e.g., low pH and low CaCO3 saturation state). Hemolymph (HL) and extrapallial fluid (EPF) samples were analyzed for pH and total dissolved inorganic carbon (CT) to calculate pCO2 and [HCO3-]. A second experiment was conducted for 2 months with three different pCO2 levels (380, 1400 and 4000 μatm). Boron isotopes (δ11B) were investigated by LA-MC-ICP-MS (Laser Ablation-Multicollector-Inductively Coupled Plasma-Mass Spectrometry) in shell portions precipitated during experimental treatment time. Additionally, elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF of specimen from the second experiment were measured via ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). Extracellular pH was not significantly different in HL and EPF but systematically lower than ambient water pH. This is due to high extracellular pCO2 values, a prerequisite for metabolic CO2 excretion. No accumulation of extracellular [HCO3-] was measured. Elemental ratios (B/Ca, Mg/Ca and Sr/Ca) in the EPF increased slightly with pH which is in accordance with increasing growth and calcification rates at higher seawater pH values. Boron isotope ratios were highly variable between different individuals but also within single shells. This corresponds to a high individual variability in fluid B/Ca ratios and may be due to high boron concentrations in the organic parts of the shell. The mean δ11B value shows no trend with pH but appears to represent internal pH (EPF) rather than ambient water pH.

The magnitude and phasing of variations in climate and ocean carbonate chemistry during Eocene Thermal Maximum 2: Insights into C cycle feedbacks

NASA Astrophysics Data System (ADS)

Harper, D. T.; Zeebe, R. E.; Hoenisch, B.; Zachos, J. C.

2016-12-01

The early Eocene features several large abrupt global warming events ("hyperthermals") that were characterized by negative δ13C excursions suggesting isotopically `light' carbon release to the atmosphere. The most prominent events, the Paleocene-Eocene Thermal Maximum (PETM) and Eocene Thermal Maximum 2 (ETM2), present an opportunity to study the operation of carbon cycle processes, and in particular negative feedbacks in the carbon cycle, such as silicate and carbonate weathering. Here we study sea surface temperature (SST) and ocean carbonate chemistry changes across ETM2, by measuring Mg/Ca, B/Ca, and δ13C in planktic foraminifera at two IODP sites (1209 in the Pacific and 1265 in the S. Atlantic). We observe a 2-3°C increase in SST in the Pacific and a 2°C increase in the Atlantic. The observed decrease in planktic B/Ca at both sites is consistent with increased atmospheric pCO2, and when scaled to the 0.3 pH unit decrease estimated for the PETM by Penman et al., 2014, the estimated pH decrease during the ETM2 is 0.15. However, reconstructions of the δ13C recovery during the ETM2 show that it is more rapid than models have been able to successfully simulate. We compare these new proxy data to LOSCAR model output, to assess whether the rapid δ13C recovery was a result of: 1) changes in the type and δ13C of weathered carbonates or δ13C of buried organic carbon during the recovery, 2) a one-time event of isotopically `light' carbon burial during the recovery phase, or 3) enhanced burial of `light' carbon due to background orbital eccentricity forcing during the recovery. Our preliminary results suggest that the phasing of the drop in the B/Ca relative to δ13C during recovery is due to the burial of organic carbon.

The application of artificial intelligence to microarray data: identification of a novel gene signature to identify bladder cancer progression.

PubMed

Catto, James W F; Abbod, Maysam F; Wild, Peter J; Linkens, Derek A; Pilarsky, Christian; Rehman, Ishtiaq; Rosario, Derek J; Denzinger, Stefan; Burger, Maximilian; Stoehr, Robert; Knuechel, Ruth; Hartmann, Arndt; Hamdy, Freddie C

2010-03-01

New methods for identifying bladder cancer (BCa) progression are required. Gene expression microarrays can reveal insights into disease biology and identify novel biomarkers. However, these experiments produce large datasets that are difficult to interpret. To develop a novel method of microarray analysis combining two forms of artificial intelligence (AI): neurofuzzy modelling (NFM) and artificial neural networks (ANN) and validate it in a BCa cohort. We used AI and statistical analyses to identify progression-related genes in a microarray dataset (n=66 tumours, n=2800 genes). The AI-selected genes were then investigated in a second cohort (n=262 tumours) using immunohistochemistry. We compared the accuracy of AI and statistical approaches to identify tumour progression. AI identified 11 progression-associated genes (odds ratio [OR]: 0.70; 95% confidence interval [CI], 0.56-0.87; p=0.0004), and these were more discriminate than genes chosen using statistical analyses (OR: 1.24; 95% CI, 0.96-1.60; p=0.09). The expression of six AI-selected genes (LIG3, FAS, KRT18, ICAM1, DSG2, and BRCA2) was determined using commercial antibodies and successfully identified tumour progression (concordance index: 0.66; log-rank test: p=0.01). AI-selected genes were more discriminate than pathologic criteria at determining progression (Cox multivariate analysis: p=0.01). Limitations include the use of statistical correlation to identify 200 genes for AI analysis and that we did not compare regression identified genes with immunohistochemistry. AI and statistical analyses use different techniques of inference to determine gene-phenotype associations and identify distinct prognostic gene signatures that are equally valid. We have identified a prognostic gene signature whose members reflect a variety of carcinogenic pathways that could identify progression in non-muscle-invasive BCa. 2009 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Effects of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride.

PubMed

Daryaei, A; Jones, E E; Ghazalibiglar, H; Glare, T R; Falloon, R E

2016-04-01

The goal was to determine the effect of temperature, light and incubation period on production, germination and bioactivity of Trichoderma atroviride LU132 against Rhizoctonia solani. The incubation temperatures of 20, 25 or 30°C were assessed on the production of T. atroviride conidia under constant light over a 25 and 50 days periods. The resulting conidia were also studied for germination and bioactivity. Conidium production was maximum at 25°C after 20 days. The second peak of conidium production occurred at 45-50 days. Incubation at 25°C after 15 days showed optimum production of T. atroviride LU132. Conidia produced at 30°C gave the greatest germination and bioactivity in comparison with incubation at 20 or 25°C. This study indicates that the temperature at which conidia of T. atroviride are produced affects germination and bioactivity. Formulations based on production of the high conidia yield may not result in optimal bioactivity and there is a trade-off between quantity and quality of T. atroviride LU132 conidia. Conidium production was shown to be a continuous process, and increased under a dark/light regime. This is the first report of bimodal conidium production in a Trichoderma biological control agent (BCA), which is likely to be on 20 days cycle, and is dependent on colony age rather than abiotic factors. Conidia produced after 15 days are likely to be the most suitable for use in commercial production of this strain as a BCA. Most studies on Trichoderma-based BCA have only shown the effect of culture conditions on the high conidia yield regardless of conidium quality. This study is the first report on conidium quality affected by principal culture conditions for Trichoderma biological control formulations. © 2016 The Society for Applied Microbiology.

Proliferation of the biocontrol agent Fusarium oxysporum f. sp. strigae and its impact on indigenous rhizosphere fungal communities in maize under different agro-ecologies.

PubMed

Zimmermann, Judith; Musyoki, Mary K; Cadisch, Georg; Rasche, Frank

2016-06-01

Our objectives were to (1) monitor the proliferation of the biocontrol agent (BCA) Fusarium oxysporum f. sp. strigae strain "Foxy-2", an effective soil-borne BCA against the parasitic weed Striga hermonthica , in the rhizosphere of maize under different agro-ecologies, and (2) investigate its impact on indigenous rhizosphere fungal community abundance and composition. Field experiments were conducted in Busia and Homa Bay districts in western Kenya during two cropping seasons to account for effects of soil type, climate, growth stage and seasonality. Maize seeds were coated with or without "Foxy-2" and soils were artificially infested with S. hermonthica seeds. One treatment with nitrogen rich organic residues ( Tithonia diversifolia ) was established to compensate hypothesized resource competition between "Foxy-2" and the indigenous fungal community. Rhizosphere soil samples collected at three growth stages (i.e., EC30, EC60, EC90) of maize were subjected to abundance measurement of "Foxy-2" and total indigenous fungi using quantitative polymerase chain reaction (qPCR) analysis. Terminal restriction fragment length polymorphism (TRFLP) analysis was used to assess potential alterations in the fungal community composition in response to "Foxy-2" presence. "Foxy-2" proliferated stronger in the soils with a sandy clay texture (Busia) than in those with a loamy sand texture (Homa Bay) and revealed slightly higher abundance in the second season. "Foxy-2" had, however, only a transient suppressive effect on total indigenous fungal abundance which ceased in the second season and was further markedly compensated after addition of T. diversifolia residues. Likewise, community structure of the indigenous fungal community was mainly altered by maize growth stages, but not by "Foxy-2". In conclusion, no adverse effects of "Foxy-2" inoculation on indigenous fungal rhizosphere communities were observed corroborating the safety of this BCA under the given agro-ecologies.

A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein

PubMed Central

Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.

2014-01-01

The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

Recombinant blood group proteins for use in antibody screening and identification tests.

PubMed

Seltsam, Axel; Blasczyk, Rainer

2009-11-01

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

Rapid kinetic BRET measurements to monitor G protein activation by GPCR and non-GPCR proteins.

PubMed

Maziarz, Marcin; Garcia-Marcos, Mikel

2017-01-01

Heterotrimeric G proteins are central hubs of signal transduction whose activity is controlled by G protein-coupled receptors (GPCRs) as well as by a complex network of regulatory proteins. Recently, bioluminescence resonance energy transfer (BRET)-based assays have been used to monitor real-time activation of heterotrimeric G proteins in cells. Here we describe the use of a previously established BRET assay to monitor G protein activation upon GPCR stimulation and its adaptation to measure G protein activation by non-GPCR proteins, such as by cytoplasmic guanine nucleotide exchange factors (GEFs) like GIV/Girdin. The BRET assay monitors the release of free Gβγ from Gα-Gβγ heterotrimers as a readout of G protein activation, which is readily observable upon agonist stimulation of GPCRs. To control the signal input for non-GPCR activators, we describe the use of a chemically induced dimerization strategy to promote rapid membrane translocation of proteins containing the Gα-binding and -activating (GBA) motif found in some nonreceptor GEFs. The assay described here allows the kinetic measurement of G protein activation with subsecond temporal resolution and to compare the levels of activation induced by GPCR agonists vs those induced by the membrane recruitment of nonreceptor G protein signaling activators. © 2017 Elsevier Inc. All rights reserved.

Prevalence and its associated psychological variables of symptoms of depression and anxiety among ovarian cancer patients in China: a cross-sectional study.

PubMed

Liu, Chun Li; Liu, Li; Zhang, Yi; Dai, Xiao Ze; Wu, Hui

2017-08-17

It is well known that cancer patients tend to have high levels of perceived stress and symptoms of depression and anxiety. However, there is less study on the association between perceived stress and symptoms of depression and anxiety among ovarian cancer patients in China. And the mediating effect of hope and resilience needs to be further studied. In this study, we aim to examine the prevalence of depression and anxiety symptoms, to analyze the association between perceived stress and symptoms of depression and anxiety, and to test whether hope and resilience mediate the association of perceived stress with symptoms of depression and anxiety among Chinese patients with ovarian cancer. A total of 220 questionnaires were distributed and collected from the First Affiliated Hospital of China Medical University and Shengjing Hospital of China Medical University. All participants in this study were ovarian cancer inpatients. After excluding the incomplete questionnaires, 198 questionnaires were valid for the analysis. Qualified patients were asked to response to the questionnaires including Hospital anxiety and depression scale (HADS), perceived stress scale (PSS-10), and the Herth hope scale and the resilience scale. Hierarchical regression analyses were used to test the associations among perceived stress, symptoms of depression and anxiety, and hope and resilience. Bootstrapping method was conducted to examine whether the indirect effect of hope and resilience was significant respectively. The prevalence of symptoms of depression and anxiety in ovarian cancer patients was 47.0% and 51.5% respectively. Perceived stress correlated significantly with symptoms of depression (r = 0.709, P < 0.01) and anxiety (r = 0.660, P < 0.01). Hope (a*b = 0.155, BCa 95% CI: 0.094, 0.223) partly mediated the association between perceived stress and symptoms of depression; similarly, hope (a*b = 0.129, BCa 95% CI: 0.048, 0.205) partly mediated the effect of perceived stress on symptoms of anxiety. However, resilience (a*b = 0.004, BCa 95% CI: -0.030, 0.040) did not mediate the association between perceived stress and symptoms of depression. And resilience (a*b = 0.041,BCa 95% CI: -0.013, 0.098) did not mediate the association between perceived stress and symptoms of anxiety. The present study suggests that perceived stress might be one of the impact factors of symptoms of depression and anxiety, while hope might ease symptoms of depression and anxiety. In view of the role of hope, medical workers and patient caregivers should pay more attention to hope, and then to intervene perceived stress among patients with ovarian cancer.

Functional and Biochemical Endothelial Profiling In Vivo in a Murine Model of Endothelial Dysfunction; Comparison of Effects of 1-Methylnicotinamide and Angiotensin-converting Enzyme Inhibitor

PubMed Central

Bar, Anna; Olkowicz, Mariola; Tyrankiewicz, Urszula; Kus, Edyta; Jasinski, Krzysztof; Smolenski, Ryszard T.; Skorka, Tomasz; Chlopicki, Stefan

2017-01-01

Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo. Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR-/-) in vivo, in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1–7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR-/- mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate®FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T1) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1–7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR-/- mice, Ach induced a vasoconstriction in BCA that amounted to –7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1–7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1–9) and Ang-(1–7) and a proportional decrease in Ang II and its active metabolites. Finally, MNA and perindopril treatment resulted in an increase in L-arginine/ADMA ratio by 107% (MNA) and 140% (perindopril), as compared to non-treated mice. Functional and biochemical endothelial profiling in ApoE/LDLR-/- mice in vivo revealed that 2-month treatment with MNA (100 mg/kg/day) displayed a similar profile of vasoprotective effect as 2-month treatment with perindopril (10 mg/kg/day): i.e., the improvement in endothelial function that was associated with the beneficial changes in ACE/Ang II-ACE2/Ang (1–7) balance and in L-arginine/ADMA ratio in plasma. PMID:28443021

[Recombinant expression of Schistosoma japonicum fructose-1, 6-bisphos- phate aldolase and its expression in different developmental stages of S. japonicum].

PubMed

Yan, Ke; Zhong, Zheng-rong; Xu, Yun-xia; Ding, Shu-qin; Hu, Jian-guo; Xu, Yuan-hong; Luo, Qing-lie; Shen, Ji-long

2015-06-01

To clone, express and purify Schistosoma japonicum fructose-1, 6-bisphosphate aldolase (SjFBPA) in E. coli and observe its expression in different developmental stages of S. japonicum. FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid, and inducibly expressed with IPTG in E. coli BL21. SDS-PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA (rSjFBPA). Then, rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS- PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore, SjFBPA mRNA was ana- lyzed in different developmental stages of S. japonicum by RT-PCR. SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro- tein could specifically reactive to the anti-His-tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT-PCR showed that SjFBPA mRNA was expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum. SjFBPA is successfully recombined and expressed in a prokaryotic system, and SjFBPA mRNA is expressed in cercaria, schistosomulum, adult worm and egg of S. japonicum.

Perturbation Theory/Machine Learning Model of ChEMBL Data for Dopamine Targets: Docking, Synthesis, and Assay of New l-Prolyl-l-leucyl-glycinamide Peptidomimetics.

PubMed

Ferreira da Costa, Joana; Silva, David; Caamaño, Olga; Brea, José M; Loza, Maria Isabel; Munteanu, Cristian R; Pazos, Alejandro; García-Mera, Xerardo; González-Díaz, Humbert

2018-06-25

Predicting drug-protein interactions (DPIs) for target proteins involved in dopamine pathways is a very important goal in medicinal chemistry. We can tackle this problem using Molecular Docking or Machine Learning (ML) models for one specific protein. Unfortunately, these models fail to account for large and complex big data sets of preclinical assays reported in public databases. This includes multiple conditions of assays, such as different experimental parameters, biological assays, target proteins, cell lines, organism of the target, or organism of assay. On the other hand, perturbation theory (PT) models allow us to predict the properties of a query compound or molecular system in experimental assays with multiple boundary conditions based on a previously known case of reference. In this work, we report the first PTML (PT + ML) study of a large ChEMBL data set of preclinical assays of compounds targeting dopamine pathway proteins. The best PTML model found predicts 50000 cases with accuracy of 70-91% in training and external validation series. We also compared the linear PTML model with alternative PTML models trained with multiple nonlinear methods (artificial neural network (ANN), Random Forest, Deep Learning, etc.). Some of the nonlinear methods outperform the linear model but at the cost of a notable increment of the complexity of the model. We illustrated the practical use of the new model with a proof-of-concept theoretical-experimental study. We reported for the first time the organic synthesis, chemical characterization, and pharmacological assay of a new series of l-prolyl-l-leucyl-glycinamide (PLG) peptidomimetic compounds. In addition, we performed a molecular docking study for some of these compounds with the software Vina AutoDock. The work ends with a PTML model predictive study of the outcomes of the new compounds in a large number of assays. Therefore, this study offers a new computational methodology for predicting the outcome for any compound in new assays. This PTML method focuses on the prediction with a simple linear model of multiple pharmacological parameters (IC 50 , EC 50 , K i , etc.) for compounds in assays involving different cell lines used, organisms of the protein target, or organism of assay for proteins in the dopamine pathway.

Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.

PubMed

Cheow, Lih Feng; Viswanathan, Ramya; Chin, Chee-Sing; Jennifer, Nancy; Jones, Robert C; Guccione, Ernesto; Quake, Stephen R; Burkholder, William F

2014-10-07

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.

Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins.

PubMed

Haynes, Lia M; Miao, Congrong; Harcourt, Jennifer L; Montgomery, Joel M; Le, Mai Quynh; Dryga, Sergey A; Kamrud, Kurt I; Rivers, Bryan; Babcock, Gregory J; Oliver, Jennifer Betts; Comer, James A; Reynolds, Mary; Uyeki, Timothy M; Bausch, Daniel; Ksiazek, Thomas; Thomas, William; Alterson, Harold; Smith, Jonathan; Ambrosino, Donna M; Anderson, Larry J

2007-03-01

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.

A Business Case Analysis (BCA) of the One Box - One Wire (OB1) Joint Combined Technology Demonstration (JCTD)

DTIC Science & Technology

2009-03-01

Framework ( IATF ) as their guidance for information assurance. The IATF defines what high 5 OB1 is compatible with and intends to use legacy...standardized levels of security. The OB1 team using the IATF as guidance defines high robustness as proving to the maximum extent possible, that

77 FR 51831 - OMB Sequestration Update Report to the President and Congress for Fiscal Year 2013

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-27

... OFFICE OF MANAGEMENT AND BUDGET OMB Sequestration Update Report to the President and Congress for Fiscal Year 2013 AGENCY: Executive Office of the President, Office of Management and Budget. ACTION.... SUMMARY: Public Law 112-25, the Budget Control Act of 2011 (BCA, ``the Act'') amended the Balanced Budget...

The response of belowground carbon allocation in forests to global change

Treesearch

Christian P. Giardina; Mark Coleman; Dan Binkley; Jessica Hancock; John S. King; Erik Lilleskov; Wendy M. Loya; Kurt S. Pregitzer; Michael G. Ryan; Carl Trettin

2005-01-01

Belowground carbon allocation (BCA) in forests regulates soil organic matter formation and influences biotic and abiotic properties of soil such as bulk density, cation exchange capacity, and water holding capacity. On a global scale, the total quantity of carbon allocated belowground by terrestrial plants is enormous, exceeding by an order of magnitude the quantity of...

Advancing Grassroots Climate Change Awareness in Botswana: BCA Campus Greenhouse Gas Baseline Inventory

ERIC Educational Resources Information Center

Batisani, Nnyaladzi; Ndiane, Abijah

2014-01-01

Purpose: This paper aims to report on the results of a case study in Botswana, aimed at raising awareness on climate issues. Higher-education institutions play a leading role in sustainability efforts, as their research role often lays the groundwork for social transformation. Design/methodology/approach: The Clean Air-Cool Planet (CACP) campus…

Black Coaches and Their Perspectives on the Black Coaches and Administrators: A Qualitative Analysis

ERIC Educational Resources Information Center

Kelly, Daniel G., II; Pastore, Donna L.; Hodge, Samuel R.; Seifried, Chad

2015-01-01

The purpose of this study was to analyze Black male collegiate coaches' understandings and perspectives about building a coaching career in NCAA Division I athletics and the function of the Black Coaches and Administrators (BCA) as a mechanism to help facilitate more coaching opportunities. The participants were seven Black male assistant coaches…

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

PubMed

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Incision of trivalent chromium [Cr(III)]-induced DNA damage by Bacillus caldotenax UvrABC endonuclease.

PubMed

O'Brien, Travis J; Jiang, Guohui; Chun, Gina; Mandel, H George; Westphal, Craig S; Kahen, Kaveh; Montaser, Akbar; States, J Christopher; Patierno, Steven R

2006-11-07

Some hexavalent chromium [Cr(VI)]-containing compounds are lung carcinogens. Once within cells, Cr(VI) is reduced to trivalent chromium [Cr(III)] which displays an affinity for both DNA bases and the phosphate backbone. A diverse array of genetic lesions is produced by Cr including Cr-DNA monoadducts, DNA interstrand crosslinks (ICLs), DNA-Cr-protein crosslinks (DPCs), abasic sites, DNA strand breaks and oxidized bases. Despite the large amount of information available on the genotoxicity of Cr, little is known regarding the molecular mechanisms involved in the removal of these lesions from damaged DNA. Recent work indicates that nucleotide excision repair (NER) is involved in the processing of Cr-DNA adducts in human and rodent cells. In order to better understand this process at the molecular level and begin to identify the Cr-DNA adducts processed by NER, the incision of CrCl(3) [Cr(III)]-damaged plasmid DNA was studied using a thermal-resistant UvrABC NER endonuclease from Bacillus caldotenax (Bca). Treatment of plasmid DNA with Cr(III) (as CrCl(3)) increased DNA binding as a function of dose. For example, at a Cr(III) concentration of 1 microM we observed approximately 2 Cr(III)-DNA adducts per plasmid. At this same concentration of Cr(III) we found that approximately 17% of the plasmid DNA contained ICLs ( approximately 0.2 ICLs/plasmid). When plasmid DNA treated with Cr(III) (1 microM) was incubated with Bca UvrABC we observed approximately 0.8 incisions/plasmid. The formation of endonuclease IV-sensitive abasic lesions or Fpg-sensitive oxidized DNA bases was not detected suggesting that the incision of Cr(III)-damaged plasmid DNA by UvrABC was not related to the generation of oxidized DNA damage. Taken together, our data suggest that a sub-fraction of Cr(III)-DNA adducts is recognized and processed by the prokaryotic NER machinery and that ICLs are not necessarily the sole lesions generated by Cr(III) that are substrates for NER.

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

PubMed Central

Boogaard, Ivette; Smith, Melanie; Pulli, Kristiina; Szynol, Agnieszka; Albertus, Faywell; Lamers, Marieke B. A. C.; Dijkstra, Sipke; Kordt, Daniel; Reindl, Wolfgang; Herrmann, Frank; McAllister, George; Fischer, David F.; Munoz-Sanjuan, Ignacio

2014-01-01

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples. PMID:24816435

Optimization of a Biomimetic Apatite Nanoparticle Delivery System for Non-viral Gene Transfection---a Simulated Body Fluid Approach

NASA Astrophysics Data System (ADS)

Das, Debobrato

Current methods for gene delivery utilize nanocarriers such as liposomes and viral vectors that may produce in vivo toxicity, immunogenicity, or mutagenesis. Moreover, these common high-cost systems have a low efficacy of gene-vehicle transport across the cell plasma membrane followed by inadequate release and weak intracellular stability of the genetic sequence. Thus, this study aims to maximize gene transfection while minimizing cytotoxicity by utilizing supersaturated blood-plasma ions derived from simulated body fluids (SBF). With favorable electrostatic interactions to create biocompatible calcium-phosphate nanoparticles (NPs) derived from biomimetic apatite (BA), results suggest that the SBF system, though naturally sensitive to reaction conditions, after optimization can serve as a tunable and versatile platform for the delivery of various types of nucleic acids. From a systematic exploration of the effects of nucleation pH, incubation temperature, and time on transfection efficiency, the study proposes distinct characteristic trends in SBF BA-NP morphology, cellular uptake, cell viability, and gene modulation. Specifically, with aggressive nucleation and growth of BA-NPs in solution (observed via scanning electron microscopy), the ensuing microenvironment imposes a more toxic cellular interaction (indicated by alamarBlue and BCA assays), limiting particle uptake (fluorescence experiments) and subsequent gene knockdown (quantitative loss of function assays). Controlled precipitation of BA-NPs function to increase particle accessibility by surrounding cells, and subsequently enhance uptake and transfection efficiency. By closely examining such trends, an optimal fabrication condition of pH 6.5-37C can be observed where particle growth is more tamed and less chaotic, providing improved, favorable cellular interactions that increase cell uptake and consequently maximize gene transfection, without compromising cellular viability.

Analysis of protein stability and ligand interactions by thermal shift assay.

PubMed

Huynh, Kathy; Partch, Carrie L

2015-02-02

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

A Database of Reaction Monitoring Mass Spectrometry Assays for Elucidating Therapeutic Response in Cancer

PubMed Central

Remily-Wood, Elizabeth R.; Liu, Richard Z.; Xiang, Yun; Chen, Yi; Thomas, C. Eric; Rajyaguru, Neal; Kaufman, Laura M.; Ochoa, Joana E.; Hazlehurst, Lori; Pinilla-Ibarz, Javier; Lancet, Jeffrey; Zhang, Guolin; Haura, Eric; Shibata, David; Yeatman, Timothy; Smalley, Keiran S.M.; Dalton, William S.; Huang, Emina; Scott, Ed; Bloom, Gregory C.; Eschrich, Steven A.; Koomen, John M.

2012-01-01

Purpose The Quantitative Assay Database (QuAD), http://proteome.moffitt.org/QUAD/, facilitates widespread implementation of quantitative mass spectrometry in cancer biology and clinical research through sharing of methods and reagents for monitoring protein expression and modification. Experimental Design Liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM) assays are developed using SDS-PAGE fractionated lysates from cancer cell lines. Pathway maps created using GeneGO Metacore provide the biological relationships between proteins and illustrate concepts for multiplexed analysis; each protein can be selected to examine assay development at the protein and peptide level. Results The coupling of SDS-PAGE and LC-MRM screening has been used to detect 876 peptides from 218 cancer-related proteins in model systems including colon, lung, melanoma, leukemias, and myeloma, which has led to the development of 95 quantitative assays including stable-isotope labeled peptide standards. Methods are published online and peptide standards are made available to the research community. Protein expression measurements for heat shock proteins, including a comparison with ELISA and monitoring response to the HSP90 inhibitor, 17-DMAG, are used to illustrate the components of the QuAD and its potential utility. Conclusions and Clinical Relevance This resource enables quantitative assessment of protein components of signaling pathways and biological processes and holds promise for systematic investigation of treatment responses in cancer. PMID:21656910

Protein biomarker validation via proximity ligation assays.

PubMed

Blokzijl, A; Nong, R; Darmanis, S; Hertz, E; Landegren, U; Kamali-Moghaddam, M

2014-05-01

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.

Lipid-binding analysis using a fat blot assay.

PubMed

Munnik, Teun; Wierzchowiecka, Magdalena

2013-01-01

Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose membrane-immobilized lipids. The assay is adapted from the method by Dowler et al. (Sci STKE 129:pl6, 2002) and provides qualitative and quantitative information on the relative affinity with which a protein binds to a particular lipid. To perform a Fat Blot assay, serial dilutions of different phospholipids are spotted onto a nitrocellulose membrane. These membranes are then incubated with a lipid-binding protein possessing a GST (or other epitope) tag. The membranes are washed and the protein, which is bound to the membrane by virtue of its interaction with the lipid's head group, is detected by immunoblotting with an antibody against GST (or other epitope). The procedure only requires a few micrograms of protein and is quick, simple and cheap to perform.

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

A high-throughput assay of membrane protein stability.

PubMed

Postis, Vincent L G; Deacon, Sarah E; Roach, Peter C J; Wright, Gareth S A; Xia, Xiaobing; Ingram, Jean C; Hadden, Jonathan M; Henderson, Peter J F; Phillips, Simon E V; McPherson, Michael J; Baldwin, Stephen A

2008-12-01

The preparation of purified, detergent-solubilized membrane proteins in a monodisperse and stable form is usually a prerequisite for investigation not only of their function but also for structural studies by X-ray crystallography and other approaches. Typically, it is necessary to explore a wide range of conditions, including detergent type, buffer pH, and the presence of additives such as glycerol, in order to identify those optimal for stability. Given the difficulty of expressing and purifying membrane proteins in large amounts, such explorations must ideally be performed on as small a scale as practicable. To achieve this objective in the UK Membrane Protein Structure Initiative, we have developed a rapid, economical, light-scattering assay of membrane protein aggregation that allows the testing of 48 buffer conditions in parallel on 6 protein targets, requiring less than 2 mg protein for each target. Testing of the assay on a number of unrelated membrane transporters has shown that it is of generic applicability. Proteins of sufficient purity for this plate-based assay are first rapidly prepared using simple affinity purification procedures performed in batch mode. Samples are then transferred by microdialysis into each of the conditions to be tested. Finally, attenuance at 340 nm is monitored in a 384-well plate using a plate reader. Optimal conditions for protein stability identified in the assay can then be exploited for the tailored purification of individual targets in as stable a form as possible.

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

PubMed

Li, Jian-Feng; Bush, Jenifer; Xiong, Yan; Li, Lei; McCormack, Matthew

2011-01-01

Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

Quantification of protein concentration using UV absorbance and Coomassie dyes.

PubMed

Noble, James E

2014-01-01

The measurement of a solubilized protein concentration in solution is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantification assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction coefficient. Where multiple samples need measurement, and/or the sample volume and concentration is limited, preparations of the Coomassie dye commonly known as the Bradford assay can be used. © 2014 Elsevier Inc. All rights reserved.

S-Nitrosothiol measurements in biological systems⋄

PubMed Central

Gow, Andrew; Doctor, Allan; Mannick, Joan; Gaston, Benjamin

2007-01-01

S-Nitrosothiol (SNO) cysteine modifications are regulated signaling reactions that dramatically affect, and are affected by, protein conformation. The lability of the S-NO bond can make SNO-modified proteins cumbersome to measure accurately. Here, we review methodologies for detecting SNO modifications in biology. There are three caveats. 1) Many assays for biological SNOs are used near the limit of detection: standard curves must be in the biologically relevant concentration range. 2) The assays that are most reliable are those that modify SNO protein or peptide chemistry the least. 3) Each result should be quantitatively validated using more than one assay. Improved assays are needed and are in development. PMID:17379583

A semi-automated multiplex high-throughput assay for measuring IgG antibodies against Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) domains in small volumes of plasma.

PubMed

Cham, Gerald K K; Kurtis, Jonathan; Lusingu, John; Theander, Thor G; Jensen, Anja T R; Turner, Louise

2008-06-12

The level of antibodies against PfEMP1 is routinely quantified by the conventional microtitre enzyme-linked immunosorbent assay (ELISA). However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlex100 (BioRad, Hercules, CA, USA) system which can quantify multiple antibodies simultaneously in a small plasma volume. A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the Baculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex100 system against pooled human hyper-immune plasma before and after lyophilization. The bead-based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were > or = 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether they were performed on individual beads or on multiplexed beads; lyophilization had no impact on the assay performance. Spearman's rank correlation coefficients (Rho) were > or = 0.97, (P < 0.0001) for all comparisons. Importantly, the reactivity of protein-coupled non-lyophilized beads decreased with long term storage at 4 degrees C in the dark. Using this lyophilized multiplex assay, antibody reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma.

Split-luciferase complementary assay: applications, recent developments, and future perspectives.

PubMed

Azad, Taha; Tashakor, Amin; Hosseinkhani, Saman

2014-09-01

Bioluminescent systems are considered as potent reporter systems for bioanalysis since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochemical events are mostly accomplished through large protein machines. These molecular complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biological activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochemical metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in analytical biochemistry, cell communication and cell signaling, molecular biology, and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.

Clinical protein mass spectrometry.

PubMed

Scherl, Alexander

2015-06-15

Quantitative protein analysis is routinely performed in clinical chemistry laboratories for diagnosis, therapeutic monitoring, and prognosis. Today, protein assays are mostly performed either with non-specific detection methods or immunoassays. Mass spectrometry (MS) is a very specific analytical method potentially very well suited for clinical laboratories. Its unique advantage relies in the high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein. On the other hand, protein MS is a relatively young technique, demanding specialized personnel and expensive instrumentation. Many scientists and opinion leaders predict MS to replace immunoassays for routine protein analysis, but there are only few protein MS applications routinely used in clinical chemistry laboratories today. The present review consists of a didactical introduction summarizing the pros and cons of MS assays compared to immunoassays, the different instrumentations, and various MS protein assays that have been proposed and/or are used in clinical laboratories. An important distinction is made between full length protein analysis (top-down method) and peptide analysis after enzymatic digestion of the proteins (bottom-up method) and its implication for the protein assay. The document ends with an outlook on what type of analyses could be used in the future, and for what type of applications MS has a clear advantage compared to immunoassays. Copyright © 2015 Elsevier Inc. All rights reserved.

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

PubMed Central

Kennedy, Jacob J.; Abbatiello, Susan E.; Kim, Kyunggon; Yan, Ping; Whiteaker, Jeffrey R.; Lin, Chenwei; Kim, Jun Seok; Zhang, Yuzheng; Wang, Xianlong; Ivey, Richard G.; Zhao, Lei; Min, Hophil; Lee, Youngju; Yu, Myeong-Hee; Yang, Eun Gyeong; Lee, Cheolju; Wang, Pei; Rodriguez, Henry; Kim, Youngsoo; Carr, Steven A.; Paulovich, Amanda G.

2014-01-01

The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort. PMID:24317253

Protein Science by DNA Sequencing: How Advances in Molecular Biology Are Accelerating Biochemistry.

PubMed

Higgins, Sean A; Savage, David F

2018-01-09

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This Perspective briefly outlines the technical innovations in DNA manipulation that allow massively parallel protein biochemistry and then summarizes the methods currently available for library construction and the functional assays of protein variants. Areas in need of future innovation are highlighted with a particular focus on assay development and the use of computational analysis with machine learning to effectively traverse the sequence-function landscape. Finally, applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering are presented.

Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.

Assessing protein oxidation by inorganic nanoparticles with enzyme-linked immunosorbent assay (ELISA).

PubMed

Sun, Wenjie; Luna-Velasco, Antonia; Sierra-Alvarez, Reyes; Field, Jim A

2013-03-01

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme-linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn(2)O(3), and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS-dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Copyright © 2012 Wiley Periodicals, Inc.

A cell death assay for assessing the mitochondrial targeting of proteins.

PubMed

Camara Teixeira, Daniel; Cordonier, Elizabeth L; Wijeratne, Subhashinee S K; Huebbe, Patricia; Jamin, Augusta; Jarecke, Sarah; Wiebe, Matthew; Zempleni, Janos

2018-06-01

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Protein-Protein Interaction Assays with Effector-GFP Fusions in Nicotiana benthamiana.

PubMed

Petre, Benjamin; Win, Joe; Menke, Frank L H; Kamoun, Sophien

2017-01-01

Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector-green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation-liquid chromatography-tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

PubMed

Sameshima, Tomoya; Miyahisa, Ikuo; Homma, Misaki; Aikawa, Katsuji; Hixon, Mark S; Matsui, Junji

2014-12-15

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

PubMed

Liu, Bing-Hsien; Lin, Yu-Chen; Ho, Chia-Shin; Yang, Che-Chuan; Chang, Yun-Tsui; Chang, Jui-Feng; Li, Chun-Yuan; Cheng, Cheng-Shun; Huang, Jiun-Yan; Lee, Yen-Fu; Hsu, Ming-Hung; Lin, Feng-Chun; Wang, Hao-Ching; Lo, Chu-Fang; Yang, Shieh-Yueh; Wang, Han-Ching

2015-01-01

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

PubMed

Luzy, Jean-Philippe; Chen, Huixiong; Gril, Brunilde; Liu, Wang-Qing; Vidal, Michel; Perdereau, Dominique; Burnol, Anne-Françoise; Garbay, Christiane

2008-02-01

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

A Mutation in the PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin

PubMed Central

Passalacqua, Karla D.; Satola, Sarah W.; Crispell, Emily K.

2012-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediate S. aureus (VISA). In this study, we characterized two isogenic USA300 S. aureus isolates collected sequentially from a single patient with endocarditis where the S. aureus isolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein of S. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII, sarT, and saeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquired in vivo. Creation of de novo mutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype. PMID:22850507

Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory

ERIC Educational Resources Information Center

Mascotti, David P.; Waner, Mark J.

2010-01-01

A protein-ligand binding, guided-inquiry laboratory project with potential application across the advanced undergraduate curriculum is described. At the heart of the project are fluorescence and spectrophotometric assays utilizing biotin-4-fluorescein and streptavidin. The use of the same stock solutions for an assay that may be examined by two…

A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

PubMed

Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

2018-01-01

In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

Population dynamics of mottled sculpin (PISCES) in a variable environment: information theoretic approaches

Treesearch

Gary D. Grossman; Robert E Ratajczak; J. Todd Petty; Mark D. Hunter; James T. Peterson; Gael Grenouillet

2006-01-01

We used strong inference with Akaike's Information Criterion (AIC) to assess the processes capable of explaining long-term (1984-1995) variation in the per capita rate of change of mottled sculpin (Cottus bairdi) populations in the Coweeta Creek drainage (USA). We sampled two fourth- and one fifth-order sites (BCA [uppermost], BCB, and CC [lowermost])...

78 FR 47778 - The Boeing Company Boeing Commercial Aircraft (BCA) Auburn, Washington; The Boeing Company Boeing...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-06

... Building 535 Garden Avenue North, Renton, Washington and 10-18 Building 635 Park Avenue North, Renton... include workers at the 10-16 Building 535 Garden Avenue North, Renton, Washington and 10-18 Building 635... Garden Avenue North and 10-18 Building 635 Park Avenue North, Renton, Washington (TA-W-82,705C), The...

Biochanin A (an isoflavone produced by red clover) promotes weight gain of steers grazed in mixed grass pastures and fed dried-distillers grains

USDA-ARS?s Scientific Manuscript database

Biochanin A (BCA) is an isoflavone produced by red clover (Trifloium pratense L.) that can inhibit hyper-ammonia producing bacteria (HAB) to reduce deamination in the rumen and increase the feed amino acids available for gastric digestion. An in vitro experiment was conducted to evaluate the effect...

Before the Emergency: A Framework for Evaluating Emergency Preparedness Alternatives at Higher Education Institutions

DTIC Science & Technology

2010-09-01

Operations and Procedures • Logistics and Facilities • Training • Exercises, Evaluation and Corrective Actions • Crisis Communications ...Assessment Team BCA Benefit-cost analysis CEO Chief Executive Officer CERT Community Emergency Response Team CFR Code of Federal Regulations...CHDS Center for Homeland Defense and Security CPG 101 Comprehensive Preparedness Guidelines 101 CPP Community Preparedness and Participation CPW

78 FR 20291 - Fisheries of the United States; Billfish Conservation Act of 2012 Implementing Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-04

.... 121227743-3275-01] RIN 0648-BC86 Fisheries of the United States; Billfish Conservation Act of 2012... request public comment on potential issues related to the implementation of the Billfish Conservation Act... Conservation Act of 2012 (BCA), Public Law 112-183, 16 U.S.C. 1827a, was signed into law on October 5, 2012...

Long-Term Implications of the 2013 Future Years Defense Program

DTIC Science & Technology

2012-07-01

difficult for the department to manage because it would need to be achieved in only nine months (between the cut’s tak - ing effect in January 2013 and the...Estimate of DoD’s Funding Under the BCA Caps After Automatic Reductionse 469 472 475 477 480 483 485 487 489 493 d Nominal Dollars 2013 Dollars 2022

Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification

PubMed Central

Zhi, Wenbo; Wang, Meiyao

2014-01-01

The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrated the feasibility of using the selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG) have good linear ranges, generally 103 – 104. There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R2 is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins at 0.2 – 2 fmol level, which are comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R2 = 0.928 and 0.851 respectively). The correlation between SRM/MRM and ELISA for CP is less desirable (R2 = 0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides (R2 = 0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or Luminex beads are not available. PMID:21594933

Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

PubMed

Duellman, Tyler; Burnett, John; Yang, Jay

2015-03-15

Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32 h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. Copyright © 2014 Elsevier Inc. All rights reserved.

A Colorimetric Microplate Assay for DNA-Binding Activity of His-Tagged MutS Protein.

PubMed

Banasik, Michał; Sachadyn, Paweł

2016-09-01

A simple microplate method was designed for rapid testing DNA-binding activity of proteins. The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein (approximately 0.5-10 pmol) and DNA (0.1-10 pmol) and a relatively short time of analysis (up to 60 min). The method is very simple to apply and convenient to test different buffer conditions of DNA-protein binding. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader. The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.

Light assisted drying (LAD) for protein stabilization: optical characterization of samples

NASA Astrophysics Data System (ADS)

Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

2018-02-01

Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

Total protein measurement in canine cerebrospinal fluid: agreement between a turbidimetric assay and 2 dye-binding methods and determination of reference intervals using an indirect a posteriori method.

PubMed

Riond, B; Steffen, F; Schmied, O; Hofmann-Lehmann, R; Lutz, H

2014-03-01

In veterinary clinical laboratories, qualitative tests for total protein measurement in canine cerebrospinal fluid (CSF) have been replaced by quantitative methods, which can be divided into dye-binding assays and turbidimetric methods. There is a lack of validation data and reference intervals (RIs) for these assays. The aim of the present study was to assess agreement between the turbidimetric benzethonium chloride method and 2 dye-binding methods (Pyrogallol Red-Molybdate method [PRM], Coomassie Brilliant Blue [CBB] technique) for measurement of total protein concentration in canine CSF. Furthermore, RIs were determined for all 3 methods using an indirect a posteriori method. For assay comparison, a total of 118 canine CSF specimens were analyzed. For RIs calculation, clinical records of 401 canine patients with normal CSF analysis were studied and classified according to their final diagnosis in pathologic and nonpathologic values. The turbidimetric assay showed excellent agreement with the PRM assay (mean bias 0.003 g/L [-0.26-0.27]). The CBB method generally showed higher total protein values than the turbidimetric assay and the PRM assay (mean bias -0.14 g/L for turbidimetric and PRM assay). From 90 of 401 canine patients, nonparametric reference intervals (2.5%, 97.5% quantile) were calculated (turbidimetric assay and PRM method: 0.08-0.35 g/L (90% CI: 0.07-0.08/0.33-0.39); CBB method: 0.17-0.55 g/L (90% CI: 0.16-0.18/0.52-0.61). Total protein concentration in canine CSF specimens remained stable for up to 6 months of storage at -80°C. Due to variations among methods, RIs for total protein concentration in canine CSF have to be calculated for each method. The a posteriori method of RIs calculation described here should encourage other veterinary laboratories to establish RIs that are laboratory-specific. ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

PubMed

Fernández-Sierra, Mónica; Quiñones, Edwin

2015-03-15

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect. Copyright © 2015 Elsevier Inc. All rights reserved.

In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

PubMed

Machida, Kazuya

2017-01-01

Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

PubMed

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

MRM assay for quantitation of complement components in human blood plasma - a feasibility study on multiple sclerosis.

PubMed

Rezeli, Melinda; Végvári, Akos; Ottervald, Jan; Olsson, Tomas; Laurell, Thomas; Marko-Varga, György

2011-12-10

As a proof-of-principle study, a multiple reaction monitoring (MRM) assay was developed for quantitation of proteotypic peptides, representing seven plasma proteins associated with inflammation (complement components and C-reactive protein). The assay development and the sample analysis were performed on a linear ion trap mass spectrometer. We were able to quantify 5 of the 7 target proteins in depleted plasma digests with reasonable reproducibility over a 2 orders of magnitude linear range (RSD≤25%). The assay panel was utilized for the analysis of a small multiple sclerosis sample cohort with 10 diseased and 8 control patients. Copyright © 2011 Elsevier B.V. All rights reserved.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads. PMID:22688388

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

PubMed

Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J

2014-01-07

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

A high-throughput differential filtration assay to screen and select detergents for membrane proteins

PubMed Central

Vergis, James M.; Purdy, Michael D.; Wiener, Michael C.

2015-01-01

Structural studies on integral membrane proteins are routinely performed on protein–detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours. PMID:20667442

Use of recombinant salmonella flagellar hook protein (flgk) for detection of anti-salmonella antibodies in chickens by automated capillary immunoassay

USDA-ARS?s Scientific Manuscript database

Background: Conventional immunoblot assays are a very useful tool for specific protein identification, but are tedious, labor-intensive and time-consuming. An automated capillary electrophoresis-based immunoblot assay called "Simple Western" has recently been developed that enables the protein sepa...

Novel Cell-Based Assays for Detecting Low Levels of Active Ricin Following Decontamination

DTIC Science & Technology

2011-12-01

fluorescent protein, are powerful tools, which have been used for detection assays for ricin protein ( DeWet , 1987). Zhao et al. (2005) have reported a...toxic Type 2 Ribosome-Inactivating Proteins. FEBS Lett. 2004, 563, pp 219–222. DeWet , J.R. et al. Firefly Luciferase Gene: Structure and

AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

EPA Science Inventory

An ELISA assay for heme oxygenase (HO-l )

Abstract

A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression.

PubMed

Yu, Yanbin; Piddington, Christopher; Fitzpatrick, Dan; Twomey, Brian; Xu, Ren; Swanson, Steven J; Jing, Shuqian

2006-10-20

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.

A modified method for determining tannin-protein precipitation capacity using accelerated solvent extraction (ASE) and microplate gel filtration.

PubMed

McArt, Scott H; Spalinger, Donald E; Kennish, John M; Collins, William B

2006-06-01

The protein precipitation assay used by Robbins et al., (1987) Ecology 68:98-107 has been shown to predict successfully the reduction in protein availability to some ruminants due to tannins. The procedure, however, is expensive and laborious, which limits its utility, especially for quantitative ecological or nutritional applications where large numbers of assays may be required. We have modified the method to decrease its cost and increase laboratory efficiency by: (1) automating the extraction by using Accelerated Solvent Extraction (ASE); and (2) by scaling and automating the precipitation reaction, chromatography, and spectrometry with microplate gel filtration and an automated UV-VIS microplate spectrometer. ASE extraction is shown to be as effective at extracting tannins as the hot methanol technique. Additionally, the microplate assay is sensitive and precise. We show that the results from the new technique correspond in a nearly 1:1 relationship to the results of the previous technique. Hence, this method could reliably replace the older method with no loss in relevance to herbivore protein digestion. Moreover, the ASE extraction technique should be applicable to other tannin-protein precipitation assays and possibly other phenolic assays.

Dye-binding protein assay using a long-wave-absorbing cyanine probe.

PubMed

Zheng, Hong; Mao, Yu Xia; Li, Dong Hui; Zhu, Chang Qing

2003-07-01

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.

A novel assay reveals preferential binding between Rabs, kinesins, and specific endosomal subpopulations

PubMed Central

Bentley, Marvin; Decker, Helena; Luisi, Julie

2015-01-01

Identifying the proteins that regulate vesicle trafficking is a fundamental problem in cell biology. In this paper, we introduce a new assay that involves the expression of an FKBP12-rapamycin–binding domain–tagged candidate vesicle-binding protein, which can be inducibly linked to dynein or kinesin. Vesicles can be labeled by any convenient method. If the candidate protein binds the labeled vesicles, addition of the linker drug results in a predictable, highly distinctive change in vesicle localization. This assay generates robust and easily interpretable results that provide direct experimental evidence of binding between a candidate protein and the vesicle population of interest. We used this approach to compare the binding of Kinesin-3 family members with different endosomal populations. We found that KIF13A and KIF13B bind preferentially to early endosomes and that KIF1A and KIF1Bβ bind preferentially to late endosomes and lysosomes. This assay may have broad utility for identifying the trafficking proteins that bind to different vesicle populations. PMID:25624392

Pull-down Assay to Characterize Ca2+/Calmodulin Binding to Plant Receptor Kinases.

PubMed

Kaufmann, Christine; Sauter, Margret

2017-01-01

Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca 2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca 2+ .

Molecular and Clinical Aspects of Protein Aggregation Assays in Neurodegenerative Diseases.

PubMed

Villar-Piqué, Anna; Schmitz, Matthias; Candelise, Niccolò; Ventura, Salvador; Llorens, Franc; Zerr, Inga

2018-02-10

The presence of protein deposits is a common pathological hallmark in patients suffering from neurodegenerative conditions and other proteinopathies. Deciphering the molecular basis of protein misfolding and aggregation is a crucial step towards the full comprehension of the factors that trigger the onset of these diseases and for the development of efficient therapeutical strategies. In this regard, in vitro aggregation assays for misfolded proteins offer an excellent tool to study pathological processes of protein deposition under controlled conditions, where confounders can be easily discriminated. These methods are generally cost-effective and have been proved useful in many fields, including drug discovery and clinical diagnostics. Here, we review the bases of in vitro aggregation and seeding assays, recapitulate their main applications and offer a critical evaluation of their limitations. Comprehending the molecular mechanisms behind these assays and combining them with in vivo or cell-based experiments will maximize their potential and allow the necessary improvement to overcome some of the current drawbacks.

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

Cancer.gov

The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

Using an enzymatic galactose assay to detect lactose glycation extents of two proteins caseinate and soybean protein isolate via the Maillard reaction.

PubMed

Wang, Xiao-Peng; Zhao, Xin-Huai

2017-06-01

Glycation of food proteins via the Maillard reaction has been widely studied in the recent years; however, the amount of saccharide connected to proteins is usually not determined. An enzymatic galactose assay was proposed firstly in this study to detect lactose glycation extents of caseinate and soybean protein isolate (SPI) during the Maillard reaction at two temperatures and different times. The separated glycated proteins were hydrolysed to release galactose necessary for the enzymatic assay and glycation calculation. Caseinate and SPI both obtained the highest lactose glycation extents at 100 °C or 121 °C by a reaction time of 180 or 20 min. Short- and long-time reaction resulted in lower glycation extents. During the reaction, three chemical indices (absorbences at 294/490 nm and fluorescence intensities) of reaction mixtures increased continually, but another index reactable NH 2 of glycated proteins showed the opposite trend. In general, changing profiles of the four indices were inconsistent with those profiles of lactose glycation extents of glycated proteins, implying practical limitation of the four indices in studies. This proposed enzymatic assay could directly detect lactose glycation of the two proteins, and thus was more useful than the four chemical indices to monitor glycation of the two proteins. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Hexahistidine (6xHis) fusion-based assays for protein-protein interactions.

PubMed

Puckett, Mary C

2015-01-01

Fusion-protein tags provide a useful method to study protein-protein interactions. One widely used fusion tag is hexahistidine (6xHis). This tag has unique advantages over others due to its small size and the relatively low abundance of naturally occurring consecutive histidine repeats. 6xHis tags can interact with immobilized metal cations to provide for the capture of proteins and protein complexes of interest. In this chapter, a description of the benefits and uses of 6xHis-fusion proteins as well as a detailed method for performing a 6xHis-pulldown assay are described.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Solid-phase assays for small molecule screening using sol-gel entrapped proteins.

PubMed

Lebert, Julie M; Forsberg, Erica M; Brennan, John D

2008-04-01

With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput screening of small molecule libraries and mixtures.

Assay Development Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

Typical steps involved in the development of a  mass spectrometry-based targeted assay include: (1) selection of surrogate or signature peptides corresponding to the targeted protein or modification of interest; (2) iterative optimization of instrument and method parameters for optimal detection of the selected peptide; (3) method development for protein extraction from biological matrices such as tissue, whole cell lysates, or blood plasma/serum and proteolytic digestion of proteins (usually with trypsin); (4) evaluation of the assay in the intended biological matrix to determine if e

Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry

PubMed Central

Wider, Diana

2017-01-01

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits. PMID:29220385

Secreted dual reporter assay with Gaussia luciferase and the red fluorescent protein mCherry.

PubMed

Wider, Diana; Picard, Didier

2017-01-01

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.

Dominance of serotype Ia among group B Streptococci causing invasive infections in nonpregnant adults in Portugal.

PubMed

Martins, E R; Melo-Cristino, J; Ramirez, M

2012-04-01

The population of group B streptococci (GBS) associated with invasive infections in nonpregnant adults from 2001 to 2008 was analyzed in isolates submitted from 24 hospital laboratories in Portugal (n = 225). The isolates were characterized by antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and surface protein gene profiling. GBS invasive cases were found more frequently among men in all age groups. In addition, serotype Ia was the most frequent in our collection, whereas serotype V is dominant elsewhere. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and surface protein gene eps and by ST24 and bca, similarly to neonatal invasive infections in Portugal, indicating that the same genetic lineages can be responsible for both vaginal colonization and invasive disease in all age groups. In contrast, the hypervirulent serotype III/ST17 neonatal lineage was responsible for a minority of infections. Serotype V isolates were distributed into two genetic lineages, one defined by ST1 and surface protein gene alp3 and macrolide resistant, and another presenting with ST2 and eps and fully susceptible to all antimicrobials tested. The erm(TR) gene was the most frequently found among erythromycin-resistant isolates, while the bovine-associated tet(O) gene was found in a minority of tetracycline-resistant isolates. Our data emphasize the importance of local identification of the genetic lineages responsible for GBS invasive infections in nonpregnant adults. The dominance of serotype Ia in invasive disease in Portugal highlights the importance of this serotype in GBS pathogenesis.

Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.

PubMed

Bryant, Anne-Marie; Davis, Jenny; Cai, Shuowei; Singh, Bal Ram

2013-02-01

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

Quantitatively and Kinetically Identifying Binding Motifs of Amelogenin Proteins to Mineral Crystals Through Biochemical and Spectroscopic Assays

PubMed Central

Zhu, Li; Hwang, Peter; Witkowska, H. Ewa; Liu, Haichuan; Li, Wu

2014-01-01

Tooth enamel is the hardest tissue in vertebrate animals. Consisting of millions of carbonated hydroxyapatite crystals, this highly mineralized tissue develops from a protein matrix in which amelogenin is the predominant component. The enamel matrix proteins are eventually and completely degraded and removed by proteinases to form mineral-enriched tooth enamel. Identification of the apatite-binding motifs in amelogenin is critical for understanding the amelogenin–crystal interactions and amelogenin–proteinases interactions during tooth enamel biomineralization. A stepwise strategy is introduced to kinetically and quantitatively identify the crystal-binding motifs in amelogenin, including a peptide screening assay, a competitive adsorption assay, and a kinetic-binding assay using amelogenin and gene-engineered amelogenin mutants. A modified enzyme-linked immunosorbent assay on crystal surfaces is also applied to compare binding amounts of amelogenin and its mutants on different planes of apatite crystals. We describe the detailed protocols for these assays and provide the considerations for these experiments in this chapter. PMID:24188774

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

PubMed

Petrovic, Voin; Olaisen, Camilla; Sharma, Animesh; Nepal, Anala; Bugge, Steffen; Sundby, Eirik; Hoff, Bård Helge; Slupphaug, Geir; Otterlei, Marit

2017-04-15

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Method for estimating protein binding capacity of polymeric systems.

PubMed

Sharma, Vaibhav; Blackwood, Keith A; Haddow, David; Hook, Lilian; Mason, Chris; Dye, Julian F; García-Gareta, Elena

2015-01-01

Composite biomaterials made from synthetic and protein-based polymers are extensively researched in tissue engineering. To successfully fabricate a protein-polymer composite, it is critical to understand how strongly the protein binds to the synthetic polymer, which occurs through protein adsorption. Currently, there is no cost-effective and simple method for characterizing this interfacial binding. To characterize this interfacial binding, we introduce a simple three-step method that involves: 1) synthetic polymer surface characterisation, 2) a quick, inexpensive and robust novel immuno-based assay that uses protein extraction compounds to characterize protein binding strength followed by 3) an in vitro 2D model of cell culture to confirm the results of the immuno-based assay. Fibrinogen, precursor of fibrin, was adsorbed (test protein) on three different polymeric surfaces: silicone, poly(acrylic acid)-coated silicone and poly(allylamine)-coated silicone. Polystyrene surface was used as a reference. Characterisation of the different surfaces revealed different chemistry and roughness. The novel immuno-based assay showed significantly stronger binding of fibrinogen to both poly(acrylic acid) and poly(allylamine) coated silicone. Finally, cell studies showed that the strength of the interaction between the protein and the polymer had an effect on cell growth. This novel immuno-based assay is a valuable tool in developing composite biomaterials of synthetic and protein-based polymers with the potential to be applied in other fields of research where protein adsorption onto surfaces plays an important role.

Protein microarray with horseradish peroxidase chemiluminescence for quantification of serum α-fetoprotein.

PubMed

Zhao, Yuanshun; Zhang, Yonghong; Lin, Dongdong; Li, Kang; Yin, Chengzeng; Liu, Xiuhong; Jin, Boxun; Sun, Libo; Liu, Jinhua; Zhang, Aiying; Li, Ning

2015-10-01

To develop and evaluate a protein microarray assay with horseradish peroxidase (HRP) chemiluminescence for quantification of α-fetoprotein (AFP) in serum from patients with hepatocellular carcinoma (HCC). A protein microarray assay for AFP was developed. Serum was collected from patients with HCC and healthy control subjects. AFP was quantified using protein microarray and enzyme-linked immunosorbent assay (ELISA). Serum AFP concentrations determined via protein microarray were positively correlated (r = 0.973) with those determined via ELISA in patients with HCC (n = 60) and healthy control subjects (n = 30). Protein microarray showed 80% sensitivity and 100% specificity for HCC diagnosis. ELISA had 83.3% sensitivity and 100% specificity. Protein microarray effectively distinguished between patients with HCC and healthy control subjects (area under ROC curve 0.974; 95% CI 0.000, 1.000). Protein microarray is a rapid, simple and low-cost alternative to ELISA for detecting AFP in human serum. © The Author(s) 2015.

Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

PubMed

Ngo, Tony; Coleman, James L J; Smith, Nicola J

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

Methodological requirements for valid tissue-based biomarker studies that can be used in clinical practice.

PubMed

True, Lawrence D

2014-03-01

Paralleling the growth of ever more cost efficient methods to sequence the whole genome in minute fragments of tissue has been the identification of increasingly numerous molecular abnormalities in cancers--mutations, amplifications, insertions and deletions of genes, and patterns of differential gene expression, i.e., overexpression of growth factors and underexpression of tumor suppressor genes. These abnormalities can be translated into assays to be used in clinical decision making. In general terms, the result of such an assay is subject to a large number of variables regarding the characteristics of the available sample, particularities of the used assay, and the interpretation of the results. This review discusses the effects of these variables on assays of tissue-based biomarkers, classified by macromolecule--DNA, RNA (including micro RNA, messenger RNA, long noncoding RNA, protein, and phosphoprotein). Since the majority of clinically applicable biomarkers are immunohistochemically detectable proteins this review focuses on protein biomarkers. However, the principles outlined are mostly applicable to any other analyte. A variety of preanalytical variables impacts on the results obtained, including analyte stability (which is different for different analytes, i.e., DNA, RNA, or protein), period of warm and of cold ischemia, fixation time, tissue processing, sample storage time, and storage conditions. In addition, assay variables play an important role, including reagent specificity (notably but not uniquely an issue concerning antibodies used in immunohistochemistry), technical components of the assay, quantitation, and assay interpretation. Finally, appropriateness of an assay for clinical application is an important issue. Reference is made to publicly available guidelines to improve on biomarker development in general and requirements for clinical use in particular. Strategic goals are formulated in order to improve on the quality of biomarker reporting, including issues of analyte quality, experimental detail, assay efficiency and precision, and assay appropriateness.

Micro-flow injection system for the urinary protein assay.

PubMed

Nishihama, Syouhei; Imabayashi, Hisano; Matoba, Tomoko; Toya, Chika; Watanabe, Kosuke; Yoshizuka, Kazuharu

2008-02-15

A urinary protein assay has been investigated, employing a micro-flow injection analysis (muFIA) combined with an adsorptive separation of protein from analyte. The adsorptive separation part of protein in the artificial urine with ceramic hydroxyapatite is integrated on the muFIA chip, since the interference of other components coexisting in urine occurs in the conventional FIA system. The typical FI peak can be obtained following the adsorption-elution process of the protein prior to the detection, and the protein concentration in artificial urine can be quantitatively determined.

Exploring physical and chemical factors influencing the properties of recombinant prion protein and the real-time quaking-induced conversion (RT-QuIC) assay.

PubMed

Cheng, Keding; Sloan, Angela; Avery, Kristen M; Coulthart, Michael; Carpenter, Michael; Knox, J David

2014-01-01

Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP(d)) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots*

PubMed Central

Chambers, Andrew G.; Percy, Andrew J.; Yang, Juncong; Borchers, Christoph H.

2015-01-01

The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R2 value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1–7.5% CV and 9.5–11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from −20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications. PMID:26342038

ApoHRP-based assay to measure intracellular regulatory heme.

PubMed

Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A; Dhahbi, Joseph M

2015-02-01

The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β (Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.

High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling.

PubMed

Ashby, Jonathan; Duan, Yaokai; Ligans, Erik; Tamsi, Michael; Zhong, Wenwan

2015-02-17

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

The Practice of Scaling down Practical Assessment Components of Agriculture in Junior Secondary Schools Curriculum: A Synthesis of Teachers Perceptions

ERIC Educational Resources Information Center

Hulela, Keba

2017-01-01

This ethnographic research study aimed at investigating factors that contributed to the decline in the number of practical assessed projects in junior secondary agricultural education assessment in Botswana. Participant-observation technique was used to gather data in the form of field notes from in-service teachers at BCA and in-school teachers…

Fact Sheet: Selected Highlights of the FY2017 Military Construction Appropriations Bills

DTIC Science & Technology

2016-08-18

heavily influenced by the statutorily mandated discretionary spending caps established by P.L. 114-74, the Bipartisan Budget Act of 2015 (BBA). A...established by the Budget Control Act (BCA) of 2011 (P.L. 112-25). New budget authority (funding not previously appropriated) for military...Military Construction and Family Housing New Budget Authority ................................ 3 Tables Table 1. Summary Military Construction

Increasing the Effectiveness of Navy Retention Compensation by Evaluating Different Auction Compensation Formats

DTIC Science & Technology

2015-06-01

ABBREVIATIONS ACCP aviation career continuation pay BCA Budget Control Act CO commanding officer DOD Department of Defense MILPERS Military Personnel...healthcare costs, overhaul retirement benefits, set a limit on pay increases, and eliminate acquisition programs. Ensuring an optimum level of...Congressional Budget Office, 2014). This appropriation houses the basic pay , allowances and retention bonuses that are offered to service members

Contribution of biocontrol agents to sustainable agriculture: do insights from microbiome research and BCA “omics” pay off

USDA-ARS?s Scientific Manuscript database

By the year 2050 there will be 9 to 11 billion people on earth to feed using the same amount or less land and water as is currently available for agricultural production. The United Nations estimated that global food production will need to increase by 70% by 2050. Currently, about one-third of all ...

Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

PubMed

Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

2014-03-15

Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. Copyright © 2013 Elsevier B.V. All rights reserved.

Technical and Clinical Success and Long-Term Durability of Endovascular Treatment for Atherosclerotic Aortic Arch Branch Origin Obstruction: Evaluation of 144 Procedures.

PubMed

van de Weijer, M A J; Vonken, E J P A; de Vries, J-P P M; Moll, F L; Vos, J A; de Borst, G J

2015-07-01

Endovascular treatment of atherosclerotic obstruction of aortic arch branch origins (AABO) has largely replaced open surgery, but long-term outcome data are lacking. This study evaluated mid-term and long-term results of these procedures. Retrospective cohort study. Patients underwent endovascular treatment for symptomatic atherosclerotic stenosis of AABO between 1995 and 2012. Technical success was defined as uncomplicated revascularization and residual stenosis ≤30%. The primary end point was freedom from restenosis ≥50% on Duplex ultrasonography or magnetic resonance angiography. Secondary end points were freedom from target lesion revascularization or recurrent symptoms. 144 lesions were treated in 114 patients (75 female; mean age 66.3 years), by percutaneous transluminal angioplasty (PTA) in 20 patients and PTA and stent in 117 patients (brachiocephalic artery [BCA] 9/54; left common carotid artery [LCCA] 0/7; left subclavian artery [LSA] 11/56). The lesion could not be passed in four patients, and in three patients the intervention was terminated before angioplasty. The 30-day technical success was 94.4%, without deaths or strokes. Mean follow-up was 52.0 months (range 2-163 months). Restenosis-free survival was 95.6%, 92.9%, 87.6%, and 83.2% at 12, 24, 48, and 60 months, respectively. Log-rank test showed no significant difference between PTA only and PTA with additional stent placement at any point (p = .375), nor between BCA (n = 51), LCCA (n = 6), or LSA (n = 57). During follow-up, 27 patients (23.7%) became symptomatic (15 BCA, 1 LCCA, and 11 LSA); 19 patients with a restenosis of the target lesion (mean 56.7 months). Symptom-free survival was 94.7%, 92.0%, 82.3%, and 77.9% at 12, 24, 48, and 60 months, respectively. Endovascular treatment of aortic arch branch origin obstruction is safe and efficacious in experienced hands and can be considered as the preferred treatment, with good mid-term durability. Recurrent symptomatic lesions can be treated safely by renewed endovascular means. Copyright © 2015 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Indigenous Pseudomonas spp. Strains from the Olive (Olea europaea L.) Rhizosphere as Effective Biocontrol Agents against Verticillium dahliae: From the Host Roots to the Bacterial Genomes

PubMed Central

Gómez-Lama Cabanás, Carmen; Legarda, Garikoitz; Ruano-Rosa, David; Pizarro-Tobías, Paloma; Valverde-Corredor, Antonio; Niqui, José L.; Triviño, Juan C.; Roca, Amalia; Mercado-Blanco, Jesús

2018-01-01

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive (Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae, were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the “Pseudomonas mandelii subgroup,” within the “Pseudomonas fluorescens group,” Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the “Pseudomonas aeruginosa group,” Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs. PMID:29527195

Indigenous Pseudomonas spp. Strains from the Olive (Olea europaea L.) Rhizosphere as Effective Biocontrol Agents against Verticillium dahliae: From the Host Roots to the Bacterial Genomes.

PubMed

Gómez-Lama Cabanás, Carmen; Legarda, Garikoitz; Ruano-Rosa, David; Pizarro-Tobías, Paloma; Valverde-Corredor, Antonio; Niqui, José L; Triviño, Juan C; Roca, Amalia; Mercado-Blanco, Jesús

2018-01-01

The use of biological control agents (BCA), alone or in combination with other management measures, has gained attention over the past decades, driven by the need to seek for sustainable and eco-friendly alternatives to confront plant pathogens. The rhizosphere of olive ( Olea europaea L.) plants is a source of bacteria with potential as biocontrol tools against Verticillium wilt of olive (VWO) caused by Verticillium dahliae Kleb. A collection of bacterial isolates from healthy nursery-produced olive (cultivar Picual, susceptible to VWO) plants was generated based on morphological, biochemical and metabolic characteristics, chemical sensitivities, and on their in vitro antagonistic activity against several olive pathogens. Three strains (PIC25, PIC105, and PICF141) showing high in vitro inhibition ability of pathogens' growth, particularly against V. dahliae , were eventually selected. Their effectiveness against VWO caused by the defoliating pathotype of V. dahliae was also demonstrated, strain PICF141 being the rhizobacteria showing the best performance as BCA. Genotypic and phenotypic traits traditionally associated with plant growth promotion and/or biocontrol abilities were evaluated as well (e.g., phytase, xylanase, catalase, cellulase, chitinase, glucanase activities, and siderophore and HCN production). Multi-locus sequence analyses of conserved genes enabled the identification of these strains as Pseudomonas spp. Strain PICF141 was affiliated to the " Pseudomonas mandelii subgroup," within the " Pseudomonas fluorescens group," Pseudomonas lini being the closest species. Strains PIC25 and PIC105 were affiliated to the " Pseudomonas aeruginosa group," Pseudomonas indica being the closest relative. Moreover, we identified P. indica (PIC105) for the first time as a BCA. Genome sequencing and in silico analyses allowed the identification of traits commonly associated with plant-bacteria interactions. Finally, the root colonization ability of these olive rhizobacteria was assessed, providing valuable information for the future development of formulations based on these strains. A set of actions, from rhizosphere isolation to genome analysis, is proposed and discussed for selecting indigenous rhizobacteria as effective BCAs.

Molecular basis of 'hypoxic' breast cancer cell radio-sensitization: phytochemicals converge on radiation induced Rel signaling.

PubMed

Aravindan, Sheeja; Natarajan, Mohan; Herman, Terence S; Awasthi, Vibhudutta; Aravindan, Natarajan

2013-03-04

Heterogeneously distributed hypoxic areas are a characteristic property of locally advanced breast cancers (BCa) and generally associated with therapeutic resistance, metastases, and poor patient survival. About 50% of locally advanced BCa, where radiotherapy is less effective are suggested to be due to hypoxic regions. In this study, we investigated the potential of bioactive phytochemicals in radio-sensitizing hypoxic BCa cells. Hypoxic (O2-2.5%; N2-92.5%; CO2-5%) MCF-7 cells were exposed to 4 Gy radiation (IR) alone or after pretreatment with Curcumin (CUR), curcumin analog EF24, neem leaf extract (NLE), Genistein (GEN), Resveratrol (RES) or raspberry extract (RSE). The cells were examined for inhibition of NFκB activity, transcriptional modulation of 88 NFκB signaling pathway genes, activation and cellular localization of radio-responsive NFκB related mediators, eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2, -5 and associated induction of cell death. EMSA revealed that cells exposed to phytochemicals showed complete suppression of IR-induced NFκB. Relatively, cells exposed EF24 revealed a robust inhibition of IR-induced NFκB. QPCR profiling showed induced expression of 53 NFκB signaling pathway genes after IR. Conversely, 53, 50, 53, 53, 53 and 53 of IR-induced genes were inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. In addition, 25, 29, 24, 16, 11 and 21 of 35 IR-suppressed genes were further inhibited with EF24, NLE, CUR, GEN, RES and RSE respectively. Immunoblotting revealed a significant attenuating effect of IR-modulated radio-responsive eNos, Erk1/2, SOD2, Akt1/2/3, p50, p65, pIκBα, TNFα, Birc-1, -2 and -5 with EF24, NLE, CUR, GEN, RES or RSE. Annexin V-FITC staining showed a consistent and significant induction of IR-induced cell death with these phytochemicals. Notably, EF24 robustly conferred IR-induced cell death. Together, these data identifies the potential hypoxic cell radio-sensitizers and further implies that the induced radio-sensitization may be exerted by selectively targeting IR-induced NFκB signaling.

The pH of chemistry assays plays an important role in monoclonal immunoglobulin interferences.

PubMed

Alberti, Michael O; Drake, Thomas A; Song, Lu

2015-12-01

Immunoglobulin paraproteins can interfere with multiple chemistry assays. We want to investigate the mechanisms of immunoglobulin interference. Serum samples containing paraproteins from the index patient and eight additional patients were used to investigate the interference with the creatinine and total protein assays on the Beckman Coulter AU5400/2700 analyzer, and to determine the effects of pH and ionic strength on the precipitation of different immunoglobulins in these patient samples. The paraprotein interference with the creatinine and total protein assays was caused by the precipitation of IgM paraprotein in the index patient's samples under alkaline assay conditions. At extremely high pH (12-13) and extremely low pH (1-2) and low ionic strength, paraprotein formed large aggregates in samples from the index patient but not from other patients. The pH and ionic strength are the key factors that contribute to protein aggregation and precipitation which interfere with the creatinine and total protein measurements on AU5400/2700. The different amino acid sequence of each monoclonal paraprotein will determine the pH and ionic strength at which the paraprotein will precipitate.

Nonaminoglycoside compounds induce readthrough of nonsense mutations

PubMed Central

Damoiseaux, Robert; Nahas, Shareef; Gao, Kun; Hu, Hailiang; Pollard, Julianne M.; Goldstine, Jimena; Jung, Michael E.; Henning, Susanne M.; Bertoni, Carmen

2009-01-01

Large numbers of genetic disorders are caused by nonsense mutations for which compound-induced readthrough of premature termination codons (PTCs) might be exploited as a potential treatment strategy. We have successfully developed a sensitive and quantitative high-throughput screening (HTS) assay, protein transcription/translation (PTT)–enzyme-linked immunosorbent assay (ELISA), for identifying novel PTC-readthrough compounds using ataxia-telangiectasia (A-T) as a genetic disease model. This HTS PTT-ELISA assay is based on a coupled PTT that uses plasmid templates containing prototypic A-T mutated (ATM) mutations for HTS. The assay is luciferase independent. We screened ∼34,000 compounds and identified 12 low-molecular-mass nonaminoglycosides with potential PTC-readthrough activity. From these, two leading compounds consistently induced functional ATM protein in ATM-deficient cells containing disease-causing nonsense mutations, as demonstrated by direct measurement of ATM protein, restored ATM kinase activity, and colony survival assays for cellular radiosensitivity. The two compounds also demonstrated readthrough activity in mdx mouse myotube cells carrying a nonsense mutation and induced significant amounts of dystrophin protein. PMID:19770270

A biotin-drug extraction and acid dissociation (BEAD) procedure to eliminate matrix and drug interference in a protein complex anti-drug antibody (ADA) isotype specific assay.

PubMed

Niu, Hongmei; Klem, Thomas; Yang, Jinsong; Qiu, Yongchang; Pan, Luying

2017-07-01

Monitoring anti-drug antibody (ADA) responses in patients receiving protein therapeutics treatment is an important safety assessment for regulatory agencies, drug manufacturers, clinicians and patients. Recombinant human IGF-1/IGFBP-3 (rhIGF-1/rhIGFBP-3) is a 1:1 formulation of naturally occurring protein complex. The individual IGF-1 and IGFBP-3 proteins have multiple binding partners in serum matrix with high binding affinity to each other, which presents challenges in ADA assay development. We have developed a biotin-drug extraction with acid dissociation (BEAD) procedure followed by an electrochemiluminescence (ECL) direct assay to overcome matrix and drug interference. The method utilizes two step acid dissociation and excess biotin-drug to extract total ADA, which are further captured by soluble biotin-drug and detected in an ECL semi-homogeneous direct assay format. The pre-treatment method effectively eliminates interference by serum matrix and free drug, and enhances assay sensitivity. The assays passed acceptance criteria for all validation parameters, and have been used for clinical sample Ab testing. This method principle exemplifies a new approach for anti-isotype ADA assays, and could be an effective strategy for neutralizing antibody (NAb), pharmacokinetic (PK) and biomarker analysis in need of overcoming interference factors. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of a high-throughput relative chemical stability assay to screen therapeutic protein formulations by assessment of conformational stability and correlation to aggregation propensity.

PubMed

Rizzo, Joseph M; Shi, Shuai; Li, Yunsong; Semple, Andrew; Esposito, Jessica J; Yu, Shenjiang; Richardson, Daisy; Antochshuk, Valentyn; Shameem, Mohammed

2015-05-01

In this study, an automated high-throughput relative chemical stability (RCS) assay was developed in which various therapeutic proteins were assessed to determine stability based on the resistance to denaturation post introduction to a chaotrope titration. Detection mechanisms of both intrinsic fluorescence and near UV circular dichroism (near-UV CD) are demonstrated. Assay robustness was investigated by comparing multiple independent assays and achieving r(2) values >0.95 for curve overlays. The complete reversibility of the assay was demonstrated by intrinsic fluorescence, near-UV CD, and biologic potency. To highlight the method utility, we compared the RCS assay with differential scanning calorimetry and dynamic scanning fluorimetry methodologies. Utilizing C1/2 values obtained from the RCS assay, formulation rank-ordering of 12 different mAb formulations was performed. The prediction of long-term stability on protein aggregation is obtained by demonstrating a good correlation with an r(2) of 0.83 between RCS and empirical aggregation propensity data. RCS promises to be an extremely useful tool to aid in candidate formulation development efforts based on the complete reversibility of the method to allow for multiple assessments without protein loss and the strong correlation between the C1/2 data obtained and accelerated stability under stressed conditions. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system.

PubMed

Colombini, S; Broderick, G A; Clayton, M K

2011-04-01

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3 mg of N/mL of inoculum) and incubated with 10 mL of ruminal inoculum and 5 mL of buffer; fermentations were stopped after 2 h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t=0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (kd) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters Km (Michaelis constant) and kd (Vmax/Km), where Vmax=maximum velocity, were estimated directly; kd values were adjusted (Akd) for the fraction FD0 using the equation Akd=kd-FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean=0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins.

PubMed

Carmichael, W W; An, J

1999-01-01

Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).

Tandem assays of protein and glucose with functionalized core/shell particles based on magnetic separation and surface-enhanced Raman scattering.

PubMed

Kong, Xianming; Yu, Qian; Lv, Zhongpeng; Du, Xuezhong

2013-10-11

Tandem assays of protein and glucose in combination with mannose-functionalized Fe3 O4 @SiO2 and Ag@SiO2 tag particles have promising potential in effective magnetic separation and highly sensitive and selective SERS assays of biomaterials. It is for the first time that tandem assay of glucose is developed using SERS based on the Con A-sandwiched microstructures between the functionalized magnetic and tag particles. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of molecular chaperones using a Xenopus oocyte protein refolding assay.

PubMed

Heikkila, John J; Kaldis, Angelo; Abdulle, Rashid

2006-01-01

Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates as a source of molecular chaperones involved in folding. In this chapter, we describe the production and purification of a Xenopus laevis recombinant small Hsp, Hsp30C, and an in vivo luciferase (LUC) refolding assay employing microinjected Xenopus oocytes. This assay tests whether LUC can be maintained in a folding-competent state when heat denatured in the presence of a small Hsp or other molecular chaperone. For example, micro-injection of heat-denatured LUC alone into oocytes resulted in minimal reactivation of enzyme activity. However, LUC heat denatured in the presence of Hsp30C resulted in 100% recovery of enzyme activity after microinjection. The in vivo oocyte refolding system is more sensitive and requires less molecular chaperone than in vitro refolding assays. Also, this protocol is not limited to testing Xenopus molecular chaperones because small Hsps from other organisms have been used successfully.

Development of a Targeted Urine Proteome Assay for kidney diseases.

PubMed

Cantley, Lloyd G; Colangelo, Christopher M; Stone, Kathryn L; Chung, Lisa; Belcher, Justin; Abbott, Thomas; Cantley, Jennifer L; Williams, Kenneth R; Parikh, Chirag R

2016-01-01

Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM. The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A green fluorescent protein-based assay for high-throughput ligand-binding studies of a mycobacterial biotin protein ligase.

PubMed

Bond, Thomas E H; Sorenson, Alanna E; Schaeffer, Patrick M

2017-12-01

Biotin protein ligase (BirA) has been identified as an emerging drug target in Mycobacterium tuberculosis due to its essential metabolic role. Indeed, it is the only enzyme capable of covalently attaching biotin onto the biotin carboxyl carrier protein subunit of the acetyl-CoA carboxylase. Despite recent interest in this protein, there is still a gap in cost-effective high-throughput screening assays for rapid identification of mycobacterial BirA-targeting inhibitors. We present for the first time the cloning, expression, purification of mycobacterial GFP-tagged BirA and its application for the development of a high-throughput assay building on the principle of differential scanning fluorimetry of GFP-tagged proteins. The data obtained in this study reveal how biotin and ATP significantly increase the thermal stability (ΔT m =+16.5°C) of M. tuberculosis BirA and lead to formation of a high affinity holoenzyme complex (K obs =7.7nM). The new findings and mycobacterial BirA high-throughput assay presented in this work could provide an efficient platform for future anti-tubercular drug discovery campaigns. Copyright © 2017 Elsevier GmbH. All rights reserved.

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

PubMed Central

Kang, Hong-Jun; Vassilopoulos, Athanassios

2016-01-01

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context. PMID:26966987

Quantification of systemic and local immune responses to individual rotavirus proteins during rotavirus infection in mice.

PubMed Central

Ishida, S; Feng, N; Tang, B; Gilbert, J M; Greenberg, H B

1996-01-01

The purpose of the present study was to develop a quantitative assay that could be used to measure the local and systemic immune responses to specific rotavirus proteins following rotavirus infection of adult mice. To measure these responses, we used an immunocytochemical staining assay of Spodoptera frugiperda (Sf-9) cells which were infected with recombinant baculovirus expressing selected rotavirus proteins. The specificity of the assay was documented by using a series of monoclonal antibodies to individual rotavirus proteins. We observed that the assay had high levels of sensitivity and specificity for a series of VP7- and VP4-specific neutralizing monoclonal antibodies which recognized conformation-dependent epitopes on their target proteins. We also studied immunoglobulin G (IgG) immune responses in serum and IgA immune responses in the stools of mice infected with wild-type murine rotavirus strain EHPw. In both sera and stools, the most immunogenic proteins were VP6 and VP4. VP2 was less immunogenic than VP6 or VP4, and the immune responses to VP7, NSP2, and NSP4 were very low in serum and undetectable in stools. PMID:8784572

ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

PubMed Central

Vänelid, Johan; Siegbahn, Agneta; Ericsson, Olle; Fredriksson, Simon; Bäcklin, Christofer; Gut, Marta; Heath, Simon; Gut, Ivo Glynne; Wallentin, Lars; Gustafsson, Mats G.; Kamali-Moghaddam, Masood; Landegren, Ulf

2011-01-01

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. PMID:21980495

Identification of RNAIII-binding proteins in Staphylococcus aureus using tethered RNAs and streptavidin aptamers based pull-down assay.

PubMed

Zhang, Xu; Zhu, Qing; Tian, Tian; Zhao, Changlong; Zang, Jianye; Xue, Ting; Sun, Baolin

2015-05-15

It has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. In Staphylococcus aureus, many sRNAs have been identified and some of them have been functionally studied. Since it is difficult to identify RNA-binding proteins (RBPs), very little has been known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified 81 proteins, and expressed heterologously 9 of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay, and the result indicates that proteins CshA, RNase J2, Era, Hu, WalR, Pyk, and FtsZ can bind to RNAIII. This study suggests that some proteins can bind to RNA III in S. aureus, and may be involved in RNA III function. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, which should facilitate the identification and functional study of RBPs in diverse bacterial species.

Assay of Deoxyhypusine Synthase Activity

PubMed Central

Wolff, Edith C.; Lee, Seung Bum; Park, Myung Hee

2011-01-01

Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (<50 amino acids). Optimum conditions for the reaction and four detection methods for the product, deoxyhypusine-containing eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [3H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [3H] deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [3H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter. PMID:21318875

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

PubMed

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.

PubMed

Nong, Rachel Yuan; Wu, Di; Yan, Junhong; Hammond, Maria; Gu, Gucci Jijuan; Kamali-Moghaddam, Masood; Landegren, Ulf; Darmanis, Spyros

2013-06-01

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Ehwang; Gao, Yuqian; Wu, Chaochao

Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

A Micro-Polyethylene Glycol Precipitation Assay as a Relative Solubility Screening Tool for Monoclonal Antibody Design and Formulation Development.

PubMed

Toprani, Vishal M; Joshi, Sangeeta B; Kueltzo, Lisa A; Schwartz, Richard M; Middaugh, C Russell; Volkin, David B

2016-08-01

Adequate protein solubility is an important prerequisite for development, manufacture, and administration of biotherapeutic drug candidates, especially for high-concentration protein formulations. A previously established method for determining the relative apparent solubility (thermodynamic activity) of proteins using polyethylene glycol (PEG) precipitation is adapted for screening and comparing monoclonal antibody (mAb) candidates where only limited quantities (≤1 mg) are available. This micro-PEG assay is used to evaluate various broadly neutralizing mAb candidates to HIV-1 viral spike (gp120 and gp41 glycoproteins). Using ∼1 mg of VRC01-WT mAb per assay, the precision of the micro-PEG assay was established. A series of 7 different broadly neutralizing mAbs to the HIV-1 viral spike proteins were compared by curve shape (%PEG vs. protein concentration), %PEGmidpoint determinations, and extrapolated apparent solubility values. Numerous formulation conditions were then evaluated for their relative effects on the VRC01-WT mAb. The PEGmidpt and apparent solubility values of VRC01-WT mAb decreased as the solution pH increased and increased as NaCl and arginine were added. A final optimization of the micro-PEG assay established that amounts as low as 0.1-0.2 mg can be used. Thus, the micro-PEG assay has significant potential as a relative solubility screening tool during candidate selection and early formulation development. Copyright © 2016 American Pharmacists Association®. All rights reserved.

A solid-phase assay for studying direct binding of progranulin to TNFR and progranulin antagonism of TNF/TNFR interactions.

PubMed

Tian, Qingyun; Zhao, Shuai; Liu, Chuanju

2014-01-01

The discovery that TNF receptors (TNFR) serve as the binding receptors for progranulin (PGRN) reveals the significant role of PGRN in inflammatory and autoimmune diseases, including inflammatory arthritis. Herein we describe a simple, antibody-free analytical assay, i.e., a biotin-based solid-phase binding assay, to examine the direct interaction of PGRN/TNFR and the PGRN inhibition of TNF/TNFR interactions. Briefly, a 96-well high-binding microplate is first coated with the first protein (protein A), and after blocking, the coated microplate is incubated with the biotin-labeled second protein (protein B) in the absence or presence of the third protein (protein C). Finally the streptavidin conjugated with a detecting enzyme is added, followed by a signal measurement. Also discussed in this chapter are the advantages of the strategy, key elements to obtain reliable results, and discrepancies among various PGRN proteins in view of the binding activity with TNFR.

Development of a Highly Automated and Multiplexed Targeted Proteome Pipeline and Assay for 112 Rat Brain Synaptic Proteins

PubMed Central

Colangelo, Christopher M.; Ivosev, Gordana; Chung, Lisa; Abbott, Thomas; Shifman, Mark; Sakaue, Fumika; Cox, David; Kitchen, Rob R.; Burton, Lyle; Tate, Stephen A; Gulcicek, Erol; Bonner, Ron; Rinehart, Jesse; Nairn, Angus C.; Williams, Kenneth R.

2015-01-01

We present a comprehensive workflow for large scale (>1000 transitions/run) label-free LC-MRM proteome assays. Innovations include automated MRM transition selection, intelligent retention time scheduling (xMRM) that improves Signal/Noise by >2-fold, and automatic peak modeling. Improvements to data analysis include a novel Q/C metric, Normalized Group Area Ratio (NGAR), MLR normalization, weighted regression analysis, and data dissemination through the Yale Protein Expression Database. As a proof of principle we developed a robust 90 minute LC-MRM assay for Mouse/Rat Post-Synaptic Density (PSD) fractions which resulted in the routine quantification of 337 peptides from 112 proteins based on 15 observations per protein. Parallel analyses with stable isotope dilution peptide standards (SIS), demonstrate very high correlation in retention time (1.0) and protein fold change (0.94) between the label-free and SIS analyses. Overall, our first method achieved a technical CV of 11.4% with >97.5% of the 1697 transitions being quantified without user intervention, resulting in a highly efficient, robust, and single injection LC-MRM assay. PMID:25476245

Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

PubMed

Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

2016-08-01

Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

PubMed

Lam, Rosanna Y Y; Lin, Zhi-Xiu; Sviderskaya, Elena V; Cheng, Christopher H K

2014-08-21

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol.

Biosensors: Viruses for ultrasensitive assays

NASA Astrophysics Data System (ADS)

Donath, Edwin

2009-04-01

A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

Method for detection of antibodies for metallic elements

DOEpatents

Barrick, C.W.; Clarke, S.M.; Nordin, C.W.

1993-11-30

An apparatus and method for detecting antibodies specific to non-protein antigens. The apparatus is an immunological plate containing a plurality of plastic projections coated with a non-protein material. Assays utilizing the plate are capable of stabilizing the non-protein antigens with detection levels for antibodies specific to the antigens on a nanogram level. A screening assay with the apparatus allows for early detection of exposure to non-protein materials. Specifically metallic elements are detected. 10 figures.

BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

PubMed Central

Kuo, P-C; Huang, C-W; Lee, C-I; Chang, H-W; Hsieh, S-W; Chung, Y-P; Lee, M-S; Huang, C-S; Tsao, L-P; Tsao, Y-P; Chen, S-L

2015-01-01

Background: We showed previously that breast carcinoma amplified sequence 2 (BCAS2) functions as a negative regulator of p53. We also found that BCAS2 is a potential AR-associated protein. AR is essential for the growth and survival of prostate carcinoma. Therefore we characterised the correlation between BCAS2 and AR. Methods: Protein interactions were examined by GST pull-down assay and co-immunoprecipitation. Clinical prostate cancer (PCa) specimens were evaluated by immunohistochemical assay. AR transcriptional activity and LNCaP cell growth were assessed by luciferase assay and MTT assay, respectively. Results: BCAS2 expression was significantly increased in PCa. BCAS2 stabilised AR protein through both hormone-dependent and -independent manners. There are at least two mechanisms for BCAS2-mediated AR protein upregulation: One is p53-dependent. The p53 is suppressed by BCAS2 that results in increasing AR mRNA and protein expression. The other is via p53-independent inhibition of proteasome degradation. As BCAS2 can form a complex with AR and HSP90, it may function with HSP90 to stabilise AR protein from being degraded by proteasome. Conclusions: In this study, we show that BCAS2 is a novel AR-interacting protein and characterise the correlation between BCAS2 and PCa. Thus we propose that BCAS2 could be a diagnostic marker and therapeutic target for PCa. PMID:25461807

Evaluation of the sensitizing potential of food proteins using two mouse models.

PubMed

Smit, Joost; Zeeuw-Brouwer, Mary-Lène de; van Roest, Manon; de Jong, Govardus; van Bilsen, Jolanda

2016-11-16

The current methodology to identify allergenic food proteins is effective in identifying those that are likely to cross-react with known allergens. However, most assays show false positive results for low/non-allergens. Therefore, an ex vivo/in vitro DC-T cell assay and an in vivo mouse model were used to distinguish known allergenic food proteins (Ara h 1, β-Lactoglobulin, Pan b 1, bovine serum albumin, whey protein isolate) from low/non allergenic food proteins (soy lipoxygenase, gelatin, beef tropomyosin, rubisco, Sola t 1). CD4+ T cells from protein/alum-immunized mice were incubated with corresponding protein-pulsed bone marrow-derived DC and analyzed for cytokine release. All known allergens induced Th2 responses in vitro, whereas soy lipoxygenase, gelatin or beef tropomyosin did not. Sola t 1 and rubisco induced a more generalized T cell response due to endotoxin contamination, indicating the endotoxin-sensitivity of the DC-T assay. To analyze responses in vivo, mice were orally sensitized on days 0 and 7. Known allergens induced IgE and mMCP-1 release upon oral challenge at day 16, whereas the low/non-allergens did not. Both the DC-T cell assay and the mouse model were able to distinguish 5 known allergens from 5 low/non-allergens and may be useful to identify novel allergenic food proteins. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.

2011-09-10

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenousmore » nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.« less

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PubMed

Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

Quantification of SAA1 and SAA2 in lung cancer plasma using the isotype-specific PRM assays.

PubMed

Kim, Yeoun Jin; Gallien, Sebastien; El-Khoury, Victoria; Goswami, Panchali; Sertamo, Katriina; Schlesser, Marc; Berchem, Guy; Domon, Bruno

2015-09-01

The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross-reactivity. On the other hand, when mass spectrometry (MS)-based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype-specific (SAA1α,  SAA 1β,  SAA1γ,  SAA2α,  SAA2β), protein-specific (SAA1 or SAA2), and pan SAA (SAA1 and SAA2) were chosen to differentiate SAAs in lung cancer plasma samples using a panel of PRM assays. The measurement of specific isotypes, leveraging the analytical performance of PRM, allowed to quantify the allelic variants of both target proteins. The isotypes detected were corroborated with the genetic information obtained from the same samples. The combination of SAA2α and SAA2β assays representing the total SAA2 concentration demonstrated a superior analytical outcome than the previously used assay on the common peptide when applied to the detection of lung cancer. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermal precipitation fluorescence assay for protein stability screening.

PubMed

Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma*

PubMed Central

Abbatiello, Susan E.; Schilling, Birgit; Mani, D. R.; Zimmerman, Lisa J.; Hall, Steven C.; MacLean, Brendan; Albertolle, Matthew; Allen, Simon; Burgess, Michael; Cusack, Michael P.; Gosh, Mousumi; Hedrick, Victoria; Held, Jason M.; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kinsinger, Christopher R.; Lyssand, John; Makowski, Lee; Mesri, Mehdi; Rodriguez, Henry; Rudnick, Paul; Sadowski, Pawel; Sedransk, Nell; Shaddox, Kent; Skates, Stephen J.; Kuhn, Eric; Smith, Derek; Whiteaker, Jeffery R.; Whitwell, Corbin; Zhang, Shucha; Borchers, Christoph H.; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel C.; MacCoss, Michael J.; Neubert, Thomas A.; Paulovich, Amanda G.; Regnier, Fred E.; Tempst, Paul; Carr, Steven A.

2015-01-01

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma. PMID:25693799

Implications of AM for the Navy Supply Chain

DTIC Science & Technology

2016-12-01

Cornell University and Queens University of Canada. He is the co-chair of the America Makes Working Group for Additive Manufacturing Qualification and...strategic deployment of additive manufacturing (AM) ma- chines throughout the supply chain, coupled with the right business model, is an imperative need in...60 Table 1. Additive Manufacturing Business Model Factors to develop a standard BCA template, taking into consideration the parameters in Table 1

Overseas Contingency Operations Funding: Background and Status

DTIC Science & Technology

2017-02-07

emergency” or “Overseas Contingency Operation/ Global War on Terror” (OCO/GWOT) requirement in annual agency budget requests—or both. Funds designated as...to be excluded from budget control limits. The BCA added the designation “Overseas Contingency Operation/ Global War on Terror” to the BBEDCA...been largely provided through supplemental appropriation acts or has been designated as an “emergency” or “overseas contingency operation/ global war on

Defense: FY2013 Authorization and Appropriations

DTIC Science & Technology

2012-07-13

overturn several cost- cutting initiatives incorporated in the Administration’s budget, including proposed reductions in the Air Force Reserve and the...additional $1.2 trillion, the BCA will trigger automatic reductions that would cut the Administration’s current DOD base budget plan by whatever amount is...needed to cover the defense share of the shortfall between whatever cuts Congress does agree to and the required total reduction of $2.1 trillion