Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma.

PubMed

Kong, Qian; Li, Wen-Jing; Huang, Hua-Rong; Zhong, Ying-Qiang; Fang, Jian-Pei

2015-05-01

Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. For fold-change>2 and p < 0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.

Dielectrophoretic Isolation and Detection of cfc-DNA Nanoparticulate Biomarkers and Virus from Blood

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; McCanna, James; Krishnan, Rajaram; Rassenti, Laura; Kipps, Thomas J.; Heller, Michael J.

2015-01-01

Dielectrophoretic (DEP) microarray devices allow important cellular nanoparticulate biomarkers and virus to be rapidly isolated, concentrated and detected directly from clinical and biological samples. A variety of sub-micron nanoparticulate entities including cell free circulating (cfc) DNA, mitochondria and virus can be isolated into DEP high-field areas on microelectrodes, while blood cells and other micron-size entities become isolated into DEP low-field areas between the microelectrodes. The nanoparticulate entities are held in the DEP high-field areas while cells are washed away along with proteins and other small molecules which are not affected by the DEP electric fields. DEP carried out on 20 µL of whole blood obtained from Chronic Lymphocytic Leukemia (CLL) patients showed a considerable amount of SYBR Green stained DNA fluorescent material concentrated in the DEP high-field regions. Whole blood obtained from healthy individuals showed little or no fluorescent DNA materials in the DEP high-field regions. Fluorescent T7 bacteriophage virus could be isolated directly from blood samples, and fluorescently stained mitochondria could be isolated from biological buffer samples. Using newer DEP microarray devices, high molecular weight (hmw) DNA could be isolated from serum and detected at levels as low as 8–16 ng/mL. PMID:23436471

Rapid detection of cancer related DNA nanoparticulate biomarkers and nanoparticles in whole blood

NASA Astrophysics Data System (ADS)

Heller, Michael J.; Krishnan, Raj; Sonnenberg, Avery

2010-08-01

The ability to rapidly detect cell free circulating (cfc) DNA, cfc-RNA, exosomes and other nanoparticulate disease biomarkers as well as drug delivery nanoparticles directly in blood is a major challenge for nanomedicine. We now show that microarray and new high voltage dielectrophoretic (DEP) devices can be used to rapidly isolate and detect cfc-DNA nanoparticulates and nanoparticles directly from whole blood and other high conductance samples (plasma, serum, urine, etc.). At DEP frequencies of 5kHz-10kHz both fluorescent-stained high molecular weight (hmw) DNA, cfc-DNA and fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP highfield regions over the microelectrodes, while blood cells move to the DEP low field-regions. The blood cells can then be removed by a simple fluidic wash while the DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260ng/ml and the nanoparticles at <9.5 x 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Disease specific cfc-DNA materials could also be detected directly in blood from patients with Chronic Lymphocytic Leukemia (CLL) and confirmed by PCR genotyping analysis.

Blood-based biomarkers used to predict disease activity in Crohn's disease and ulcerative colitis.

PubMed

Burakoff, Robert; Pabby, Vikas; Onyewadume, Louisa; Odze, Robert; Adackapara, Cheryl; Wang, Wei; Friedman, Sonia; Hamilton, Matthew; Korzenik, Joshua; Levine, Jonathan; Makrauer, Frederick; Cheng, Changming; Smith, Hai Choo; Liew, Choong-Chin; Chao, Samuel

2015-05-01

Identifying specific genes that are differentially expressed during inflammatory bowel disease flares may help stratify disease activity. The aim of this study was to identify panels of genes to be able to distinguish disease activity in Crohn's disease (CD) and ulcerative colitis (UC). Patients were grouped into categories based on disease and severity determined by histological grading. Whole blood was collected by PAXgene Blood RNA collection tubes, (PreAnalytiX) and gene expression analysis using messenger RNA was conducted. Logistic regression was performed on multiple combinations of common probe sets, and data were evaluated in terms of discrimination by computing the area under the receiving operator characteristic curve (ROC-AUC). Nine inactive CD, 8 mild CD, 10 moderate-to-severe CD, 9 inactive UC, 8 mild UC, 10 moderate-to-severe UC, and 120 controls were hybridized to Affymetrix U133 Plus 2 microarrays. Panels of 6 individual genes discriminated the stages of disease activity: CD with mild severity {ROC-AUC, 0.89 (95% confidence interval [CI], 0.84%-0.95%)}, CD with moderate-to-severe severity (ROC-AUC 0.98 [95% CI, 0.97-1.0]), UC with mild severity (ROC-AUC 0.92 [95% CI, 0.87-0.96]), and UC with moderate-to-severe severity (ROC-AUC 0.99 [95% CI, 0.97-1.0]). Validation by real-time reverse transcription-PCR confirmed the Affymetrix microarray data. The specific whole blood gene panels reliably distinguished CD and UC and determined the activity of disease, with high sensitivity and specificity in our cohorts of patients. This simple serological test has the potential to become a biomarker to determine the activity of disease.

Novel approach for deriving genome wide SNP analysis data from archived blood spots

PubMed Central

2012-01-01

Background The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. Findings DNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. Conclusions DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies. PMID:22974252

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

PubMed Central

Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

2013-01-01

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

Whole Blood Gene Expression Profile Associated with Spontaneous Preterm Birth in Women with Threatened Preterm Labor

PubMed Central

Heng, Yujing Jan; Pennell, Craig Edward; Chua, Hon Nian; Perkins, Jonathan Edward; Lye, Stephen James

2014-01-01

Threatened preterm labor (TPTL) is defined as persistent premature uterine contractions between 20 and 37 weeks of gestation and is the most common condition that requires hospitalization during pregnancy. Most of these TPTL women continue their pregnancies to term while only an estimated 5% will deliver a premature baby within ten days. The aim of this work was to study differential whole blood gene expression associated with spontaneous preterm birth (sPTB) within 48 hours of hospital admission. Peripheral blood was collected at point of hospital admission from 154 women with TPTL before any medical treatment. Microarrays were utilized to investigate differential whole blood gene expression between TPTL women who did (n = 48) or did not have a sPTB (n = 106) within 48 hours of admission. Total leukocyte and neutrophil counts were significantly higher (35% and 41% respectively) in women who had sPTB than women who did not deliver within 48 hours (p<0.001). Fetal fibronectin (fFN) test was performed on 62 women. There was no difference in the urine, vaginal and placental microbiology and histopathology reports between the two groups of women. There were 469 significant differentially expressed genes (FDR<0.05); 28 differentially expressed genes were chosen for microarray validation using qRT-PCR and 20 out of 28 genes were successfully validated (p<0.05). An optimal random forest classifier model to predict sPTB was achieved using the top nine differentially expressed genes coupled with peripheral clinical blood data (sensitivity 70.8%, specificity 75.5%). These differentially expressed genes may further elucidate the underlying mechanisms of sPTB and pave the way for future systems biology studies to predict sPTB. PMID:24828675

Aging: a portrait from gene expression profile in blood cells.

PubMed

Calabria, Elisa; Mazza, Emilia Maria Cristina; Dyar, Kenneth Allen; Pogliaghi, Silvia; Bruseghini, Paolo; Morandi, Carlo; Salvagno, Gian Luca; Gelati, Matteo; Guidi, Gian Cesare; Bicciato, Silvio; Schiaffino, Stefano; Schena, Federico; Capelli, Carlo

2016-08-01

The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43±3 and 68±4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process.

Bone health nutraceuticals alter microarray mRNA gene expression: A randomized, parallel, open-label clinical study.

PubMed

Lin, Yumei; Kazlova, Valentina; Ramakrishnan, Shyam; Murray, Mary A; Fast, David; Chandra, Amitabh; Gellenbeck, Kevin W

2016-01-15

Dietary intake of fruits and vegetables has been suggested to have a role in promoting bone health. More specifically, the polyphenols they contain have been linked to physiological effects related to bone mineral density and bone metabolism. In this research, we use standard microarray analyses of peripheral whole blood from post-menopausal women treated with two fixed combinations of plant extracts standardized to polyphenol content to identify differentially expressed genes relevant to bone health. In this 28-day open-label study, healthy post-menopausal women were randomized into three groups, each receiving one of three investigational fixed combinations of plant extracts: an anti-resorptive (AR) combination of pomegranate fruit (Punica granatum L.) and grape seed (Vitis vinifera L.) extracts; a bone formation (BF) combination of quercetin (Dimorphandra mollis Benth) and licorice (Glycyrrhiza glabra L.) extracts; and a fixed combination of all four plant extracts (AR plus BF). Standard microarray analysis was performed on peripheral whole blood samples taken before and after each treatment. Annotated genes were analyzed for their association to bone health by comparison to a gene library. The AR combination down-regulated a number of genes involved in reduction of bone resorption including cathepsin G (CTSG) and tachykinin receptor 1 (TACR1). The AR combination also up-regulated genes associated with formation of extracellular matrix including heparan sulfate proteoglycan 2 (HSPG2) and hyaluronoglucosaminidase 1 (HYAL1). In contrast, treatment with the BF combination resulted in up-regulation of bone morphogenetic protein 2 (BMP-2) and COL1A1 (collagen type I α1) genes which are linked to bone and collagen formation while down-regulating genes linked to osteoclastogenesis. Treatment with a combination of all four plant extracts had a distinctly different effect on gene expression than the results of the AR and BF combinations individually. These results could be due to multiple feedback systems balancing activities of osteoblasts and osteoclasts. In summary, this ex-vivo microarray study indicated that the pomegranate, grape seed, quercetin and licorice combinations of plant extracts modulated gene expression for both osteoclastic and osteogenic processes. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Molecular methods for septicemia diagnosis.

PubMed

Marco, Francesc

2017-11-01

Septicemia remains a major cause of hospital mortality. Blood culture remains the best approach to identify the etiological microorganisms when a bloodstream infection is suspected but it takes long time because it relies on bacterial or fungal growth. The introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology, DNA hybridization, microarrays or rapid PCR-based test significantly reduce the time to results. Tests for direct detection in whole blood samples are highly desirable because of their potential to identify bloodstream pathogens without waiting for blood cultures to become positive. Nonetheless, limitations of current molecular diagnostic methods are substantial. This article reviews these new molecular approaches (LightCycler SeptiFast, Magicplex sepsis real time, Septitest, VYOO, PCR/ESI-MS analysis, T2Candida). Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Detection of total and A1c-glycosylated hemoglobin in human whole blood using sandwich immunoassays on polydimethylsiloxane-based antibody microarrays.

PubMed

Chen, Huang-Han; Wu, Chih-Hsing; Tsai, Mei-Ling; Huang, Yi-Jing; Chen, Shu-Hui

2012-10-16

The percentage of glycosylated hemoglobin A1c (%GHbA1c) in human whole blood indicates the average plasma glucose concentration over a prolonged period of time and is used to diagnose diabetes. However, detecting GHbA1c in the whole blood using immunoassays has limited detection sensitivity due to its low percentage in total hemoglobin (tHb) and interference from various glycan moieties in the sample. We have developed a sandwich immunoassay using an antibody microarray on a polydimethylsiloxane (PDMS) substrate modified with fluorinated compounds to detect tHb and glycosylated hemoglobin A1c (GHbA1c) in human whole blood without sample pretreatment. A polyclonal antibody against hemoglobin (Hb) immobilized on PDMS is used as a common capture probe to enrich all forms of Hb followed by detection via monoclonal anti-Hb and specific monoclonal anti-GHbA1c antibodies for tHb and GHbA1c detection, respectively. This method prevents the use of glycan binding molecules and dramatically reduces the background interference, yielding a detection limit of 3.58 ng/mL for tHb and 0.20 ng/mL for GHbA1c. The fluorinated modification on PDMS is superior to the glass substrate and eliminates the need for the blocking step which is required in commercial enzyme linked immunosorbent assay (ELISA) kits. Moreover, the detection sensitivity for GHbA1c is 4-5 orders of magnitude higher, but the required sample amount is 25 times less than the commercial method. On the basis of patient sample data, a good linear correlation between %GHbA1c values determined by our method and the certified high performance liquid chromatography (HPLC) standard method is shown with R(2) > 0.98, indicating the great promise of the developed method for clinical applications.

Statistical analysis of an RNA titration series evaluates microarray precision and sensitivity on a whole-array basis

PubMed Central

Holloway, Andrew J; Oshlack, Alicia; Diyagama, Dileepa S; Bowtell, David DL; Smyth, Gordon K

2006-01-01

Background Concerns are often raised about the accuracy of microarray technologies and the degree of cross-platform agreement, but there are yet no methods which can unambiguously evaluate precision and sensitivity for these technologies on a whole-array basis. Results A methodology is described for evaluating the precision and sensitivity of whole-genome gene expression technologies such as microarrays. The method consists of an easy-to-construct titration series of RNA samples and an associated statistical analysis using non-linear regression. The method evaluates the precision and responsiveness of each microarray platform on a whole-array basis, i.e., using all the probes, without the need to match probes across platforms. An experiment is conducted to assess and compare four widely used microarray platforms. All four platforms are shown to have satisfactory precision but the commercial platforms are superior for resolving differential expression for genes at lower expression levels. The effective precision of the two-color platforms is improved by allowing for probe-specific dye-effects in the statistical model. The methodology is used to compare three data extraction algorithms for the Affymetrix platforms, demonstrating poor performance for the commonly used proprietary algorithm relative to the other algorithms. For probes which can be matched across platforms, the cross-platform variability is decomposed into within-platform and between-platform components, showing that platform disagreement is almost entirely systematic rather than due to measurement variability. Conclusion The results demonstrate good precision and sensitivity for all the platforms, but highlight the need for improved probe annotation. They quantify the extent to which cross-platform measures can be expected to be less accurate than within-platform comparisons for predicting disease progression or outcome. PMID:17118209

Blood miRNAs as sensitive and specific biological indicators of environmental and occupational exposure to volatile organic compound (VOC).

PubMed

Song, Mi-Kyung; Ryu, Jae-Chun

2015-10-01

To date, there is still shortage of highly sensitive and specific minimally invasive biomarkers for assessment of environmental toxicants exposure. Because of the significance of microRNA (miRNA) in various diseases, circulating miRNAs in blood may be unique biomarkers for minimally invasive prediction of toxicants exposure. We identified and validated characteristic miRNA expression profiles of human whole blood in workers exposed to volatile organic compounds (VOCs) and compared the usefulness of miRNA indicator of VOCs with the effectiveness of the already used urinary biomarkers of occupational exposure. Using a microarray based approach we screened and detected deregulated miRNAs in their expression in workers exposed to VOCs (toluene [TOL], xylene [XYL] and ethylbenzene [EBZ]). Total 169 workers from four dockyards were enrolled in current study, and 50 subjects of them were used for miRNA microarray analysis. We identified 467 miRNAs for TOL, 211 miRNAs for XYL, and 695 miRNAs for XYL as characteristic discernible exposure indicator, which could discerned each VOC from the control group with higher accuracy, sensitivity, and specificity than urinary biomarkers. Current observations from this study point out that the altered levels of circulating miRNAs can be a reliable novel, minimally invasive biological indicator of occupational exposure to VOCs. Copyright © 2015 Elsevier GmbH. All rights reserved.

Assessing copy number from exome sequencing and exome array CGH based on CNV spectrum in a large clinical cohort.

PubMed

Retterer, Kyle; Scuffins, Julie; Schmidt, Daniel; Lewis, Rachel; Pineda-Alvarez, Daniel; Stafford, Amanda; Schmidt, Lindsay; Warren, Stephanie; Gibellini, Federica; Kondakova, Anastasia; Blair, Amanda; Bale, Sherri; Matyakhina, Ludmila; Meck, Jeanne; Aradhya, Swaroop; Haverfield, Eden

2015-08-01

Detection of copy-number variation (CNV) is important for investigating many genetic disorders. Testing a large clinical cohort by array comparative genomic hybridization provides a deep perspective on the spectrum of pathogenic CNV. In this context, we describe a bioinformatics approach to extract CNV information from whole-exome sequencing and demonstrate its utility in clinical testing. Exon-focused arrays and whole-genome chromosomal microarray analysis were used to test 14,228 and 14,000 individuals, respectively. Based on these results, we developed an algorithm to detect deletions/duplications in whole-exome sequencing data and a novel whole-exome array. In the exon array cohort, we observed a positive detection rate of 2.4% (25 duplications, 318 deletions), of which 39% involved one or two exons. Chromosomal microarray analysis identified 3,345 CNVs affecting single genes (18%). We demonstrate that our whole-exome sequencing algorithm resolves CNVs of three or more exons. These results demonstrate the clinical utility of single-exon resolution in CNV assays. Our whole-exome sequencing algorithm approaches this resolution but is complemented by a whole-exome array to unambiguously identify intragenic CNVs and single-exon changes. These data illustrate the next advancements in CNV analysis through whole-exome sequencing and whole-exome array.Genet Med 17 8, 623-629.

Development and characterization of a disposable plastic microarray printhead.

PubMed

Griessner, Matthias; Hartig, Dave; Christmann, Alexander; Pohl, Carsten; Schellhase, Michaela; Ehrentreich-Förster, Eva

2011-06-01

During the last decade microarrays have become a powerful analytical tool. Commonly microarrays are produced in a non-contact manner using silicone printheads. However, silicone printheads are expensive and not able to be used as a disposable. Here, we show the development and functional characterization of 8-channel plastic microarray printheads that overcome both disadvantages of their conventional silicone counterparts. A combination of injection-molding and laser processing allows us to produce a high quantity of cheap, customizable and disposable microarray printheads. The use of plastics (e.g., polystyrene) minimizes the need for surface modifications required previously for proper printing results. Time-consuming regeneration processes, cleaning procedures and contaminations caused by residual samples are avoided. The utilization of plastic printheads for viscous liquids, such as cell suspensions or whole blood, is possible. Furthermore, functional parts within the plastic printhead (e.g., particle filters) can be included. Our printhead is compatible with commercially available TopSpot devices but provides additional economic and technical benefits as compared to conventional TopSpot printheads, while fulfilling all requirements demanded on the latter. All in all, this work describes how the field of traditional microarray spotting can be extended significantly by low cost plastic printheads.

Longitudinal Analysis of Whole Blood Transcriptomes to Explore Molecular Signatures Associated With Acute Renal Allograft Rejection

PubMed Central

Shin, Heesun; Günther, Oliver; Hollander, Zsuzsanna; Wilson-McManus, Janet E.; Ng, Raymond T.; Balshaw, Robert; Keown, Paul A.; McMaster, Robert; McManus, Bruce M.; Isbel, Nicole M.; Knoll, Greg; Tebbutt, Scott J.

2014-01-01

In this study, we explored a time course of peripheral whole blood transcriptomes from kidney transplantation patients who either experienced an acute rejection episode or did not in order to better delineate the immunological and biological processes measureable in blood leukocytes that are associated with acute renal allograft rejection. Using microarrays, we generated gene expression data from 24 acute rejectors and 24 nonrejectors. We filtered the data to obtain the most unambiguous and robustly expressing probe sets and selected a subset of patients with the clearest phenotype. We then performed a data-driven exploratory analysis using data reduction and differential gene expression analysis tools in order to reveal gene expression signatures associated with acute allograft rejection. Using a template-matching algorithm, we then expanded our analysis to include time course data, identifying genes whose expression is modulated leading up to acute rejection. We have identified molecular phenotypes associated with acute renal allograft rejection, including a significantly upregulated signature of neutrophil activation and accumulation following transplant surgery that is common to both acute rejectors and nonrejectors. Our analysis shows that this expression signature appears to stabilize over time in nonrejectors but persists in patients who go on to reject the transplanted organ. In addition, we describe an expression signature characteristic of lymphocyte activity and proliferation. This lymphocyte signature is significantly downregulated in both acute rejectors and nonrejectors following surgery; however, patients who go on to reject the organ show a persistent downregulation of this signature relative to the neutrophil signature. PMID:24526836

Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis

PubMed Central

Pan, Nanan; Sun, Ning; Wang, Qiujun; Fan, Jingxue; Zhou, Ping

2015-01-01

The incidence of osteoporosis is high in postmenopausal women due to altered estrogen levels and continuous calcium loss that occurs with aging. Recent studies have shown that microRNAs (miRNAs) are involved in the development of osteoporosis. These miRNAs may be used as potential biomarkers to identify women at a high risk for developing the disease. In this study, whole blood samples were collected from 48 postmenopausal Chinese women with osteopenia or osteoporosis and pooled into six groups according to individual T-scores. A miRNA microarray analysis was performed on pooled blood samples to identify potential miRNA biomarkers for postmenopausal osteoporosis. Five miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, and -590-5p) were identified in the microarray analysis. These dysregulated miRNAs were subjected to a pathway analysis investigating whether they were involved in regulating osteoporosis-related pathways. Among them, only miR-194-5p was enriched in multiple osteoporosis-related pathways. Enhanced miR-194-5p expression in women with osteoporosis was confirmed by quantitative reverse transcription–polymerase chain reaction analysis. For external validation, a significant correlation between the expression of miR-194-5p and T-scores was found in an independent patient collection comprised of 24 postmenopausal women with normal bone mineral density, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (p < 0.05). Taken together, the present findings suggest that miR-194-5p may be a viable miRNA biomarker for postmenopausal osteoporosis. PMID:26038726

Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis.

PubMed

Meng, Jia; Zhang, Dapeng; Pan, Nanan; Sun, Ning; Wang, Qiujun; Fan, Jingxue; Zhou, Ping; Zhu, Wenliang; Jiang, Lihong

2015-01-01

The incidence of osteoporosis is high in postmenopausal women due to altered estrogen levels and continuous calcium loss that occurs with aging. Recent studies have shown that microRNAs (miRNAs) are involved in the development of osteoporosis. These miRNAs may be used as potential biomarkers to identify women at a high risk for developing the disease. In this study, whole blood samples were collected from 48 postmenopausal Chinese women with osteopenia or osteoporosis and pooled into six groups according to individual T-scores. A miRNA microarray analysis was performed on pooled blood samples to identify potential miRNA biomarkers for postmenopausal osteoporosis. Five miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, and -590-5p) were identified in the microarray analysis. These dysregulated miRNAs were subjected to a pathway analysis investigating whether they were involved in regulating osteoporosis-related pathways. Among them, only miR-194-5p was enriched in multiple osteoporosis-related pathways. Enhanced miR-194-5p expression in women with osteoporosis was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. For external validation, a significant correlation between the expression of miR-194-5p and T-scores was found in an independent patient collection comprised of 24 postmenopausal women with normal bone mineral density, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (p < 0.05). Taken together, the present findings suggest that miR-194-5p may be a viable miRNA biomarker for postmenopausal osteoporosis.

Whole blood miRNA expression analysis reveals miR-3613-3p as a potential biomarker for dedifferentiated liposarcoma.

PubMed

Fricke, A; Cimniak, A F V; Ullrich, P V; Becherer, C; Bickert, C; Pfeifer, D; Heinz, J; Stark, G B; Bannasch, H; Braig, D; Eisenhardt, S U

2018-04-09

Liposarcoma constitute about 13% of all soft tissue sarcoma and are associated with a high risk of metastases. As the preoperative differentiation between benign and malign lipomatous tumors is restricted to magnetic resonance imaging, computed tomography and biopsy, we performed a miRNA array to distinguish dedifferentiated liposarcoma patients from healthy controls and lipoma patients. Blood samples of patients with dedifferentiated liposarcoma, healthy controls and lipoma patients were collected. Whole blood RNA was extracted and samples of patients with dedifferentiated liposarcoma (n= 6) and of healthy donors (n= 4) were analyzed using an Affymetrix GeneChip miRNA Array v. 4.0. qRT-PCR was carried out to confirm the most differentially expressed miRNA; being further analyzed in an independent cohort of healthy controls as well as in lipoma patients. As shown by the microarray, two miRNAs (miR-3613-3p, miR-4668-5p) were shown to be significantly upregulated (fold change: > 2.5; p< 0.05) in patients with dedifferentiated liposarcoma (n= 6) as compared to healthy controls (n= 4). miR-3613-3p was further validated by qRT-PCR to be significantly upregulated in dedifferentiated liposarcoma patients compared to an independent cohort of healthy controls (n= 3) and lipoma patients (n= 5). We identified a specific whole blood miRNA (miR-3613-3p) that may serve to distinguish between dedifferentiated liposarcoma patients and healthy controls, thus potentially serving as a specific biomarker for dedifferentiated liposarcoma.

Biomarkers of the Hedgehog/Smoothened pathway in healthy volunteers

PubMed Central

Kadam, Sunil K; Patel, Bharvin K R; Jones, Emma; Nguyen, Tuan S; Verma, Lalit K; Landschulz, Katherine T; Stepaniants, Sergey; Li, Bin; Brandt, John T; Brail, Leslie H

2012-01-01

The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway. PMID:22611475

Gene expression profiling to identify the toxicities and potentially relevant human disease outcomes associated with environmental heavy metal exposure.

PubMed

Korashy, Hesham M; Attafi, Ibraheem M; Famulski, Konrad S; Bakheet, Saleh A; Hafez, Mohammed M; Alsaad, Abdulaziz M S; Al-Ghadeer, Abdul Rahman M

2017-02-01

Heavy metals are the most commonly encountered toxic substances that increase susceptibility to various diseases after prolonged exposure. We have previously shown that healthy volunteers living near a mining area had significant contamination with heavy metals associated with significant changes in the expression of some detoxifying genes, xenobiotic metabolizing enzymes, and DNA repair genes. However, alterations of most of the molecular target genes associated with diseases are still unknown. Thus, the aims of this study were to (a) evaluate the gene expression profile and (b) identify the toxicities and potentially relevant human disease outcomes associated with long-term human exposure to environmental heavy metals in mining area using microarray analysis. For this purpose, 40 healthy male volunteers who were residents of a heavy metal-polluted area (Mahd Al-Dhahab city, Saudi Arabia) and 20 healthy male volunteers who were residents of a non-heavy metal-polluted area were included in the study. Total RNA was isolated from whole blood using PAXgene Blood RNA tubes and then reversed transcribed and hybridized to the gene array using the Affymetrix U219 GeneChip. Microarray analysis showed about 2129 genes were identified and differentially altered, among which a shared set of 425 genes was differentially expressed in the heavy metal-exposed groups. Ingenuity pathway analysis revealed that the most altered gene-regulated diseases in heavy metal-exposed groups included hematological and developmental disorders and mostly renal and urological diseases. Quantitative real-time polymerase chain reaction closely matched the microarray data for some genes tested. Importantly, changes in gene-related diseases were attributed to alterations in the genes encoded for protein synthesis. Renal and urological diseases were the diseases that were most frequently associated with the heavy metal-exposed group. Therefore, there is a need for further studies to validate these genes, which could be used as early biomarkers to prevent renal injury. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of Gene Expression Changes in PHA-M Stimulated Lymphocytes - Unraveling PHA Activity as Prerequisite for Dicentric Chromosome Analysis.

PubMed

Beinke, C; Port, M; Ullmann, R; Gilbertz, K; Majewski, M; Abend, M

2018-06-01

Dicentric chromosome analysis (DCA) is the gold standard for individual radiation dose assessment. However, DCA is limited by the time-consuming phytohemagglutinin (PHA)-mediated lymphocyte activation. In this study using human peripheral blood lymphocytes, we investigated PHA-associated whole genome gene expression changes to elucidate this process and sought to identify suitable gene targets as a means of meeting our long-term objective of accelerating cell cycle kinetics to reduce DCA culture time. Human peripheral whole blood from three healthy donors was separately cultured in RPMI/FCS/antibiotics with BrdU and PHA-M. Diluted whole blood samples were transferred into PAXgene tubes at 0, 12, 24 and 36 h culture time. RNA was isolated and aliquots were used for whole genome gene expression screening. Microarray results were validated using qRT-PCR and differentially expressed genes [significantly (FDR corrected) twofold different from the 0 h value reference] were analyzed using several bioinformatic tools. The cell cycle positions and DNA-synthetic activities of lymphocytes were determined by analyzing the correlated total DNA content and incorporated BrdU level with flow cytometry after continued BrdU incubation. From 42,545 transcripts of the whole genome microarray 47.6%, on average, appeared expressed. The number of differentially expressed genes increased linearly from 855 to 2,858 and 4,607 at 12, 24 and 36 h after PHA addition, respectively. Approximately 2-3 times more up- than downregulated genes were observed with several hundred genes differentially expressed at each time point. Earliest enrichment was observed for gene sets related to the nucleus (12 h) followed by genes assigned to intracellular structures such as organelles (24 h) and finally genes related to the membrane and the extracellular matrix were enriched (36 h). Early gene expression changes at 12 h, in particular, were associated with protein classes such as chemokines/cytokines (e.g., CXCL1, CXCL2) and chaperones. Genes coding for biological processes involved in cell cycle control (e.g., MYBL2, RBL1, CCNA, CCNE) and DNA replication (e.g., POLA, POLE, MCM) appeared enriched at 24 h and later, but many more biological processes (42 altogether) showed enrichment as well. Flow cytometry data fit together with gene expression and bioinformatic analyses as cell cycle transition into S phase was observed with interindividual differences from 12 h onward, whereas progression into G 2 as well as into the second G 1 occurred from 36 h onward after activation. Gene set enrichment analysis over time identifies, in particular, two molecular categories of PHA-responsive gene targets (cytokine and cell cycle control genes). Based on that analysis target genes for cell cycle acceleration in lymphocytes have been identified ( CDKN1A/B/C, RBL-1/RBL-2, E2F2, Deaf-1), and it remains undetermined whether the time expenditure for DCA can be reduced by influencing gene expression involved in the regulatory circuits controlling PHA-associated cell cycle entry and/or progression at a specific early cell cycle phase.

A Peripheral Blood Signature of Vasodilator-Responsive Pulmonary Arterial Hypertension

PubMed Central

Hemnes, Anna R.; Trammell, Aaron W.; Archer, Stephen L.; Rich, Stuart; Yu, Chang; Nian, Hui; Penner, Niki; Funke, Mitchell; Wheeler, Lisa; Robbins, Ivan M.; Austin, Eric D.; Newman, John H.; West, James

2014-01-01

Background Heterogeneity in response to treatment of pulmonary arterial hypertension (PAH) is a major challenge to improving outcome in this disease. Although vasodilator responsive PAH (VR-PAH) accounts for a minority of cases, VR-PAH has a pronounced response to calcium channel blockers and better survival than non-responsive PAH (VN-PAH). We hypothesized that VR-PAH has a different molecular etiology from VN-PAH that can be detected in the peripheral blood. Methods and Results Microarrays of cultured lymphocytes from VR-PAH and VN-PAH patients followed at Vanderbilt University were performed with quantitative PCR performed on peripheral blood for the 25 most different genes. We developed a decision tree to identify VR-PAH patients based on the results with validation in a second VR-PAH cohort from the University of Chicago. We found broad differences in gene expression patterns on microarray analysis including cell-cell adhesion factors, cytoskeletal and rho/GTPase genes. 13/25 genes tested in whole blood were significantly different: EPDR1, DSG2, SCD5, P2RY5, MGAT5, RHOQ, UCHL1, ZNF652, RALGPS2, TPD52, MKNL1, RAPGEF2 and PIAS1. Seven decision trees were built using expression levels of two genes as the primary genes: DSG2, a desmosomal cadherin involved in Wnt/β-catenin signaling, and RHOQ, which encodes a cytoskeletal protein involved in insulin-mediated signaling. These trees correctly identified 5/5 VR-PAH in the validation cohort. Conclusions VR-PAH and VN-PAH can be differentiated using RNA expression patterns in peripheral blood. These differences may reflect different molecular etiologies of the two PAH phenotypes. This biomarker methodology may identify PAH patients that have a favorable treatment response. PMID:25361553

Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

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

Gardner, S; Jaing, C

2012-03-27

The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interimmore » report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.« less

Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

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

Katika, Madhumohan R.; Department of Health Risk Analysis and Toxicology, Maastricht University; Netherlands Toxicogenomics Centre

Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examinedmore » gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human PBMCs were exposed to DON. ► Whole-genome microarray experiments were performed. ► Microarray data indicates that DON affects ribosome and RNA/protein synthesis. ► DON treatment induces ER stress, calcium mediated signaling, NFAT and NF-κB. ► Exposure to DON induces T cell activation, oxidative stress and apoptosis.« less

Pathway analysis in blood cells of pigs infected with classical swine fever virus: comparison of pigs that develop a chronic form of infection or recover.

PubMed

Hulst, Marcel; Loeffen, Willie; Weesendorp, Eefke

2013-02-01

Infection of pigs with CSFV can lead to either acute disease, resulting in death or recovery, or chronic disease. The mechanisms by which CSFV manipulates the pig's first line of defence to establish a chronic infection are poorly understood. Therefore, pigs were infected with moderately virulent CSFV, and whole blood was collected on a regular basis during a period of 18 days. Using whole-genome microarrays, time-dependent changes in gene expression were recorded in blood cells of chronically diseased pigs and pigs that recovered. Bioinformatics analysis of regulated genes indicated that different immunological pathways were regulated in chronically diseased pigs compared to recovered pigs. In recovered pigs, antiviral defence mechanisms were rapidly activated, whereas in chronically diseased pigs, several genes with the potential to inhibit NF-κB- and IRF3/7-mediated transcription of type I interferons were up-regulated. Compared to recovered pigs, chronically diseased pigs failed to activate NK or cytotoxic T-cell pathways, and they showed decreased gene activity in antigen-presenting monocytes/macrophages. Remarkably, in chronically diseased pigs, genes related to the human autoimmune disease systemic lupus erythematosus (SLE) were up-regulated during the whole period of 18 days. CSFV pathology in kidney and skin resembles that of SLE. Furthermore, enzymes involved in the degradation of 1,25-dihydroxyvitamin D3 and of tryptophan to kynurenines were expressed at different levels in chronically diseased and recovered pigs. Both of these chemical processes may affect the functions of T helper/regulatory cells that are crucial for tempering the inflammatory response after a viral infection.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

PubMed

Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

2016-03-02

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus. Copyright © 2016 Elsevier B.V. All rights reserved.

mRNA transcripts as molecular biomarkers in medicine and nutrition

PubMed Central

Sunde, Roger A.

2010-01-01

In medicine, mRNA transcripts are being developed as molecular biomarkers for the diagnosis and treatment of a number of diseases. These biomarkers offer early and more accurate prediction and diagnosis of disease and disease progression, and ability to identify individuals at risk. Use of microarrays also offers opportunity to identify orthogonal (uncorrelated) biomarkers not known to be linked with conventional biomarkers. Investigators are increasingly using blood as a surrogate tissue for biopsy and analysis; total RNA isolated from whole blood is predominantly from erythroid cells, and whole blood mRNA share more than 80% of the transcriptome with major tissues. Thus blood mRNA biomarkers for individualized disease prediction and diagnosis are an exciting area in medicine; mRNA biomarkers in nutrition have potential application that parallel these opportunities. Assessment of selenium (Se) status and requirements is one area where tissue mRNA levels have been used successfully. Selenoprotein-H and selenoprotein-W as well as glutathione peroxidase-1 (Gpx1) mRNAs are highly down-regulated in Se deficiency in rat liver, and the minimum dietary Se requirement is 0.06–0.07 μg Se/g based on these biomarkers, similar to requirements determined using conventional biomarkers. Blood Gpx1 mRNA can also be used to determine Se requirements in rats, showing that blood mRNA has potential for assessment of nutrient status. Future research is needed to develop mRNA biomarker panels for all nutrients that will discriminate between deficient, marginal, adequate, and supernutritional individuals and populations, and differentiate between individuals that will benefit versus be adversely affected by nutrient supplementation. PMID:20303730

Retinal cell responses to elevated intraocular pressure: a gene array comparison between the whole retina and retinal ganglion cell layer.

PubMed

Guo, Ying; Cepurna, William O; Dyck, Jennifer A; Doser, Tom A; Johnson, Elaine C; Morrison, John C

2010-06-01

To determine and compare gene expression patterns in the whole retina and retinal ganglion cell layer (RGCL) in a rodent glaucoma model. IOP was unilaterally elevated in Brown Norway rats (N = 26) by injection of hypertonic saline and monitored for 5 weeks. A cDNA microarray was used on whole retinas from one group of eyes with extensive optic nerve injury and on RGCL isolated by laser capture microdissection (LCM) from another group with comparable injury, to determine the significantly up- or downregulated genes and gene categories in both groups. Expression changes of selected genes were examined by quantitative reverse transcription-PCR (qPCR) to verify microarray results. Microarray analysis of the whole retina identified 632 genes with significantly changed expression (335 up, 297 down), associated with 9 upregulated and 3 downregulated biological processes. In contrast, the RGCL microarray yielded 3726 genes with significantly changed expression (2003 up, 1723 down), including 60% of those found in whole retina. Thirteen distinct upregulated biological processes were identified in the RGCL, dominated by protein synthesis. Among 11 downregulated processes, axon extension and dendrite morphogenesis and generation of precursor metabolism and energy were uniquely identified in the RGCL. qPCR confirmed significant changes in 6 selected messages in whole retina and 11 in RGCL. Increased Atf3, the most upregulated gene in the RGCL, was confirmed by immunohistochemistry of RGCs. Isolation of RGCL by LCM allows a more refined detection of gene response to elevated pressure and improves the potential of determining cellular mechanisms in RGCs and their supporting cells that could be targets for enhancing RGC survival.

[Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

PubMed

Wang, Hong; Bi, Yongyi; Tao, Ning; Wang, Chunhong

2005-08-01

To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

Automation of complex assays: pharmacogenetics of warfarin dosing.

PubMed

Wu, Whei-Kuo; Hujsak, Paul G; Kureshy, Fareed

2007-10-01

AutoGenomics, Inc. (Carlsbad, CA, USA) have developed a multiplex microarray assay for genotyping both VKORC1 and CYP2C9 using the INFINITI(™) Analyzer. Multiple alleles in each DNA sample are analyzed by polymerase chain reaction amplification, followed by detection primer extension using the INFINITI Analyzer. The INFINITI Analyzer performs single-nucleotide polymorphism (SNP) analysis using universal oligonucleotides immobilized on the biochip. To genotype broader ethnic groups, genomic DNA from whole blood was tested for nine SNPs for VKORC1 and six for CYP2C9 genotypes. Information related to all 15 SNPs is needed to determine dosing of population of diverse ethnic origin. The INFINITI system provides genotyping information for same day dosing of warfarin.

A Versatile Microarray Platform for Capturing Rare Cells

NASA Astrophysics Data System (ADS)

Brinkmann, Falko; Hirtz, Michael; Haller, Anna; Gorges, Tobias M.; Vellekoop, Michael J.; Riethdorf, Sabine; Müller, Volkmar; Pantel, Klaus; Fuchs, Harald

2015-10-01

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs) - about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube-CTC chip

NASA Astrophysics Data System (ADS)

Khosravi, Farhad; Trainor, Patrick J.; Lambert, Christopher; Kloecker, Goetz; Wickstrom, Eric; Rai, Shesh N.; Panchapakesan, Balaji

2016-11-01

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 μl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ˜90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%-100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (˜12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube-CTC chip study.

Early Whole Blood Transcriptional Signatures Are Associated with Severity of Lung Inflammation in Cynomolgus Macaques with Mycobacterium tuberculosis Infection.

PubMed

Gideon, Hannah P; Skinner, Jason A; Baldwin, Nicole; Flynn, JoAnne L; Lin, Philana Ling

2016-12-15

Whole blood transcriptional profiling offers great diagnostic and prognostic potential. Although studies identified signatures for pulmonary tuberculosis (TB) and transcripts that predict the risk for developing active TB in humans, the early transcriptional changes immediately following Mycobacterium tuberculosis infection have not been evaluated. We evaluated the gene expression changes in the cynomolgus macaque model of TB, which recapitulates all clinical aspects of human M. tuberculosis infection, using a human microarray and analytics platform. We performed genome-wide blood transcriptional analysis on 38 macaques at 11 postinfection time points during the first 6 mo of M. tuberculosis infection. Of 6371 differentially expressed transcripts between preinfection and postinfection, the greatest change in transcriptional activity occurred 20-56 d postinfection, during which fluctuation of innate and adaptive immune response-related transcripts was observed. Modest transcriptional differences between active TB and latent infection were observed over the time course with substantial overlap. The pattern of module activity previously published for human active TB was similar in macaques with active disease. Blood transcript activity was highly correlated with lung inflammation (lung [ 18 F]fluorodeoxyglucose [FDG] avidity) measured by positron emission tomography and computed tomography at early time points postinfection. The differential signatures between animals with high and low lung FDG were stronger than between clinical outcomes. Analysis of preinfection signatures of macaques revealed that IFN signatures could influence eventual clinical outcomes and lung FDG avidity, even before infection. Our data support that transcriptional changes in the macaque model are translatable to human M. tuberculosis infection and offer important insights into early events of M. tuberculosis infection. Copyright © 2016 by The American Association of Immunologists, Inc.

A pilot study of gene expression analysis in workers with hand-arm vibration syndrome.

PubMed

Maeda, Setsuo; Yu, Xiaozhong; Wang, Rui-Sheng; Sakakibara, Hisataka

2008-04-01

The purpose of this pilot study was to examine differences in gene expressions by cDNA microarray analysis of hand-arm vibration syndrome (HAVS) patients. Vein blood samples were collected and total RNA was extracted. All blood samples were obtained in the morning in one visit after a standard light breakfast. We performed microarray analysis with the labeled cDNA prepared by reverse transcription from RNA samples, using the Human CHIP version 1 (DNA Chip Research Inc, Yokohama, Japan). There are 2,976 genes on the chip, and these genes were selected from a cDNA library prepared with human peripheral white blood cells (WBC). Different gene levels between the HAVS patients and controls, and between groups of HAVS with different levels of symptoms, were indicated by the randomized variance model. The most up-regulated genes were analyzed for their possible functions and association with the occurrence of HAVS. From the results of this pilot study, although the results were obtained a limited number of subjects, it would appear that cDNA microarray analysis of HAVS patients has potential as a new objective method of HAVS diagnosis. Further research is needed to examine the gene expression with increased numbers of patients at different stages of HAVS.

A microarray whole-genome gene expression dataset in a rat model of inflammatory corneal angiogenesis.

PubMed

Mukwaya, Anthony; Lindvall, Jessica M; Xeroudaki, Maria; Peebo, Beatrice; Ali, Zaheer; Lennikov, Anton; Jensen, Lasse Dahl Ejby; Lagali, Neil

2016-11-22

In angiogenesis with concurrent inflammation, many pathways are activated, some linked to VEGF and others largely VEGF-independent. Pathways involving inflammatory mediators, chemokines, and micro-RNAs may play important roles in maintaining a pro-angiogenic environment or mediating angiogenic regression. Here, we describe a gene expression dataset to facilitate exploration of pro-angiogenic, pro-inflammatory, and remodelling/normalization-associated genes during both an active capillary sprouting phase, and in the restoration of an avascular phenotype. The dataset was generated by microarray analysis of the whole transcriptome in a rat model of suture-induced inflammatory corneal neovascularisation. Regions of active capillary sprout growth or regression in the cornea were harvested and total RNA extracted from four biological replicates per group. High quality RNA was obtained for gene expression analysis using microarrays. Fold change of selected genes was validated by qPCR, and protein expression was evaluated by immunohistochemistry. We provide a gene expression dataset that may be re-used to investigate corneal neovascularisation, and may also have implications in other contexts of inflammation-mediated angiogenesis.

Identification of candidate genes in osteoporosis by integrated microarray analysis.

PubMed

Li, J J; Wang, B Q; Fei, Q; Yang, Y; Li, D

2016-12-01

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation.Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594-601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1. © 2016 Fei et al.

Transcriptome analysis supports viral infection and fluoride toxicity as contributors to chronic kidney disease of unknown etiology (CKDu) in Sri Lanka.

PubMed

Sayanthooran, Saravanabavan; Gunerathne, Lishanthe; Abeysekera, Tilak D J; Magana-Arachchi, Dhammika N

2018-05-28

Chronic kidney disease of unknown etiology (CKDu), having epidemic characteristics, is being diagnosed increasingly in certain tropical regions of the world, mainly Latin America and Sri Lanka. They have been observed primarily in farming communities and current hypotheses point toward many environmental and occupational triggers. CKDu does not have common etiologies of chronic kidney disease (CKD) such as hypertension, diabetes, or autoimmune disease. We aimed to understand the molecular processes underlying CKDu in Sri Lanka using transcriptome analysis. RNA extracted from whole blood was reverse transcribed and used for microarray analysis using the Human HT-12 v.4 array (Illumina). Pathway analysis was carried out using ingenuity pathway analysis (IPA-Qiagen). Microarray results were validated using real-time PCR of five selected genes. Pathways related to innate immune response, including interferon signaling, inflammasome signaling and TREM1 signaling had the most significant positive activation z scores, where as EIF2 signaling and mTOR signaling had the most significant negative activation z scores. Pathways previously linked to fluoride toxicity; G-protein activation, Cdc42 signaling, Rac signaling and RhoA signaling were activated in CKDu patients. The most significantly activated biological functions were cell death, cell movement and antimicrobial response. Significant toxicological functions were mitochondrial dysfunction, oxidative stress and apoptosis. Based on the molecular pathway analysis in CKDu patients and review of literature, viral infections and fluoride toxicity appear to be contributing to the molecular mechanisms underlying CKDu.

Gene expression analysis in rat lungs after intratracheal exposure to nanoparticles doped with cadmium

NASA Astrophysics Data System (ADS)

Coccini, Teresa; Fabbri, Marco; Roda, Elisa; Grazia Sacco, Maria; Manzo, Luigi; Gribaldo, Laura

2011-07-01

Silica nanoparticles (NPs) incorporating cadmium (Cd) have been developed for a range of potential application including drug delivery devices. Occupational Cd inhalation has been associated with emphysema, pulmonary fibrosis and lung tumours. Mechanistically, Cd can induce oxidative stress and mediate cell-signalling pathways that are involved in inflammation.This in vivo study aimed at investigating pulmonary molecular effects of NPs doped with Cd (NP-Cd, 1 mg/animal) compared to soluble CdCl2 (400 μg/animal), in Sprague Dawley rats treated intra-tracheally, 7 and 30 days after administration. NPs of silica containing Cd salt were prepared starting from commercial nano-size silica powder (HiSil™ T700 Degussa) with average pore size of 20 nm and surface area of 240 m2/g. Toxicogenomic analysis was performed by the DNA microarray technology (using Agilent Whole Rat Genome Microarray 4×44K) to evaluate changes in gene expression of the entire genome. These findings indicate that the whole genome analysis may represent a valuable approach to assess the whole spectrum of biological responses to cadmium containing nanomaterials.

Alteration of peripheral blood monocyte gene expression in humans following diesel exhaust inhalation

PubMed Central

Pettit, Ashley P.; Brooks, Andrew; Laumbach, Robert; Fiedler, Nancy; Wang, Qi; Strickland, Pamela Ohman; Madura, Kiran; Zhang, Junfeng; Kipen, Howard M.

2013-01-01

Context Epidemiologic associations between acutely increased cardiorespiratory morbidity and mortality and particulate air pollution are well established, but the effects of acute pollution exposure on human gene expression changes are not well understood. Objective In order to identify potential mechanisms underlying epidemiologic associations between air pollution and morbidity, we explored changes in gene expression in humans following inhalation of fresh diesel exhaust (DE), a model for particulate air pollution. Materials and methods Fourteen ethnically homogeneous (white males), young, healthy subjects underwent 60-min inhalation exposures on 2 separate days with clean filtered air (CA) or freshly generated and diluted DE at a concentration of 300 μg/m3 PM2.5. Prior to and 24 h following each session, whole blood was sampled and fractionated for peripheral blood mononuclear cell (PBMC) isolation, RNA extraction, and generation of cDNA, followed by hybridization with Agilent Whole Human Genome (4X44K) arrays. Results Oxidative stress and the ubiquitin proteasome pathway, as well as the coagulation system, were among hypothesized pathways identified by analysis of differentially expressed genes. Nine genes from these pathways were validated using real-time polymerase chain reaction (PCR) to compare fold change in expression between DE exposed and CA days. Quantitative gene fold changes generated by real-time PCR were directionally consistent with the fold changes from the microarray analysis. Discussion and conclusion Changes in gene expression connected with key oxidative stress, protein degradation, and coagulation pathways are likely to underlie observed physiologic and clinical outcomes and suggest specific avenues and sensitive time points for further physiologic exploration. PMID:22369193

Microarray Technology for the Diagnosis of Fetal Chromosomal Aberrations: Which Platform Should We Use?

PubMed Central

Karampetsou, Evangelia; Morrogh, Deborah; Chitty, Lyn

2014-01-01

The advantage of microarray (array) over conventional karyotype for the diagnosis of fetal pathogenic chromosomal anomalies has prompted the use of microarrays in prenatal diagnostics. In this review we compare the performance of different array platforms (BAC, oligonucleotide CGH, SNP) and designs (targeted, whole genome, whole genome, and targeted, custom) and discuss their advantages and disadvantages in relation to prenatal testing. We also discuss the factors to consider when implementing a microarray testing service for the diagnosis of fetal chromosomal aberrations. PMID:26237396

mRNAs and miRNAs in whole blood associated with lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma following multi-walled carbon nanotube inhalation exposure in mice

PubMed Central

Snyder-Talkington, Brandi N.; Dong, Chunlin; Sargent, Linda M.; Porter, Dale W.; Staska, Lauren M.; Hubbs, Ann F.; Raese, Rebecca; McKinney, Walter; Chen, Bean T.; Battelli, Lori; Lowry, David T.; Reynolds, Steven H.; Castranova, Vincent; Qian, Yong; Guo, Nancy L.

2015-01-01

Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fibrosis, and the promotion of lung adenocarcinoma; however, the molecular basis behind these pathologies is unknown. This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma. Six-week-old, male, B6C3F1 mice received a single intraperitoneal injection of either the DNA-damaging agent methylcholanthrene (MCA, 10 μg/g body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg/m³, 5 hours/day, 5 days/week) or filtered air (control) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for the development of pathological changes in the lung, and whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. Numerous mRNAs and miRNAs in the blood were significantly up- or down-regulated in animals developing pathological changes in the lung after MCA/corn oil administration followed by MWCNT/air inhalation, including fcrl5 and miR-122-5p in the presence of hyperplasia, mthfd2 and miR-206-3p in the presence of fibrosis, fam178a and miR-130a-3p in the presence of bronchiolo-alveolar adenoma, and il7r and miR-210-3p in the presence of bronchiolo-alveolar adenocarcinoma, among others. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT-induced lung pathological changes. PMID:25926378

A functional genomics tool for the Pacific bluefin tuna: Development of a 44K oligonucleotide microarray from whole-genome sequencing data for global transcriptome analysis.

PubMed

Yasuike, Motoshige; Fujiwara, Atushi; Nakamura, Yoji; Iwasaki, Yuki; Nishiki, Issei; Sugaya, Takuma; Shimizu, Akio; Sano, Motohiko; Kobayashi, Takanori; Ototake, Mitsuru

2016-02-01

Bluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis, has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.

PubMed

Scheidl, Stefan J; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

2002-03-01

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

PubMed Central

Wang, Denong; Tang, Jin; Liu, Shaoyi

2015-01-01

Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gpHAE3, was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation. PMID:26539555

Use of whole genome expression analysis in the toxicity screening of nanoparticles

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

Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at; Meindl, Claudia; Wagner, Karin

2014-10-15

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays formore » NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.« less

DNA Microarray Detection of 18 Important Human Blood Protozoan Species

PubMed Central

Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

2016-01-01

Background Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. Methodology/Principal Findings The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Conclusions/Significance Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species. PMID:27911895

Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-Linked Immunosorbent Assay

PubMed Central

Caciula, Adrian; Price, Adam; Thakkar, Riddhi; Ng, James; Chauhan, Lokendra V.; Jain, Komal; Che, Xiaoyu; Espinosa, Diego A.; Montoya Cruz, Magelda; Balmaseda, Angel; Sullivan, Eric H.; Patel, Jigar J.; Jarman, Richard G.; Rakeman, Jennifer L.; Egan, Christina T.; Reusken, Chantal B. E. M.; Koopmans, Marion P. G.; Harris, Eva; Tokarz, Rafal; Briese, Thomas

2018-01-01

ABSTRACT Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection). PMID:29511073

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

PubMed Central

Grigoryev, Yevgeniy A.; Kurian, Sunil M.; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L.; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M.; Kantor, Aaron B.; Marsh, Christopher; Salomon, Daniel R.

2010-01-01

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant. PMID:20976225

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

PubMed

Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

2010-10-14

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles.

PubMed

Bălăcescu, Loredana; Bălăcescu, O; Crişan, N; Fetica, B; Petruţ, B; Bungărdean, Cătălina; Rus, Meda; Tudoran, Oana; Meurice, G; Irimie, Al; Dragoş, N; Berindan-Neagoe, Ioana

2011-01-01

Prostate cancer represents the first leading cause of cancer among western male population, with different clinical behavior ranging from indolent to metastatic disease. Although many molecules and deregulated pathways are known, the molecular mechanisms involved in the development of prostate cancer are not fully understood. The aim of this study was to explore the molecular variation underlying the prostate cancer, based on microarray analysis and bioinformatics approaches. Normal and prostate cancer tissues were collected by macrodissection from prostatectomy pieces. All prostate cancer specimens used in our study were Gleason score 7. Gene expression microarray (Agilent Technologies) was used for Whole Human Genome evaluation. The bioinformatics and functional analysis were based on Limma and Ingenuity software. The microarray analysis identified 1119 differentially expressed genes between prostate cancer and normal prostate, which were up- or down-regulated at least 2-fold. P-values were adjusted for multiple testing using Benjamini-Hochberg method with a false discovery rate of 0.01. These genes were analyzed with Ingenuity Pathway Analysis software and were established 23 genetic networks. Our microarray results provide new information regarding the molecular networks in prostate cancer stratified as Gleason 7. These data highlighted gene expression profiles for better understanding of prostate cancer progression.

Nonlinear matching measure for the analysis of on-off type DNA microarray images

NASA Astrophysics Data System (ADS)

Kim, Jong D.; Park, Misun; Kim, Jongwon

2003-07-01

In this paper, we propose a new nonlinear matching measure for automatic analysis of the on-off type DNA microarray images in which the hybridized spots are detected by the template matching method. The targeting spots of HPV DNA chips are designed for genotyping the human papilloma virus(HPV). The proposed measure is obtained by binarythresholding over the whole template region and taking the number of white pixels inside the spotted area. This measure is evaluated in terms of the accuracy of the estimated marker location to show better performance than the normalized covariance.

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray

PubMed Central

Bayer, D K; Martinez, C A; Sorte, H S; Forbes, L R; Demmler-Harrison, G J; Hanson, I C; Pearson, N M; Noroski, L M; Zaki, S R; Bellini, W J; Leduc, M S; Yang, Y; Eng, C M; Patel, A; Rodningen, O K; Muzny, D M; Gibbs, R A; Campbell, I M; Shaw, C A; Baker, M W; Zhang, V; Lupski, J R; Orange, J S; Seeborg, F O; Stray-Pedersen, A

2014-01-01

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8+ T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20–30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5–10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients. PMID:25046553

Fabrication of Carbohydrate Microarrays by Boronate Formation.

PubMed

Adak, Avijit K; Lin, Ting-Wei; Li, Ben-Yuan; Lin, Chun-Cheng

2017-01-01

The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; Skowronski, Elaine A.; Manouchehri, Sareh; Rassenti, Laura; Ghia, Emanuela M.; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.

2014-01-01

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 µL of CLL blood and 5 µL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard). PMID:24723219

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma.

PubMed

Sonnenberg, Avery; Marciniak, Jennifer Y; Skowronski, Elaine A; Manouchehri, Sareh; Rassenti, Laura; Ghia, Emanuela M; Widhopf, George F; Kipps, Thomas J; Heller, Michael J

2014-07-01

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 μL of CLL blood and 5 μL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Treatment of obstructive sleep apnea alters cancer-associated transcriptional signatures in circulating leukocytes.

PubMed

Gharib, Sina A; Seiger, Ashley N; Hayes, Amanda L; Mehra, Reena; Patel, Sanjay R

2014-04-01

Obstructive sleep apnea (OSA) has been associated with a number of chronic disorders that may improve with effective therapy. However, the molecular pathways affected by continuous positive airway pressure (CPAP) treatment are largely unknown. We sought to assess the system-wide consequences of CPAP therapy by transcriptionally profiling peripheral blood leukocytes (PBLs). Subjects in whom severe OSA was diagnosed were treated with CPAP, and whole-genome expression measurement of PBLs was performed at baseline and following therapy. We used gene set enrichment analysis (GSEA) to identify pathways that were differentially enriched. Network analysis was then applied to highlight key drivers of processes influenced by CPAP. Eighteen subjects with significant OSA underwent CPAP therapy and microarray analysis of their PBLs. Treatment with CPAP improved apnea-hypopnea index (AHI), daytime sleepiness, and blood pressure, but did not affect anthropometric measures. GSEA revealed a number of enriched gene sets, many of which were involved in neoplastic processes and displayed downregulated expression patterns in response to CPAP. Network analysis identified several densely connected genes that are important modulators of cancer and tumor growth. Effective therapy of OSA with CPAP is associated with alterations in circulating leukocyte gene expression. Functional enrichment and network analyses highlighted transcriptional suppression in cancer-related pathways, suggesting potentially novel mechanisms linking OSA with neoplastic signatures.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Blood transcriptomic comparison of individuals with and without autism spectrum disorder: A combined-samples mega-analysis.

PubMed

Tylee, Daniel S; Hess, Jonathan L; Quinn, Thomas P; Barve, Rahul; Huang, Hailiang; Zhang-James, Yanli; Chang, Jeffrey; Stamova, Boryana S; Sharp, Frank R; Hertz-Picciotto, Irva; Faraone, Stephen V; Kong, Sek Won; Glatt, Stephen J

2017-04-01

Blood-based microarray studies comparing individuals affected with autism spectrum disorder (ASD) and typically developing individuals help characterize differences in circulating immune cell functions and offer potential biomarker signal. We sought to combine the subject-level data from previously published studies by mega-analysis to increase the statistical power. We identified studies that compared ex vivo blood or lymphocytes from ASD-affected individuals and unrelated comparison subjects using Affymetrix or Illumina array platforms. Raw microarray data and clinical meta-data were obtained from seven studies, totaling 626 affected and 447 comparison subjects. Microarray data were processed using uniform methods. Covariate-controlled mixed-effect linear models were used to identify gene transcripts and co-expression network modules that were significantly associated with diagnostic status. Permutation-based gene-set analysis was used to identify functionally related sets of genes that were over- and under-expressed among ASD samples. Our results were consistent with diminished interferon-, EGF-, PDGF-, PI3K-AKT-mTOR-, and RAS-MAPK-signaling cascades, and increased ribosomal translation and NK-cell related activity in ASD. We explored evidence for sex-differences in the ASD-related transcriptomic signature. We also demonstrated that machine-learning classifiers using blood transcriptome data perform with moderate accuracy when data are combined across studies. Comparing our results with those from blood-based studies of protein biomarkers (e.g., cytokines and trophic factors), we propose that ASD may feature decoupling between certain circulating signaling proteins (higher in ASD samples) and the transcriptional cascades which they typically elicit within circulating immune cells (lower in ASD samples). These findings provide insight into ASD-related transcriptional differences in circulating immune cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Blood Transcriptomic Comparison of Individuals with and without Autism Spectrum Disorder: A Combined-Samples Mega-Analysis

PubMed Central

Tylee, Daniel S.; Hess, Jonathan L.; Quinn, Thomas P.; Barve, Rahul; Huang, Hailiang; Zhang-James, Yanli; Chang, Jeffrey; Stamova, Boryana S.; Sharp, Frank R.; Hertz-Picciotto, Irva; Faraone, Stephen V.; Kong, Sek Won; Glatt, Stephen J.

2017-01-01

Blood-based microarray studies comparing individuals affected with autism spectrum disorder (ASD) and typically developing individuals help characterize differences in circulating immune cell functions and offer potential biomarker signal. We sought to combine the subject-level data from previously published studies by mega-analysis to increase the statistical power. We identified studies that compared ex-vivo blood or lymphocytes from ASD-affected individuals and unrelated comparison subjects using Affymetrix or Illumina array platforms. Raw microarray data and clinical meta-data were obtained from seven studies, totaling 626 affected and 447 comparison subjects. Microarray data were processed using uniform methods. Covariate-controlled mixed-effect linear models were used to identify gene transcripts and co-expression network modules that were significantly associated with diagnostic status. Permutation-based gene-set analysis was used to identify functionally related sets of genes that were over- and under-expressed among ASD samples. Our results were consistent with diminished interferon-, EGF-, PDGF-, PI3K-AKT-mTOR-, and RAS-MAPK-signaling cascades, and increased ribosomal translation and NK-cell related activity in ASD. We explored evidence for sex-differences in the ASD-related transcriptomic signature. We also demonstrated that machine-learning classifiers using blood transcriptome data perform with moderate accuracy when data are combined across studies. Comparing our results with those from blood-based studies of protein biomarkers (e.g., cytokines and trophic factors), we propose that ASD may feature decoupling between certain circulating signaling proteins (higher in ASD samples) and the transcriptional cascades which they typically elicit within circulating immune cells (lower in ASD samples). These findings provide insight into ASD-related transcriptional differences in circulating immune cells. PMID:27862943

An overview of the Progenika ID CORE XT: an automated genotyping platform based on a fluidic microarray system.

PubMed

Goldman, Mindy; Núria, Núria; Castilho, Lilian M

2015-01-01

Automated testing platforms facilitate the introduction of red cell genotyping of patients and blood donors. Fluidic microarray systems, such as Luminex XMAP (Austin, TX), are used in many clinical applications, including HLA and HPA typing. The Progenika ID CORE XT (Progenika Biopharma-Grifols, Bizkaia, Spain) uses this platform to analyze 29 polymorphisms determining 37 antigens in 10 blood group systems. Once DNA has been extracted, processing time is approximately 4 hours. The system is highly automated and includes integrated analysis software that produces a file and a report with genotype and predicted phenotype results.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

mRNA Expression Profiling of Laser Microbeam Microdissected Cells from Slender Embryonic Structures

PubMed Central

Scheidl, Stefan J.; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

2002-01-01

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-β1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions. PMID:11891179

Isolation of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays - Application in Aerospace Medicine

DTIC Science & Technology

2004-01-01

of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays  Application in...VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND KINETIC RT-PCR ASSAYS  APPLICATION IN AEROSPACE MEDICINE INTRODUCTION Extraction of cellular

Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

DTIC Science & Technology

2013-01-01

heparin was purchased from Innovative Research (Novi, MI). Goat and horse whole blood was provided by our Veterinary Medicine Division (USAMRIID, Fort...quinquefasciatus (10) BA House NA Human Culex Th9-0122 Ae. aegypti (1) BA House DENV-3 DNP Aedes Th9-0164 Cx. tritaeniorhynchus (24) LT Farm JEV NA...Culex Th9-0167 Ae. albopictus (1) LT Farm NA NA Aedes Th9-0175 Cx. tritaeniorhynchus (25) LT Farm JEV NA Culex Th9-0235 Cx. tritaeniorhynchus (25) BA

Association of Body Mass Index with DNA Methylation and Gene Expression in Blood Cells and Relations to Cardiometabolic Disease: A Mendelian Randomization Approach

PubMed Central

Joehanes, Roby; Liu, Chunyu; Aslibekyan, Stella; Demerath, Ellen W.; Guan, Weihua; Zhi, Degui; Willinger, Christine; Courchesne, Paul; Multhaup, Michael; Irvin, Marguerite R.; Schadt, Eric E.; Bressler, Jan; North, Kari; Sundström, Johan; Gustafsson, Stefan; Shah, Sonia; McRae, Allan F.; Harris, Sarah E.; Gibson, Jude; Redmond, Paul; Corley, Janie; Starr, John M.; Visscher, Peter M.; Wray, Naomi R.; Krauss, Ronald M.; Feinberg, Andrew; Fornage, Myriam; Pankow, James S.; Lind, Lars; Fox, Caroline; Ingelsson, Erik; Arnett, Donna K.; Boerwinkle, Eric; Liang, Liming; Levy, Daniel; Deary, Ian J.

2017-01-01

Background The link between DNA methylation, obesity, and adiposity-related diseases in the general population remains uncertain. Methods and Findings We conducted an association study of body mass index (BMI) and differential methylation for over 400,000 CpGs assayed by microarray in whole-blood-derived DNA from 3,743 participants in the Framingham Heart Study and the Lothian Birth Cohorts, with independent replication in three external cohorts of 4,055 participants. We examined variations in whole blood gene expression and conducted Mendelian randomization analyses to investigate the functional and clinical relevance of the findings. We identified novel and previously reported BMI-related differential methylation at 83 CpGs that replicated across cohorts; BMI-related differential methylation was associated with concurrent changes in the expression of genes in lipid metabolism pathways. Genetic instrumental variable analysis of alterations in methylation at one of the 83 replicated CpGs, cg11024682 (intronic to sterol regulatory element binding transcription factor 1 [SREBF1]), demonstrated links to BMI, adiposity-related traits, and coronary artery disease. Independent genetic instruments for expression of SREBF1 supported the findings linking methylation to adiposity and cardiometabolic disease. Methylation at a substantial proportion (16 of 83) of the identified loci was found to be secondary to differences in BMI. However, the cross-sectional nature of the data limits definitive causal determination. Conclusions We present robust associations of BMI with differential DNA methylation at numerous loci in blood cells. BMI-related DNA methylation and gene expression provide mechanistic insights into the relationship between DNA methylation, obesity, and adiposity-related diseases. PMID:28095459

Common Marker Genes Identified from Various Sample Types for Systemic Lupus Erythematosus.

PubMed

Bing, Peng-Fei; Xia, Wei; Wang, Lan; Zhang, Yong-Hong; Lei, Shu-Feng; Deng, Fei-Yan

2016-01-01

Systemic lupus erythematosus (SLE) is a complex auto-immune disease. Gene expression studies have been conducted to identify SLE-related genes in various types of samples. It is unknown whether there are common marker genes significant for SLE but independent of sample types, which may have potentials for follow-up translational research. The aim of this study is to identify common marker genes across various sample types for SLE. Based on four public microarray gene expression datasets for SLE covering three representative types of blood-born samples (monocyte; peripheral blood mononuclear cell, PBMC; whole blood), we utilized three statistics (fold-change, FC; t-test p value; false discovery rate adjusted p value) to scrutinize genes simultaneously regulated with SLE across various sample types. For common marker genes, we conducted the Gene Ontology enrichment analysis and Protein-Protein Interaction analysis to gain insights into their functions. We identified 10 common marker genes associated with SLE (IFI6, IFI27, IFI44L, OAS1, OAS2, EIF2AK2, PLSCR1, STAT1, RNASE2, and GSTO1). Significant up-regulation of IFI6, IFI27, and IFI44L with SLE was observed in all the studied sample types, though the FC was most striking in monocyte, compared with PBMC and whole blood (8.82-251.66 vs. 3.73-74.05 vs. 1.19-1.87). Eight of the above 10 genes, except RNASE2 and GSTO1, interact with each other and with known SLE susceptibility genes, participate in immune response, RNA and protein catabolism, and cell death. Our data suggest that there exist common marker genes across various sample types for SLE. The 10 common marker genes, identified herein, deserve follow-up studies to dissert their potentials as diagnostic or therapeutic markers to predict SLE or treatment response.

Using pathway modules as targets for assay development in xenobiotic screening

EPA Science Inventory

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...

Unique Proteins Expressed by Blood Vessels in Skeletal Sites Colonized by Breast Cancer Cells

DTIC Science & Technology

2006-08-01

fluorescent labeled acetylated LDL at an accelerated rate (3). After one week in culture BVECs and MVECs were harvested. Total RNA was extracted from...both cell types using the Qiagen RNeasy kit (Valencia, CA). Microarray labeling, hybridization and analysis was conducted on the RNA by the Penn...State University DNA Microarray Facility under the direction of Dr. Craig Praul. Briefly, RNA obtained from three separate isolations of BVECs and

Microarray-Based Analysis of Subnanogram Quantities of Microbial Community DNAs by Using Whole-Community Genome Amplification†

PubMed Central

Wu, Liyou; Liu, Xueduan; Schadt, Christopher W.; Zhou, Jizhong

2006-01-01

Microarray technology provides the opportunity to identify thousands of microbial genes or populations simultaneously, but low microbial biomass often prevents application of this technology to many natural microbial communities. We developed a whole-community genome amplification-assisted microarray detection approach based on multiple displacement amplification. The representativeness of amplification was evaluated using several types of microarrays and quantitative indexes. Representative detection of individual genes or genomes was obtained with 1 to 100 ng DNA from individual or mixed genomes, in equal or unequal abundance, and with 1 to 500 ng community DNAs from groundwater. Lower concentrations of DNA (as low as 10 fg) could be detected, but the lower template concentrations affected the representativeness of amplification. Robust quantitative detection was also observed by significant linear relationships between signal intensities and initial DNA concentrations ranging from (i) 0.04 to 125 ng (r2 = 0.65 to 0.99) for DNA from pure cultures as detected by whole-genome open reading frame arrays, (ii) 0.1 to 1,000 ng (r2 = 0.91) for genomic DNA using community genome arrays, and (iii) 0.01 to 250 ng (r2 = 0.96 to 0.98) for community DNAs from ethanol-amended groundwater using 50-mer functional gene arrays. This method allowed us to investigate the oligotrophic microbial communities in groundwater contaminated with uranium and other metals. The results indicated that microorganisms containing genes involved in contaminant degradation and immobilization are present in these communities, that their spatial distribution is heterogeneous, and that microbial diversity is greatly reduced in the highly contaminated environment. PMID:16820490

Blood cells transcriptomics as source of potential biomarkers of articular health improvement: effects of oral intake of a rooster combs extract rich in hyaluronic acid.

PubMed

Sánchez, Juana; Bonet, M Luisa; Keijer, Jaap; van Schothorst, Evert M; Mölller, Ingrid; Chetrit, Carles; Martinez-Puig, Daniel; Palou, Andreu

2014-09-01

The aim of the study was to explore peripheral blood gene expression as a source of biomarkers of joint health improvement related to glycosaminoglycan (GAG) intake in humans. Healthy individuals with joint discomfort were enrolled in a randomized, double-blind, placebo-controlled intervention study in humans. Subjects ate control yoghurt or yoghurt supplemented with a recently authorized novel food in Europe containing hyaluronic acid (65 %) from rooster comb (Mobilee™ as commercial name) for 90 days. Effects on functional quality-of-life parameters related to joint health were assessed. Whole-genome microarray analysis of peripheral blood samples from a subset of 20 subjects (10 placebo and 10 supplemented) collected pre- and post-intervention was performed. Mobilee™ supplementation reduced articular pain intensity and synovial effusion and improved knee muscular strength indicators as compared to placebo. About 157 coding genes were differentially expressed in blood cells between supplemented and placebo groups post-intervention, but not pre-intervention (p < 0.05; fold change ≥1.2). Among them, a reduced gene expression of glucuronidase-beta (GUSB), matrix metallopeptidase 23B (MMP23B), xylosyltransferase II (XYLT2), and heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) was found in the supplemented group. Correlation analysis indicated a direct relationship between blood cell gene expression of MMP23B, involved in the breakdown of the extracellular matrix, and pain intensity, and an inverse relationship between blood cell gene expression of HS6ST1, responsible for 6-O-sulfation of heparan sulfate, and indicators of knee muscular strength. Expression levels of specific genes in blood cells, in particular genes related to GAG metabolism and extracellular matrix dynamics, are potential biomarkers of beneficial effects on articular health.

Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

PubMed

Lee, Patrick K H; Men, Yujie; Wang, Shanquan; He, Jianzhong; Alvarez-Cohen, Lisa

2015-02-03

Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugar-dependent response

PubMed Central

Zinke, Ingo; Schütz, Christina S.; Katzenberger, Jörg D.; Bauer, Matthias; Pankratz, Michael J.

2002-01-01

We have identified genes regulated by starvation and sugar signals in Drosophila larvae using whole-genome microarrays. Based on expression profiles in the two nutrient conditions, they were organized into different categories that reflect distinct physiological pathways mediating sugar and fat metabolism, and cell growth. In the category of genes regulated in sugar-fed, but not in starved, animals, there is an upregulation of genes encoding key enzymes of the fat biosynthesis pathway and a downregulation of genes encoding lipases. The highest and earliest activated gene upon sugar ingestion is sugarbabe, a zinc finger protein that is induced in the gut and the fat body. Identification of potential targets using microarrays suggests that sugarbabe functions to repress genes involved in dietary fat breakdown and absorption. The current analysis provides a basis for studying the genetic mechanisms underlying nutrient signalling. PMID:12426388

Microarray analysis of Mycobacterium tuberculosis-infected monocytes reveals IL26 as a new candidate gene for tuberculosis susceptibility.

PubMed

Guerra-Laso, José M; Raposo-García, Sara; García-García, Silvia; Diez-Tascón, Cristina; Rivero-Lezcano, Octavio M

2015-02-01

Differences in the activity of monocytes/macrophages, important target cells of Mycobacterium tuberculosis, might influence tuberculosis progression. With the purpose of identifying candidate genes for tuberculosis susceptibility we infected monocytes from both healthy elderly individuals (a tuberculosis susceptibility group) and elderly tuberculosis patients with M. tuberculosis, and performed a microarray experiment. We detected 78 differentially expressed transcripts and confirmed these results by quantitative PCR of selected genes. We found that monocytes from tuberculosis patients showed similar expression patterns for these genes, regardless of whether they were obtained from younger or older patients. Only one of the detected genes corresponded to a cytokine: IL26, a member of the interleukin-10 (IL-10) cytokine family which we found to be down-regulated in infected monocytes from tuberculosis patients. Non-infected monocytes secreted IL-26 constitutively but they reacted strongly to M. tuberculosis infection by decreasing IL-26 production. Furthermore, IL-26 serum concentrations appeared to be lower in the tuberculosis patients. When whole blood was infected, IL-26 inhibited the observed pathogen-killing capability. Although lymphocytes expressed IL26R, the receptor mRNA was not detected in either monocytes or neutrophils, suggesting that the inhibition of anti-mycobacterial activity may be mediated by lymphocytes. Additionally, IL-2 concentrations in infected blood were lower in the presence of IL-26. The negative influence of IL-26 on the anti-mycobacterial activity and its constitutive presence in both serum and monocyte supernatants prompt us to propose IL26 as a candidate gene for tuberculosis susceptibility. © 2014 John Wiley & Sons Ltd.

Effects of oral contraception with ethinylestradiol and drospirenone on oxidative stress in women 18-35 years old.

PubMed

De Groote, Donat; Perrier d'Hauterive, Sophie; Pintiaux, Axelle; Balteau, Bénédicte; Gerday, Colette; Claesen, Jürgen; Foidart, Jean-Michel

2009-08-01

Oral contraceptives (OCs) with estrogens and progestins may affect oxidative stress (OS) status. A group of 32 women using oral contraceptives (OCU) containing 0.03 mg ethinylestradiol and 3 mg drospirenone have been compared to a matched control group of 30 noncontraception users (NCU). Blood levels of antioxidants, trace elements and markers of lipid peroxidation were assessed by biochemical methods. A microarray analysis of whole blood mRNA levels of 200 genes involved in OS-dependant pathway was also performed. Levels of zinc, vitamin E and antibodies to oxidized low-density lipoproteins (LDLs) were not significantly different between the two groups. On the other hand, significant increases in the mean levels of lipid peroxides (+176%, p<.001), oxidized LDLs (+145%, p<.002), copper (+103%, p<.001), Cu/Zn ratio (+100%, p<.001) and a significant decrease in the mean level of beta-carotene (-41%, p<.01) were observed in the OCU compared to NCU. There was a highly significant positive correlation between the lipid peroxide levels and the copper-to-zinc ratio. From the 200 genes tested by microarray, one coding for HSP70 was significantly up-regulated (log(2) fold change=+ 0.45, p<.02) and one coding for inducible nitric oxide synthase significantly down-regulated (log(2) fold change=-0.24, p<.05) in the OCU compared to the NCU. The recently introduced combination of ethinylestradiol and drospirenone induced the heightening of lipid peroxidation correlated with high levels of copper, a situation that could be associated with increased cardiovascular risk.

Whole Blood Transcriptomics Is Relevant to Identify Molecular Changes in Response to Genetic Selection for Feed Efficiency and Nutritional Status in the Pig.

PubMed

Jégou, Maëva; Gondret, Florence; Vincent, Annie; Tréfeu, Christine; Gilbert, Hélène; Louveau, Isabelle

2016-01-01

The molecular mechanisms underlying feed efficiency need to be better understood to improve animal efficiency, a research priority to support a competitive and sustainable livestock production. This study was undertaken to determine whether pig blood transcriptome was affected by differences in feed efficiency and by ingested nutrients. Growing pigs from two lines divergently selected for residual feed intake (RFI) and fed isoproteic and isocaloric diets contrasted in energy source and nutrients were considered. Between 74 and 132 days of age, pigs (n = 12 by diet and by line) received a regular diet rich in cereals and low in fat (LF) or a diet where cereals where partially substituted by lipids and fibers (HF). At the end of the feeding trial, the total number of white blood cells was not affected by the line or by the diet, whereas the red blood cell number was higher (P<0.001) in low RFI than in high RFI pigs. Analysis of the whole blood transcriptome using a porcine microarray reveals a higher number of probes differentially expressed (DE) between RFI lines than between diets (2,154 versus 92 probes DE, P<0.01). This corresponds to 528 overexpressed genes and 477 underexpressed genes in low RFI pigs compared with high RFI pigs, respectively. Overexpressed genes were predominantly associated with translational elongation. Underexpressed genes were mainly involved in the immune response, regulation of inflammatory response, anti-apoptosis process, and cell organization. These findings suggest that selection for RFI has affected the immune status and defense mechanisms of pigs. Genes DE between diets were mainly related to the immune system and lipid metabolism. Altogether, this study demonstrates the usefulness of the blood transcriptome to identify the main biological processes affected by genetic selection and feeding strategies.

Whole Blood Transcriptomics Is Relevant to Identify Molecular Changes in Response to Genetic Selection for Feed Efficiency and Nutritional Status in the Pig

PubMed Central

Jégou, Maëva; Gondret, Florence; Vincent, Annie; Tréfeu, Christine; Gilbert, Hélène; Louveau, Isabelle

2016-01-01

The molecular mechanisms underlying feed efficiency need to be better understood to improve animal efficiency, a research priority to support a competitive and sustainable livestock production. This study was undertaken to determine whether pig blood transcriptome was affected by differences in feed efficiency and by ingested nutrients. Growing pigs from two lines divergently selected for residual feed intake (RFI) and fed isoproteic and isocaloric diets contrasted in energy source and nutrients were considered. Between 74 and 132 days of age, pigs (n = 12 by diet and by line) received a regular diet rich in cereals and low in fat (LF) or a diet where cereals where partially substituted by lipids and fibers (HF). At the end of the feeding trial, the total number of white blood cells was not affected by the line or by the diet, whereas the red blood cell number was higher (P<0.001) in low RFI than in high RFI pigs. Analysis of the whole blood transcriptome using a porcine microarray reveals a higher number of probes differentially expressed (DE) between RFI lines than between diets (2,154 versus 92 probes DE, P<0.01). This corresponds to 528 overexpressed genes and 477 underexpressed genes in low RFI pigs compared with high RFI pigs, respectively. Overexpressed genes were predominantly associated with translational elongation. Underexpressed genes were mainly involved in the immune response, regulation of inflammatory response, anti-apoptosis process, and cell organization. These findings suggest that selection for RFI has affected the immune status and defense mechanisms of pigs. Genes DE between diets were mainly related to the immune system and lipid metabolism. Altogether, this study demonstrates the usefulness of the blood transcriptome to identify the main biological processes affected by genetic selection and feeding strategies. PMID:26752050

Molecular Classifiers for Acute Kidney Transplant Rejection in Peripheral Blood by Whole Genome Gene Expression Profiling

PubMed Central

Kurian, S. M.; Williams, A. N.; Gelbart, T.; Campbell, D.; Mondala, T. S.; Head, S. R.; Horvath, S.; Gaber, L.; Thompson, R.; Whisenant, T.; Lin, W.; Langfelder, P.; Robison, E. H.; Schaffer, R. L.; Fisher, J. S.; Friedewald, J.; Flechner, S. M.; Chan, L. K.; Wiseman, A. C.; Shidban, H.; Mendez, R.; Heilman, R.; Abecassis, M. M.; Marsh, C. L.; Salomon, D. R.

2015-01-01

There are no minimally invasive diagnostic metrics for acute kidney transplant rejection (AR), especially in the setting of the common confounding diagnosis, acute dysfunction with no rejection (ADNR). Thus, though kidney transplant biopsies remain the gold standard, they are invasive, have substantial risks, sampling error issues and significant costs and are not suitable for serial monitoring. Global gene expression profiles of 148 peripheral blood samples from transplant patients with excellent function and normal histology (TX; n = 46), AR (n = 63) and ADNR (n = 39), from two independent cohorts were analyzed with DNA microarrays. We applied a new normalization tool, frozen robust multi-array analysis, particularly suitable for clinical diagnostics, multiple prediction tools to discover, refine and validate robust molecular classifiers and we tested a novel one-by-one analysis strategy to model the real clinical application of this test. Multiple three-way classifier tools identified 200 highest value probesets with sensitivity, specificity, positive predictive value, negative predictive value and area under the curve for the validation cohort ranging from 82% to 100%, 76% to 95%, 76% to 95%, 79% to 100%, 84% to 100% and 0.817 to 0.968, respectively. We conclude that peripheral blood gene expression profiling can be used as a minimally invasive tool to accurately reveal TX, AR and ADNR in the setting of acute kidney transplant dysfunction. PMID:24725967

Interim Report on SNP analysis and forensic microarray probe design for South American hemorrhagic fever viruses, tick-borne encephalitis virus, henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever viruses, Rift Valley fever

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

Jaing, C; Gardner, S

The goal of this project is to develop forensic genotyping assays for select agent viruses, enhancing the current capabilities for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the whole genomemore » wide SNP analysis and microarray probe design for forensics characterization of South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.« less

Blood Gene Signatures of Chagas Cardiomyopathy With or Without Ventricular Dysfunction.

PubMed

Ferreira, Ludmila Rodrigues Pinto; Ferreira, Frederico Moraes; Nakaya, Helder Imoto; Deng, Xutao; Cândido, Darlan da Silva; de Oliveira, Lea Campos; Billaud, Jean-Noel; Lanteri, Marion C; Rigaud, Vagner Oliveira-Carvalho; Seielstad, Mark; Kalil, Jorge; Fernandes, Fabio; Ribeiro, Antonio Luiz P; Sabino, Ester Cerdeira; Cunha-Neto, Edecio

2017-02-01

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects 7 million people in Latin American areas of endemicity. About 30% of infected patients will develop chronic Chagas cardiomyopathy (CCC), an inflammatory cardiomyopathy characterized by hypertrophy, fibrosis, and myocarditis. Further studies are necessary to understand the molecular mechanisms of disease progression. Transcriptome analysis has been increasingly used to identify molecular changes associated with disease outcomes. We thus assessed the whole-blood transcriptome of patients with Chagas disease. Microarray analysis was performed on blood samples from 150 subjects, of whom 30 were uninfected control patients and 120 had Chagas disease (1 group had asymptomatic disease, and 2 groups had CCC with either a preserved or reduced left ventricular ejection fraction [LVEF]). Each Chagas disease group displayed distinct gene expression and functional pathway profiles. The most different expression patterns were between CCC groups with a preserved or reduced LVEF. A more stringent analysis indicated that 27 differentially expressed genes, particularly those related to natural killer (NK)/CD8+ T-cell cytotoxicity, separated the 2 groups. NK/CD8+ T-cell cytotoxicity could play a role in determining Chagas disease progression. Understanding genes associated with disease may lead to improved insight into CCC pathogenesis and the identification of prognostic factors for CCC progression. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

PubMed

El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

2015-01-30

In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

[Diagnosis of a case with Williams-Beuren syndrome with nephrocalcinosis using chromosome microarray analysis].

PubMed

Jin, S J; Liu, M; Long, W J; Luo, X P

2016-12-02

Objective: To explore the clinical phenotypes and the genetic cause for a boy with unexplained growth retardation, nephrocalcinosis, auditory anomalies and multi-organ/system developmental disorders. Method: Routine G-banding and chromosome microarray analysis were applied to a child with unexplained growth retardation, nephrocalcinosis, auditory anomalies and multi-organ/system developmental disorders treated in the Department of Pediatrics of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology in September 2015 and his parents to conduct the chromosomal karyotype analysis and the whole genome scanning. Deleted genes were searched in the Decipher and NCBI databases, and their relationships with the clinical phenotypes were analyzed. Result: A six-month-old boy was refered to us because of unexplained growth retardation and feeding intolerance.The affected child presented with abnormal manifestation such as special face, umbilical hernia, growth retardation, hypothyroidism, congenital heart disease, right ear sensorineural deafness, hypercalcemia and nephrocalcinosis. The child's karyotype was 46, XY, 16qh + , and his parents' karyotypes were normal. Chromosome microarray analysis revealed a 1 436 kb deletion on the 7q11.23(72701098_74136633) region of the child. This region included 23 protein-coding genes, which were reported to be corresponding to Williams-Beuren syndrome and its certain clinical phenotypes. His parents' results of chromosome microarray analysis were normal. Conclusion: A boy with characteristic manifestation of Williams-Beuren syndrome and rare nephrocalcinosis was diagnosed using chromosome microarray analysis. The deletion on the 7q11.23 might be related to the clinical phenotypes of Williams-Beuren syndrome, yet further studies are needed.

Clearance of a persistent Picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

USDA-ARS?s Scientific Manuscript database

Immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in distinct microanatomic compartments of the bovine nasopharynx were investigated using quantitative RT-PCR and whole transcriptome microarray. Analysis of tissue samples obtained during ...

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1 are enriched for innate immunity genes.

PubMed

Welker, Noah C; Habig, Jeffrey W; Bass, Brenda L

2007-07-01

We describe the first microarray analysis of a whole animal containing a mutation in the Dicer gene. We used adult Caenorhabditis elegans and, to distinguish among different roles of Dicer, we also performed microarray analyses of animals with mutations in rde-4 and rde-1, which are involved in silencing by siRNA, but not miRNA. Surprisingly, we find that the X chromosome is greatly enriched for genes regulated by Dicer. Comparison of all three microarray data sets indicates the majority of Dicer-regulated genes are not dependent on RDE-4 or RDE-1, including the X-linked genes. However, all three data sets are enriched in genes important for innate immunity and, specifically, show increased expression of innate immunity genes.

Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1 are enriched for innate immunity genes

PubMed Central

Welker, Noah C.; Habig, Jeffrey W.; Bass, Brenda L.

2007-01-01

We describe the first microarray analysis of a whole animal containing a mutation in the Dicer gene. We used adult Caenorhabditis elegans and, to distinguish among different roles of Dicer, we also performed microarray analyses of animals with mutations in rde-4 and rde-1, which are involved in silencing by siRNA, but not miRNA. Surprisingly, we find that the X chromosome is greatly enriched for genes regulated by Dicer. Comparison of all three microarray data sets indicates the majority of Dicer-regulated genes are not dependent on RDE-4 or RDE-1, including the X-linked genes. However, all three data sets are enriched in genes important for innate immunity and, specifically, show increased expression of innate immunity genes. PMID:17526642

Rapid evaluation of fibrinogen levels using the CG02N whole blood coagulation analyzer.

PubMed

Hayakawa, Mineji; Gando, Satoshi; Ono, Yuichi; Mizugaki, Asumi; Katabami, Kenichi; Maekawa, Kunihiko; Miyamoto, Daisuke; Wada, Takeshi; Yanagida, Yuichiro; Sawamura, Atsushi

2015-04-01

Rapid evaluation of fibrinogen (Fbg) levels is essential for maintaining homeostasis in patients with massive bleeding during severe trauma and major surgery. This study evaluated the accuracy of fibrinogen levels measured by the CG02N whole blood coagulation analyzer (A&T Corporation, Kanagawa, Japan) using heparinized blood drawn for blood gas analysis (whole blood-Fbg). A total of 100 matched pairs of heparinized blood samples and citrated blood samples were simultaneously collected from patients in the intensive care unit. Whole blood-Fbg results were compared with those of citrated plasma (standard-Fbg). The whole blood coagulation analyzer measured fibrinogen levels within 2 minutes. Strong correlations between standard-Fbg and whole blood-Fbg were observed (ρ = 0.91, p < 0.001). Error grid analysis showed that 88% of the values were clinically acceptable, and 12% were in a range with possible effects on clinical decision-making; none were in a clinically dangerous range without appropriate treatment. Using a fibrinogen cutoff value of 1.5 g/L for standard-Fbg, the area under the receiver operating characteristic curve of whole blood-Fbg was 0.980 (95% confidence interval 0.951-1.000, p < 0.001). The whole blood coagulation analyzer can rapidly measure fibrinogen levels in heparinized blood and could be useful in critical care settings where excessive bleeding is a concern. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Robust Segmentation of Overlapping Cells in Histopathology Specimens Using Parallel Seed Detection and Repulsive Level Set

PubMed Central

Qi, Xin; Xing, Fuyong; Foran, David J.; Yang, Lin

2013-01-01

Automated image analysis of histopathology specimens could potentially provide support for early detection and improved characterization of breast cancer. Automated segmentation of the cells comprising imaged tissue microarrays (TMA) is a prerequisite for any subsequent quantitative analysis. Unfortunately, crowding and overlapping of cells present significant challenges for most traditional segmentation algorithms. In this paper, we propose a novel algorithm which can reliably separate touching cells in hematoxylin stained breast TMA specimens which have been acquired using a standard RGB camera. The algorithm is composed of two steps. It begins with a fast, reliable object center localization approach which utilizes single-path voting followed by mean-shift clustering. Next, the contour of each cell is obtained using a level set algorithm based on an interactive model. We compared the experimental results with those reported in the most current literature. Finally, performance was evaluated by comparing the pixel-wise accuracy provided by human experts with that produced by the new automated segmentation algorithm. The method was systematically tested on 234 image patches exhibiting dense overlap and containing more than 2200 cells. It was also tested on whole slide images including blood smears and tissue microarrays containing thousands of cells. Since the voting step of the seed detection algorithm is well suited for parallelization, a parallel version of the algorithm was implemented using graphic processing units (GPU) which resulted in significant speed-up over the C/C++ implementation. PMID:22167559

DNA microarray unravels rapid changes in transcriptome of MK-801 treated rat brain

PubMed Central

Kobayashi, Yuka; Kulikova, Sofya P; Shibato, Junko; Rakwal, Randeep; Satoh, Hiroyuki; Pinault, Didier; Masuo, Yoshinori

2015-01-01

AIM: To investigate the impact of MK-801 on gene expression patterns genome wide in rat brain regions. METHODS: Rats were treated with an intraperitoneal injection of MK-801 [0.08 (low-dose) and 0.16 (high-dose) mg/kg] or NaCl (vehicle control). In a first series of experiment, the frontoparietal electrocorticogram was recorded 15 min before and 60 min after injection. In a second series of experiments, the whole brain of each animal was rapidly removed at 40 min post-injection, and different regions were separated: amygdala, cerebral cortex, hippocampus, hypothalamus, midbrain and ventral striatum on ice followed by DNA microarray (4 × 44 K whole rat genome chip) analysis. RESULTS: Spectral analysis revealed that a single systemic injection of MK-801 significantly and selectively augmented the power of baseline gamma frequency (30-80 Hz) oscillations in the frontoparietal electroencephalogram. DNA microarray analysis showed the largest number (up- and down- regulations) of gene expressions in the cerebral cortex (378), midbrain (376), hippocampus (375), ventral striatum (353), amygdala (301), and hypothalamus (201) under low-dose (0.08 mg/kg) of MK-801. Under high-dose (0.16 mg/kg), ventral striatum (811) showed the largest number of gene expression changes. Gene expression changes were functionally categorized to reveal expression of genes and function varies with each brain region. CONCLUSION: Acute MK-801 treatment increases synchrony of baseline gamma oscillations, and causes very early changes in gene expressions in six individual rat brain regions, a first report. PMID:26629322

Comparison of Reference Values in Whole Blood of DMDmdx/J and C57BL/6J Mice Using Neutron Activation Analysis

NASA Astrophysics Data System (ADS)

Metairon, S.; Zamboni, C. B.; Suzuki, M. F.; Júnior, C. R. B.; Sant'Anna, O. A.

2011-08-01

The Br, Ca, Cl, K, Na and S concentrations in whole blood of DMDmdx/J and C57BL/6J mice were determined using Neutron Activation Analysis technique. Reference values obtained from twenty one whole blood samples of these strains were analyzed in the IEA-R1 nuclear reactor at IPEN (São Paulo, Brasil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood as well as to evaluate the performance of treatments in muscular dystrophy.

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways. PMID:16608515

Oxidative stress and reduced responsiveness of challenged circulating leukocytes following pulmonary instillation of metal-rich particulate matter in rats

PubMed Central

2014-01-01

Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically. PMID:25123171

Oxidative stress and reduced responsiveness of challenged circulating leukocytes following pulmonary instillation of metal-rich particulate matter in rats.

PubMed

Erdely, Aaron; Antonini, James M; Young, Shih-Houng; Kashon, Michael L; Gu, Ja K; Hulderman, Tracy; Salmen, Rebecca; Meighan, Terence; Roberts, Jenny R; Zeidler-Erdely, Patti C

2014-08-15

Welding fume is an exposure that consists of a mixture of metal-rich particulate matter with gases (ozone, carbon monoxide) and/or vapors (VOCs). Data suggests that welders are immune compromised. Given the inability of pulmonary leukocytes to properly respond to a secondary infection in animal models, the question arose whether the dysfunction persisted systemically. Our aim was to evaluate the circulating leukocyte population in terms of cellular activation, presence of oxidative stress, and functionality after a secondary challenge, following welding fume exposure. Rats were intratracheally instilled (ITI) with PBS or 2 mg of welding fume collected from a stainless steel weld. Rats were sacrificed 4 and 24 h post-exposure and whole blood was collected. Whole blood was used for cellular differential counts, RNA isolation with subsequent microarray and Ingenuity Pathway Analysis, and secondary stimulation with LPS utilizing TruCulture technology. In addition, mononuclear cells were isolated 24 h post-exposure to measure oxidative stress by flow cytometry and confocal microscopy. Welding fume exposure had rapid effects on the circulating leukocyte population as identified by relative mRNA expression changes. Instillation of welding fume reduced inflammatory protein production of circulating leukocytes when challenged with the secondary stimulus LPS. The effects were not related to transcription, but were observed in conjunction with oxidative stress. These findings support previous studies of an inadequate pulmonary immune response following a metal-rich exposure and extend those findings showing leukocyte dysfunction occurs systemically.

Moving Toward Integrating Gene Expression Profiling into High-throughput Testing:A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium

EPA Science Inventory

Microarray profiling of chemical-induced effects is being increasingly used in medium and high-throughput formats. In this study, we describe computational methods to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), ...

Derivation of an artificial gene to improve classification accuracy upon gene selection.

PubMed

Seo, Minseok; Oh, Sejong

2012-02-01

Classification analysis has been developed continuously since 1936. This research field has advanced as a result of development of classifiers such as KNN, ANN, and SVM, as well as through data preprocessing areas. Feature (gene) selection is required for very high dimensional data such as microarray before classification work. The goal of feature selection is to choose a subset of informative features that reduces processing time and provides higher classification accuracy. In this study, we devised a method of artificial gene making (AGM) for microarray data to improve classification accuracy. Our artificial gene was derived from a whole microarray dataset, and combined with a result of gene selection for classification analysis. We experimentally confirmed a clear improvement of classification accuracy after inserting artificial gene. Our artificial gene worked well for popular feature (gene) selection algorithms and classifiers. The proposed approach can be applied to any type of high dimensional dataset. Copyright © 2011 Elsevier Ltd. All rights reserved.

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

PubMed

Kim, Chang Sup; Seo, Jeong Hyun; Cha, Hyung Joon

2012-08-07

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Gene Expression Profiling of Gastric Cancer

PubMed Central

Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

2015-01-01

Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.

PubMed

Shi, Xiang Yang; Dumenyo, C Korsi; Hernandez-Martinez, Rufina; Azad, Hamid; Cooksey, Donald A

2007-11-01

Many virulence genes in plant bacterial pathogens are coordinately regulated by "global" regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Transcriptome and secretome analyses of Phanerochaete chrysosporium reveal complex patterns of gene expression

Treesearch

Amber J. Vanden Wymelenberg; Jill A. Gaskell; Michael D. Mozuch; Philip J. Kersten; Grzegorz Sabat; Diego Martinez; Daniel Cullen

2009-01-01

The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or...

The low-abundance transcriptome reveals novel biomarkers, specific intracellular pathways and targetable genes associated with advanced gastric cancer.

PubMed

Bizama, Carolina; Benavente, Felipe; Salvatierra, Edgardo; Gutiérrez-Moraga, Ana; Espinoza, Jaime A; Fernández, Elmer A; Roa, Iván; Mazzolini, Guillermo; Sagredo, Eduardo A; Gidekel, Manuel; Podhajcer, Osvaldo L

2014-02-15

Studies on the low-abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low-abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low-abundance transcriptome and the whole transcriptome. Analysis of the low-abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type-specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF-C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low-abundance transcriptome provides useful insights into the molecular basis and treatment of cancer. © 2013 UICC.

Whole blood genome-wide expression profiling and network analysis suggest MELAS master regulators.

PubMed

Mende, Susanne; Royer, Loic; Herr, Alexander; Schmiedel, Janet; Deschauer, Marcus; Klopstock, Thomas; Kostic, Vladimir S; Schroeder, Michael; Reichmann, Heinz; Storch, Alexander

2011-07-01

The heteroplasmic mitochondrial DNA (mtDNA) mutation A3243G causes the mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome as one of the most frequent mitochondrial diseases. The process of reconfiguration of nuclear gene expression profile to accommodate cellular processes to the functional status of mitochondria might be a key to MELAS disease manifestation and could contribute to its diverse phenotypic presentation. To determine master regulatory protein networks and disease-modifying genes in MELAS syndrome. Analyses of whole blood transcriptomes from 10 MELAS patients using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses. Hierarchical cluster analysis elucidated that the relative abundance of mutant mtDNA molecules is decisive for the nuclear gene expression response. Further analyses confirmed not only transcription factors already known to be involved in mitochondrial diseases (such as TFAM), but also detected the hypoxia-inducible factor 1 complex, nuclear factor Y and cAMP responsive element-binding protein-related transcription factors as novel master regulators for reconfiguration of nuclear gene expression in response to the MELAS mutation. Correlation analyses of gene alterations and clinico-genetic data detected significant correlations between A3243G-induced nuclear gene expression changes and mutant mtDNA load as well as disease characteristics. These potential disease-modifying genes influencing the expression of the MELAS phenotype are mainly related to clusters primarily unrelated to cellular energy metabolism, but important for nucleic acid and protein metabolism, and signal transduction. Our data thus provide a framework to search for new pathogenetic concepts and potential therapeutic approaches to treat the MELAS syndrome.

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

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

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

DOE PAGES

Thissen, James B.; McLoughlin, Kevin; Gardner, Shea; ...

2014-06-01

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA wasmore » able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.« less

Intratumoral immune cells expressing PD-1/PD-L1 and their prognostic implications in cancer: a meta-analysis.

PubMed

Kim, Younghoon; Wen, Xianyu; Cho, Nam Yun; Kang, Gyeong Hoon

2018-05-01

The prognostic value of immune cells expressing programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) in cancer are controversial, and the potential differential impact of using tissue microarrays and whole tissue sections to assess the positivity of immune cells has not been addressed. The current study included 30 eligible studies with 7251 patients that evaluated the relationship between tumor-infiltrating lymphocytes expressing PD-1/PD-L1 and overall survival and disease-free survival, or progression-free survival. Subgroup analysis was based on the tissue type of cancer and the type of tissue sampling (tissue microarray or whole tissue section). In the meta-analysis, PD-1-positive and PD-L1-positive tumor-infiltrating lymphocytes had a positive effect on disease-free survival or progression-free survival (hazard ratio [HR] 0.732; 95% confidence interval [CI] 0.565, 0.947; and HR 0.727; 95% CI 0.584, 0.905, respectively). PD-L1-positive tumor-infiltrating lymphocytes had a positive impact on overall survival in studies using tissue microarray (HR 0.586; 95% CI 0.476, 0.721), but had a poor impact when only whole tissue sections were considered (HR 1.558; 95% CI 1.232, 1.969). Lung cancer was associated with good overall survival and disease-free survival (HR 0.639; 95% CI 0.491, 0.831; and HR 0.693; 95% CI 0.538, 0.891, respectively) for PD-1-positive tumor-infiltrating lymphocytes, and colorectal cancer showed favorable disease-free survival (HR 0.471; 95% CI 0.308, 0.722) for PD-L1-positive tumor-infiltrating lymphocytes. Immune cells expressing PD-1 and PD-L1 within tumors are associated with the prognosis. However, the correlation may vary among different tumor types and by the type of tissue sampling used for the assessment.

Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms.

PubMed

Lopez, G H; Morrison, J; Condon, J A; Wilson, B; Martin, J R; Liew, Y-W; Flower, R L; Hyland, C A

2015-10-01

Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fy(a) /Fy(b) polymorphism, for weak Fy(b) antigen, and for the red cell null Fy(a-b-) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. Samples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a-b-) n = 47 (one with anti-Fy3 antibody). Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fy(b) expression discrepant with Fy(b-) (Group 1 and 3). For three, SNP genotyping predicted Fy(a) , discrepant with Fy(a-b-) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null. Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status. © 2015 International Society of Blood Transfusion.

RNA Expression Profiles from Blood for the Diagnosis of Stroke and its Causes

PubMed Central

Sharp, Frank R; Jickling, Glen C; Stamova, Boryana; Tian, Yingfang; Zhan, Xinhua; Ander, Bradley P; Cox, Christopher; Kuczynski, Beth; Liu, DaZhi

2013-01-01

A blood test to detect stroke and its causes would be particularly useful in babies, young children, and patients in intensive care units, and for emergencies when imaging is difficult to obtain or unavailable. Using whole genome microarrays, we first showed specific gene expression profiles in rats 24 hours after ischemic and hemorrhagic stroke, hypoxia, and hypoglycemia. These proof-of-principle studies revealed that groups of genes (called gene profiles) can distinguish ischemic stroke patients from controls 3 hours to 24 hours after the strokes. In addition, gene expression profiles have been developed that distinguish stroke due to large-vessel atherosclerosis from cardioembolic stroke. These profiles will be useful for predicting the causes of cryptogenic stroke. Our results in adults suggest similar diagnostic tools could be developed for children. PMID:21636778

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

PubMed

Haas, Christian S; Creighton, Chad J; Pi, Xiujun; Maine, Ira; Koch, Alisa E; Haines, G Kenneth; Ling, Song; Chinnaiyan, Arul M; Holoshitz, Joseph

2006-07-01

To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

BRIC-17 Mapping Spaceflight-Induced Hypoxic Signaling and Response in Plants

NASA Technical Reports Server (NTRS)

Gilroy, Simon; Choi, Won-Gyu; Swanson, Sarah

2012-01-01

Goals of this work are: (1) Define global changes in gene expression patterns in Arabidopsis plants grown in microgravity using whole genome microarrays (2) Compare to mutants resistant to low oxygen challenge using whole genome microarrays Also measuring root and shoot size Outcomes from this research are: (1) Provide fundamental information on plant responses to the stresses inherent in spaceflight (2) Potential for informing on genetic strategies to engineer plants for optimal growth in space

Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.

PubMed

Zhang, Linsheng; Znoyko, Iya; Costa, Luciano J; Conlin, Laura K; Daber, Robert D; Self, Sally E; Wolff, Daynna J

2011-12-01

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. The methods currently used for monitoring CLL and determining conditions for treatment are limited in their ability to predict disease progression, patient survival, and response to therapy. Although clonal diversity and the acquisition of new chromosomal abnormalities during the disease course (clonal evolution) have been associated with disease progression, their prognostic potential has been underappreciated because cytogenetic and fluorescence in situ hybridization (FISH) studies have a restricted ability to detect genomic abnormalities and clonal evolution. We hypothesized that whole genome analysis using high resolution single nucleotide polymorphism (SNP) microarrays would be useful to detect diversity and infer clonal evolution to offer prognostic information. In this study, we used the Infinium Omni1 BeadChip (Illumina, San Diego, CA) array for the analysis of genetic variation and percent mosaicism in 25 non-selected CLL patients to explore the prognostic value of the assessment of clonal diversity in patients with CLL. We calculated the percentage of mosaicism for each abnormality by applying a mathematical algorithm to the genotype frequency data and by manual determination using the Simulated DNA Copy Number (SiDCoN) tool, which was developed from a computer model of mosaicism. At least one genetic abnormality was identified in each case, and the SNP data was 98% concordant with FISH results. Clonal diversity, defined as the presence of two or more genetic abnormalities with differing percentages of mosaicism, was observed in 12 patients (48%), and the diversity correlated with the disease stage. Clonal diversity was present in most cases of advanced disease (Rai stages III and IV) or those with previous treatment, whereas 9 of 13 patients without detected clonal diversity were asymptomatic or clinically stable. In conclusion, SNP microarray studies with simultaneous evaluation of genomic alterations and mosaic distribution of clones can be used to assess apparent clonal evolution via analysis of clonal diversity. Since clonal evolution in CLL is strongly correlated with disease progression, whole genome SNP microarray analysis provides a new comprehensive and reliable prognostic tool for CLL patients. Copyright © 2011 Elsevier Inc. All rights reserved.

Preparing Platelet-Rich Plasma with Whole Blood Harvested Intraoperatively During Spinal Fusion.

PubMed

Shen, Bin; Zhang, Zheng; Zhou, Ning-Feng; Huang, Yu-Feng; Bao, Yu-Jie; Wu, De-Sheng; Zhang, Ya-Dong

2017-07-22

BACKGROUND Platelet-rich plasma (PRP) has gained growing popularity in use in spinal fusion procedures in the last decade. Substantial intraoperative blood loss is frequently accompanied with spinal fusion, and it is unknown whether blood harvested intraoperatively qualifies for PRP preparation. MATERIAL AND METHODS Whole blood was harvested intraoperatively and venous blood was collected by venipuncture. Then, we investigated the platelet concentrations in whole blood and PRP, the concentration of growth factors in PRP, and the effects of PRP on the proliferation and viability of human bone marrow-derived mesenchymal stem cells (HBMSCs). RESULTS Our results revealed that intraoperatively harvested whole blood and whole blood collected by venipuncture were similar in platelet concentration. In addition, PRP formulations prepared from both kinds of whole blood were similar in concentration of platelet and growth factors. Additional analysis showed that the similar concentrations of growth factors resulted from the similar platelet concentrations of whole blood and PRP between the two groups. Moreover, these two kinds of PRP formulations had similar effects on promoting cell proliferation and enhancing cell viability. CONCLUSIONS Therefore, intraoperatively harvested whole blood may be a potential option for preparing PRP spinal fusion.

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

PubMed

McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

2015-07-01

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

Interpretation of Genomic Data Questions and Answers

PubMed Central

Simon, Richard

2008-01-01

Using a question and answer format we describe important aspects of using genomic technologies in cancer research. The main challenges are not managing the mass of data, but rather the design, analysis and accurate reporting of studies that result in increased biological knowledge and medical utility. Many analysis issues address the use of expression microarrays but are also applicable to other whole genome assays. Microarray based clinical investigations have generated both unrealistic hyperbole and excessive skepticism. Genomic technologies are tremendously powerful and will play instrumental roles in elucidating the mechanisms of oncogenesis and in devlopingan era of predictive medicine in which treatments are tailored to individual tumors. Achieving these goals involves challenges in re-thinking many paradigms for the conduct of basic and clinical cancer research and for the organization of interdisciplinary collaboration. PMID:18582627

Transcriptomic analysis of persistent infection with foot-and-mouth disease virus in cattle suggests impairment of cell-mediated immunity in the nasopharynx

USDA-ARS?s Scientific Manuscript database

In order to investigate the mechanisms of persistent foot-and-mouth disease virus (FMDV) infection in cattle, transcriptome alterations associated with the FMDV carrier state were characterized using a bovine whole-transcriptome microarray. Eighteen cattle (8 vaccinated with a recombinant FMDV A vac...

Global transcriptional analysis of psoriatic skin and blood confirms known disease-associated pathways and highlights novel genomic "hot spots" for differentially expressed genes.

PubMed

Coda, Alvin B; Icen, Murat; Smith, Jason R; Sinha, Animesh A

2012-07-01

There are major gaps in our knowledge regarding the exact mechanisms and genetic basis of psoriasis. To investigate the pathogenesis of psoriasis, gene expression in 10 skin (5 lesional, 5 nonlesional) and 11 blood (6 psoriatic, 5 nonpsoriatic) samples were examined using Affymetrix HG-U95A microarrays. We detected 535 (425 upregulated, 110 downregulated) DEGs in lesional skin at 1% false discovery rate (FDR). Combining nine microarray studies comparing lesional and nonlesional psoriatic skin, 34.5% of dysregulated genes were overlapped in multiple studies. We further identified 20 skin and 2 blood associated transcriptional "hot spots" at specified genomic locations. At 5% FDR, 11.8% skin and 10.4% blood DEGs in our study mapped to one of the 12 PSORS loci. DEGs that overlap with PSORS loci may offer prioritized targets for downstream genetic fine mapping studies. Novel DEG "hot spots" may provide new targets for defining susceptibility loci in future studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Whole transcriptome profiling reveals major cell types in the cellular immune response against acute and chronic active Epstein-Barr virus infection.

PubMed

Zhong, Huaqing; Hu, Xinran; Janowski, Andrew B; Storch, Gregory A; Su, Liyun; Cao, Lingfeng; Yu, Jinsheng; Xu, Jin

2017-12-19

Epstein-Barr virus (EBV) is a common human pathogen that infects over 95% of the population worldwide. In the present study, the whole transcriptome microarray data were generated from peripheral blood mononuclear cells from Chinese children with acute infectious mononucleosis (AIM) and chronic active EBV infection (CAEBV) that were also compared with a publicly available microarray dataset from a study of American college students with AIM. Our study characterized for the first time a broad spectrum of molecular signatures in AIM and CAEBV. The key findings from the transcriptome profiling were validated with qPCR and flow cytometry assays. The most important finding in our study is the discovery of predominant γδ TCR expression and γδ T cell expansion in AIM. This finding, in combination with the striking up-regulation of CD3, CD8 and CD94, suggests that CD8+ T cells and CD94+ NK cells may play a major role in AIM. Moreover, the unique up-regulation of CD64A/B and its significant correlation with the monocyte marker CD14 was observed in CAEBV and that implies an important role of monocytes in CAEBV. In conclusion, our study reveals major cell types (particularly γδ T cells) in the host cellular immune response against AIM and CAEBV.

Comprehensive Analysis of DNA Methylation Data with RnBeads

PubMed Central

Walter, Jörn; Lengauer, Thomas; Bock, Christoph

2014-01-01

RnBeads is a software tool for large-scale analysis and interpretation of DNA methylation data, providing a user-friendly analysis workflow that yields detailed hypertext reports (http://rnbeads.mpi-inf.mpg.de). Supported assays include whole genome bisulfite sequencing, reduced representation bisulfite sequencing, Infinium microarrays, and any other protocol that produces high-resolution DNA methylation data. Important applications of RnBeads include the analysis of epigenome-wide association studies and epigenetic biomarker discovery in cancer cohorts. PMID:25262207

Inkjet-printed point-of-care immunoassay on a nanoscale polymer brush enables subpicomolar detection of analytes in blood

NASA Astrophysics Data System (ADS)

Joh, Daniel Y.; Hucknall, Angus M.; Wei, Qingshan; Mason, Kelly A.; Lund, Margaret L.; Fontes, Cassio M.; Hill, Ryan T.; Blair, Rebecca; Zimmers, Zackary; Achar, Rohan K.; Tseng, Derek; Gordan, Raluca; Freemark, Michael; Ozcan, Aydogan; Chilkoti, Ashutosh

2017-08-01

The ELISA is the mainstay for sensitive and quantitative detection of protein analytes. Despite its utility, ELISA is time-consuming, resource-intensive, and infrastructure-dependent, limiting its availability in resource-limited regions. Here, we describe a self-contained immunoassay platform (the “D4 assay”) that converts the sandwich immunoassay into a point-of-care test (POCT). The D4 assay is fabricated by inkjet printing assay reagents as microarrays on nanoscale polymer brushes on glass chips, so that all reagents are “on-chip,” and these chips show durable storage stability without cold storage. The D4 assay can interrogate multiple analytes from a drop of blood, is compatible with a smartphone detector, and displays analytical figures of merit that are comparable to standard laboratory-based ELISA in whole blood. These attributes of the D4 POCT have the potential to democratize access to high-performance immunoassays in resource-limited settings without sacrificing their performance.

Progestins Upregulate FKBP51 Expression in Human Endometrial Stromal Cells to Induce Functional Progesterone and Glucocorticoid Withdrawal: Implications for Contraceptive- Associated Abnormal Uterine Bleeding.

PubMed

Guzeloglu Kayisli, Ozlem; Kayisli, Umit A; Basar, Murat; Semerci, Nihan; Schatz, Frederick; Lockwood, Charles J

2015-01-01

Use of long-acting progestin only contraceptives (LAPCs) offers a discrete and highly effective family planning method. Abnormal uterine bleeding (AUB) is the major side effect of, and cause for, discontinuation of LAPCs. The endometria of LAPC-treated women display abnormally enlarged, fragile blood vessels, decreased endometrial blood flow and oxidative stress. To understanding to mechanisms underlying AUB, we propose to identify LAPC-modulated unique gene cluster(s) in human endometrial stromal cells (HESCs). Protein and RNA isolated from cultured HESCs treated 7 days with estradiol (E2) or E2+ medroxyprogesterone acetate (MPA) or E2+ etonogestrel (ETO) or E2+ progesterone (P4) were analyzed by quantitative Real-time (q)-PCR and immunoblotting. HSCORES were determined for immunostained-paired endometria of pre-and 3 months post-Depot MPA (DMPA) treated women and ovariectomized guinea pigs (GPs) treated with placebo or E2 or MPA or E2+MPA for 21 days. In HESCs, whole genome analysis identified a 67 gene group regulated by all three progestins, whereas a 235 gene group was regulated by E2+ETO and E2+MPA, but not E2+P4. Ingenuity pathway analysis identified glucocorticoid receptor (GR) activation as one of upstream regulators of the 235 MPA and ETO-specific genes. Among these, microarray results demonstrated significant enhancement of FKBP51, a repressor of PR/GR transcriptional activity, by both MPA and ETO. q-PCR and immunoblot analysis confirmed the microarray results. In endometria of post-DMPA versus pre-DMPA administered women, FKBP51 expression was significantly increased in endometrial stromal and glandular cells. In GPs, E2+MPA or MPA significantly increased FKBP51 immunoreactivity in endometrial stromal and glandular cells versus placebo- and E2-administered groups. MPA or ETO administration activates GR signaling and increases endometrial FKBP51 expression, which could be one of the mechanisms causing AUB by inhibiting PR and GR-mediated transcription. The resultant PR and/or GR-mediated functional withdrawal may contribute to associated endometrial inflammation, aberrant angiogenesis, and bleeding.

Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants

PubMed Central

Walter, Andreas; Knapp, Brigitte A.; Farbmacher, Theresa; Ebner, Christian; Insam, Heribert; Franke‐Whittle, Ingrid H.

2012-01-01

Summary To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full‐scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real‐time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year. PMID:22950603

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

PubMed Central

Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

2014-01-01

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

Prediction of Microbial Infection of Cultured Cells Using DNA Microarray Gene-Expression Profiles of Host Responses

PubMed Central

Park, Yu Rang; Chung, Tae Su; Lee, Young Joo; Song, Yeong Wook; Lee, Eun Young; Sohn, Yeo Won; Song, Sukgil; Park, Woong Yang

2012-01-01

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI). PMID:23091307

Automated detection system of single nucleotide polymorphisms using two kinds of functional magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Liu, Hongna; Li, Song; Wang, Zhifei; Li, Zhiyang; Deng, Yan; Wang, Hua; Shi, Zhiyang; He, Nongyue

2008-11-01

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome wide codominant SNPs identification. Therefore, large-scale codominant SNPs identification, especially for those associated with complex diseases, has induced the need for completely high-throughput and automated SNP genotyping method. Herein, we present an automated detection system of SNPs based on two kinds of functional magnetic nanoparticles (MNPs) and dual-color hybridization. The amido-modified MNPs (NH 2-MNPs) modified with APTES were used for DNA extraction from whole blood directly by electrostatic reaction, and followed by PCR, was successfully performed. Furthermore, biotinylated PCR products were captured on the streptavidin-coated MNPs (SA-MNPs) and interrogated by hybridization with a pair of dual-color probes to determine SNP, then the genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes. This system provided a rapid, sensitive and highly versatile automated procedure that will greatly facilitate the analysis of different known SNPs in human genome.

Differences in iron concentration in whole blood of animal models using NAA

NASA Astrophysics Data System (ADS)

Bahovschi, V.; Zamboni, C. B.; Lopes Silva, L. F. F.; Metairon, S.; Medeiros, I. M. M. A.

2015-07-01

In this study Neutron Activation Analysis technique (NAA) was applied to determine Fe concentration in whole blood samples of several animal models such as: mice (Mus musculus), Golden Hamster (Mesocricetus auratus), Wistar rats, Albinic Rabbits of New Zealand, Golden Retriever dogs and Crioulabreed horses. These results were compared with human whole blood estimation to check their similarities.

Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes specifically overexpressed in tumor epithelia.

PubMed

Badea, Liviu; Herlea, Vlad; Dima, Simona Olimpia; Dumitrascu, Traian; Popescu, Irinel

2008-01-01

The precise details of pancreatic ductal adenocarcinoma (PDAC) pathogenesis are still insufficiently known, requiring the use of high-throughput methods. However, PDAC is especially difficult to study using microarrays due to its strong desmoplastic reaction, which involves a hyperproliferating stroma that effectively "masks" the contribution of the minoritary neoplastic epithelial cells. Thus it is not clear which of the genes that have been found differentially expressed between normal and whole tumor tissues are due to the tumor epithelia and which simply reflect the differences in cellular composition. To address this problem, laser microdissection studies have been performed, but these have to deal with much smaller tissue sample quantities and therefore have significantly higher experimental noise. In this paper we combine our own large sample whole-tissue study with a previously published smaller sample microdissection study by Grützmann et al. to identify the genes that are specifically overexpressed in PDAC tumor epithelia. The overlap of this list of genes with other microarray studies of pancreatic cancer as well as with the published literature is impressive. Moreover, we find a number of genes whose over-expression appears to be inversely correlated with patient survival: keratin 7, laminin gamma 2, stratifin, platelet phosphofructokinase, annexin A2, MAP4K4 and OACT2 (MBOAT2), which are all specifically upregulated in the neoplastic epithelia, rather than the tumor stroma. We improve on other microarray studies of PDAC by putting together the higher statistical power due to a larger number of samples with information about cell-type specific expression and patient survival.

Whole gene expression profile in blood reveals multiple pathways deregulation in R6/2 mouse model

PubMed Central

2013-01-01

Background Huntington Disease (HD) is a progressive neurological disorder, with pathological manifestations in brain areas and in periphery caused by the ubiquitous expression of mutant Huntingtin protein. Transcriptional dysregulation is considered a key molecular mechanism responsible of HD pathogenesis but, although numerous studies investigated mRNA alterations in HD, so far none evaluated a whole gene expression profile in blood of R6/2 mouse model. Findings To discover novel pathogenic mechanisms and potential peripheral biomarkers useful to monitor disease progression or drug efficacy, a microarray study was performed in blood of R6/2 at manifest stage and wild type littermate mice. This approach allowed to propose new peripheral molecular processes involved in HD and to suggest different panels of candidate biomarkers. Among the discovered deregulated processes, we focused on specific ones: complement and coagulation cascades, PPAR signaling, cardiac muscle contraction, and dilated cardiomyopathy pathways. Selected genes derived from these pathways were additionally investigated in other accessible tissues to validate these matrices as source of biomarkers, and in brain, to link central and peripheral disease manifestations. Conclusions Our findings validated the skeletal muscle as suitable source to investigate peripheral transcriptional alterations in HD and supported the hypothesis that immunological alteration may contribute to neurological degeneration. Moreover, the identification of altered signaling in mouse blood enforce R6/2 transgenic mouse as a powerful HD model while suggesting novel disease biomarkers for pre-clinical investigation. PMID:24252798

Effect of solvent/detergent-treated pooled plasma on fibrinolysis in reconstituted whole blood.

PubMed

Saadah, Nicholas H; van der Meer, Pieter F; Brinkman, Herm Jan M; de Korte, Dirk; Bontekoe, Ido J; Korsten, Herbert H; Middelburg, Rutger A; van der Bom, Johanna G; Schipperus, Martin R

2017-10-01

Hyperfibrinolysis has been observed in patients heavily transfused with solvent/detergent-treated pooled plasma (S/D plasma). We compared coagulation and fibrinolytic variables in blood containing S/D plasma with blood containing fresh-frozen plasma (FFP), with and without α2-antiplasmin or tranexamic acid (TXA) supplementation. Whole blood samples were reconstituted from red blood cells, platelet (PLT) concentrates, and varying mixtures of FFP and S/D plasma. Hematocrit and PLT count of reconstituted whole blood samples were varied. For a subset of runs, α2-antiplasmin or TXA was added to S/D plasma whole blood samples. Thromboelastography (TEG) analysis was performed to assess 50% clot lysis time (CLT 50% ), maximum amplitude (MA), and initial clotting time (R-time). The change in CLT 50% of whole blood as the plasma compartment transitions from FFP to S/D plasma was -52% (95% confidence interval [CI], -60% to -45%; p < 0.001). PLT count strengthened the effect, leading to an additional change in CLT 50% of -8% (95% CI, -14% to -2%; p = 0.012) as PLT count increased from 10 × 10 9 to 150 × 10 9 /L. MA and R-time were not associated with fraction of S/D plasma in whole blood. α2-Antiplasmin and TXA restored clot lysis time in S/D plasma whole blood. Whole blood with S/D plasma has shorter clot lysis times in vitro compared to whole blood with FFP. α2-Antiplasmin and TXA restore clot lysis time of S/D plasma whole blood to that of FFP whole blood. Clinicians should be aware of the decreased clot lysis time associated with S/D plasma transfusion. © 2017 AABB.

Genomic analysis, cytokine expression, and microRNA profiling reveal biomarkers of human dietary zinc depletion and homeostasis.

PubMed

Ryu, Moon-Suhn; Langkamp-Henken, Bobbi; Chang, Shou-Mei; Shankar, Meena N; Cousins, Robert J

2011-12-27

Implementation of zinc interventions for subjects suspected of being zinc-deficient is a global need, but is limited due to the absence of reliable biomarkers. To discover molecular signatures of human zinc deficiency, a combination of transcriptome, cytokine, and microRNA analyses was applied to a dietary zinc depletion/repletion protocol with young male human subjects. Concomitant with a decrease in serum zinc concentration, changes in buccal and blood gene transcripts related to zinc homeostasis occurred with zinc depletion. Microarray analyses of whole blood RNA revealed zinc-responsive genes, particularly, those associated with cell cycle regulation and immunity. Responses of potential signature genes of dietary zinc depletion were further assessed by quantitative real-time PCR. The diagnostic properties of specific serum microRNAs for dietary zinc deficiency were identified by acute responses to zinc depletion, which were reversible by subsequent zinc repletion. Depression of immune-stimulated TNFα secretion by blood cells was observed after low zinc consumption and may serve as a functional biomarker. Our findings introduce numerous novel candidate biomarkers for dietary zinc status assessment using a variety of contemporary technologies and which identify changes that occur prior to or with greater sensitivity than the serum zinc concentration which represents the current zinc status assessment marker. In addition, the results of gene network analysis reveal potential clinical outcomes attributable to suboptimal zinc intake including immune function defects and predisposition to cancer. These demonstrate through a controlled depletion/repletion dietary protocol that the illusive zinc biomarker(s) can be identified and applied to assessment and intervention strategies.

Image-derived input function with factor analysis and a-priori information.

PubMed

Simončič, Urban; Zanotti-Fregonara, Paolo

2015-02-01

Quantitative PET studies often require the cumbersome and invasive procedure of arterial cannulation to measure the input function. This study sought to minimize the number of necessary blood samples by developing a factor-analysis-based image-derived input function (IDIF) methodology for dynamic PET brain studies. IDIF estimation was performed as follows: (a) carotid and background regions were segmented manually on an early PET time frame; (b) blood-weighted and tissue-weighted time-activity curves (TACs) were extracted with factor analysis; (c) factor analysis results were denoised and scaled using the voxels with the highest blood signal; (d) using population data and one blood sample at 40 min, whole-blood TAC was estimated from postprocessed factor analysis results; and (e) the parent concentration was finally estimated by correcting the whole-blood curve with measured radiometabolite concentrations. The methodology was tested using data from 10 healthy individuals imaged with [(11)C](R)-rolipram. The accuracy of IDIFs was assessed against full arterial sampling by comparing the area under the curve of the input functions and by calculating the total distribution volume (VT). The shape of the image-derived whole-blood TAC matched the reference arterial curves well, and the whole-blood area under the curves were accurately estimated (mean error 1.0±4.3%). The relative Logan-V(T) error was -4.1±6.4%. Compartmental modeling and spectral analysis gave less accurate V(T) results compared with Logan. A factor-analysis-based IDIF for [(11)C](R)-rolipram brain PET studies that relies on a single blood sample and population data can be used for accurate quantification of Logan-V(T) values.

A microarray-based genotyping and genetic mapping approach for highly heterozygous outcrossing species enables localization of a large fraction of the unassembled Populus trichocarpa genome sequence.

PubMed

Drost, Derek R; Novaes, Evandro; Boaventura-Novaes, Carolina; Benedict, Catherine I; Brown, Ryan S; Yin, Tongming; Tuskan, Gerald A; Kirst, Matias

2009-06-01

Microarrays have demonstrated significant power for genome-wide analyses of gene expression, and recently have also revolutionized the genetic analysis of segregating populations by genotyping thousands of loci in a single assay. Although microarray-based genotyping approaches have been successfully applied in yeast and several inbred plant species, their power has not been proven in an outcrossing species with extensive genetic diversity. Here we have developed methods for high-throughput microarray-based genotyping in such species using a pseudo-backcross progeny of 154 individuals of Populus trichocarpa and P. deltoides analyzed with long-oligonucleotide in situ-synthesized microarray probes. Our analysis resulted in high-confidence genotypes for 719 single-feature polymorphism (SFP) and 1014 gene expression marker (GEM) candidates. Using these genotypes and an established microsatellite (SSR) framework map, we produced a high-density genetic map comprising over 600 SFPs, GEMs and SSRs. The abundance of gene-based markers allowed us to localize over 35 million base pairs of previously unplaced whole-genome shotgun (WGS) scaffold sequence to putative locations in the genome of P. trichocarpa. A high proportion of sampled scaffolds could be verified for their placement with independently mapped SSRs, demonstrating the previously un-utilized power that high-density genotyping can provide in the context of map-based WGS sequence reassembly. Our results provide a substantial contribution to the continued improvement of the Populus genome assembly, while demonstrating the feasibility of microarray-based genotyping in a highly heterozygous population. The strategies presented are applicable to genetic mapping efforts in all plant species with similarly high levels of genetic diversity.

Alteration of Gene Expression Profile in Kidney of Spontaneously Hypertensive Rats Treated with Protein Hydrolysate of Blue Mussel (Mytilus edulis) by DNA Microarray Analysis

PubMed Central

Feng, Junli; Dai, Zhiyuan; Zhang, Yanping; Meng, Lu; Ye, Jian; Ma, Xuting

2015-01-01

Marine organisms are rich sources of bioactive components, which are often reported to have antihypertensive effects. However, the underlying mechanisms have yet to be fully identified. The aim of this study was to investigate the antihypertensive effect of enzymatic hydrolysis of blue mussel protein (HBMP) in rats. Peptides with in vitro ACE inhibitory activity were purified from HBMP by ultrafiltration, gel filtration chromatography and reversed-phase high performance liquid chromatography. And the amino acid sequences of isolated peptides were estimated to be Val-Trp, Leu-Gly-Trp, and Met-Val-Trp-Thr. To study its in vivo action, spontaneously hypertensive rats (SHRs) were orally administration with high- or low-dose of HBMP for 28 days. Major components of the renin-angiotensin (RAS) system in serum of SHRs from different groups were analyzed, and gene expression profiling were performed in the kidney of SHRs, using the Whole Rat Genome Oligonucleotide Microarray. Results indicated although genes involved in RAS system were not significantly altered, those related to blood coagulation system, cytokine and growth factor, and fatty acids metabolism were remarkablely changed. Several genes which were seldom reported to be implicated in pathogenesis of hypertension also showed significant expression alterations after oral administration of HBMP. These data provided valuable information for our understanding of the molecular mechanisms that underlie the potential antihypertensive activities of HBMP, and will contribute towards increased value-added utilization of blue mussel protein. PMID:26517713

Whole blood analysis rotor assembly having removable cellular sedimentation bowl

DOEpatents

Burtis, C.A.; Johnson, W.F.

1975-08-26

A rotor assembly for performing photometric analyses using whole blood samples is described. Following static loading of a gross blood sample within a centrally located, removable, cell sedimentation bowl, the red blood cells in the gross sample are centrifugally separated from the plasma, the plasm displaced from the sedimentation bowl, and measured subvolumes of plasma distributed to respective sample analysis cuvettes positioned in an annular array about the rotor periphery. Means for adding reagents to the respective cuvettes are also described. (auth)

Development and assessment of whole-genome oligonucleotide microarrays to analyze an anaerobic microbial community and its responses to oxidative stress.

PubMed

Scholten, Johannes C M; Culley, David E; Nie, Lei; Munn, Kyle J; Chow, Lely; Brockman, Fred J; Zhang, Weiwen

2007-06-29

The application of DNA microarray technology to investigate multiple-species microbial communities presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri, and Methanospirillum hungatei, and their application to analyze global gene expression in a four-species microbial community in response to oxidative stress. In order to minimize the possibility of cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. This study showed that S. fumaroxidans and M. hungatei responded to the oxidative stress with up-regulation of several genes known to be involved in reactive oxygen species (ROS) detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. However, D. vulgaris seemed to be less sensitive to the oxidative stress as a member of a four-species community, since no gene involved in ROS detoxification was up-regulated. Our work demonstrated the successful application of microarrays to a multiple-species microbial community, and our preliminary results indicated that this approach could provide novel insights on the metabolism within microbial communities.

Ubiquitous sialometabolism present among oral fusobacteria.

PubMed

Yoneda, Saori; Loeser, Brandon; Feng, Joseph; Dmytryk, John; Qi, Fengxia; Merritt, Justin

2014-01-01

Fusobacterium nucleatum is a ubiquitous member of the human oral flora and is associated with the development of periodontitis and a variety of other types of polymicrobial infections of the mucosa. In the oral cavity, this species is one of the few that is prevalent in both healthy and diseased subgingival plaque. Using microarray analysis, we examined the transcriptional response of F. nucleatum subspecies nucleatum to whole blood in order to identify some of the genetic responses that might occur during the transition from health to disease. From these studies, we identified a sialic acid catabolism operon that was induced by the presence of blood. We subsequently confirmed that this operon was inducible by the presence of synthetic sialic acid, but we found no evidence suggesting sialic acid was used as a major carbon source. However, this organism was found to possess a de novo synthesized surface sialylation ability that is widely conserved among the various F. nucleatum subspecies as well as in F. periodonticum. We provide evidence that fusobacterial sialylation does occur in the oral cavity irrespective of health status. Interestingly, only a minority of fusobacterial cells exhibit surface sialylation within dental plaque, whereas most cells are uniformly sialylated when grown in pure culture. The implications of these results are discussed.

Method and apparatus for automated processing and aliquoting of whole blood samples for analysis in a centrifugal fast analyzer

DOEpatents

Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

1988-01-01

A rotor and disc assembly for use in a centrifugal fast analyzer. The assembly is designed to process multiple samples of whole blood followed by aliquoting of the resultant serum into precisely measured samples for subsequent chemical analysis. The assembly requires minimal operator involvement with no mechanical pipetting. The system comprises (1) a whole blood sample disc, (2) a serum sample disc, (3) a sample preparation rotor, and (4) an analytical rotor. The blood sample disc and serum sample disc are designed with a plurality of precision bore capillary tubes arranged in a spoked array. Samples of blood are loaded into the blood sample disc in capillary tubes filled by capillary action and centrifugally discharged into cavities of the sample preparation rotor where separation of serum and solids is accomplished. The serum is loaded into the capillaries of the serum sample disc by capillary action and subsequently centrifugally expelled into cuvettes of the analytical rotor for analysis by conventional methods.

Ultrafiltration and Microarray for Detection of Microbial Source Tracking Marker and Pathogen Genes in Riverine and Marine Systems

PubMed Central

Li, Xiang; Harwood, Valerie J.; Nayak, Bina

2016-01-01

Pathogen identification and microbial source tracking (MST) to identify sources of fecal pollution improve evaluation of water quality. They contribute to improved assessment of human health risks and remediation of pollution sources. An MST microarray was used to simultaneously detect genes for multiple pathogens and indicators of fecal pollution in freshwater, marine water, sewage-contaminated freshwater and marine water, and treated wastewater. Dead-end ultrafiltration (DEUF) was used to concentrate organisms from water samples, yielding a recovery efficiency of >95% for Escherichia coli and human polyomavirus. Whole-genome amplification (WGA) increased gene copies from ultrafiltered samples and increased the sensitivity of the microarray. Viruses (adenovirus, bocavirus, hepatitis A virus, and human polyomaviruses) were detected in sewage-contaminated samples. Pathogens such as Legionella pneumophila, Shigella flexneri, and Campylobacter fetus were detected along with genes conferring resistance to aminoglycosides, beta-lactams, and tetracycline. Nonmetric dimensional analysis of MST marker genes grouped sewage-spiked freshwater and marine samples with sewage and apart from other fecal sources. The sensitivity (percent true positives) of the microarray probes for gene targets anticipated in sewage was 51 to 57% and was lower than the specificity (percent true negatives; 79 to 81%). A linear relationship between gene copies determined by quantitative PCR and microarray fluorescence was found, indicating the semiquantitative nature of the MST microarray. These results indicate that ultrafiltration coupled with WGA provides sufficient nucleic acids for detection of viruses, bacteria, protozoa, and antibiotic resistance genes by the microarray in applications ranging from beach monitoring to risk assessment. PMID:26729716

Adult and cord blood endothelial progenitor cells have different gene expression profiles and immunogenic potential.

PubMed

Nuzzolo, Eugenia R; Capodimonti, Sara; Martini, Maurizio; Iachininoto, Maria G; Bianchi, Maria; Cocomazzi, Alessandra; Zini, Gina; Leone, Giuseppe; Larocca, Luigi M; Teofili, Luciana

2014-01-01

Endothelial colony-forming cells (ECFC) are endowed with vascular regenerative ability in vivo and in vitro. In this study we compared the genotypic profile and the immunogenic potential of adult and cord blood ECFC, in order to explore the feasibility of using them as a cell therapy product. ECFC were obtained from cord blood samples not suitable for haematopoietic stem cell transplantation and from adult healthy blood donors after informed consent. Genotypes were analysed by commercially available microarray assays and results were confirmed by real-time polymerase chain reaction analysis. HLA antigen expression was evaluated by flow-cytometry. Immunogenic capacity was investigated by evaluating the activation of allogeneic lymphocytes and monocytes in co-cultures with ECFC. Microarray assays revealed that the genetic profile of cord blood and adult ECFC differed in about 20% of examined genes. We found that cord blood ECFC were characterised by lower pro-inflammatory and pro-thrombotic gene expression as compared to adult ECFC. Furthermore, whereas cord blood and adult ECFCs expressed similar amount of HLA molecules both at baseline and after incubation with γ-interferon, cord blood ECFC elicited a weaker expression of pro-inflammatory cytokine genes. Finally, we observed no differences in the amount of HLA antigens expressed among cord blood ECFC, adult ECFC and mesenchymal cells. Our observations suggest that cord blood ECFC have a lower pro-inflammatory and pro-thrombotic profile than adult ECFC. These preliminary data offer level-headed evidence to use cord blood ECFC as a cell therapy product in vascular diseases.

Transcriptomic profile of host response in mouse brain after exposure to plant toxin abrin.

PubMed

Bhaskar, A S Bala; Gupta, Nimesh; Rao, P V Lakshmana

2012-09-04

Abrin toxin is a plant glycoprotein, which is similar in structure and properties to ricin and is obtained from the seeds of Abrus precatorius (jequirity bean). Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 μg/kg, and has caused death after accidental and intentional poisoning. Abrin is a potent biological toxin warfare agent. There are no chemical antidotes available against the toxin. Neurological symptoms like delirium, hallucinations, reduced consciousness and generalized seizures were reported in human poisoning cases. Death of a patient with symptoms of acute demyelinating encephalopathy with gastrointestinal bleeding due to ingestion of abrin seeds was reported in India. The aim of this study was to examine both dose and time-dependent transcriptional responses induced by abrin in the adult mouse brain. Mice (n=6) were exposed to 1 and 2 LD50 (2.83 and 5.66 μg/kg respectively) dose of abrin by intraperitoneal route and observed over 3 days. A subset of animals (n=3) were sacrificed at 1 and 2 day intervals for microarray and histopathology analysis. None of the 2 LD50 exposed animals survived till 3 days. The histopathological analysis showed the severe damage in brain and the infiltration of inflammatory cells in a dose and time dependent manner. The abrin exposure resulted in the induction of rapid immune and inflammatory response in brain. Clinical biochemistry parameters like lactate dehydrogenase, aspartate aminotransferase, urea and creatinine showed significant increase at 2-day 2 LD50 exposure. The whole genome microarray data revealed the significant regulation of various pathways like MAPK pathway, cytokine-cytokine receptor interaction, calcium signaling pathway, Jak-STAT signaling pathway and natural killer cell mediated toxicity. The comparison of differential gene expression at both the doses showed dose dependent effects of abrin toxicity. The real-time qRT-PCR analysis of selected genes supported the microarray data. This is the first report on host-gene response using whole genome microarray in an animal model after abrin exposure. The data generated provides leads for developing suitable medical counter measures against abrin poisoning. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Coagulation dynamics of a blood sample by multiple scattering analysis

NASA Astrophysics Data System (ADS)

Faivre, Magalie; Peltié, Philippe; Planat-Chrétien, Anne; Cosnier, Marie-Line; Cubizolles, Myriam; Nougier, Christophe; Négrier, Claude; Pouteau, Patrick

2011-05-01

We report a new technique to measure coagulation dynamics on whole-blood samples. The method relies on the analysis of the speckle figure resulting from a whole-blood sample mixed with coagulation reagent and introduced in a thin chamber illuminated with a coherent light. A dynamic study of the speckle reveals a typical behavior due to coagulation. We compare our measured coagulation times to a reference method obtained in a medical laboratory.

Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome

PubMed Central

Danger, Richard; Royer, Pierre-Joseph; Reboulleau, Damien; Durand, Eugénie; Loy, Jennifer; Tissot, Adrien; Lacoste, Philippe; Roux, Antoine; Reynaud-Gaubert, Martine; Gomez, Carine; Kessler, Romain; Mussot, Sacha; Dromer, Claire; Brugière, Olivier; Mornex, Jean-François; Guillemain, Romain; Dahan, Marcel; Knoop, Christiane; Botturi, Karine; Foureau, Aurore; Pison, Christophe; Koutsokera, Angela; Nicod, Laurent P.; Brouard, Sophie; Magnan, Antoine; Jougon, J.

2018-01-01

Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01) and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS. PMID:29375549

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

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

Gene expression profiling to characterize sediment toxicity – a pilot study using Caenorhabditis elegans whole genome microarrays

PubMed Central

Menzel, Ralph; Swain, Suresh C; Hoess, Sebastian; Claus, Evelyn; Menzel, Stefanie; Steinberg, Christian EW; Reifferscheid, Georg; Stürzenbaum, Stephen R

2009-01-01

Background Traditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants. Results In this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment. Conclusion This study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments. PMID:19366437

Hsa-circRNA11783-2 in peripheral blood is correlated with coronary artery disease and type 2 diabetes mellitus.

PubMed

Li, Xuejie; Zhao, Zhenzhou; Jian, Dongdong; Li, Wentao; Tang, Haiyu; Li, Muwei

2017-11-01

The purpose of this study was to identify the expression characteristics of circular RNAs in the peripheral blood of coronary artery disease patients and type 2 diabetes mellitus patients. Circular RNA in the peripheral blood from 6 control individuals, 6 coronary artery disease patients, 6 type 2 diabetes mellitus patients and 6 coronary artery disease combined with type 2 diabetes mellitus patients was collected for microarray analysis, and a further independent cohort consisting of 20 normal individuals, 20 type 2 diabetes mellitus subjects and 20 coronary artery disease subjects was used to verify the expression of five circular RNAs chosen for further analysis. The findings were then tested in a third cohort using quantitative real-time polymerase chain reaction. In total, 40 circular RNAs differentially expressed between the three experimental groups and the control group were identified by microarray analysis: 13 were upregulated in the experimental groups, while 27 were downregulated. Of the five circular RNAs chosen for further analysis, three were significantly downregulated in the experimental groups. The crude odds ratios and adjusted odds ratios of hsa-circRNA11783-2 showed significant differences in both the coronary artery disease group and type 2 diabetes mellitus group. We then verified hsa-circRNA11783-2 in the third cohort, and it remained closely related to both coronary artery disease and type 2 diabetes mellitus. Hsa-circRNA11783-2 is closely related to both coronary artery disease and type 2 diabetes mellitus.

Microarray analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice

PubMed Central

Garg, Rohini; Tyagi, Akhilesh K.; Jain, Mukesh

2012-01-01

Hormones exert pleiotropic effects on plant growth and development throughout the life cycle. Many of these effects are mediated at molecular level via altering gene expression. In this study, we investigated the exogenous effect of plant hormones, including auxin, cytokinin, abscisic acid, ethylene, salicylic acid and jasmonic acid, on the transcription of rice genes at whole genome level using microarray. Our analysis identified a total of 4171 genes involved in several biological processes, whose expression was altered significantly in the presence of different hormones. Further, 28% of these genes exhibited overlapping transcriptional responses in the presence of any two hormones, indicating crosstalk among plant hormones. In addition, we identified genes showing only a particular hormone-specific response, which can be used as hormone-specific markers. The results of this study will facilitate further studies in hormone biology in rice. PMID:22827941

WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data

PubMed Central

Yi, Ming; Horton, Jay D; Cohen, Jonathan C; Hobbs, Helen H; Stephens, Robert M

2006-01-01

Background Analysis of High Throughput (HTP) Data such as microarray and proteomics data has provided a powerful methodology to study patterns of gene regulation at genome scale. A major unresolved problem in the post-genomic era is to assemble the large amounts of data generated into a meaningful biological context. We have developed a comprehensive software tool, WholePathwayScope (WPS), for deriving biological insights from analysis of HTP data. Result WPS extracts gene lists with shared biological themes through color cue templates. WPS statistically evaluates global functional category enrichment of gene lists and pathway-level pattern enrichment of data. WPS incorporates well-known biological pathways from KEGG (Kyoto Encyclopedia of Genes and Genomes) and Biocarta, GO (Gene Ontology) terms as well as user-defined pathways or relevant gene clusters or groups, and explores gene-term relationships within the derived gene-term association networks (GTANs). WPS simultaneously compares multiple datasets within biological contexts either as pathways or as association networks. WPS also integrates Genetic Association Database and Partial MedGene Database for disease-association information. We have used this program to analyze and compare microarray and proteomics datasets derived from a variety of biological systems. Application examples demonstrated the capacity of WPS to significantly facilitate the analysis of HTP data for integrative discovery. Conclusion This tool represents a pathway-based platform for discovery integration to maximize analysis power. The tool is freely available at . PMID:16423281

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow

PubMed Central

Berry, Nadine Kaye; Bain, Nicole L; Enjeti, Anoop K; Rowlings, Philip

2014-01-01

Aim To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). Methods A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Results Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. Conclusions We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH. PMID:23969274

Genomic profiling of plasma cell disorders in a clinical setting: integration of microarray and FISH, after CD138 selection of bone marrow.

PubMed

Berry, Nadine Kaye; Bain, Nicole L; Enjeti, Anoop K; Rowlings, Philip

2014-01-01

To evaluate the role of whole genome comparative genomic hybridisation microarray (array-CGH) in detecting genomic imbalances as compared to conventional karyotype (GTG-analysis) or myeloma specific fluorescence in situ hybridisation (FISH) panel in a diagnostic setting for plasma cell dyscrasia (PCD). A myeloma-specific interphase FISH (i-FISH) panel was carried out on CD138 PC-enriched bone marrow (BM) from 20 patients having BM biopsies for evaluation of PCD. Whole genome array-CGH was performed on reference (control) and neoplastic (test patient) genomic DNA extracted from CD138 PC-enriched BM and analysed. Comparison of techniques demonstrated a much higher detection rate of genomic imbalances using array-CGH. Genomic imbalances were detected in 1, 19 and 20 patients using GTG-analysis, i-FISH and array-CGH, respectively. Genomic rearrangements were detected in one patient using GTG-analysis and seven patients using i-FISH, while none were detected using array-CGH. I-FISH was the most sensitive method for detecting gene rearrangements and GTG-analysis was the least sensitive method overall. All copy number aberrations observed in GTG-analysis were detected using array-CGH and i-FISH. We show that array-CGH performed on CD138-enriched PCs significantly improves the detection of clinically relevant and possibly novel genomic abnormalities in PCD, and thus could be considered as a standard diagnostic technique in combination with IGH rearrangement i-FISH.

Application of Microchip for Biomarker Analysis

NASA Astrophysics Data System (ADS)

Kataoka, Masatoshi; Yatsushiro, Shouki; Yamamura, Shouhei; Abe, Hiroko

Microchip technologies have received considerable attention, due to their competitive advantages, especially in regards to reduced sample and reagent consumption, analysis time, and easy operation. This approach has been successfully used to analyze DNA, amino acids, proteins, and carbohydrates. In the present study, we showed the potential of microchip technologies for the biomarker analysis, blood carbohydrate analysis on microchip electrophoresis, quantitative analysis of protein with antigen-antibody reaction on microchip, and the detection of malaria-infected erythrocyte with a cell microarray chip.

Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium

PubMed Central

2009-01-01

Background Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood. Results We successfully adapted the SCOTS method (Selective Capture of Transcribed Sequences) on the model Rickettsiales ER to capture mRNAs. Southern Blots and RT-PCR revealed an enrichment of ER's cDNAs and a diminution of ribosomal contaminants after three rounds of capture. qRT-PCR and whole-genome ER microarrays hybridizations demonstrated that SCOTS method introduced only a limited bias on gene expression. Indeed, we confirmed the differential gene expression between poorly and highly expressed genes before and after SCOTS captures. The comparative gene expression obtained from ER microarrays data, on samples before and after SCOTS at 96 hpi was significantly correlated (R2 = 0.7). Moreover, SCOTS method is crucial for microarrays analysis of ER, especially for early time points post-infection. There was low detection of transcripts for untreated samples whereas 24% and 70.7% were revealed for SCOTS samples at 24 and 96 hpi respectively. Conclusions We conclude that this SCOTS method has a key importance for the transcriptomic analysis of ER and can be potentially used for other Rickettsiales. This study constitutes the first step for further gene expression analyses that will lead to a better understanding of both ER pathogenicity and the adaptation of obligate intracellular bacteria to their environment. PMID:20034374

A HaemAtlas: characterizing gene expression in differentiated human blood cells.

PubMed

Watkins, Nicholas A; Gusnanto, Arief; de Bono, Bernard; De, Subhajyoti; Miranda-Saavedra, Diego; Hardie, Debbie L; Angenent, Will G J; Attwood, Antony P; Ellis, Peter D; Erber, Wendy; Foad, Nicola S; Garner, Stephen F; Isacke, Clare M; Jolley, Jennifer; Koch, Kerstin; Macaulay, Iain C; Morley, Sarah L; Rendon, Augusto; Rice, Kate M; Taylor, Niall; Thijssen-Timmer, Daphne C; Tijssen, Marloes R; van der Schoot, C Ellen; Wernisch, Lorenz; Winzer, Thilo; Dudbridge, Frank; Buckley, Christopher D; Langford, Cordelia F; Teichmann, Sarah; Göttgens, Berthold; Ouwehand, Willem H

2009-05-07

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.

A HaemAtlas: characterizing gene expression in differentiated human blood cells

PubMed Central

Gusnanto, Arief; de Bono, Bernard; De, Subhajyoti; Miranda-Saavedra, Diego; Hardie, Debbie L.; Angenent, Will G. J.; Attwood, Antony P.; Ellis, Peter D.; Erber, Wendy; Foad, Nicola S.; Garner, Stephen F.; Isacke, Clare M.; Jolley, Jennifer; Koch, Kerstin; Macaulay, Iain C.; Morley, Sarah L.; Rendon, Augusto; Rice, Kate M.; Taylor, Niall; Thijssen-Timmer, Daphne C.; Tijssen, Marloes R.; van der Schoot, C. Ellen; Wernisch, Lorenz; Winzer, Thilo; Dudbridge, Frank; Buckley, Christopher D.; Langford, Cordelia F.; Teichmann, Sarah; Göttgens, Berthold; Ouwehand, Willem H.

2009-01-01

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. PMID:19228925

Quantification of Rifapentine, a Potent Antituberculosis Drug, from Dried Blood Spot Samples Using Liquid Chromatographic-Tandem Mass Spectrometric Analysis

PubMed Central

Parsons, Teresa L.; Marzinke, Mark A.; Hoang, Thuy; Bliven-Sizemore, Erin; Weiner, Marc; Mac Kenzie, William R.; Dorman, Susan E.

2014-01-01

The quantification of antituberculosis drug concentrations in multinational trials currently requires the collection of modest blood volumes, centrifugation, aliquoting of plasma, freezing, and keeping samples frozen during shipping. We prospectively enrolled healthy individuals into the Tuberculosis Trials Consortium Study 29B, a phase I dose escalation study of rifapentine, a rifamycin under evaluation in tuberculosis treatment trials. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying rifapentine in whole blood on dried blood spots (DBS) to facilitate pharmacokinetic/pharmacodynamic analyses in clinical trials. Paired plasma and whole-blood samples were collected by venipuncture, and whole blood was spotted on Whatman protein saver 903 cards. The methods were optimized for plasma and then validated for DBS. The analytical measuring range for quantification of rifapentine and its metabolite was 50 to 80,000 ng/ml in whole-blood DBS. The analyte was stable on the cards for 11 weeks with a desiccant at room temperature and protected from light. The method concordance for paired plasma and whole-blood DBS samples was determined after correcting for participant hematocrit or population-based estimates of bias from Bland-Altman plots. The application of either correction factor resulted in acceptable correlation between plasma and whole-blood DBS (Passing-Bablok regression corrected for hematocrit; y = 0.98x + 356). Concentrations of rifapentine may be determined from whole-blood DBS collected via venipuncture after normalization in order to account for the dilutional effects of red blood cells. Additional studies are focused on the application of this methodology to capillary blood collected by finger stick. The simplicity of processing, storage, shipping, and low blood volume makes whole-blood DBS attractive for rifapentine pharmacokinetic evaluations, especially in international and pediatric trials. PMID:25182637

Proteins and antibodies in serum, plasma, and whole blood-size characterization using asymmetrical flow field-flow fractionation (AF4).

PubMed

Leeman, Mats; Choi, Jaeyeong; Hansson, Sebastian; Storm, Matilda Ulmius; Nilsson, Lars

2018-05-29

The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood.

Gene expression profile in cardiovascular disease and preeclampsia: a meta-analysis of the transcriptome based on raw data from human studies deposited in Gene Expression Omnibus.

PubMed

Sitras, V; Fenton, C; Acharya, G

2015-02-01

Cardiovascular disease (CVD) and preeclampsia (PE) share common clinical features. We aimed to identify common transcriptomic signatures involved in CVD and PE in humans. Meta-analysis of individual raw microarray data deposited in GEO, obtained from blood samples of patients with CVD versus controls and placental samples from women with PE versus healthy women with uncomplicated pregnancies. Annotation of cases versus control samples was taken directly from the microarray documentation. Genes that showed a significant differential expression in the majority of experiments were selected for subsequent analysis. Hypergeometric gene list analysis was performed using Bioconductor GOstats package. Bioinformatic analysis was performed in PANTHER. Seven studies in CVD and 5 studies in PE were eligible for meta-analysis. A total of 181 genes were found to be differentially expressed in microarray studies investigating gene expression in blood samples obtained from patients with CVD compared to controls and 925 genes were differentially expressed between preeclamptic and healthy placentas. Among these differentially expressed genes, 22 were common between CVD and PE. Bioinformatic analysis of these genes revealed oxidative stress, p-53 pathway feedback, inflammation mediated by chemokines and cytokines, interleukin signaling, B-cell activation, PDGF signaling, Wnt signaling, integrin signaling and Alzheimer disease pathways to be involved in the pathophysiology of both CVD and PE. Metabolism, development, response to stimulus, immune response and cell communication were the associated biologic processes in both conditions. Gene set enrichment analysis showed the following overlapping pathways between CVD and PE: TGF-β-signaling, apoptosis, graft-versus-host disease, allograft rejection, chemokine signaling, steroid hormone synthesis, type I and II diabetes mellitus, VEGF signaling, pathways in cancer, GNRH signaling, Huntingtons disease and Notch signaling. CVD and PE share same common traits in their gene expression profile indicating common pathways in their pathophysiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminatedmore » the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.« less

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel.

PubMed

Nam, Jeonghun; Lim, Hyunjung; Kim, Dookon; Shin, Sehyun

2011-10-07

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.

Importance of correlation between gene expression levels: application to the type I interferon signature in rheumatoid arthritis.

PubMed

Reynier, Frédéric; Petit, Fabien; Paye, Malick; Turrel-Davin, Fanny; Imbert, Pierre-Emmanuel; Hot, Arnaud; Mougin, Bruno; Miossec, Pierre

2011-01-01

The analysis of gene expression data shows that many genes display similarity in their expression profiles suggesting some co-regulation. Here, we investigated the co-expression patterns in gene expression data and proposed a correlation-based research method to stratify individuals. Using blood from rheumatoid arthritis (RA) patients, we investigated the gene expression profiles from whole blood using Affymetrix microarray technology. Co-expressed genes were analyzed by a biclustering method, followed by gene ontology analysis of the relevant biclusters. Taking the type I interferon (IFN) pathway as an example, a classification algorithm was developed from the 102 RA patients and extended to 10 systemic lupus erythematosus (SLE) patients and 100 healthy volunteers to further characterize individuals. We developed a correlation-based algorithm referred to as Classification Algorithm Based on a Biological Signature (CABS), an alternative to other approaches focused specifically on the expression levels. This algorithm applied to the expression of 35 IFN-related genes showed that the IFN signature presented a heterogeneous expression between RA, SLE and healthy controls which could reflect the level of global IFN signature activation. Moreover, the monitoring of the IFN-related genes during the anti-TNF treatment identified changes in type I IFN gene activity induced in RA patients. In conclusion, we have proposed an original method to analyze genes sharing an expression pattern and a biological function showing that the activation levels of a biological signature could be characterized by its overall state of correlation.

Evaluation of a microarray-based genotyping assay for the rapid detection of cytochrome P450 2C19 *2 and *3 polymorphisms from whole blood using nanoparticle probes.

PubMed

Buchan, Blake W; Peterson, Jess F; Cogbill, Christopher H; Anderson, Dennis K; Ledford, Joellen S; White, Mary N; Quigley, Neil B; Jannetto, Paul J; Ledeboer, Nathan A

2011-10-01

Numerous drugs such as clopidogrel have been developed to reduce coagulation or inhibit platelet function. The hepatic cytochrome P450 (CYP) pathway is involved in the conversion of clopidogrel to its active metabolite. A recent black-box warning was included in the clopidogrel package insert indicating a significant clinical link between specific CYP2C19 genetic variants and poor metabolism of clopidogrel. Of these variants, *2 and *3 are the most common and are associated with complete loss of enzyme activity. In patients who are carriers of a CYP2C19 *2 or *3 allele, the conversion of clopidogrel to its active metabolite may be reduced, which can lead to ischemic events and negative consequence for the patient. We examined the ability of the Verigene CLO assay (Nanosphere, Northbrook, IL) to identify CYP2C19 *2 and *3 polymorphisms in 1,286 unique whole blood samples. The Verigene CLO assay accurately identified homozygous and heterozygous *2 and *3 phenotypes with a specificity of 100% and a final call rate of 99.7%. The assay is fully automated and can produce a result in approximately 3.5 hours.

Preliminary profiling of blood transcriptome in a rat model of hemorrhagic shock.

PubMed

Braga, D; Barcella, M; D'Avila, F; Lupoli, S; Tagliaferri, F; Santamaria, M H; DeLano, F A; Baselli, G; Schmid-Schönbein, G W; Kistler, E B; Aletti, F; Barlassina, C

2017-08-01

Hemorrhagic shock is a leading cause of morbidity and mortality worldwide. Significant blood loss may lead to decreased blood pressure and inadequate tissue perfusion with resultant organ failure and death, even after replacement of lost blood volume. One reason for this high acuity is that the fundamental mechanisms of shock are poorly understood. Proteomic and metabolomic approaches have been used to investigate the molecular events occurring in hemorrhagic shock but, to our knowledge, a systematic analysis of the transcriptomic profile is missing. Therefore, a pilot analysis using paired-end RNA sequencing was used to identify changes that occur in the blood transcriptome of rats subjected to hemorrhagic shock after blood reinfusion. Hemorrhagic shock was induced using a Wigger's shock model. The transcriptome of whole blood from shocked animals shows modulation of genes related to inflammation and immune response (Tlr13, Il1b, Ccl6, Lgals3), antioxidant functions (Mt2A, Mt1), tissue injury and repair pathways (Gpnmb, Trim72) and lipid mediators (Alox5ap, Ltb4r, Ptger2) compared with control animals. These findings are congruent with results obtained in hemorrhagic shock analysis by other authors using metabolomics and proteomics. The analysis of blood transcriptome may be a valuable tool to understand the biological changes occurring in hemorrhagic shock and a promising approach for the identification of novel biomarkers and therapeutic targets. Impact statement This study provides the first pilot analysis of the changes occurring in transcriptome expression of whole blood in hemorrhagic shock (HS) rats. We showed that the analysis of blood transcriptome is a useful approach to investigate pathways and functional alterations in this disease condition. This pilot study encourages the possible application of transcriptome analysis in the clinical setting, for the molecular profiling of whole blood in HS patients.

Integrative network analysis unveils convergent molecular pathways in Parkinson's disease and diabetes.

PubMed

Santiago, Jose A; Potashkin, Judith A

2013-01-01

Shared dysregulated pathways may contribute to Parkinson's disease and type 2 diabetes, chronic diseases that afflict millions of people worldwide. Despite the evidence provided by epidemiological and gene profiling studies, the molecular and functional networks implicated in both diseases, have not been fully explored. In this study, we used an integrated network approach to investigate the extent to which Parkinson's disease and type 2 diabetes are linked at the molecular level. Using a random walk algorithm within the human functional linkage network we identified a molecular cluster of 478 neighboring genes closely associated with confirmed Parkinson's disease and type 2 diabetes genes. Biological and functional analysis identified the protein serine-threonine kinase activity, MAPK cascade, activation of the immune response, and insulin receptor and lipid signaling as convergent pathways. Integration of results from microarrays studies identified a blood signature comprising seven genes whose expression is dysregulated in Parkinson's disease and type 2 diabetes. Among this group of genes, is the amyloid precursor protein (APP), previously associated with neurodegeneration and insulin regulation. Quantification of RNA from whole blood of 192 samples from two independent clinical trials, the Harvard Biomarker Study (HBS) and the Prognostic Biomarker Study (PROBE), revealed that expression of APP is significantly upregulated in Parkinson's disease patients compared to healthy controls. Assessment of biomarker performance revealed that expression of APP could distinguish Parkinson's disease from healthy individuals with a diagnostic accuracy of 80% in both cohorts of patients. These results provide the first evidence that Parkinson's disease and diabetes are strongly linked at the molecular level and that shared molecular networks provide an additional source for identifying highly sensitive biomarkers. Further, these results suggest for the first time that increased expression of APP in blood may modulate the neurodegenerative phenotype in type 2 diabetes patients.

Review: nanoparticles in delivery of cardiovascular drugs.

PubMed

Arayne, M Saeed; Sultana, Najma; Qureshi, Faiza

2007-10-01

Everything in nature is built upward from the atomic level to define limits and structures to everything. Nanomedicines marked the field of medicine from nanobiotechnology, biological micro-electromechanical systems, microfluidics, biosensors, drug delivery, microarrays to tissue microengineering. Since then nanoparticles has overcome many challenges from blood brain barrier to targeting tumors. Where solid biodegradable nanoparticles were a step up liposome, targeting nanoparticles opened a whole new field for drug delivery. In this article, we attempt to discuss how the pioneered technique is serving in the drug delivery to cardiovascular system and how with the manipulation of their properties, nanoparticles can be made to fulfill desired function. Also how nanocarriers are improving molecular imaging to help improve diagnosis and treatment of cardiovascular disease is focused in this article.

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

PubMed

Rieger, Hannah; Schmidt, Patrik; Schaeffeler, Elke; Abe, Manabu; Schiffhauer, Mira; Schwab, Matthias; von Ahsen, Nicolas; Zurek, Gabriela; Kirchherr, Hartmut; Shipkova, Maria; Wieland, Eberhard

2018-04-25

Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Detection of growth hormone doping by gene expression profiling of peripheral blood.

PubMed

Mitchell, Christopher J; Nelson, Anne E; Cowley, Mark J; Kaplan, Warren; Stone, Glenn; Sutton, Selina K; Lau, Amie; Lee, Carol M Y; Ho, Ken K Y

2009-12-01

GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

Using expression genetics to study the neurobiology of ethanol and alcoholism.

PubMed

Farris, Sean P; Wolen, Aaron R; Miles, Michael F

2010-01-01

Recent simultaneous progress in human and animal model genetics and the advent of microarray whole genome expression profiling have produced prodigious data sets on genetic loci, potential candidate genes, and differential gene expression related to alcoholism and ethanol behaviors. Validated target genes or gene networks functioning in alcoholism are still of meager proportions. Genetical genomics, which combines genetic analysis of both traditional phenotypes and whole genome expression data, offers a potential methodology for characterizing brain gene networks functioning in alcoholism. This chapter will describe concepts, approaches, and recent findings in the field of genetical genomics as it applies to alcohol research. Copyright 2010 Elsevier Inc. All rights reserved.

Screening of differentially expressed genes between multiple trauma patients with and without sepsis.

PubMed

Ji, S C; Pan, Y T; Lu, Q Y; Sun, Z Y; Liu, Y Z

2014-03-17

The purpose of this study was to identify critical genes associated with septic multiple trauma by comparing peripheral whole blood samples from multiple trauma patients with and without sepsis. A microarray data set was downloaded from the Gene Expression Omnibus (GEO) database. This data set included 70 samples, 36 from multiple trauma patients with sepsis and 34 from multiple trauma patients without sepsis (as a control set). The data were preprocessed, and differentially expressed genes (DEGs) were then screened for using packages of the R language. Functional analysis of DEGs was performed with DAVID. Interaction networks were then established for the most up- and down-regulated genes using HitPredict. Pathway-enrichment analysis was conducted for genes in the networks using WebGestalt. Fifty-eight DEGs were identified. The expression levels of PLAU (down-regulated) and MMP8 (up-regulated) presented the largest fold-changes, and interaction networks were established for these genes. Further analysis revealed that PLAT (plasminogen activator, tissue) and SERPINF2 (serpin peptidase inhibitor, clade F, member 2), which interact with PLAU, play important roles in the pathway of the component and coagulation cascade. We hypothesize that PLAU is a major regulator of the component and coagulation cascade, and down-regulation of PLAU results in dysfunction of the pathway, causing sepsis.

Genomic paradigms for food-borne enteric pathogen analysis at the USFDA: case studies highlighting method utility, integration and resolution.

PubMed

Elkins, C A; Kotewicz, M L; Jackson, S A; Lacher, D W; Abu-Ali, G S; Patel, I R

2013-01-01

Modern risk control and food safety practices involving food-borne bacterial pathogens are benefiting from new genomic technologies for rapid, yet highly specific, strain characterisations. Within the United States Food and Drug Administration (USFDA) Center for Food Safety and Applied Nutrition (CFSAN), optical genome mapping and DNA microarray genotyping have been used for several years to quickly assess genomic architecture and gene content, respectively, for outbreak strain subtyping and to enhance retrospective trace-back analyses. The application and relative utility of each method varies with outbreak scenario and the suspect pathogen, with comparative analytical power enhanced by database scale and depth. Integration of these two technologies allows high-resolution scrutiny of the genomic landscapes of enteric food-borne pathogens with notable examples including Shiga toxin-producing Escherichia coli (STEC) and Salmonella enterica serovars from a variety of food commodities. Moreover, the recent application of whole genome sequencing technologies to food-borne pathogen outbreaks and surveillance has enhanced resolution to the single nucleotide scale. This new wealth of sequence data will support more refined next-generation custom microarray designs, targeted re-sequencing and "genomic signature recognition" approaches involving a combination of genes and single nucleotide polymorphism detection to distil strain-specific fingerprinting to a minimised scale. This paper examines the utility of microarrays and optical mapping in analysing outbreaks, reviews best practices and the limits of these technologies for pathogen differentiation, and it considers future integration with whole genome sequencing efforts.

Coagulation function of stored whole blood is preserved for 14 days in austere conditions: A ROTEM feasibility study during a Norwegian antipiracy mission and comparison to equal ratio reconstituted blood.

PubMed

Strandenes, Geir; Austlid, Ivar; Apelseth, Torunn O; Hervig, Tor A; Sommerfelt-Pettersen, Jan; Herzig, Maryanne C; Cap, Andrew P; Pidcoke, Heather F; Kristoffersen, Einar K

2015-06-01

Formulation of a medical preparedness plan for treating severely bleeding casualties during naval deployment is a significant challenge because of territory covered during most missions. The aim of this study was to evaluate the concept of "walking blood bank" as a supportable plan for supplying safe blood and blood products. In 2013, the Royal Norwegian Navy conducted antipiracy operations from a frigate, beginning in the Gulf of Aden and ending in the Indian Ocean. Crews were on 24-hour emergency alert in preparation for an enemy assault on the frigate. Under an approved command protocol, a "walking blood bank," using crew blood donations, was established for use on board and on missions conducted in rigid-hulled inflatable boats, during which freeze-dried plasma and leukoreduced, group O low anti-A/anti-B titer, cold-stored whole blood were stored in Golden Hour Boxes. Data demonstrating the ability to collect, store, and provide whole blood were collected to establish feasibility of implementing a whole blood-focused remote damage-control resuscitation program aboard a naval vessel. In addition, ROTEM data were collected to demonstrate feasibility of performing this analysis on a large naval vessel and to also measure hemostatic efficacy of cold-stored leukoreduced whole blood (CWB) stored during a period of 14 days. ROTEM data on CWB was compared with reconstituted whole blood. Drills simulating massive transfusion activation were conducted, in which 2 U of warm fresh whole blood with platelet sparing leukoreduction were produced in 40 minutes, followed by collection of two additional units at 15-minute increments. The ROTEM machine performed well during ship-rolling, as shown by the overlapping calculated and measured mechanical piston movements measured by the ROTEM device. Error messages were recorded in 4 (1.5%) of 267 tests. CWB yielded reproducible ROTEM results demonstrating preserved fibrinogen function and platelet function for at least 3.5 weeks and 2 weeks, respectively. The frequency of ROTEM tests were as follows: EXTEM (n = 88), INTEM (n = 85), FIBTEM (n = 82), and APTEM (n = 12). CWB results were grouped. Compared with Days 0 to 2, EXTEM maximum clot firmness was significantly reduced, beginning on Days 10 to 14; however, results through that date remained within reference ranges and were comparable with the EXTEM maximum clot firmness for the reconstituted whole blood samples containing Day 5 room temperature-stored platelets. A "walking blood bank" can provide a balanced transfusion product to support damage-control resuscitation/remote damage-control resuscitation aboard a frigate in the absence of conventional blood bank products. ROTEM analysis is feasible to monitor damage-control resuscitation and blood product quality. ROTEM analysis was possible in challenging operational conditions. Therapeutic study, level V.

Method and apparatus for automated processing and aliquoting of whole blood samples for analysis in a centrifugal fast analyzer

DOEpatents

Burtis, C.A.; Johnson, W.F.; Walker, W.A.

1985-08-05

A rotor and disc assembly for use in a centrifugal fast analyzer. The assembly is designed to process multiple samples of whole blood followed by aliquoting of the resultant serum into precisely measured samples for subsequent chemical analysis. The assembly requires minimal operator involvement with no mechanical pipetting. The system comprises: (1) a whole blood sample disc; (2) a serum sample disc; (3) a sample preparation rotor; and (4) an analytical rotor. The blood sample disc and serum sample disc are designed with a plurality of precision bore capillary tubes arranged in a spoked array. Samples of blood are loaded into the blood sample disc by capillary action and centrifugally discharged into cavities of the sample preparation rotor where separation of serum and solids is accomplished. The serum is loaded into the capillaries of the serum sample disc by capillary action and subsequently centrifugally expelled into cuvettes of the analyticaly rotor for conventional methods. 5 figs.

Gene expression analysis identify a metabolic and cell function alterations as a hallmark of obesity without metabolic syndrome in peripheral blood, a pilot study.

PubMed

de Luis, Daniel Antonio; Almansa, Raquel; Aller, Rocío; Izaola, Olatz; Romero, E

2017-06-10

Understanding molecular basis involved in overweight is an important first step in developing therapeutic pathways against excess in body weight gain. The purpose of our pilot study was to evaluate the gene expression profiles in the peripheral blood of obese patients without other metabolic complications. A sample of 17 obese patients without metabolic syndrome and 15 non obese control subjects was evaluated in a prospective way. Following 'One-Color Microarray-Based Gene Expression Analysis' protocol Version 5.7 (Agilent p/n 4140-90040), cRNA was hybridized with Whole Human Genome Oligo Microarray Kit (Agilent p/n G2519F-014850) containing 41,000+ unique human genes and transcripts. The average age of the study group was 43.6 ± 19.7 years with a sex distribution of 64.7% females and 35.3% males. No statistical differences were detected with healthy controls 41.9 ± 12.3 years with a sex distribution of 70% females and 30% males. Obese patients showed 1436 genes that were differentially expressed compared to control group. Ingenuity Pathway Analysis showed that these genes participated in 13 different categories related to metabolism and cellular functions. In the gene set of cellular function, the most important genes were C-terminal region of Nel-like molecule 1 protein (NELL1) and Pigment epithelium-derived factor (SPEDF), both genes were over-expressed. In the gene set of metabolism, insulin growth factor type 1 (IGF1), ApoA5 (apolipoprotein subtype 5), Foxo4 (Forkhead transcription factor 4), ADIPOR1 (receptor of adiponectin type 1) and AQP7 (aquaporin channel proteins7) were over expressed. Moreover, PIKFYVE (PtdIns(3) P 5-kinase), and ROCK-2 (rho-kinase II) were under expressed. We showed that PBMCs from obese subjects presented significant changes in gene expression, exhibiting 1436 differentially expressed genes compared to PBMCs from non-obese subjects. Furthermore, our data showed a number of genes involved in relevant processes implicated in metabolism, with genes presenting high fold-change values (up-regulation and down regulation) associated with lipid, carbohydrate and protein metabolism. Copyright © 2017 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

NAA TECHNIQUE FOR CLINICAL INVESTIGATION OF MICE IMMUNIZED WITH BOTHROP VENOM

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

Zamboni, C. B.; Aguiar, R. O.; Kovacs, L.

2009-06-03

In the present study Neutron Activation Analysis (NAA) technique was used to determine sodium concentration in whole blood of mice immunized with Bothrops venom. With this value it was possible to perform clinical investigation in this animal model using whole blood.

Microarray RNA expression analysis of cerebral white matter lesions reveals changes in multiple functional pathways.

PubMed

Simpson, Julie E; Hosny, Ola; Wharton, Stephen B; Heath, Paul R; Holden, Hazel; Fernando, Malee S; Matthews, Fiona; Forster, Gill; O'Brien, John T; Barber, Robert; Kalaria, Raj N; Brayne, Carol; Shaw, Pamela J; Lewis, Claire E; Ince, Paul G

2009-02-01

White matter lesions (WML) in brain aging are linked to dementia and depression. Ischemia contributes to their pathogenesis but other mechanisms may contribute. We used RNA microarray analysis with functional pathway grouping as an unbiased approach to investigate evidence for additional pathogenetic mechanisms. WML were identified by MRI and pathology in brains donated to the Medical Research Council Cognitive Function and Ageing Study Cognitive Function and Aging Study. RNA was extracted to compare WML with nonlesional white matter samples from cases with lesions (WM[L]), and from cases with no lesions (WM[C]) using RNA microarray and pathway analysis. Functional pathways were validated for selected genes by quantitative real-time polymerase chain reaction and immunocytochemistry. We identified 8 major pathways in which multiple genes showed altered RNA transcription (immune regulation, cell cycle, apoptosis, proteolysis, ion transport, cell structure, electron transport, metabolism) among 502 genes that were differentially expressed in WML compared to WM[C]. In WM[L], 409 genes were altered involving the same pathways. Genes selected to validate this microarray data all showed the expected changes in RNA levels and immunohistochemical expression of protein. WML represent areas with a complex molecular phenotype. From this and previous evidence, WML may arise through tissue ischemia but may also reflect the contribution of additional factors like blood-brain barrier dysfunction. Differential expression of genes in WM[L] compared to WM[C] indicate a "field effect" in the seemingly normal surrounding white matter.

Transcriptome-Wide Mega-Analyses Reveal Joint Dysregulation of Immunologic Genes and Transcription Regulators in Brain and Blood in Schizophrenia

PubMed Central

Hess, Jonathan L.; Tylee, Daniel S.; Barve, Rahul; de Jong, Simone; Ophoff, Roel A.; Kumarasinghe, Nishantha; Tooney, Paul; Schall, Ulrich; Gardiner, Erin; Beveridge, Natalie Jane; Scott, Rodney J.; Yasawardene, Surangi; Perera, Antionette; Mendis, Jayan; Carr, Vaughan; Kelly, Brian; Cairns, Murray; Tsuang, Ming T.; Glatt, Stephen J.

2016-01-01

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n = 315) and from ex-vivo blood tissues (n = 578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia. PMID:27450777

Transcriptome-wide mega-analyses reveal joint dysregulation of immunologic genes and transcription regulators in brain and blood in schizophrenia.

PubMed

Hess, Jonathan L; Tylee, Daniel S; Barve, Rahul; de Jong, Simone; Ophoff, Roel A; Kumarasinghe, Nishantha; Tooney, Paul; Schall, Ulrich; Gardiner, Erin; Beveridge, Natalie Jane; Scott, Rodney J; Yasawardene, Surangi; Perera, Antionette; Mendis, Jayan; Carr, Vaughan; Kelly, Brian; Cairns, Murray; Tsuang, Ming T; Glatt, Stephen J

2016-10-01

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n=315) and from ex-vivo blood tissues (n=578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia. Published by Elsevier B.V.

Depleted uranium analysis in blood by inductively coupled plasma mass spectrometry

USGS Publications Warehouse

Todorov, T.I.; Xu, H.; Ejnik, J.W.; Mullick, F.G.; Squibb, K.; McDiarmid, M.A.; Centeno, J.A.

2009-01-01

In this study we report depleted uranium (DU) analysis in whole blood samples. Internal exposure to DU causes increased uranium levels as well as change in the uranium isotopic composition in blood specimen. For identification of DU exposure we used the 235U/238U ratio in blood samples, which ranges from 0.00725 for natural uranium to 0.002 for depleted uranium. Uranium quantification and isotopic composition analysis were performed by inductively coupled plasma mass spectrometry. For method validation we used eight spiked blood samples with known uranium concentrations and isotopic composition. The detection limit for quantification was determined to be 4 ng L-1 uranium in whole blood. The data reproduced within 1-5% RSD and an accuracy of 1-4%. In order to achieve a 235U/238U ratio range of 0.00698-0.00752% with 99.7% confidence limit a minimum whole blood uranium concentration of 60 ng L??1 was required. An additional 10 samples from a cohort of veterans exposed to DU in Gulf War I were analyzed with no knowledge of their medical history. The measured 235U/ 238U ratios in the blood samples were used to identify the presence or absence of DU exposure within this patient group. ?? 2009 The Royal Society of Chemistry.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

PubMed Central

2013-01-01

Background Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis. All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort. Results Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples. Conclusions gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies. PMID:24168095

Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

NASA Technical Reports Server (NTRS)

Crucian, Brian E.; Sams, Clarence F.

2001-01-01

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent distinct monocyte subsets with unique functions. CONCLUSIONS: Whole blood culture eliminates the need to purify cell populations prior to culture and may have Significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. In this study, alterations in cytokine production are demonstrated between whole blood and PBMC activation. It is likely that whole blood activation more accurately represents the in-vivo immune balance than PBMC activation.

Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results.

PubMed

Dai, Yilin; Guo, Ling; Li, Meng; Chen, Yi-Bu

2012-06-08

Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

A Transcriptional Signature of Fatigue Derived from Patients with Primary Sjögren's Syndrome.

PubMed

James, Katherine; Al-Ali, Shereen; Tarn, Jessica; Cockell, Simon J; Gillespie, Colin S; Hindmarsh, Victoria; Locke, James; Mitchell, Sheryl; Lendrem, Dennis; Bowman, Simon; Price, Elizabeth; Pease, Colin T; Emery, Paul; Lanyon, Peter; Hunter, John A; Gupta, Monica; Bombardieri, Michele; Sutcliffe, Nurhan; Pitzalis, Costantino; McLaren, John; Cooper, Annie; Regan, Marian; Giles, Ian; Isenberg, David; Saravanan, Vadivelu; Coady, David; Dasgupta, Bhaskar; McHugh, Neil; Young-Min, Steven; Moots, Robert; Gendi, Nagui; Akil, Mohammed; Griffiths, Bridget; Wipat, Anil; Newton, Julia; Jones, David E; Isaacs, John; Hallinan, Jennifer; Ng, Wan-Fai

2015-01-01

Fatigue is a debilitating condition with a significant impact on patients' quality of life. Fatigue is frequently reported by patients suffering from primary Sjögren's Syndrome (pSS), a chronic autoimmune condition characterised by dryness of the eyes and the mouth. However, although fatigue is common in pSS, it does not manifest in all sufferers, providing an excellent model with which to explore the potential underpinning biological mechanisms. Whole blood samples from 133 fully-phenotyped pSS patients stratified for the presence of fatigue, collected by the UK primary Sjögren's Syndrome Registry, were used for whole genome microarray. The resulting data were analysed both on a gene by gene basis and using pre-defined groups of genes. Finally, gene set enrichment analysis (GSEA) was used as a feature selection technique for input into a support vector machine (SVM) classifier. Classification was assessed using area under curve (AUC) of receiver operator characteristic and standard error of Wilcoxon statistic, SE(W). Although no genes were individually found to be associated with fatigue, 19 metabolic pathways were enriched in the high fatigue patient group using GSEA. Analysis revealed that these enrichments arose from the presence of a subset of 55 genes. A radial kernel SVM classifier with this subset of genes as input displayed significantly improved performance over classifiers using all pathway genes as input. The classifiers had AUCs of 0.866 (SE(W) 0.002) and 0.525 (SE(W) 0.006), respectively. Systematic analysis of gene expression data from pSS patients discordant for fatigue identified 55 genes which are predictive of fatigue level using SVM classification. This list represents the first step in understanding the underlying pathophysiological mechanisms of fatigue in patients with pSS.

Transcriptome from circulating cells suggests dysregulated pathways associated with long-term recurrent events following first-time myocardial infarction.

PubMed

Suresh, Rahul; Li, Xing; Chiriac, Anca; Goel, Kashish; Terzic, Andre; Perez-Terzic, Carmen; Nelson, Timothy J

2014-09-01

Whole-genome gene expression analysis has been successfully utilized to diagnose, prognosticate, and identify potential therapeutic targets for high-risk cardiovascular diseases. However, the feasibility of this approach to identify outcome-related genes and dysregulated pathways following first-time myocardial infarction (AMI) remains unknown and may offer a novel strategy to detect affected expressome networks that predict long-term outcome. Whole-genome expression microarray on blood samples from normal cardiac function controls (n=21) and first-time AMI patients (n=31) within 48-hours post-MI revealed expected differential gene expression profiles enriched for inflammation and immune-response pathways. To determine molecular signatures at the time of AMI associated with long-term outcomes, transcriptional profiles from sub-groups of AMI patients with (n=5) or without (n=22) any recurrent events over an 18-month follow-up were compared. This analysis identified 559 differentially-expressed genes. Bioinformatic analysis of this differential gene-set for associated pathways revealed 1) increasing disease severity in AMI patients is associated with a decreased expression of genes involved in the developmental epithelial-to-mesenchymal transition pathway, and 2) modulation of cholesterol transport genes that include ABCA1, CETP, APOA1, and LDLR is associated with clinical outcome. Differentially regulated genes and modulated pathways were identified that were associated with recurrent cardiovascular outcomes in first-time AMI patients. This cell-based approach for risk stratification in AMI could represent a novel, non-invasive platform to anticipate modifiable pathways and therapeutic targets to optimize long-term outcome for AMI patients and warrants further study to determine the role of metabolic remodeling and regenerative processes required for optimal outcomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Time-series RNA-seq analysis package (TRAP) and its application to the analysis of rice, Oryza sativa L. ssp. Japonica, upon drought stress.

PubMed

Jo, Kyuri; Kwon, Hawk-Bin; Kim, Sun

2014-06-01

Measuring expression levels of genes at the whole genome level can be useful for many purposes, especially for revealing biological pathways underlying specific phenotype conditions. When gene expression is measured over a time period, we have opportunities to understand how organisms react to stress conditions over time. Thus many biologists routinely measure whole genome level gene expressions at multiple time points. However, there are several technical difficulties for analyzing such whole genome expression data. In addition, these days gene expression data is often measured by using RNA-sequencing rather than microarray technologies and then analysis of expression data is much more complicated since the analysis process should start with mapping short reads and produce differentially activated pathways and also possibly interactions among pathways. In addition, many useful tools for analyzing microarray gene expression data are not applicable for the RNA-seq data. Thus a comprehensive package for analyzing time series transcriptome data is much needed. In this article, we present a comprehensive package, Time-series RNA-seq Analysis Package (TRAP), integrating all necessary tasks such as mapping short reads, measuring gene expression levels, finding differentially expressed genes (DEGs), clustering and pathway analysis for time-series data in a single environment. In addition to implementing useful algorithms that are not available for RNA-seq data, we extended existing pathway analysis methods, ORA and SPIA, for time series analysis and estimates statistical values for combined dataset by an advanced metric. TRAP also produces visual summary of pathway interactions. Gene expression change labeling, a practical clustering method used in TRAP, enables more accurate interpretation of the data when combined with pathway analysis. We applied our methods on a real dataset for the analysis of rice (Oryza sativa L. Japonica nipponbare) upon drought stress. The result showed that TRAP was able to detect pathways more accurately than several existing methods. TRAP is available at http://biohealth.snu.ac.kr/software/TRAP/. Copyright © 2014 Elsevier Inc. All rights reserved.

Self-aliquoting micro-grooves in combination with laser ablation-ICP-mass spectrometry for the analysis of challenging liquids: quantification of lead in whole blood.

PubMed

Nischkauer, Winfried; Vanhaecke, Frank; Limbeck, Andreas

2016-08-01

We present a technique for the fast screening of the lead concentration in whole blood samples using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The whole blood sample is deposited on a polymeric surface and wiped across a set of micro-grooves previously engraved into the surface. The engraving of the micro-grooves was accomplished with the same laser system used for LA-ICP-MS analysis. In each groove, a part of the liquid blood is trapped, and thus, the sample is divided into sub-aliquots. These aliquots dry quasi instantly and are then investigated by means of LA-ICP-MS. For quantification, external calibration against aqueous standard solutions was relied on, with iron as an internal standard to account for varying volumes of the sample aliquots. The (208)Pb/(57)Fe nuclide ratio used for quantification was obtained via a data treatment protocol so far only used in the context of isotope ratio determination involving transient signals. The method presented here was shown to provide reliable results for Recipe ClinChek® Whole Blood Control levels I-III (nos. 8840-8842), with a repeatability of typically 3 % relative standard deviation (n = 6, for Pb at 442 μg L(-1)). Spiked and non-spiked real whole blood was analysed as well, and the results were compared with those obtained via dilution and sectorfield ICP-MS. A good agreement between both methods was observed. The detection limit (3 s) for lead in whole blood was established to be 10 μg L(-1) for the laser ablation method presented here. Graphical Abstract Micro-grooves are filled with whole blood, dried, and analyzed by laser ablation ICP-mass spectrometry. Notice that the laser moves in perpendicular direction with regard to the micro-grooves.

A novel approach using metabolomics coupled with hematological and biochemical parameters to explain the enriching-blood effect and mechanism of unprocessed Angelica sinensis and its 4 kinds of processed products.

PubMed

Ji, Peng; Wei, Yanming; Hua, Yongli; Zhang, Xiaosong; Yao, Wanling; Ma, Qi; Yuan, Ziwen; Wen, Yanqiao; Yang, Chaoxue

2018-01-30

Angelica sinensis (AS), root of Angelica sinensis (Oliv.) Diels, an important kind of Chinese traditional herbal medicine, has been used for women to enrich the blood for thousands of years. It is mainly distributed in Gansu province of China. According to Traditional Chinese medicine usage, unprocessed AS (UAS) and its 4 kinds of processed products (ASs) are all used to treat different diseases or syndromes. The difference among the enriching-blood effects of ASs is unclear. And their exact mechanisms of enriching the blood are not fully understood. In this study, our aim is to compare the enriching-blood effect and explain the related mechanism of ASs, to lay the foundation for the blood deficiency diagnosis and the rational use of ASs in the clinic. ASs were used to intervene the blood deficiency syndrome model mice induced by acetyl phenylhydrazine (APH) and cyclophosphamide (CTX). A novel approach using metabolomics coupled with hematological and biochemical parameters to explain the enriching-blood effect and mechanism of ASs was established. The blood routine examination, ATPase, glucose-6-phosphate dehydrogenase, methemoglobin, glutathion peroxidase, glutathione reductase, and erythropoietin were measured. Two biofluids (plasma and urine) obtained from mice were analyzed with GC-MS. Distinct changes in metabolite patterns of the two biofluids after mice were induced by APH and CTX, and mice were intervened with ASs were analyzed using partial least squares-discriminant analysis. Potential biomarkers were found using a novel method including variable importance in the projection (VIP) >1.0, volcano plot analysis, and significance analysis of microarray. The results of hematological, biochemical parameters and the integrated metabolomics all showed the blood deficiency syndrome model was built successfully, ASs exhibited different degree of enriching-blood effect, and AS pached with alcohol (AAS) exhibited the best enriching-blood effect. 16 metabolites in the plasma and 8 metabolites in the urine were considered as the potential biomarkers. These metabolites were involved in 7 metabolic pathways which were concerned with the different enriching-blood effect mechanisms of ASs. The correlation analysis results confirmed L-Valine (plasma), Linoleic acid (urine), L-Aspartic acid (urine) and Cholesterol (urine) were strong positive or negative associated with biochemical indicators. The enriching-blood effects of ASs are different. The pathological mechanisms of blood deficiency syndrome and the enriching-blood effect mechanism of ASs are involved in 7 metabolic pathways. L-Valine (plasma), Linoleic acid (urine), L-Aspartic acid (urine), Cholesterol (urine) are four important biomarkers being related to the enriching-blood effect of ASs. The combination of VIP, volcano plot analysis and significance analysis of microarray is suitable for screening biomarkers in metabolomics study. They can lay the foundation for clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of genetic variability of Venezuelan equine encephalitis viruses

DOE PAGES

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.; ...

2016-04-07

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Oral streptococci utilize a Siglec-like domain of serine-rich repeat adhesins to preferentially target platelet sialoglycans in human blood.

PubMed

Deng, Lingquan; Bensing, Barbara A; Thamadilok, Supaporn; Yu, Hai; Lau, Kam; Chen, Xi; Ruhl, Stefan; Sullam, Paul M; Varki, Ajit

2014-12-01

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.

Applications of nanotechnology, next generation sequencing and microarrays in biomedical research.

PubMed

Elingaramil, Sauli; Li, Xiaolong; He, Nongyue

2013-07-01

Next-generation sequencing technologies, microarrays and advances in bio nanotechnology have had an enormous impact on research within a short time frame. This impact appears certain to increase further as many biomedical institutions are now acquiring these prevailing new technologies. Beyond conventional sampling of genome content, wide-ranging applications are rapidly evolving for next-generation sequencing, microarrays and nanotechnology. To date, these technologies have been applied in a variety of contexts, including whole-genome sequencing, targeted re sequencing and discovery of transcription factor binding sites, noncoding RNA expression profiling and molecular diagnostics. This paper thus discusses current applications of nanotechnology, next-generation sequencing technologies and microarrays in biomedical research and highlights the transforming potential these technologies offer.

Absorption spectroscopy setup for determination of whole human blood and blood-derived materials spectral characteristics

NASA Astrophysics Data System (ADS)

Wróbel, M. S.; Gnyba, M.; Milewska, D.; Mitura, K.; Karpienko, K.

2015-09-01

A dedicated absorption spectroscopy system was set up using tungsten-halogen broadband source, optical fibers, sample holder, and a commercial spectrometer with CCD array. Analysis of noise present in the setup was carried out. Data processing was applied to the absorption spectra to reduce spectral noise, and improve the quality of the spectra and to remove the baseline level. The absorption spectra were measured for whole blood samples, separated components: plasma, saline, washed erythrocytes in saline and human whole blood with biomarkers - biocompatible nanodiamonds (ND). Blood samples had been derived from a number of healthy donors. The results prove a correct setup arrangement, with adequate preprocessing of the data. The results of blood-ND mixtures measurements show no toxic effect on blood cells, which proves the NDs as a potential biocompatible biomarkers.

FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny

PubMed Central

Patel, Isha R.; Gangiredla, Jayanthi; Lacher, David W.; Mammel, Mark K.; Jackson, Scott A.; Lampel, Keith A.

2016-01-01

ABSTRACT Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli. IMPORTANCE This study describes a robust, state-of-the-art platform developed from available whole-genome sequences of E. coli and Shigella spp. by distilling useful signatures for epidemiology and molecular risk assessment into one assay. The FDA-ECID microarray contains features that enable comprehensive molecular serotyping and virulence profiling along with genome-scale genotyping and SNP analysis. Hence, it is a molecular toolbox that stratifies strain identification and pathogenic potential in the contexts of epidemiology and phylogeny. We applied this tool to strains from food, environmental, and clinical sources, resulting in significantly greater phylogenetic and strain-specific resolution than previously reported for available typing methods. PMID:27037122

DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

PubMed Central

Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

2015-01-01

Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

Donor vigilance data of a blood transfusion service: A multicenter analysis.

PubMed

Burkhardt, T; Dimanski, B; Karl, R; Sievert, U; Karl, A; Hübler, C; Tonn, T; Sopvinik, I; Ertl, H; Moog, R

2015-10-01

Donor vigilance is an important part of the quality management system of blood transfusion services. The evaluation of donor side effects helps to improve the donation process and donor compliance. The aim of the present study was to evaluate donor vigilance data in whole blood and plasmapheresis donors of a blood donor service. Donors fulfilling current national and European eligibility criteria underwent whole blood and plasmapheresis donation (PCS and MCS+ (Haemonetics, Braintree, USA), A 200 (Fenwal, Round Lake, USA). Whole blood was collected at fixed and mobile sites while plasmaphereses were performed at 8 plasma centers. From 2011 to 2013 donor information was provided for gender, age, body weight, height, first and repeat donation. Donors were monitored for venipuncture and circulatory associated side effects. The total incidences of adverse events were 5004 (0.56%) in repeat donors and 2111 (2.78%) in first time donors for whole blood donation and 3323 (1.01%) and 514 (7.96%) for plasmaphereses, respectively. Circulatory associated events were 2679 (0.30%) for whole blood donation and 1624 (0.49%) for plasmaphereses. Our donor vigilance data of a blood transfusion service show that whole blood and plasmapheresis are safe with low incidences of adverse events. Repeat donation and age are predictors for low rates of adverse events. On the other hand, first time donation and female gender were associated with higher incidences of adverse events. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chromosomal microarray analysis as the first-tier test for the identification of pathogenic copy number variants in chromosome 9 pericentric regions and its challenge.

PubMed

Wang, Jia-Chi; Boyar, Fatih Z

2016-01-01

Chromosomal microarray analysis (CMA) has been recommended and practiced routinely in the large reference laboratories of U.S.A. as the first-tier test for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Using CMA as a diagnostic tool and without a routine setting of fluorescence in situ hybridization with labeled bacterial artificial chromosome probes (BAC-FISH) in the large reference laboratories becomes a challenge in the characterization of chromosome 9 pericentric region. This region has a very complex genomic structure and contains a variety of heterochromatic and euchromatic polymorphic variants. These variants were usually studied by G-banding, C-banding and BAC-FISH analysis. Chromosomal microarray analysis (CMA) was not recommended since it may lead to false positive results. Here, we presented a cohort of four cases, in which high-resolution CMA was used as the first-tier test or simultaneously with G-banding analysis on the proband to identify pathogenic copy number variants (CNVs) in the whole genome. CMA revealed large pathogenic CNVs from chromosome 9 in 3 cases which also revealed different G-banding patterns between the two chromosome 9 homologues. Although we demonstrated that high-resolution CMA played an important role in the identification of pathogenic copy number variants in chromosome 9 pericentric regions, the lack of BAC-FISH analysis or other useful tools renders significant challenges in the characterization of chromosome 9 pericentric regions. None; it is not a clinical trial, and the cases were retrospectively collected and analyzed.

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry.

PubMed

Vogeser, Michael; Spöhrer, Ute

2006-01-01

Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 microg/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods.

Motivational differences between whole blood and plasma donors already exist before their first donation experience.

PubMed

Veldhuizen, Ingrid; van Dongen, Anne

2013-08-01

The demand for plasma products has increased rapidly. It is therefore important to understand donating behavior by plasma donors. This study investigates whether motivational differences between whole blood and plasma donors already exist at the beginning of a donor career. New donors (n = 4861) were invited to fill out a questionnaire before their first donation (response, 61%). The questionnaire assessed variables from the Theory of Planned Behavior (intention, self-efficacy, attitude, and norms), conscientiousness, and donation anxiety. Three years later it was determined who became whole blood or plasma donor. Multivariable linear regression analyses for intention were fitted separately for whole blood and plasma donors. A logistic regression analysis was executed to estimate the effect of intention at the beginning of a donor career on becoming a plasma donor. Plasma donors had a higher intention, self-efficacy, attitude, and conscientiousness and a lower anxiety than whole blood donors. In plasma and whole blood donors, both self-efficacy and cognitive attitude were positively related to intention but with different strength (plasma, β = 0.47 and β = 0.30; whole blood, β = 0.57 and β = 0.17). Having a high level of intention increased the odds of becoming a plasma donor (odds ratio, 1.33; 95% confidence interval, 1.12-1.59). Motivational differences already exist between future whole blood and plasma donors before their first donation. Although a feeling of self-efficacy is necessary for all new donors, more favorable cognitions are important for future plasma donors. Recruitment strategies for plasma donors should focus on attracting the more self-confident donors by highlighting the usefulness of plasma donation. © 2012 American Association of Blood Banks.

Preliminary profiling of blood transcriptome in a rat model of hemorrhagic shock

PubMed Central

Braga, D; Barcella, M; D’Avila, F; Lupoli, S; Tagliaferri, F; Santamaria, MH; DeLano, FA; Baselli, G; Schmid-Schönbein, GW; Kistler, EB; Aletti, F

2017-01-01

Hemorrhagic shock is a leading cause of morbidity and mortality worldwide. Significant blood loss may lead to decreased blood pressure and inadequate tissue perfusion with resultant organ failure and death, even after replacement of lost blood volume. One reason for this high acuity is that the fundamental mechanisms of shock are poorly understood. Proteomic and metabolomic approaches have been used to investigate the molecular events occurring in hemorrhagic shock but, to our knowledge, a systematic analysis of the transcriptomic profile is missing. Therefore, a pilot analysis using paired-end RNA sequencing was used to identify changes that occur in the blood transcriptome of rats subjected to hemorrhagic shock after blood reinfusion. Hemorrhagic shock was induced using a Wigger’s shock model. The transcriptome of whole blood from shocked animals shows modulation of genes related to inflammation and immune response (Tlr13, Il1b, Ccl6, Lgals3), antioxidant functions (Mt2A, Mt1), tissue injury and repair pathways (Gpnmb, Trim72) and lipid mediators (Alox5ap, Ltb4r, Ptger2) compared with control animals. These findings are congruent with results obtained in hemorrhagic shock analysis by other authors using metabolomics and proteomics. The analysis of blood transcriptome may be a valuable tool to understand the biological changes occurring in hemorrhagic shock and a promising approach for the identification of novel biomarkers and therapeutic targets. Impact statement This study provides the first pilot analysis of the changes occurring in transcriptome expression of whole blood in hemorrhagic shock (HS) rats. We showed that the analysis of blood transcriptome is a useful approach to investigate pathways and functional alterations in this disease condition. This pilot study encourages the possible application of transcriptome analysis in the clinical setting, for the molecular profiling of whole blood in HS patients. PMID:28661205

Placental gene-expression profiles of intrahepatic cholestasis of pregnancy reveal involvement of multiple molecular pathways in blood vessel formation and inflammation.

PubMed

Du, QiaoLing; Pan, YouDong; Zhang, YouHua; Zhang, HaiLong; Zheng, YaJuan; Lu, Ling; Wang, JunLei; Duan, Tao; Chen, JianFeng

2014-07-07

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-associated liver disease with potentially deleterious consequences for the fetus, particularly when maternal serum bile-acid concentration >40 μM. However, the etiology and pathogenesis of ICP remain elusive. To reveal the underlying molecular mechanisms for the association of maternal serum bile-acid level and fetal outcome in ICP patients, DNA microarray was applied to characterize the whole-genome expression profiles of placentas from healthy women and women diagnosed with ICP. Thirty pregnant women recruited in this study were categorized evenly into three groups: healthy group; mild ICP, with serum bile-acid concentration ranging from 10-40 μM; and severe ICP, with bile-acid concentration >40 μM. Gene Ontology analysis in combination with construction of gene-interaction and gene co-expression networks were applied to identify the core regulatory genes associated with ICP pathogenesis, which were further validated by quantitative real-time PCR and histological staining. The core regulatory genes were mainly involved in immune response, VEGF signaling pathway and G-protein-coupled receptor signaling, implying essential roles of immune response, vasculogenesis and angiogenesis in ICP pathogenesis. This implication was supported by the observed aggregated immune-cell infiltration and deficient blood vessel formation in ICP placentas. Our study provides a system-level insight into the placental gene-expression profiles of women with mild or severe ICP, and reveals multiple molecular pathways in immune response and blood vessel formation that might contribute to ICP pathogenesis.

Passing the anaerobic threshold is associated with substantial changes in the gene expression profile in white blood cells.

PubMed

Sakharov, Dmitry A; Maltseva, Diana V; Riabenko, Evgeniy A; Shkurnikov, Maxim U; Northoff, Hinnak; Tonevitsky, Alexander G; Grigoriev, Anatoly I

2012-03-01

High and moderate intensity endurance exercise alters gene expression in human white blood cells (WBCs), but the understanding of how this effect occurs is limited. To increase our knowledge of the nature of this process, we investigated the effects of passing the anaerobic threshold (AnT) on the gene expression profile in WBCs of athletes. Nineteen highly trained skiers participated in a treadmill test with an incremental step protocol until exhaustion (ramp test to exhaustion, RTE). The average total time to exhaustion was 14:40 min and time after AnT was 4:50 min. Two weeks later, seven of these skiers participated in a moderate treadmill test (MT) at 80% peak O(2) uptake for 30 min, which was slightly below their AnTs. Blood samples were obtained before and immediately after both tests. RTE was associated with substantially greater leukocytosis and acidosis than MT. Gene expression in WBCs was measured using whole genome microarray expression analysis before and immediately after each test. A total of 310 upregulated genes were found after RTE, and 69 genes after MT of which 64 were identical to RTE. Both tests influenced a variety of known gene pathways related to inflammation, stress response, signal transduction and apoptosis. A large group of differentially expressed previously unknown small nucleolar RNA and small Cajal body RNA was found. In conclusion, a 15-min test to exhaustion was associated with substantially greater changes of gene expression than a 30-min test just below the AnT.

An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

PubMed Central

Li, Siyang; Plouffe, Brian D.; Belov, Arseniy M.; Ray, Somak; Wang, Xianzhe; Murthy, Shashi K.; Karger, Barry L.; Ivanov, Alexander R.

2015-01-01

Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. PMID:25755294

Whole-body vibration and blood flow and muscle oxygenation: a meta-analysis.

PubMed

Games, Kenneth E; Sefton, JoEllen M; Wilson, Alan E

2015-05-01

The use and popularity of whole-body vibration (WBV) has increased in recent years, but there is a lack of consensus in the literature about the effectiveness of the treatment. To quantitatively examine the effects of WBV on muscle oxygenation and peripheral blood flow in healthy adults. We searched Web of Science and PubMed databases and reference lists from relevant articles using the key terms whole body vibration, whole-body vibration, WBV, blood flow, peripheral blood flow, oxygenation, muscle oxygenation, circulation, circulatory, near infrared spectroscopy, NIRS, and power Doppler. Key terms were searched using single word and combination searches. No date range was specified. Criteria for inclusion were (1) use of a commercially available WBV device, (2) a human research model, (3) a pre-WBV condition and at least 1 WBV experimental condition, and (4) reporting of unstandardized means and standard deviations of muscle oxygenation or peripheral blood flow. Means, standard deviations, and sample sizes were extracted from the text, tables, and figures of included studies. A total of 35 and 90 data points were extracted for the muscle-oxygenation and blood-flow meta-analyses, respectively. Data for each meta-analysis were combined and analyzed using meta-analysis software. Weighted, random-effects meta-analyses using the Hedges g metric were completed for muscle oxygenation and blood flow. We then conducted follow-up analyses using the moderator variables of vibration type, vibration time, vibration frequency, measurement location, and sample type. We found 18 potential articles. Further examination yielded 10 studies meeting the inclusion criteria. Whole-body vibration was shown to positively influence peripheral blood flow. Additionally, the moderators of vibration type and frequency altered the influence of WBV on blood flow. Overall, WBV did not alter muscle oxygenation; however, when the measurement site was considered, muscle oxygenation increased or decreased depending on the location. Acute bouts of WBV increase peripheral blood flow but do not alter skeletal muscle oxygenation. Vibration type appears to be the most important factor influencing both muscle oxygenation and peripheral blood flow.

Whole-Body Vibration and Blood Flow and Muscle Oxygenation: A Meta-Analysis

PubMed Central

Games, Kenneth E.; Sefton, JoEllen M.; Wilson, Alan E.

2015-01-01

Context: The use and popularity of whole-body vibration (WBV) has increased in recent years, but there is a lack of consensus in the literature about the effectiveness of the treatment. Objective: To quantitatively examine the effects of WBV on muscle oxygenation and peripheral blood flow in healthy adults. Data Sources: We searched Web of Science and PubMed databases and reference lists from relevant articles using the key terms whole body vibration, whole-body vibration, WBV, blood flow, peripheral blood flow, oxygenation, muscle oxygenation, circulation, circulatory, near infrared spectroscopy, NIRS, and power Doppler. Key terms were searched using single word and combination searches. No date range was specified. Study Selection: Criteria for inclusion were (1) use of a commercially available WBV device, (2) a human research model, (3) a pre-WBV condition and at least 1 WBV experimental condition, and (4) reporting of unstandardized means and standard deviations of muscle oxygenation or peripheral blood flow. Data Extraction: Means, standard deviations, and sample sizes were extracted from the text, tables, and figures of included studies. A total of 35 and 90 data points were extracted for the muscle-oxygenation and blood-flow meta-analyses, respectively. Data for each meta-analysis were combined and analyzed using meta-analysis software. Weighted, random-effects meta-analyses using the Hedges g metric were completed for muscle oxygenation and blood flow. We then conducted follow-up analyses using the moderator variables of vibration type, vibration time, vibration frequency, measurement location, and sample type. Data Synthesis: We found 18 potential articles. Further examination yielded 10 studies meeting the inclusion criteria. Whole-body vibration was shown to positively influence peripheral blood flow. Additionally, the moderators of vibration type and frequency altered the influence of WBV on blood flow. Overall, WBV did not alter muscle oxygenation; however, when the measurement site was considered, muscle oxygenation increased or decreased depending on the location. Conclusions: Acute bouts of WBV increase peripheral blood flow but do not alter skeletal muscle oxygenation. Vibration type appears to be the most important factor influencing both muscle oxygenation and peripheral blood flow. PMID:25974682

Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

PubMed

Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

2018-02-01

Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

Whole-exome analysis of foetal autopsy tissue reveals a frameshift mutation in OBSL1, consistent with a diagnosis of 3-M Syndrome.

PubMed

Marshall, Christian R; Farrell, Sandra A; Cushing, Donna; Paton, Tara; Stockley, Tracy L; Stavropoulos, Dimitri J; Ray, Peter N; Szego, Michael; Lau, Lynette; Pereira, Sergio L; Cohn, Ronald D; Wintle, Richard F; Abuzenadah, Adel M; Abu-Elmagd, Muhammad; Scherer, Stephen W

2015-01-01

We report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants. Whole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings. Our study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a recurrent set of clinical symptoms.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

PubMed

Darias, M J; Zambonino-Infante, J L; Hugot, K; Cahu, C L; Mazurais, D

2008-01-01

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

Oligonucleotide microarray analysis of gene expression profiles followed by real-time reverse-transcriptase polymerase chain reaction assay in chronic active Epstein-Barr virus infection.

PubMed

Ito, Yoshinori; Shibata-Watanabe, Yukiko; Ushijima, Yoko; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kojima, Seiji; Kimura, Hiroshi

2008-03-01

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by recurrent infectious mononucleosis-like symptoms and has high mortality and morbidity. To clarify the mechanisms of CAEBV, the gene-expression profiles of peripheral blood obtained from patients with CAEBV were investigated. Twenty genes were differentially expressed in 4 patients with CAEBV. This microarray result was verified using a real-time reverse-transcriptase polymerase chain reaction assay in a larger group of patients with CAEBV. Eventually, 3 genes were found to be significantly upregulated: guanylate binding protein 1, tumor necrosis factor-induced protein 6, and guanylate binding protein 5. These genes may be associated with the inflammatory reaction or with cell proliferation.

Examining Radiation-Induced In Vivo and In Vitro Gene Expression Changes of the Peripheral Blood in Different Laboratories for Biodosimetry Purposes: First RENEB Gene Expression Study.

PubMed

Abend, M; Badie, C; Quintens, R; Kriehuber, R; Manning, G; Macaeva, E; Njima, M; Oskamp, D; Strunz, S; Moertl, S; Doucha-Senf, S; Dahlke, S; Menzel, J; Port, M

2016-02-01

The risk of a large-scale event leading to acute radiation exposure necessitates the development of high-throughput methods for providing rapid individual dose estimates. Our work addresses three goals, which align with the directive of the European Union's Realizing the European Network of Biodosimetry project (EU-RENB): 1. To examine the suitability of different gene expression platforms for biodosimetry purposes; 2. To perform this examination using blood samples collected from prostate cancer patients (in vivo) and from healthy donors (in vitro); and 3. To compare radiation-induced gene expression changes of the in vivo with in vitro blood samples. For the in vitro part of this study, EDTA-treated whole blood was irradiated immediately after venipuncture using single X-ray doses (1 Gy/min(-1) dose rate, 100 keV). Blood samples used to generate calibration curves as well as 10 coded (blinded) samples (0-4 Gy dose range) were incubated for 24 h in vitro, lysed and shipped on wet ice. For the in vivo part of the study PAXgene tubes were used and peripheral blood (2.5 ml) was collected from prostate cancer patients before and 24 h after the first fractionated 2 Gy dose of localized radiotherapy to the pelvis [linear accelerator (LINAC), 580 MU/min, exposure 1-1.5 min]. Assays were run in each laboratory according to locally established protocols using either microarray platforms (2 laboratories) or qRT-PCR (2 laboratories). Report times on dose estimates were documented. The mean absolute difference of estimated doses relative to the true doses (Gy) were calculated. Doses were also merged into binary categories reflecting aspects of clinical/diagnostic relevance. For the in vitro part of the study, the earliest report time on dose estimates was 7 h for qRT-PCR and 35 h for microarrays. Methodological variance of gene expression measurements (CV ≤10% for technical replicates) and interindividual variance (≤twofold for all genes) were low. Dose estimates based on one gene, ferredoxin reductase (FDXR), using qRT-PCR were as precise as dose estimates based on multiple genes using microarrays, but the precision decreased at doses ≥2 Gy. Binary dose categories comprising, for example, unexposed compared with exposed samples, could be completely discriminated with most of our methods. Exposed prostate cancer blood samples (n = 4) could be completely discriminated from unexposed blood samples (n = 4, P < 0.03, two-sided Fisher's exact test) without individual controls. This could be performed by introducing an in vitro-to-in vivo correction factor of FDXR, which varied among the laboratories. After that the in vitro-constructed calibration curves could be used for dose estimation of the in vivo exposed prostate cancer blood samples within an accuracy window of ±0.5 Gy in both contributing qRT-PCR laboratories. In conclusion, early and precise dose estimates can be performed, in particular at doses ≤2 Gy in vitro. Blood samples of prostate cancer patients exposed to 0.09-0.017 Gy could be completely discriminated from pre-exposure blood samples with the doses successfully estimated using adjusted in vitro-constructed calibration curves.

Discrimination of lymphoma using laser-induced breakdown spectroscopy conducted on whole blood samples

PubMed Central

Chen, Xue; Li, Xiaohui; Yang, Sibo; Yu, Xin; Liu, Aichun

2018-01-01

Lymphoma is a significant cancer that affects the human lymphatic and hematopoietic systems. In this work, discrimination of lymphoma using laser-induced breakdown spectroscopy (LIBS) conducted on whole blood samples is presented. The whole blood samples collected from lymphoma patients and healthy controls are deposited onto standard quantitative filter papers and ablated with a 1064 nm Q-switched Nd:YAG laser. 16 atomic and ionic emission lines of calcium (Ca), iron (Fe), magnesium (Mg), potassium (K) and sodium (Na) are selected to discriminate the cancer disease. Chemometric methods, including principal component analysis (PCA), linear discriminant analysis (LDA) classification, and k nearest neighbor (kNN) classification are used to build the discrimination models. Both LDA and kNN models have achieved very good discrimination performances for lymphoma, with an accuracy of over 99.7%, a sensitivity of over 0.996, and a specificity of over 0.997. These results demonstrate that the whole-blood-based LIBS technique in combination with chemometric methods can serve as a fast, less invasive, and accurate method for detection and discrimination of human malignancies. PMID:29541503

Comparison of three chemometrics methods for near-infrared spectra of glucose in the whole blood

NASA Astrophysics Data System (ADS)

Zhang, Hongyan; Ding, Dong; Li, Xin; Chen, Yu; Tang, Yuguo

2005-01-01

Principal Component Regression (PCR), Partial Least Square (PLS) and Artificial Neural Networks (ANN) methods are used in the analysis for the near infrared (NIR) spectra of glucose in the whole blood. The calibration model is built up in the spectrum band where there are the glucose has much more spectral absorption than the water, fat, and protein with these methods and the correlation coefficients of the model are showed in this paper. Comparing these results, a suitable method to analyze the glucose NIR spectrum in the whole blood is found.

Differential blood-based diagnosis between benign prostatic hyperplasia and prostate cancer: miRNA as source for biomarkers independent of PSA level, Gleason score, or TNM status.

PubMed

Leidinger, Petra; Hart, Martin; Backes, Christina; Rheinheimer, Stefanie; Keck, Bastian; Wullich, Bernd; Keller, Andreas; Meese, Eckart

2016-08-01

Since the benefit of prostate-specific antigen (PSA) screening remains controversial, new non-invasive biomarkers for prostate carcinoma (PCa) are still required. There is evidence that microRNAs (miRNAs) in whole peripheral blood can separate patients with localized prostate cancer from healthy individuals. However, the potential of blood-based miRNAs for the differential diagnosis of PCa and benign prostatic hyperplasia (BPH) has not been tested. We compared the miRNome from blood of PCa and BPH patients and further investigated the influence of the tumor volume, tumor-node-metastasis (TNM) classification, Gleason score, pretreatment risk status, and the pretreatment PSA value on the miRNA pattern. By microarray approach, we identified seven miRNAs that were significantly deregulated in PCa patients compared to BPH patients. Using quantitative real time PCR (qRT-PCR), we confirmed downregulation of hsa-miR-221* (now hsa-miR-221-5p) and hsa-miR-708* (now hsa-miR-708-3p) in PCa compared to BPH. Clinical parameters like PSA level, Gleason score, or TNM status seem to have only limited impact on the overall abundance of miRNAs in patients' blood, suggesting a no influence of these factors on the expression of deregulated miRNAs.

HEMORHEOLOGY INDEX CHANGES IN A RAT ACUTE BLOOD STASIS MODEL: A SYSTEMATIC REVIEW AND META-ANALYSIS

PubMed Central

Zhang, Jun-Xiu; Feng, Yu; Zhang, Yin; Liu, Yi; Li, Shao-Dan; Yang, Ming-Hui

2017-01-01

Background: Blood stasis has received increasing attention in research related to traditional Chinese medicine (TCM) and integrative Chinese and Western medicine. More than 90% of research studies use hemorheology indexes to evaluate the establishment of animal blood stasis models rather than pathological methods, as hemorheology index evaluations of blood stasis were short of the consolidated standard. The aim of this study was to evaluate the accuracy of hemorheology indexes in rat models of acute blood stasis (ABS) based on studies in which the ABS model had been confirmed by pathological methods. Materials and Methods: We searched the Chinese National Knowledge Infrastructure database (CNKI), Chinese Medical Journal Database (CMJD), Chinese Biology Medicine disc (CBM), Wanfang database, and PubMed for studies of rat blood stasis models; the search identified 18 studies of rat ABS models induced by subcutaneous injection of epinephrine combined with an ice bath. Each included study received a modified Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) score list and methodological quality assessment, then data related to whole blood viscosity, plasma viscosity, platelet aggregation rate, erythrocyte aggregation index, and fibrinogen concentration were extracted. Extracted data were analyzed using Revman 5.3; heterogeneity was tested using Egger’s test. Results: A total of 343 studies of rat blood stasis were reviewed. Eighteen studies were included in this meta-analysis; the mean CAMARADES score was 3.5. The rat ABS model revealed a significant increase in whole blood viscosity (medium shear rate), whole blood viscosity (high shear rate), plasma viscosity, platelet aggregation rate, erythrocyte aggregation index, and fibrinogen concentration compared to controls, with weighted mean differences (WMD) of 2.42 mPa/s (95% confidence interval (CI) = 1.73 - 3.10); 1.76 mPa/s (95% CI = 1.28 - 2.24); 0.39 mPa/s (95% CI = 0.24 - 0.55); 13.66% (95% CI = 9.78 - 17.55); 0.84 (95% CI = 0.53 - 1.16); and 1.22 g/L (95% CI = 0.76 - 1.67), respectively. Subgroup analysis showed that whole blood viscosity, plasma viscosity, and the platelet aggregation rate test methods were more sensitive when measured at 0-24 h than at 24-72 h after induction of blood stasis. Conclusions: Rat blood stasis studies have incomplete experimental design and quality controls, and thus need an integrated improvement. Meta-analysis of included studies indicated that the unified hemorheology index of whole blood viscosity (medium and high shear rate), platelet aggregation rate, erythrocyte aggregation rate, and fibrinogen concentration might be used for assessment of rat ABS models independent of pathology methods. PMID:28638872

Assessment and Comparison of Vitreous Humor as an Alternative Matrix for Forensic Toxicology Screening by GC-MS.

PubMed

Metushi, Imir G; Fitzgerald, Robert L; McIntyre, Iain M

2016-05-01

Alternative specimens have been occasionally considered as substitutes for whole blood for postmortem toxicology testing. We studied the applicability of vitreous humor, and evaluated whether it would be suitable to replace (or augment) whole blood for routine drug screening. Results showed that from 51 autopsy cases, we were able to identify an aggregate of 209 findings in whole blood compared with 169 in vitreous. The total number of compounds identified was 71 for whole blood and 60 for vitreous humor. Quantitative analysis showed that whole-blood concentrations of trazodone were several fold higher than vitreous humor concentrations (1.42 ± 0.57 vs. 0.15 ± 0.05 mg/L, respectively) and similar results were also obtained for diazepam (0.37 ± 0.06 vs. 0.13 ± 0.01, respectively). For other drugs such as oxycodone, hydrocodone and doxylamine, a trend suggesting higher concentrations in vitreous humor vs. whole blood was observed; however, this was not significant. Our results are consistent with the limited work of other investigators, and suggest that vitreous humor could be an appropriate matrix for drug screening in postmortem toxicology. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

S100A9+ MDSC and TAM-mediated EGFR-TKI resistance in lung adenocarcinoma: the role of RELB.

PubMed

Feng, Po-Hao; Yu, Chih-Teng; Chen, Kuan-Yuan; Luo, Ching-Shan; Wu, Shen Ming; Liu, Chien-Ying; Kuo, Lu Wei; Chan, Yao-Fei; Chen, Tzu-Tao; Chang, Chih-Cheng; Lee, Chun-Nin; Chuang, Hsiao-Chi; Lin, Chiou-Feng; Han, Chia-Li; Lee, Wei-Hwa; Lee, Kang-Yun

2018-01-26

Monocytic myeloid-derived suppressor cells (MDSCs), particularly the S100A9+ subset, has been shown initial clinical relevance. However, its role in EGFR-mutated lung adenocarcinoma, especially to EGFR-tyrosine kinase inhibitor (EGFR-TKI) is not clear. In a clinical setting of EGFR mutated lung adenocarcinoma, a role of the MDSC apart from T cell suppression was also investigated. Blood monocytic S100A9 + MDSC counts were higher in lung cancer patients than healthy donors, and were associated with poor treatment response and shorter progression-free survival (PFS). S100A9 + MDSCs in PBMC were well correlated to tumor infiltrating CD68 + and S100A9 + cells, suggesting an origin of TAMs. Patient's MDMs, mostly from S100A9 + MDSC, similar to primary alveolar macrophages from patients, both expressed S100A9 and CD206, attenuated EGFR-TKI cytotoxicity. Microarray analysis identified up-regulation of the RELB signaling genes, confirmed by Western blotting and functionally by RELB knockdown. In conclusion, blood S100A9 + MDSC is a predictor of poor treatment response to EGFR-TKI, possibly via its derived TAMs through activation of the non-canonical NF-κB RELB pathway. Patients with activating EGFR mutation lung adenocarcinoma receiving first line EGFR TKIs were prospectively enrolled. Peripheral blood mononuclear cells (PBMCs) were collected for MDSCs analysis and for monocyte-derived macrophages (MDMs) and stored tissue for TAM analysis by IHC. A transwell co-culture system of MDMs/macrophages and H827 cells was used to detect the effect of macrophages on H827 and microarray analysis to explore the underlying molecular mechanisms, functionally confirmed by RNA interference.

Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

PubMed Central

Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

2013-01-01

Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

21 CFR 640.1 - Whole Blood.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for transfusion... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs...

21 CFR 640.1 - Whole Blood.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for transfusion...

21 CFR 640.1 - Whole Blood.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for transfusion...

21 CFR 640.1 - Whole Blood.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for transfusion...

21 CFR 640.1 - Whole Blood.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Whole Blood. 640.1 Section 640.1 Food and Drugs... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Whole Blood § 640.1 Whole Blood. The proper name of this product shall be Whole Blood. Whole Blood is defined as blood collected from human donors for transfusion...

Design of a new therapy for patients with chronic kidney disease: use of microarrays for selective hemoadsorption of uremic wastes and toxins to improve homeostasis.

PubMed

Shahidi Bonjar, Mohammad Rashid; Shahidi Bonjar, Leyla

2015-01-01

The hypothesis proposed here would provide near to optimum homeostasis for patients with chronic kidney disease (CKD) without the need for hemodialysis. This strategy has not been described previously in the scientific literature. It involves a targeted therapy that may prevent progression of the disease and help to improve the well-being of CKD patients. It proposes a nanotechnological device, ie, a microarray-oriented homeostasis provider (MOHP), to improve homeostasis in CKD patients. MOHP would be an auxiliary kidney aid, and would improve the filtration functions that impaired kidneys cannot perform by their own. MOHP is composed of two main computer-oriented components, ie, a quantitative microarray detector (QMD) and a homeostasis-oriented microarray column (HOMC). QMD detects and HOMC selectively removes defined quantities of uremic wastes, toxins and any other metabolites which is programmed for. The QMD and HOMC would accomplish this with the help of a peristaltic blood pump that would circulate blood aseptically in an extracorporeal closed circuit. During the passage of blood through the QMD, this microarray detector would quantitatively monitor all of the blood compounds that accumulate in the blood of a patient with impaired glomerular filtration, including small-sized, middle-sized and large-sized molecules. The electronic information collected by QMD would be electronically transmitted to the HOMC, which would adjust the molecules to the concentrations they are electronically programmed for and/or receive from QMD. This process of monitoring and removal of waste continues until the programmed homeostasis criteria are reached. Like a conventional kidney machine, MOHP can be used in hospitals and homes under the supervision of a trained technician. The main advantages of this treatment would include improved homeostasis, a reduced likelihood of side effects and of the morbidity resulting from CKD, slower progression of kidney impairment, prevention of end-stage renal failure, a decreased need for hemodialysis therapy, avoidance of dialysis-related side effects later on in the patient's life, improved quality of life and increased life expectancy.

Experience With Rapid Microarray-Based Diagnostic Technology and Antimicrobial Stewardship for Patients With Gram-Positive Bacteremia.

PubMed

Neuner, Elizabeth A; Pallotta, Andrea M; Lam, Simon W; Stowe, David; Gordon, Steven M; Procop, Gary W; Richter, Sandra S

2016-11-01

OBJECTIVE To describe the impact of rapid diagnostic microarray technology and antimicrobial stewardship for patients with Gram-positive blood cultures. DESIGN Retrospective pre-intervention/post-intervention study. SETTING A 1,200-bed academic medical center. PATIENTS Inpatients with blood cultures positive for Staphylococcus aureus, Enterococcus faecalis, E. faecium, Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. anginosus, Streptococcus spp., and Listeria monocytogenes during the 6 months before and after implementation of Verigene Gram-positive blood culture microarray (BC-GP) with an antimicrobial stewardship intervention. METHODS Before the intervention, no rapid diagnostic technology was used or antimicrobial stewardship intervention was undertaken, except for the use of peptide nucleic acid fluorescent in situ hybridization and MRSA agar to identify staphylococcal isolates. After the intervention, all Gram-positive blood cultures underwent BC-GP microarray and the antimicrobial stewardship intervention consisting of real-time notification and pharmacist review. RESULTS In total, 513 patients with bacteremia were included in this study: 280 patients with S. aureus, 150 patients with enterococci, 82 patients with stretococci, and 1 patient with L. monocytogenes. The number of antimicrobial switches was similar in the pre-BC-GP (52%; 155 of 300) and post-BC-GP (50%; 107 of 213) periods. The time to antimicrobial switch was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 48±41 hours versus 75±46 hours, respectively (P<.001). The most common antimicrobial switch was de-escalation and time to de-escalation, was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 53±41 hours versus 82±48 hours, respectively (P<.001). There was no difference in mortality or hospital length of stay as a result of the intervention. CONCLUSIONS The combination of a rapid microarray diagnostic test with an antimicrobial stewardship intervention improved time to antimicrobial switch, especially time to de-escalation to optimal therapy, in patients with Gram-positive blood cultures. Infect Control Hosp Epidemiol 2016;1-6.

Induction of Attachment-Independent Biofilm Formation and Repression of hfq Expression by Low-Fluid-Shear Culture of Staphylococcus aureus ▿

PubMed Central

Castro, Sarah L.; Nelman-Gonzalez, Mayra; Nickerson, Cheryl A.; Ott, C. Mark

2011-01-01

The opportunistic pathogen Staphylococcus aureus encounters a wide variety of fluid shear levels within the human host, and they may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. By using rotating-wall vessel bioreactors to create a physiologically relevant, low-fluid-shear environment, S. aureus was evaluated for cellular responses that could impact its colonization and virulence. S. aureus cells grown in a low-fluid-shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear-cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole-genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed downregulation of the RNA chaperone Hfq, which parallels low-fluid-shear responses of certain Gram-negative organisms. This is the first study to report an Hfq association with fluid shear in a Gram-positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggest S. aureus responds to a low-fluid-shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during the initial host-pathogen interaction. PMID:21803898

Analysis of High-Throughput ELISA Microarray Data

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

White, Amanda M.; Daly, Don S.; Zangar, Richard C.

Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

Development of an objective gene expression panel as an alternative to self-reported symptom scores in human influenza challenge trials.

PubMed

Muller, Julius; Parizotto, Eneida; Antrobus, Richard; Francis, James; Bunce, Campbell; Stranks, Amanda; Nichols, Marshall; McClain, Micah; Hill, Adrian V S; Ramasamy, Adaikalavan; Gilbert, Sarah C

2017-06-08

Influenza challenge trials are important for vaccine efficacy testing. Currently, disease severity is determined by self-reported scores to a list of symptoms which can be highly subjective. A more objective measure would allow for improved data analysis. Twenty-one volunteers participated in an influenza challenge trial. We calculated the daily sum of scores (DSS) for a list of 16 influenza symptoms. Whole blood collected at baseline and 24, 48, 72 and 96 h post challenge was profiled on Illumina HT12v4 microarrays. Changes in gene expression most strongly correlated with DSS were selected to train a Random Forest model and tested on two independent test sets consisting of 41 individuals profiled on a different microarray platform and 33 volunteers assayed by qRT-PCR. 1456 probes are significantly associated with DSS at 1% false discovery rate. We selected 19 genes with the largest fold change to train a random forest model. We observed good concordance between predicted and actual scores in the first test set (r = 0.57; RMSE = -16.1%) with the greatest agreement achieved on samples collected approximately 72 h post challenge. Therefore, we assayed samples collected at baseline and 72 h post challenge in the second test set by qRT-PCR and observed good concordance (r = 0.81; RMSE = -36.1%). We developed a 19-gene qRT-PCR panel to predict DSS, validated on two independent datasets. A transcriptomics based panel could provide a more objective measure of symptom scoring in future influenza challenge studies. Trial registration Samples were obtained from a clinical trial with the ClinicalTrials.gov Identifier: NCT02014870, first registered on December 5, 2013.

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

Nasopharyngeal teratoma, congenital diaphragmatic hernia and Dandy-Walker malformation - a yet uncharacterized syndrome.

PubMed

Gupta, N; Shastri, S; Singh, P K; Jana, M; Mridha, A; Verma, G; Kabra, M

2016-11-01

An association of congenital diaphragmatic hernia, dandy walker malformation and nasopharyngeal teratoma is very rare. Here, we report a fourth case with this association where chromosomal microarray and whole exome sequencing (WES) was performed to understand the underlying genetic basis. Findings of few variants especially a novel variation in HIRA provided some insights. An association of congenital diaphragmatic hernia, dandy walker malformation and nasopharyngeal teratoma is very rare. Here, we report a fourth case with this association where chromosomal microarray and whole exome sequencing (WES) was performed to understand the underlying genetic basis. Findings of few variants especially a novel variation in HIRA provided some insights. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Whole genome mRNA transcriptomics analysis reveals different modes of action of the diarrheic shellfish poisons okadaic acid and dinophysis toxin-1 versus azaspiracid-1 in Caco-2 cells.

PubMed

Bodero, Marcia; Hoogenboom, Ron L A P; Bovee, Toine F H; Portier, Liza; de Haan, Laura; Peijnenburg, Ad; Hendriksen, Peter J M

2018-02-01

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the CMV Status

PubMed Central

Pérez-Bercoff, Lena; Valentini, Davide; Gaseitsiwe, Simani; Mahdavifar, Shahnaz; Schutkowski, Mike; Poiret, Thomas; Pérez-Bercoff, Åsa; Ljungman, Per; Maeurer, Markus J.

2014-01-01

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution. PMID:24740411

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

PubMed Central

Uehara, Yuriko; Oda, Katsutoshi; Ikeda, Yuji; Koso, Takahiro; Tsuji, Shingo; Yamamoto, Shogo; Asada, Kayo; Sone, Kenbun; Kurikawa, Reiko; Makii, Chinami; Hagiwara, Otoe; Tanikawa, Michihiro; Maeda, Daichi; Hasegawa, Kosei; Nakagawa, Shunsuke; Wada-Hiraike, Osamu; Kawana, Kei; Fukayama, Masashi; Fujiwara, Keiichi; Yano, Tetsu; Osuga, Yutaka; Fujii, Tomoyuki; Aburatani, Hiroyuki

2015-01-01

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis. PMID:26043110

Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

PubMed

Shea, A A; Bernhards, R C; Cote, C K; Chase, C J; Koehler, J W; Klimko, C P; Ladner, J T; Rozak, D A; Wolcott, M J; Fetterer, D P; Kern, S J; Koroleva, G I; Lovett, S P; Palacios, G F; Toothman, R G; Bozue, J A; Worsham, P L; Welkos, S L

2017-01-01

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated "Smooth" and "Rough", under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants' genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection.

Whole Blood Gene Expression Profiles to Assess Pathogenesis and Disease Severity in Infants with Respiratory Syncytial Virus Infection

PubMed Central

Mejias, Asuncion; Dimo, Blerta; Suarez, Nicolas M.; Garcia, Carla; Suarez-Arrabal, M. Carmen; Jartti, Tuomas; Blankenship, Derek; Jordan-Villegas, Alejandro; Ardura, Monica I.; Xu, Zhaohui; Banchereau, Jacques; Chaussabel, Damien; Ramilo, Octavio

2013-01-01

Background Respiratory syncytial virus (RSV) is the leading cause of viral lower respiratory tract infection (LRTI) and hospitalization in infants. Mostly because of the incomplete understanding of the disease pathogenesis, there is no licensed vaccine, and treatment remains symptomatic. We analyzed whole blood transcriptional profiles to characterize the global host immune response to acute RSV LRTI in infants, to characterize its specificity compared with influenza and human rhinovirus (HRV) LRTI, and to identify biomarkers that can objectively assess RSV disease severity. Methods and Findings This was a prospective observational study over six respiratory seasons including a cohort of infants hospitalized with RSV (n = 135), HRV (n = 30), and influenza (n = 16) LRTI, and healthy age- and sex-matched controls (n = 39). A specific RSV transcriptional profile was identified in whole blood (training cohort, n = 45 infants; Dallas, Texas, US) and validated in three different cohorts (test cohort, n = 46, Dallas, Texas, US; validation cohort A, n = 16, Turku, Finland; validation cohort B, n = 28, Columbus, Ohio, US) with high sensitivity (94% [95% CI 87%–98%]) and specificity (98% [95% CI 88%–99%]). It classified infants with RSV LRTI versus HRV or influenza LRTI with 95% accuracy. The immune dysregulation induced by RSV (overexpression of neutrophil, inflammation, and interferon genes, and suppression of T and B cell genes) persisted beyond the acute disease, and immune dysregulation was greatly impaired in younger infants (<6 mo). We identified a genomic score that significantly correlated with outcomes of care including a clinical disease severity score and, more importantly, length of hospitalization and duration of supplemental O2. Conclusions Blood RNA profiles of infants with RSV LRTI allow specific diagnosis, better understanding of disease pathogenesis, and assessment of disease severity. This study opens new avenues for biomarker discovery and identification of potential therapeutic or preventive targets, and demonstrates that large microarray datasets can be translated into a biologically meaningful context and applied to the clinical setting. Please see later in the article for the Editors' Summary PMID:24265599

Chemiluminescence microarrays in analytical chemistry: a critical review.

PubMed

Seidel, Michael; Niessner, Reinhard

2014-09-01

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

Computational synchronization of microarray data with application to Plasmodium falciparum.

PubMed

Zhao, Wei; Dauwels, Justin; Niles, Jacquin C; Cao, Jianshu

2012-06-21

Microarrays are widely used to investigate the blood stage of Plasmodium falciparum infection. Starting with synchronized cells, gene expression levels are continually measured over the 48-hour intra-erythrocytic cycle (IDC). However, the cell population gradually loses synchrony during the experiment. As a result, the microarray measurements are blurred. In this paper, we propose a generalized deconvolution approach to reconstruct the intrinsic expression pattern, and apply it to P. falciparum IDC microarray data. We develop a statistical model for the decay of synchrony among cells, and reconstruct the expression pattern through statistical inference. The proposed method can handle microarray measurements with noise and missing data. The original gene expression patterns become more apparent in the reconstructed profiles, making it easier to analyze and interpret the data. We hypothesize that reconstructed gene expression patterns represent better temporally resolved expression profiles that can be probabilistically modeled to match changes in expression level to IDC transitions. In particular, we identify transcriptionally regulated protein kinases putatively involved in regulating the P. falciparum IDC. By analyzing publicly available microarray data sets for the P. falciparum IDC, protein kinases are ranked in terms of their likelihood to be involved in regulating transitions between the ring, trophozoite and schizont developmental stages of the P. falciparum IDC. In our theoretical framework, a few protein kinases have high probability rankings, and could potentially be involved in regulating these developmental transitions. This study proposes a new methodology for extracting intrinsic expression patterns from microarray data. By applying this method to P. falciparum microarray data, several protein kinases are predicted to play a significant role in the P. falciparum IDC. Earlier experiments have indeed confirmed that several of these kinases are involved in this process. Overall, these results indicate that further functional analysis of these additional putative protein kinases may reveal new insights into how the P. falciparum IDC is regulated.

Micro-array isolation of circulating tumor cells (CTCs): the droplet biopsy chip

NASA Astrophysics Data System (ADS)

Panchapakesan, B.

2017-08-01

We present a new method for circulating tumor cell capture based on micro-array isolation from droplets. Called droplet biopsy, our technique uses a 76-element array of carbon nanotube devices functionalized with anti-EpCAM and antiHer2 antibodies for immunocapture of spiked breast cancer cells in the blood. This droplet biopsy chip can enable capture of CTCs based on both positive and negative selection strategy. Negative selection is achieved through depletion of contaminating leukocytes through the differential settling of blood into layers. We report 55%-100% cancer cell capture yield in this first droplet biopsy chip study. The droplet biopsy is an enabling idea where one can capture CTCs based on multiple biomarkers in a single blood sample.

Analysis of Hemoglobin A1c from Dried Blood Spot Samples with the Tina-quant® II Immunoturbidimetric Method

PubMed Central

Jones, Trevor G.; Warber, Kimbrough D.; Roberts, Billy D.

2010-01-01

Background Hemoglobin A1c (HbA1c) has been endorsed as a tool for the diagnosis of diabetes. This test requires instrumentation that may not be available in underdeveloped areas. Dried blood spot (DBS) samples collected by finger stick procedures offer a mechanism to transport samples to laboratories that do measure HbA1c. Methods Whole blood (ethylenediaminetetraacetic acid) was applied to Ahlstrom 226 filter paper. These DBS samples were compared to whole blood samples using the Roche Tina-quant® II immunoturbidometric assay. Hemoglobin A1c stability on DBS was assessed at three temperatures—4, 25, and 40°C—for up to 9 days. A 44-day study was also done for DBS at 20–25°C. Results The Tina-quant® II DBS method showed excellent agreement with whole blood HbA1c results (r2 = 0.99) with a slight positive mean bias of 0.08 ± 0.04% HbA1c (95% confidence interval). The variation in HbA1c on DBS samples subjected to different temperatures and times did not exceed 5.6%. Conclusions Dried blood spot samples represent an alternative to whole blood for HbA1c by measurement when transporting whole blood is not feasible. PMID:20307383

Does whole blood coagulation analysis reflect developmental haemostasis?

PubMed

Ravn, Hanne Berg; Andreasen, Jo Bønding; Hvas, Anne-Mette

2017-04-01

: Developmental haemostasis has been well documented over the last 3 decades and age-dependent reference ranges have been reported for a number of plasmatic coagulation parameters. With the increasing use of whole blood point-of-care tests like rotational thromboelastometry (ROTEM) and platelet function tests, an evaluation of age-dependent changes is warranted for these tests as well. We obtained blood samples from 149 children, aged 1 day to 5.9 years, and analysed conventional plasmatic coagulation tests, including activated partial prothrombin time, prothrombin time, and fibrinogen (functional). Whole blood samples were analysed using ROTEM to assess overall coagulation capacity and Multiplate analyzer to evaluate platelet aggregation. Age-dependent changes were analysed for all variables. We found age-dependent differences in all conventional coagulation tests (all P values < 0.05), but there was no sign of developmental changes in whole blood coagulation assessment when applying ROTEM, apart from clotting time in the EXTEM assay (P < 0.03). Despite marked differences in mean platelet aggregation between age groups, data did not reach statistical significance. Citrate-anticoagulated blood showed significantly reduced platelet aggregation compared with blood anticoagulated with heparin or hirudin (all P values < 0.003). We confirmed previous developmental changes in conventional plasmatic coagulation test. However, these age-dependent changes were not displayed in whole blood monitoring using ROTEM or Multiplate analyzer. Type of anticoagulant had a significant influence on platelet aggregation across all age groups.

Chromosomal microarray analysis of Bulgarian patients with epilepsy and intellectual disability.

PubMed

Peycheva, Valentina; Kamenarova, Kunka; Ivanova, Neviana; Stamatov, Dimitar; Avdjieva-Tzavella, Daniela; Alexandrova, Iliana; Zhelyazkova, Sashka; Pacheva, Iliana; Dimova, Petya; Ivanov, Ivan; Litvinenko, Ivan; Bozhinova, Veneta; Tournev, Ivailo; Simeonov, Emil; Mitev, Vanyo; Jordanova, Albena; Kaneva, Radka

2018-08-15

High resolution chromosomal microarray analysis (CMA) has facilitated the identification of small chromosomal rearrangements throughout the genome, associated with various neurodevelopmental phenotypes, including ID/DD. Recently, it became evident that intellectual disability (ID)/developmental delay (DD) can occur with associated co-morbidities like epileptic seizures, autism and additional congenital anomalies. These observations require whole genome approach in order to detect the genetic causes of these complex disorders. In this study, we examined 92 patients of Bulgarian origin at age between 1 and 22 years with ID, generalized epilepsy, autistic signs and congenital anomalies. CMA was carried out using SurePrint G3 Human CGH Microarray Kit, 4 × 180 K and SurePrint G3 Unrestricted CGH ISCA v2, 4 × 180 K oligo platforms. Referral indications for selection of the patients were the presence of generalized refractory seizures disorders and co-morbid ID. Clearly pathogenic copy number variations (CNVs) were detected in eight patients (8.7%) from our cohort. Additionally, possibly pathogenic rearrangements of unclear clinical significance were detected in six individuals (6.5%), which make for an overall diagnostic yield of 15.2% among our cohort of patients. We report here the patients with clearly pathogenic CNVs, discuss the potential causality of the possibly pathogenic CNVs and make genotype - phenotype correlations. One novel possibly pathogenic heterozygous deletion in 15q22.31 region was detected in a case with ID/DD. Additionally, whole APBA2 gene duplication in 15q13.1 was found in three generations of a family with epilepsy, ID and psychiatric abnormalities. The results from this study allow us to define the genetic diagnosis in a subset of Bulgarian patients and improve the genetic counseling of the affected families. To our knowledge, this is the first aCGH evaluation of a Bulgarian cohort of children with epilepsy and ID so far. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of a point-of-care glucose and β-hydroxybutyrate meter operated in various environmental conditions in prepartum and postpartum sheep.

PubMed

Hornig, Katlin J; Byers, Stacey R; Callan, Robert J; Holt, Timothy; Field, Megan; Han, Hyungchul

2013-08-01

To compare β-hydroxybutyrate (BHB) and glucose concentrations measured with a dual-purpose point-of-care (POC) meter designed for use in humans and a laboratory biochemical analyzer (LBA) to determine whether the POC meter would be reliable for on-farm measurement of blood glucose and BHB concentrations in sheep in various environmental conditions and nutritional states. 36 pregnant mixed-breed ewes involved in a maternal feed restriction study. Blood samples were collected from each sheep at multiple points throughout gestation and lactation to allow for tracking of gradually increasing metabolic hardship. Whole blood glucose and BHB concentrations were measured with the POC meter and compared with serum results obtained with an LBA. 464 samples were collected. Whole blood BHB concentrations measured with the POC meter compared well with LBA results, and error grid analysis showed the POC values were acceptable. Whole blood glucose concentrations measured with the POC meter had more variation, compared with LBA values, over the glucose ranges evaluated. Results of error grid analysis of POC-measured glucose concentrations were not acceptable, indicating errors likely to result in needless treatment with glucose or other supplemental energy sources in normoglycemic sheep. The POC meter was user-friendly and performed well across a wide range of conditions. The meter was adequate for detection of pregnancy toxemia in sheep via whole blood BHB concentration. Results should be interpreted with caution when the POC meter is used to measure blood glucose concentrations.

Diagnosis of Zika Virus Infection by Peptide Array and Enzyme-Linked Immunosorbent Assay.

PubMed

Mishra, Nischay; Caciula, Adrian; Price, Adam; Thakkar, Riddhi; Ng, James; Chauhan, Lokendra V; Jain, Komal; Che, Xiaoyu; Espinosa, Diego A; Montoya Cruz, Magelda; Balmaseda, Angel; Sullivan, Eric H; Patel, Jigar J; Jarman, Richard G; Rakeman, Jennifer L; Egan, Christina T; Reusken, Chantal B E M; Koopmans, Marion P G; Harris, Eva; Tokarz, Rafal; Briese, Thomas; Lipkin, W Ian

2018-03-06

Zika virus (ZIKV) is implicated in fetal stillbirth, microcephaly, intracranial calcifications, and ocular anomalies following vertical transmission from infected mothers. In adults, infection may trigger autoimmune inflammatory polyneuropathy. Transmission most commonly follows the bite of infected Aedes mosquitoes but may also occur through sexual intercourse or receipt of blood products. Definitive diagnosis through detection of viral RNA is possible in serum or plasma within 10 days of disease onset, in whole blood within 3 weeks of onset, and in semen for up to 3 months. Serological diagnosis is nonetheless critical because few patients have access to molecular diagnostics during the acute phase of infection and infection may be associated with only mild or inapparent disease that does not prompt molecular testing. Serological diagnosis is confounded by cross-reactivity of immune sera with other flaviviruses endemic in the areas where ZIKV has recently emerged. Accordingly, we built a high-density microarray comprising nonredundant 12-mer peptides that tile, with one-residue overlap, the proteomes of Zika, dengue, yellow fever, West Nile, Ilheus, Oropouche, and chikungunya viruses. Serological analysis enabled discovery of a ZIKV NS2B 20-residue peptide that had high sensitivity (96.0%) and specificity (95.9%) versus natural infection with or vaccination against dengue, chikungunya, yellow fever, West Nile, tick-borne encephalitis, or Japanese encephalitis virus in a microarray assay and an enzyme-linked immunosorbent assay (ELISA) of early-convalescent-phase sera (2 to 3 weeks after onset of symptomatic infection). IMPORTANCE The emergence of Zika virus (ZIKV) as a teratogen is a profound challenge to global public health. Molecular diagnosis of infection is straightforward during the 3-week period when patients are viremic. However, serological diagnosis thereafter of historical exposure has been confounded by cross-reactivity. Using high-density peptide arrays that tile the proteomes of a selection of flaviviruses to identify a ZIKV-specific peptide, we established two assays that enable sensitive and specific diagnosis of exposure to ZIKV. These assays may be useful in guiding clinical management of mothers at risk for potential exposure to ZIKV and enable insights into the epidemiology of ZIKV infections.

The safety of early fresh, whole blood transfusion among severely battle injured at US Marine Corps forward surgical care facilities in Afghanistan.

PubMed

Auten, Jonathan D; Lunceford, Nicole L; Horton, Jaime L; Galarneau, Mike R; Galindo, Roger M; Shepps, Craig D; Zieber, Tara J; Dewing, Chris B

2015-11-01

In Afghanistan, care of the acutely injured trauma patient commonly occurred in facilities with limited blood banking capabilities. Apheresis platelets were often not available. Component therapy consisted of 1:1 packed red blood cells and fresh frozen plasma. Fresh, whole blood transfusion often augmented therapy in the severely injured patient. This study analyzed the safety of fresh, whole blood use in a resource-limited setting. A retrospective analysis was performed on a prospectively collected data set of US battle injuries presenting to three US Marine Corps (USMC) expeditionary surgical care facilities in Helmand Province, Afghanistan, between January 2010 and July 2012. Included in the review were patients with Injury Severity Scores (ISSs) of 15 or higher receiving blood transfusions. Univariate analyses were performed, followed by multivariable logistic regression to describe the relationship between the treatment group and posttreatment complications such as trauma-induced coagulopathy, infection, mortality, venous thromboembolism, and transfusion reaction. Propensity scores were calculated and included in multivariable models to adjust for potential bias in treatment selection. A total of 61 patients were identified; all were male marines with a mean (SD) age of 23.5 (3.6) years. The group receiving fresh, whole blood was noted to have higher ISSs and lower blood pressure, pH, and base deficits on arrival. Traumatic coagulopathy was significantly less common in the group receiving fresh, whole blood (odds ratio, 0.01; 95% confidence interval, 0.00-0.18). Multivariable models found no other significant differences between the treatment groups. The early use of fresh, whole blood in a resource-limited setting seems to confer a benefit in reducing traumatic coagulopathy. This study's small sample size precludes further statement on the overall safety of fresh, whole blood use. Therapy study, level IV.

Analysis of the effects of sex hormone background on the rat choroid plexus transcriptome by cDNA microarrays.

PubMed

Quintela, Telma; Gonçalves, Isabel; Carreto, Laura C; Santos, Manuel A S; Marcelino, Helena; Patriarca, Filipa M; Santos, Cecília R A

2013-01-01

The choroid plexus (CP) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF), the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis.

Analysis of the Effects of Sex Hormone Background on the Rat Choroid Plexus Transcriptome by cDNA Microarrays

PubMed Central

Quintela, Telma; Gonçalves, Isabel; Carreto, Laura C.; Santos, Manuel A. S.; Marcelino, Helena; Patriarca, Filipa M.; Santos, Cecília R. A.

2013-01-01

The choroid plexus (CP) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF), the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis. PMID:23585832

Protein profiles associated with survival in lung adenocarcinoma

PubMed Central

Chen, Guoan; Gharib, Tarek G; Wang, Hong; Huang, Chiang-Ching; Kuick, Rork; Thomas, Dafydd G.; Shedden, Kerby A.; Misek, David E.; Taylor, Jeremy M. G.; Giordano, Thomas J.; Kardia, Sharon L. R.; Iannettoni, Mark D.; Yee, John; Hogg, Philip J.; Orringer, Mark B.; Hanash, Samir M.; Beer, David G.

2003-01-01

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P = 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer. PMID:14573703

High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains

PubMed Central

Abbey, Darren; Hickman, Meleah; Gresham, David; Berman, Judith

2011-01-01

Phenotypic diversity can arise rapidly through loss of heterozygosity (LOH) or by the acquisition of copy number variations (CNV) spanning whole chromosomes or shorter contiguous chromosome segments. In Candida albicans, a heterozygous diploid yeast pathogen with no known meiotic cycle, homozygosis and aneuploidy alter clinical characteristics, including drug resistance. Here, we developed a high-resolution microarray that simultaneously detects ∼39,000 single nucleotide polymorphism (SNP) alleles and ∼20,000 copy number variation loci across the C. albicans genome. An important feature of the array analysis is a computational pipeline that determines SNP allele ratios based upon chromosome copy number. Using the array and analysis tools, we constructed a haplotype map (hapmap) of strain SC5314 to assign SNP alleles to specific homologs, and we used it to follow the acquisition of loss of heterozygosity (LOH) and copy number changes in a series of derived laboratory strains. This high-resolution SNP/CGH microarray and the associated hapmap facilitated the phasing of alleles in lab strains and revealed detrimental genome changes that arose frequently during molecular manipulations of laboratory strains. Furthermore, it provided a useful tool for rapid, high-resolution, and cost-effective characterization of changes in allele diversity as well as changes in chromosome copy number in new C. albicans isolates. PMID:22384363

Analysis of human serum and whole blood for mineral content by ICP-MS and ICP-OES: development of a mineralomics method.

PubMed

Harrington, James M; Young, Daniel J; Essader, Amal S; Sumner, Susan J; Levine, Keith E

2014-07-01

Minerals are inorganic compounds that are essential to the support of a variety of biological functions. Understanding the range and variability of the content of these minerals in biological samples can provide insight into the relationships between mineral content and the health of individuals. In particular, abnormal mineral content may serve as an indicator of illness. The development of robust, reliable analytical methods for the determination of the mineral content of biological samples is essential to developing biological models for understanding the relationship between minerals and illnesses. This paper describes a method for the analysis of the mineral content of small volumes of serum and whole blood samples from healthy individuals. Interday and intraday precision for the mineral content of the blood (250 μL) and serum (250 μL) samples was measured for eight essential minerals--sodium (Na), calcium (Ca), magnesium (Mg), potassium (K), iron (Fe), zinc (Zn), copper (Cu), and selenium (Se)--by plasma spectrometric methods and ranged from 0.635 to 10.1% relative standard deviation (RSD) for serum and 0.348-5.98% for whole blood. A comparison of the determined ranges for ten serum samples and six whole blood samples provided good agreement with literature reference ranges. The results demonstrate that the digestion and analysis methods can be used to reliably measure the content of these minerals and potentially of other minerals.

Differential gene expression associated with dietary methylmercury (MeHg) exposure in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio)

PubMed Central

Liu, Qing; Basu, Niladri; Goetz, Giles; Jiang, Nan; Hutz, Reinhold J.; Tonellato, Peter J.; Carvan, Michael J.

2013-01-01

The objective of this study was to identify and evaluate conserved biomarkers that could be used in most species of teleost fish at most life-stages. We investigated the effects of sublethal methylmercury (MeHg) exposure on developing rainbow trout and zebrafish. Juvenile rainbow trout and young adult zebrafish were fed food with MeHg added at 0, 0.5, 5 and 50 ppm. Atomic absorption spectrometry was applied to measure whole body total Hg levels, and pathologic analysis was performed to identify MeHg-induced toxicity. Fish at six weeks were sampled from each group for microarray analysis using RNA from whole fish. MeHg-exposed trout and zebrafish did not show overt signs of toxicity or pathology, nor were significant differences seen in mortality, length, mass, or condition factor. The accumulation of MeHg in trout and zebrafish exhibited dose- and time-dependent patterns during six weeks, and zebrafish exhibited greater assimilation of total Hg than rainbow trout. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as iron ion homeostasis, glutathione transferase activity, regulation of muscle contraction, troponin I binding and calcium-dependent protein binding. Genes were selected as biomarker candidates based on their microarray data and their expression was evaluated by QPCR. Unfortunately, these genes are not good consistent biomarkers for both rainbow trout and zebrafish from QPCR evaluation using individual fish. Our conclusion is that biomarker analysis for aquatic toxicant assessment using fish needs to be based on tissue-, sex- and species-specific consideration. PMID:23529582

Determination of proflavine in rat whole blood without sample pretreatment by laser desorption postionization mass spectrometry.

PubMed

Chen, Jiaxin; Hu, Yongjun; Lu, Qiao; Wang, Pengchao; Zhan, Huaqi

2017-04-01

A novel pretreatment-free method involving laser desorption postionization (LDPI) coupled with time-of-flight mass spectrometry (MS) was developed for the monitoring of proflavine level in rat whole blood. It comprises a protocol for dosing via intravenous administration and collection of whole blood, followed by direct LDPI-MS analysis without any sample pretreatment. An intense ion signal at m/z 209 was observed from whole blood without any interference signals, except some background signals below m/z 100. The calibration curve was established with use of 9-phenylacridine as the internal standard for proflavine determination from the plotting of the peak ratios of proflavine to the internal standard, with a correlation coefficient (R 2 ) greater than 0.99. The limit of detection was estimated to be 0.48 pmol/mm 2 and the quantification range was 0.5-16.5 μg/mL for proflavine. In addition, only a minimal matrix effect was observed, as expected from considerations of the desorption and ionization mechanism. Interday and intraday accuracy and precision were calculated to be within 13% and 82-114%, respectively. Estimated concentrations of proflavine residue in whole blood were also successfully obtained at selected time points after dosing. The proposed method is simple, low cost, and sensitive, and should be seen as a complementary method for monitoring drug levels in blood. Graphical Abstract Monitoring proflavine levels in rat whole blood at different time points using laser desorption postionization mass spectrometry (LDPI-MS).

Near Infrared Optical Properties of Whole Human Blood and Blood Containing Nanoparticulates

NASA Astrophysics Data System (ADS)

Mimun, Lawrence C.; Yust, Brian; Nash, Kelly L.; Sardar, Dhiraj K.

2010-10-01

Whole human blood is optically characterized in the near infrared (NIR) with and without the addition of nanocrystals. The optical properties were obtained using the double-integrating sphere technique at the Nd excitation wavelength of 808 nm. Y2O3 and Nd^3+:Y2O3 nanoparticles were added in predetermined amounts to water, blood plasma, and whole blood samples, from which a computational analysis was conducted using the Kubelka-Munk calculational method, the Inverse Adding Doubling Method, and the Magic Light Monte Carlo Method to characterized the optical properties such as the absorption (μa) and scattering coefficients (μs) and the scattering anisotropy (g). Through comparison with control samples, the optical properties of each component (blood, plasma, and nanoparticles) can be determined individually, thus illuminating any changes due to the biological environment. The emission from the Nd^3+:Y2O3 particles through the blood is also detected thus exhibiting their usefulness as real world biological markers.

Serum reactome induced by Bordetella pertussis infection and Pertussis vaccines: qualitative differences in serum antibody recognition patterns revealed by peptide microarray analysis.

PubMed

Valentini, Davide; Ferrara, Giovanni; Advani, Reza; Hallander, Hans O; Maeurer, Markus J

2015-07-01

Pertussis (whooping cough) remains a public health problem despite extensive vaccination strategies. Better understanding of the host-pathogen interaction and the detailed B. pertussis (Bp) target recognition pattern will help in guided vaccine design. We characterized the specific epitope antigen recognition profiles of serum antibodies ('the reactome') induced by whooping cough and B. pertussis (Bp) vaccines from a case-control study conducted in 1996 in infants enrolled in a Bp vaccine trial in Sweden (Gustafsson, NEJM, 1996, 334, 349-355). Sera from children with whooping cough, vaccinated with Diphtheria Tetanus Pertussis (DTP) whole-cell (wc), acellular 5 (DPTa5), or with the 2 component (a2) vaccines and from infants receiving only DT (n=10 for each group) were tested with high-content peptide microarrays containing 17 Bp proteins displayed as linear (n=3175) peptide stretches. Slides were incubated with serum and peptide-IgG complexes detected with Cy5-labeled goat anti-human IgG and analyzed using a GenePix 4000B microarray scanner, followed by statistical analysis, using PAM (Prediction Analysis for Microarrays) and the identification of uniquely recognized peptide epitopes. 367/3,085 (11.9%) peptides were recognized in 10/10 sera from children with whooping cough, 239 (7.7%) in DTPwc, 259 (8.4%) in DTPa5, 105 (3.4%) DTPa2, 179 (5.8%) in the DT groups. Recognition of strongly recognized peptides was similar between whooping cough and DPTwc, but statistically different between whooping cough vs. DTPa5 (p<0.05), DTPa2 and DT (p<0.001 vs. both) vaccines. 6/3,085 and 2/3,085 peptides were exclusively recognized in (10/10) sera from children with whooping cough and DTPa2 vaccination, respectively. DTPwc resembles more closely the whooping cough reactome as compared to acellular vaccines. We could identify a unique recognition signature common for each vaccination group (10/10 children). Peptide microarray technology allows detection of subtle differences in epitope signature responses and may help to guide rational vaccine development by the objective description of a clinically relevant immune response that confers protection against infectious pathogens.

Whole Blood Activation Results in Enhanced Detection of T Cell and Monocyte Cytokine Production by Flow Cytometry

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Crucian, Brian E.

2001-01-01

An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a wholeblood activation culture has been described. We compared whole blood culture to standard PBMC culture and determined the individual cytokine secretion patterns for both T cells and monocytes via flow cytometry. For T cells cytokine assessment following PMA +ionomycin activation: (1) a significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture; (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. In addition, a four-color cytometric analysis was used to allow accurate phenotyping and quantitation of cytokine producing lymphocyte populations. Using this technique we found IFNgamma production to be significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines in conjunction with CD 14. The cytokine pairs used for analysis were IL-1a/IL-12, and IL-10ITNFa. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFalpha equally well in both culture systems. Monocyte production of IL-10 was significantly elevated following whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing monocytes may represent functionally different monocyte subsets with distinct functions. Whole blood culture eliminates the need to purify cell populations prior to culture and may have significant utility for the routine monitoring of the cytokine balances of the peripheral blood T cell and monocyte populations. In addition, there are distinct advantages to performing whole-blood (WB) activation as compared to PBMC activation. These advantages would include retaining all various cell-cell interactions as well as any soluble factors present in serum that influence cell activation. It is likely that the altered cytokine production observed following whole blood culture more accurately represents the in-vivo immune balance.

Microarray analysis of gene expression in West Nile virus–infected human retinal pigment epithelium

PubMed Central

Munoz-Erazo, Luis; Natoli, Ricardo; Provis, Jan Marie; Madigan, Michelle Catherine

2012-01-01

Purpose To identify key genes differentially expressed in the human retinal pigment epithelium (hRPE) following low-level West Nile virus (WNV) infection. Methods Primary hRPE and retinal pigment epithelium cell line (ARPE-19) cells were infected with WNV (multiplicity of infection 1). RNA extracted from mock-infected and WNV-infected cells was assessed for differential expression of genes using Affymetrix microarray. Quantitative real-time PCR analysis of 23 genes was used to validate the microarray results. Results Functional annotation clustering of the microarray data showed that gene clusters involved in immune and antiviral responses ranked highly, involving genes such as chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X-C motif) ligand 10 (CXCL10), and toll like receptor 3 (TLR3). In conjunction with the quantitative real-time PCR analysis, other novel genes regulated by WNV infection included indoleamine 2,3-dioxygenase (IDO1), genes involved in the transforming growth factor–β pathway (bone morphogenetic protein and activin membrane-bound inhibitor homolog [BAMBI] and activating transcription factor 3 [ATF3]), and genes involved in apoptosis (tumor necrosis factor receptor superfamily, member 10d [TNFRSF10D]). WNV-infected RPE did not produce any interferon-γ, suggesting that IDO1 is induced by other soluble factors, by the virus alone, or both. Conclusions Low-level WNV infection of hRPE cells induced expression of genes that are typically associated with the host cell response to virus infection. We also identified other genes, including IDO1 and BAMBI, that may influence the RPE and therefore outer blood-retinal barrier integrity during ocular infection and inflammation, or are associated with degeneration, as seen for example in aging. PMID:22509103

Self-driven filter-based blood plasma separator microfluidic chip for point-of-care testing.

PubMed

Madadi, Hojjat; Casals-Terré, Jasmina; Mohammadi, Mahdi

2015-05-22

There is currently a growing need for lab-on-a-chip devices for use in clinical analysis and diagnostics, especially in the area of patient care. The first step in most blood assays is plasma extraction from whole blood. This paper presents a novel, self-driven blood plasma separation microfluidic chip, which can extract more than 0.1 μl plasma from a single droplet of undiluted fresh human blood (~5 μl). This volume of blood plasma is extracted from whole blood with high purity (more than 98%) in a reasonable time frame (3 to 5 min), and without the need for any external force. This would be the first step towards the realization of a single-use, self-blood test that does not require any external force or power source to deliver and analyze a fresh whole-blood sample, in contrast to the existing time-consuming conventional blood analysis. The prototypes are manufactured in polydimethylsiloxane that has been modified with a strong nonionic surfactant (Silwet L-77) to achieve hydrophilic behavior. The main advantage of this microfluidic chip design is the clogging delay in the filtration area, which results in an increased amount of extracted plasma (0.1 μl). Moreover, the plasma can be collected in one or more 10 μm-deep channels to facilitate the detection and readout of multiple blood assays. This high volume of extracted plasma is achieved thanks to a novel design that combines maximum pumping efficiency without disturbing the red blood cells' trajectory through the use of different hydrodynamic principles, such as a constriction effect and a symmetrical filtration mode. To demonstrate the microfluidic chip's functionality, we designed and fabricated a novel hybrid microdevice that exhibits the benefits of both microfluidics and lateral flow immunochromatographic tests. The performance of the presented hybrid microdevice is validated using rapid detection of thyroid stimulating hormone within a single droplet of whole blood.

High-resolution whole-genome analysis of skull base chordomas implicates FHIT loss in chordoma pathogenesis.

PubMed

Diaz, Roberto Jose; Guduk, Mustafa; Romagnuolo, Rocco; Smith, Christian A; Northcott, Paul; Shih, David; Berisha, Fitim; Flanagan, Adrienne; Munoz, David G; Cusimano, Michael D; Pamir, M Necmettin; Rutka, James T

2012-09-01

Chordoma is a rare tumor arising in the sacrum, clivus, or vertebrae. It is often not completely resectable and shows a high incidence of recurrence and progression with shortened patient survival and impaired quality of life. Chemotherapeutic options are limited to investigational therapies at present. Therefore, adjuvant therapy for control of tumor recurrence and progression is of great interest, especially in skull base lesions where complete tumor resection is often not possible because of the proximity of cranial nerves. To understand the extent of genetic instability and associated chromosomal and gene losses or gains in skull base chordoma, we undertook whole-genome single-nucleotide polymorphism microarray analysis of flash frozen surgical chordoma specimens, 21 from the clivus and 1 from C1 to C2 vertebrae. We confirm the presence of a deletion at 9p involving CDKN2A, CDKN2B, and MTAP but at a much lower rate (22%) than previously reported for sacral chordoma. At a similar frequency (21%), we found aneuploidy of chromosome 3. Tissue microarray immunohistochemistry demonstrated absent or reduced fragile histidine triad (FHIT) protein expression in 98% of sacral chordomas and 67%of skull base chordomas. Our data suggest that chromosome 3 aneuploidy and epigenetic regulation of FHIT contribute to loss of the FHIT tumor suppressor in chordoma. The finding that FHIT is lost in a majority of chordomas provides new insight into chordoma pathogenesis and points to a potential new therapeutic target for this challenging neoplasm.

Evaluation of the efficacy of constitutional array-based comparative genomic hybridization in the diagnosis of aneuploidy using genomic and amplified DNA.

PubMed

Tan, Niap H; Palmer, Rodger; Wang, Rubin

2010-02-01

Array-based comparative genomic hybridization (array CGH) is a new molecular technique that has the potential to revolutionize cytogenetics. However, use of high resolution array CGH in the clinical setting is plagued by the problem of widespread copy number variations (CNV) in the human genome. Constitutional microarray, containing only clones that interrogate regions of known constitutional syndromes, may circumvent the dilemma of detecting CNV of unknown clinical significance. The present study investigated the efficacy of constitutional microarray in the diagnosis of trisomy. Test samples included genomic DNA from trisomic cell lines, amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. DNA amplification was achieved by means of multiple displacement amplification (MDA) over 16 h. The trisomic and sex chromosomes copy number imbalances in the genomic DNA were correctly identified by the constitutional microarrays. However, there was a failure to detect the trisomy in the amplification products of 50 ng of genomic DNA and whole genome amplification products of single cells. Using carefully selected clones, Spectral Genomics constitutional microarray was able to detect the chromosomal copy number imbalances in genomic DNA without the confounding effects of CNV. The diagnostic failure in amplified DNA samples could be attributed to the amplification process. The MDA duration of 16 h generated excessive amount of biases and shortening the duration might minimize the problem.

Classifying compound mechanism of action for linking whole cell phenotypes to molecular targets

PubMed Central

Bourne, Christina R.; Wakeham, Nancy; Bunce, Richard A.; Berlin, K. Darrell; Barrow, William W.

2013-01-01

Drug development programs have proven successful when performed at a whole cell level, thus incorporating solubility and permeability into the primary screen. However, linking those results to the target within the cell has been a major set-back. The Phenotype Microarray system, marketed and sold by Biolog, seeks to address this need by assessing the phenotype in combination with a variety of chemicals with known mechanism of action (MOA). We have evaluated this system for usefulness in deducing the MOA for three test compounds. To achieve this, we constructed a database with 21 known antimicrobials, which served as a comparison for grouping our unknown MOA compounds. Pearson correlation and Ward linkage calculations were used to generate a dendrogram that produced clustering largely by known MOA, although there were exceptions. Of the three unknown compounds, one was definitively placed as an anti-folate. The second and third compounds’ MOA were not clearly identified, likely due to unique MOA not represented within the commercial database. The availability of the database generated in this report for S. aureus ATCC 29213 will increase the accessibility of this technique to other investigators. From our analysis, the Phenotype Microarray system can group compounds with clear MOA, but distinction of unique or broadly acting MOA at this time is less clear. PMID:22434711

cDNA microarray analysis of esophageal cancer: discoveries and prospects.

PubMed

Shimada, Yutaka; Sato, Fumiaki; Shimizu, Kazuharu; Tsujimoto, Gozoh; Tsukada, Kazuhiro

2009-07-01

Recent progress in molecular biology has revealed many genetic and epigenetic alterations that are involved in the development and progression of esophageal cancer. Microarray analysis has also revealed several genetic networks that are involved in esophageal cancer. However, clinical application of microarray techniques and use of microarray data have not yet occurred. In this review, we focus on the recent developments and problems with microarray analysis of esophageal cancer.

Optimization and application of octadecyl-modified monolithic silica for solid-phase extraction of drugs in whole blood samples.

PubMed

Namera, Akira; Saito, Takeshi; Ota, Shigenori; Miyazaki, Shota; Oikawa, Hiroshi; Murata, Kazuhiro; Nagao, Masataka

2017-09-29

Monolithic silica in MonoSpin for solid-phase extraction of drugs from whole blood samples was developed to facilitate high-throughput analysis. Monolithic silica of various pore sizes and octadecyl contents were synthesized, and their effects on recovery rates were evaluated. The silica monolith M18-200 (20μm through-pore size, 10.4nm mesopore size, and 17.3% carbon content) achieved the best recovery of the target analytes in whole blood samples. The extraction proceeded with centrifugal force at 1000rpm for 2min, and the eluate was directly injected into the liquid chromatography-mass spectrometry system without any tedious steps such as evaporation of extraction solvents. Under the optimized condition, low detection limits of 0.5-2.0ngmL -1 and calibration ranges up to 1000ngmL -1 were obtained. The recoveries of the target drugs in the whole blood were 76-108% with relative standard deviation of less than 14.3%. These results indicate that the developed method based on monolithic silica is convenient, highly efficient, and applicable for detecting drugs in whole blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Peripheral blood mononuclear cells as a model to study the response of energy homeostasis-related genes to acute changes in feeding conditions.

PubMed

Caimari, Antoni; Oliver, Paula; Keijer, Jaap; Palou, Andreu

2010-04-01

Peripheral blood mononuclear cells (PBMCs) are readily accessible biological material and a potential tissue source to discover novel biomarkers of response to environmental exposures including nutrition. We analyzed whether PBMCs could reflect molecular changes that take place in response to different feeding conditions in key organs/tissues involved in energy homeostasis. We studied energy balance-related genes whose expression was altered in normoweight (control) rats and in diet-induced (cafeteria) obese rats in response to ad libitum feeding, 14-h fasting, and 6-h refeeding after fasting, using whole-genome microarray analysis. In PBMCs, the expression of the genes central to energy metabolism was altered by the feeding conditions. The number of affected genes was 75 in the control rats, but only 23 in the cafeteria obese rats. Most of these genes play a role in metabolic pathways regulated by nutritional changes, such as lipid metabolism (the metabolic pathway mainly reflected in blood cells), carbohydrate metabolism, central energy metabolism, respiratory chain/mitochondrial ATPase system, and food intake regulation. Importantly, our results showed a similar behavior to that of the mesenteric white adipose tissue. In conclusion, metabolic adaptations to acute changes in feeding conditions are reflected in the expression of genes central to energy homeostasis in PBMCs of normoweight rats, while response is impaired in cafeteria obese animals. The lower number of genes affected in obese animals indicates impaired nutritional regulation. PBMCs appear as a suitable potential model to characterize metabolic adaptations to food intake and body weight maintenance in experimental animals. These findings may also inform the development of future peripheral tissue models in the emerging field of clinical nutrigenomics.

Importing MAGE-ML format microarray data into BioConductor.

PubMed

Durinck, Steffen; Allemeersch, Joke; Carey, Vincent J; Moreau, Yves; De Moor, Bart

2004-12-12

The microarray gene expression markup language (MAGE-ML) is a widely used XML (eXtensible Markup Language) standard for describing and exchanging information about microarray experiments. It can describe microarray designs, microarray experiment designs, gene expression data and data analysis results. We describe RMAGEML, a new Bioconductor package that provides a link between cDNA microarray data stored in MAGE-ML format and the Bioconductor framework for preprocessing, visualization and analysis of microarray experiments. http://www.bioconductor.org. Open Source.

Initiation of follicular atresia: gene networks during early atresia in pig ovaries.

PubMed

Zhang, Jinbi; Liu, Yang; Yao, Wang; Li, Qifa; Liu, Hong-Lin; Pan, Zengxiang

2018-05-09

In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involve in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly up-regulated in early atretic with respect to healthy group and 308 were down-regulated. Similar expression trends were observed between microarray data and qRT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with 1) a crosstalk of cell apoptosis, autophagy, and ferroptosis rather than change of typical apoptosis markers, 2) dramatic shift of steroidogenic enzymes, 3) deficient glutathione metabolism, and 4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.

The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks.

PubMed

Adan, Aysun; Baran, Yusuf

2015-11-01

Fisetin and hesperetin, flavonoids from various plants, have several pharmaceutical activities including antioxidative, anti-inflammatory, and anticancer effects. However, studies elucidating the role and the mechanism(s) of action of fisetin and hesperetin in acute promyelocytic leukemia are absent. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by fisetin and hesperetin on human HL60 acute promyelocytic leukemia cells. The viability of HL60 cells was evaluated using the MTT assay, apoptosis by annexin V/propidium iodide (PI) staining and cell cycle distribution using flow cytometry, and changes in caspase-3 enzyme activity and mitochondrial transmembrane potential. Moreover, we performed whole-genome microarray gene expression analysis to reveal genes affected by fisetin and hesperetin that can be important for developing of future targeted therapy. Based on data obtained from microarray analysis, we also described biological networks modulated after fisetin and hesperetin treatment by KEGG and IPA analysis. Fisetin and hesperetin treatment showed a concentration- and time-dependent inhibition of proliferation and induced G2/M arrest for both agents and G0/G1 arrest for hesperetin at only the highest concentrations. There was a disruption of mitochondrial membrane potential together with increased caspase-3 activity. Furthermore, fisetin- and hesperetin-triggered apoptosis was confirmed by annexin V/PI analysis. The microarray gene profiling analysis revealed some important biological pathways including mitogen-activated protein kinases (MAPK) and inhibitor of DNA binding (ID) signaling pathways altered by fisetin and hesperetin treatment as well as gave a list of genes modulated ≥2-fold involved in cell proliferation, cell division, and apoptosis. Altogether, data suggested that fisetin and hesperetin have anticancer properties and deserve further investigation.

Electrodialytic extraction of anionic pharmaceutical compounds from a single drop of whole blood using a supported liquid membrane.

PubMed

Imoto, Yurika; Nishiyama, Hiroka; Nakamura, Yukihide; Ohira, Shin-Ichi; Toda, Kei

2018-05-01

A method to introduce target analytes to a chromatograph from a single drop of whole blood was investigated for minimally invasive monitoring of anionic pharmaceuticals. In this work, salicylate and loxoprofen were examined as organic anions. A micro ion extractor (MIE) has been developed for extraction of inorganic trace anions from whole blood, but this device is not suitable for extraction of pharmaceuticals. In the present study, we improved and optimized the MIE device for organic anion extraction. Various supported liquid membranes were evaluated for use as the ion transfer membrane, with each membrane placed between a droplet sample (donor) and an acceptor solution. A supported liquid membrane of porous polypropylene impregnated with 1-butanol was selected. In addition, the methods for electric field creation and electrode contact were examined to improve the characteristics of the MIE device. The current and extraction time were also optimized. With the optimized method, salicylate and loxoprofen were successfully extracted from a single drop of whole blood. Changes in the concentrations of these pharmaceuticals in blood over time were monitored after administration. As only 25μL of whole blood was required for analysis, repeat measurements could be conducted to monitor changes in the concentrations. This MIE will be useful for monitoring pharmaceutical concentrations in blood. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative determination of opioids in whole blood using fully automated dried blood spot desorption coupled to on-line SPE-LC-MS/MS.

PubMed

Verplaetse, Ruth; Henion, Jack

2016-01-01

Opioids are well known, widely used painkillers. Increased stability of opioids in the dried blood spot (DBS) matrix compared to blood/plasma has been described. Other benefits provided by DBS techniques include point-of-care collection, less invasive micro sampling, more economical shipment, and convenient storage. Current methodology for analysis of micro whole blood samples for opioids is limited to the classical DBS workflow, including tedious manual punching of the DBS cards followed by extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. The goal of this study was to develop and validate a fully automated on-line sample preparation procedure for the analysis of DBS micro samples relevant to the detection of opioids in finger prick blood. To this end, automated flow-through elution of DBS cards was followed by on-line solid-phase extraction (SPE) and analysis by LC-MS/MS. Selective, sensitive, accurate, and reproducible quantitation of five representative opioids in human blood at sub-therapeutic, therapeutic, and toxic levels was achieved. The range of reliable response (R(2)  ≥0.997) was 1 to 500 ng/mL whole blood for morphine, codeine, oxycodone, hydrocodone; and 0.1 to 50 ng/mL for fentanyl. Inter-day, intra-day, and matrix inter-lot accuracy and precision was less than 15% (even at lower limits of quantitation (LLOQ) level). The method was successfully used to measure hydrocodone and its major metabolite norhydrocodone in incurred human samples. Our data support the enormous potential of DBS sampling and automated analysis for monitoring opioids as well as other pharmaceuticals in both anti-doping and pain management regimens. Copyright © 2015 John Wiley & Sons, Ltd.

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array.

PubMed

Hosokawa, Masahito; Asami, Marie; Yoshino, Tomoko; Tsujimura, Noriyuki; Takahashi, Masayuki; Nakasono, Satoshi; Tanaka, Tsuyoshi; Matsunaga, Tadashi

2013-02-15

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials. Copyright © 2012 Elsevier B.V. All rights reserved.

Glucose Meters: A Review of Technical Challenges to Obtaining Accurate Results

PubMed Central

Tonyushkina, Ksenia; Nichols, James H.

2009-01-01

Glucose meters are universally utilized in the management of hypoglycemic and hyperglycemic disorders in a variety of healthcare settings. Establishing the accuracy of glucose meters, however, is challenging. Glucose meters can only analyze whole blood, and glucose is unstable in whole blood. Technical accuracy is defined as the closeness of agreement between a test result and the true value of that analyte. Truth for glucose is analysis by isotope dilution mass spectrometry, and frozen serum standards analyzed by this method are available from the National Institute of Standards and Technology. Truth for whole blood has not been established, and cells must be separated from the whole blood matrix before analysis by a method like isotope dilution mass spectrometry. Serum cannot be analyzed by glucose meters, and isotope dilution mass spectrometry is not commonly available in most hospitals and diabetes clinics to evaluate glucose meter accuracy. Consensus standards recommend comparing whole blood analysis on a glucose meter against plasma/serum centrifuged from a capillary specimen and analyzed by a clinical laboratory comparative method. Yet capillary samples may not provide sufficient volume to test by both methods, and venous samples may be used as an alternative when differences between venous and capillary blood are considered. There are thus multiple complexities involved in defining technical accuracy and no clear consensus among standards agencies and professional societies on accuracy criteria. Clinicians, however, are more concerned with clinical agreement of the glucose meter with a serum/plasma laboratory result. Acceptance criteria for clinical agreement vary across the range of glucose concentrations and depend on how the result will be used in screening or management of the patient. A variety of factors can affect glucose meter results, including operator technique, environmental exposure, and patient factors, such as medication, oxygen therapy, anemia, hypotension, and other disease states. This article reviews the challenges involved in obtaining accurate glucose meter results. PMID:20144348

Determination of trace amounts of cobalt in blood

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

Godlewska, B.; Hulanicki, A.; Abou-Shakra, F.R.

1994-11-01

The analysis of cobalt in whole blood and blood fractions has been carried out using three different analytical techniques namely, electrothermal atomic absorption spectrometry, inductively coupled plasma mass spectrometry and cathodic stripping voltammetry. This study showed that inductively coupled plasma mass spectrometry was the better equipped technique for conducting such analyses due to its low detection limits and wide linear dynamic range. The results ranged between 0.7 - 2.62 {mu}g/l for plasma, 1.02 - 2.31 {mu}g/l for serum, and 0.66 - 1.28 {mu}g/l for whole blood. The introduction of different forms of cobalt to Wistar rats resulted in a differingmore » distribution of the element between serum and whole blood. This observation suggests that there are at least two modes of Co uptake and transport depending on the administered or taken chemical form.« less

Advances in cell-free protein array methods.

PubMed

Yu, Xiaobo; Petritis, Brianne; Duan, Hu; Xu, Danke; LaBaer, Joshua

2018-01-01

Cell-free protein microarrays represent a special form of protein microarray which display proteins made fresh at the time of the experiment, avoiding storage and denaturation. They have been used increasingly in basic and translational research over the past decade to study protein-protein interactions, the pathogen-host relationship, post-translational modifications, and antibody biomarkers of different human diseases. Their role in the first blood-based diagnostic test for early stage breast cancer highlights their value in managing human health. Cell-free protein microarrays will continue to evolve to become widespread tools for research and clinical management. Areas covered: We review the advantages and disadvantages of different cell-free protein arrays, with an emphasis on the methods that have been studied in the last five years. We also discuss the applications of each microarray method. Expert commentary: Given the growing roles and impact of cell-free protein microarrays in research and medicine, we discuss: 1) the current technical and practical limitations of cell-free protein microarrays; 2) the biomarker discovery and verification pipeline using protein microarrays; and 3) how cell-free protein microarrays will advance over the next five years, both in their technology and applications.

Analysis of cyanide in whole blood of dosed cathartids

USGS Publications Warehouse

Krynitsky, A.J.; Wiemeyer, Stanley N.; Hill, E.F.; Carpenter, J.W.

1986-01-01

A gas-liquid chromatographic method was modified to quantify both unmetabolized ('free') and metabolized ('bound', i.e., thiocyanates) cyanides. The methods for both are efficient and sensitive to 0.05 ppm. Repeated freezing and thawing of whole blood from treated cathartids caused an initial increase in free cyanide concentrations, followed by a gradual decline to a plateau. Bound cyanide concentrations declined after repeated freezing and thawing.

Growth hormone regulation of metabolic gene expression in muscle: a microarray study in hypopituitary men.

PubMed

Sjögren, Klara; Leung, Kin-Chuen; Kaplan, Warren; Gardiner-Garden, Margaret; Gibney, James; Ho, Ken K Y

2007-07-01

Muscle is a target of growth hormone (GH) action and a major contributor to whole body metabolism. Little is known about how GH regulates metabolic processes in muscle or the extent to which muscle contributes to changes in whole body substrate metabolism during GH treatment. To identify GH-responsive genes that regulate substrate metabolism in muscle, we studied six hypopituitary men who underwent whole body metabolic measurement and skeletal muscle biopsies before and after 2 wk of GH treatment (0.5 mg/day). Transcript profiles of four subjects were analyzed using Affymetrix GeneChips. Serum insulin-like growth factor I (IGF-I) and procollagens I and III were measured by RIA. GH increased serum IGF-I and procollagens I and III, enhanced whole body lipid oxidation, reduced carbohydrate oxidation, and stimulated protein synthesis. It induced gene expression of IGF-I and collagens in muscle. GH reduced expression of several enzymes regulating lipid oxidation and energy production. It reduced calpain 3, increased ribosomal protein L38 expression, and displayed mixed effects on genes encoding myofibrillar proteins. It increased expression of circadian gene CLOCK, and reduced that of PERIOD. In summary, GH exerted concordant effects on muscle expression and blood levels of IGF-I and collagens. It induced changes in genes regulating protein metabolism in parallel with a whole body anabolic effect. The discordance between muscle gene expression profiles and metabolic responses suggests that muscle is unlikely to contribute to GH-induced stimulation of whole body energy and lipid metabolism. GH may regulate circadian function in skeletal muscle by modulating circadian gene expression with possible metabolic consequences.

Widespread activation of immunity and pro-inflammatory programs in peripheral blood leukocytes of HIV-infected patients with impaired lung gas exchange.

PubMed

Crothers, Kristina; Petrache, Irina; Wongtrakool, Cherry; Lee, Patty J; Schnapp, Lynn M; Gharib, Sina A

2016-04-01

HIV infection is associated with impaired lung gas transfer as indicated by a low diffusing capacity (DLCO), but the mechanisms are not well understood. We hypothesized that HIV-associated gas exchange impairment is indicative of system-wide perturbations that could be reflected by alterations in peripheral blood leukocyte (PBL) gene expression. Forty HIV-infected (HIV(+)) and uninfected (HIV(-)) men with preserved versus low DLCO were enrolled. All subjects were current smokers and those with acute illness, lung diseases other than COPD or asthma were excluded. Total RNA was extracted from PBLs and hybridized to whole-genome microarrays. Gene set enrichment analysis (GSEA) was performed between HIV(+) versus HIV(-) subjects with preserved DLCO and those with low DLCO to identify differentially activated pathways. Using pathway-based analyses, we found that in subjects with preserved DLCO, HIV infection is associated with activation of processes involved in immunity, cell cycle, and apoptosis. Applying a similar analysis to subjects with low DLCO, we identified a much broader repertoire of pro-inflammatory and immune-related pathways in HIV(+) patients relative to HIV(-) subjects, with up-regulation of multiple interleukin pathways, interferon signaling, and toll-like receptor signaling. We confirmed elevated circulating levels of IL-6 in HIV(+) patients with low DLCO relative to the other groups. Our findings reveal that PBLs of subjects with HIV infection and low DLCO are distinguished by widespread enrichment of immuno-inflammatory programs. Activation of these pathways may alter the biology of circulating leukocytes and play a role in the pathogenesis of HIV-associated gas exchange impairment. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

A multiscale, model-based analysis of the multi-tissue interplay underlying blood glucose regulation in type I diabetes.

PubMed

Wadehn, Federico; Schaller, Stephan; Eissing, Thomas; Krauss, Markus; Kupfer, Lars

2016-08-01

A multiscale model for blood glucose regulation in diabetes type I patients is constructed by integrating detailed metabolic network models for fat, liver and muscle cells into a whole body physiologically-based pharmacokinetic/pharmacodynamic (pBPK/PD) model. The blood glucose regulation PBPK/PD model simulates the distribution and metabolization of glucose, insulin and glucagon on an organ and whole body level. The genome-scale metabolic networks in contrast describe intracellular reactions. The developed multiscale model is fitted to insulin, glucagon and glucose measurements of a 48h clinical trial featuring 6 subjects and is subsequently used to simulate (in silico) the influence of geneknockouts and drug-induced enzyme inhibitions on whole body blood glucose levels. Simulations of diabetes associated gene knockouts and impaired cellular glucose metabolism, resulted in elevated whole body blood-glucose levels, but also in a metabolic shift within the cell's reaction network. Such multiscale models have the potential to be employed in the exploration of novel drug-targets or to be integrated into control algorithms for artificial pancreas systems.

The Longhorn Array Database (LAD): An Open-Source, MIAME compliant implementation of the Stanford Microarray Database (SMD)

PubMed Central

Killion, Patrick J; Sherlock, Gavin; Iyer, Vishwanath R

2003-01-01

Background The power of microarray analysis can be realized only if data is systematically archived and linked to biological annotations as well as analysis algorithms. Description The Longhorn Array Database (LAD) is a MIAME compliant microarray database that operates on PostgreSQL and Linux. It is a fully open source version of the Stanford Microarray Database (SMD), one of the largest microarray databases. LAD is available at Conclusions Our development of LAD provides a simple, free, open, reliable and proven solution for storage and analysis of two-color microarray data. PMID:12930545

Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

PubMed Central

Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

2005-01-01

The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

Recurrent pregnancy loss evaluation combined with 24-chromosome microarray of miscarriage tissue provides a probable or definite cause of pregnancy loss in over 90% of patients.

PubMed

Popescu, F; Jaslow, C R; Kutteh, W H

2018-04-01

Will the addition of 24-chromosome microarray analysis on miscarriage tissue combined with the standard American Society for Reproductive Medicine (ASRM) evaluation for recurrent miscarriage explain most losses? Over 90% of patients with recurrent pregnancy loss (RPL) will have a probable or definitive cause identified when combining genetic testing on miscarriage tissue with the standard ASRM evaluation for recurrent miscarriage. RPL is estimated to occur in 2-4% of reproductive age couples. A probable cause can be identified in approximately 50% of patients after an ASRM recommended workup including an evaluation for parental chromosomal abnormalities, congenital and acquired uterine anomalies, endocrine imbalances and autoimmune factors including antiphospholipid syndrome. Single-center, prospective cohort study that included 100 patients seen in a private RPL clinic from 2014 to 2017. All 100 women had two or more pregnancy losses, a complete evaluation for RPL as defined by the ASRM, and miscarriage tissue evaluated by 24-chromosome microarray analysis after their second or subsequent miscarriage. Frequencies of abnormal results for evidence-based diagnostic tests considered definite or probable causes of RPL (karyotyping for parental chromosomal abnormalities, and 24-chromosome microarray evaluation for products of conception (POC); pelvic sonohysterography, hysterosalpingogram, or hysteroscopy for uterine anomalies; immunological tests for lupus anticoagulant and anticardiolipin antibodies; and blood tests for thyroid stimulating hormone (TSH), prolactin and hemoglobin A1c) were evaluated. We excluded cases where there was maternal cell contamination of the miscarriage tissue or if the ASRM evaluation was incomplete. A cost analysis for the evaluation of RPL was conducted to determine whether a proposed procedure of 24-chromome microarray evaluation followed by an ASRM RPL workup (for those RPL patients who had a normal 24-chromosome microarray evaluation) was more cost-efficient than conducting ASRM RPL workups on RPL patients followed by 24-chromosome microarray analysis (for those RPL patients who had a normal RPL workup). A definite or probable cause of pregnancy loss was identified in the vast majority (95/100; 95%) of RPL patients when a 24-chromosome pair microarray evaluation of POC testing is combined with the standard ASRM RPL workup evaluation at the time of the second or subsequent loss. The ASRM RPL workup identified an abnormality and a probable explanation for pregnancy loss in only 45/100 or 45% of all patients. A definite abnormality was identified in 67/100 patients or 67% when initial testing was performed using 24-chromosome microarray analyses on the miscarriage tissue. Only 5/100 (5%) patients, who had a euploid loss and a normal ASRM RPL workup, had a pregnancy loss without a probable or definitive cause identified. All other losses were explained by an abnormal 24-chromosome microarray analysis of the miscarriage tissue, an abnormal finding of the RPL workup, or a combination of both. Results from the cost analysis indicated that an initial approach of using a 24-chromosome microarray analysis on miscarriage tissue resulted in a 50% savings in cost to the health care system and to the patient. This is a single-center study on a small group of well-characterized women with RPL. There was an incomplete follow-up on subsequent pregnancy outcomes after evaluation, however this should not affect our principal results. The maternal age of patients varied from 26 to 45 years old. More aneuploid pregnancy losses would be expected in older women, particularly over the age of 35 years old. Evaluation of POC using 24-chromosome microarray analysis adds significantly to the ASRM recommended evaluation of RPL. Genetic evaluation on miscarriage tissue obtained at the time of the second and subsequent pregnancy losses should be offered to all couples with two or more consecutive pregnancy losses. The combination of a genetic evaluation on miscarriage tissue with an evidence-based evaluation for RPL will identify a probable or definitive cause in over 90% of miscarriages. No funding was received for this study and there are no conflicts of interest to declare. Not applicable.

Microarray expression profiling in adhesion and normal peritoneal tissues.

PubMed

Ambler, Dana R; Golden, Alicia M; Gell, Jennifer S; Saed, Ghassan M; Carey, David J; Diamond, Michael P

2012-05-01

To identify molecular markers associated with adhesion and normal peritoneal tissue using microarray expression profiling. Comparative study. University hospital. Five premenopausal women. Adhesion and normal peritoneal tissue samples were obtained from premenopausal women. Ribonucleic acid was extracted using standard protocols and processed for hybridization to Affymetrix Whole Transcript Human Gene Expression Chips. Microarray data were obtained from five different patients, each with adhesion tissue and normal peritoneal samples. Real-time polymerase chain reaction was performed for confirmation using standard protocols. Gene expression in postoperative adhesion and normal peritoneal tissues. A total of 1,263 genes were differentially expressed between adhesion and normal tissues. One hundred seventy-three genes were found to be up-regulated and 56 genes were down-regulated in the adhesion tissues compared with normal peritoneal tissues. The genes were sorted into functional categories according to Gene Ontology annotations. Twenty-six up-regulated genes and 11 down-regulated genes were identified with functions potentially relevant to the pathophysiology of postoperative adhesions. We evaluated and confirmed expression of 12 of these specific genes via polymerase chain reaction. The pathogenesis, natural history, and optimal treatment of postoperative adhesive disease remains unanswered. Microarray analysis of adhesions identified specific genes with increased and decreased expression when compared with normal peritoneum. Knowledge of these genes and ontologic pathways with altered expression provide targets for new therapies to treat patients who have or are at risk for postoperative adhesions. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

PubMed Central

2011-01-01

Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. PMID:21851640

Genome-wide transcription analysis of histidine-related cataract in Atlantic salmon (Salmo salar L)

PubMed Central

Waagbø, Rune; Breck, Olav; Stavrum, Anne-Kristin; Petersen, Kjell; Olsvik, Pål A.

2009-01-01

Purpose Elevated levels of dietary histidine have previously been shown to prevent or mitigate cataract formation in farmed Atlantic salmon (Salmo salar L). The aim of this study was to shed light on the mechanisms by which histidine acts. Applying microarray analysis to the lens transcriptome, we screened for differentially expressed genes in search for a model explaining cataract development in Atlantic salmon and possible markers for early cataract diagnosis. Methods Adult Atlantic salmon (1.7 kg) were fed three standard commercial salmon diets only differing in the histidine content (9, 13, and 17 g histidine/kg diet) for four months. Individual cataract scores for both eyes were assessed by slit-lamp biomicroscopy. Lens N-acetyl histidine contents were measured by high performance liquid chromatography (HPLC). Total RNA extracted from whole lenses was analyzed using the GRASP 16K salmonid microarray. The microarray data were analyzed using J-Express Pro 2.7 and validated by quantitative real-time polymerase chain reaction (qRT–PCR). Results Fish developed cataracts with different severity in response to dietary histidine levels. Lens N-acetyl histidine contents reflected the dietary histidine levels and were negatively correlated to cataract scores. Significance analysis of microarrays (SAM) revealed 248 significantly up-regulated transcripts and 266 significantly down-regulated transcripts in fish that were fed a low level of histidine compared to fish fed a higher histidine level. Among the differentially expressed transcripts were metallothionein A and B as well as transcripts involved in lipid metabolism, carbohydrate metabolism, regulation of ion homeostasis, and protein degradation. Hierarchical clustering and correspondence analysis plot confirmed differences in gene expression between the feeding groups. The differentially expressed genes could be categorized as “early” and “late” responsive according to their expression pattern relative to progression in cataract formation. Conclusions Dietary histidine regimes affected cataract formation and lens gene expression in adult Atlantic salmon. Regulated transcripts selected from the results of this genome-wide transcription analysis might be used as possible biological markers for cataract development in Atlantic salmon. PMID:19597568

Microarray analysis identifies candidate genes for key roles in coral development

PubMed Central

Grasso, Lauretta C; Maindonald, John; Rudd, Stephen; Hayward, David C; Saint, Robert; Miller, David J; Ball, Eldon E

2008-01-01

Background Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired. Results Of 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization. Conclusion This study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemone Nematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease. PMID:19014561

Identification of genes associated with the long-gut-persistence phenotype of the probiotic Lactobacillus johnsonii strain NCC533 using a combination of genomics and transcriptome analysis.

PubMed

Denou, Emmanuel; Pridmore, Raymond David; Berger, Bernard; Panoff, Jean-Michel; Arigoni, Fabrizio; Brüssow, Harald

2008-05-01

Lactobacillus johnsonii strains NCC533 and ATCC 33200 (the type strain of this species) differed significantly in gut residence time (12 versus 5 days) after oral feeding to mice. Genes affecting the long gut residence time of the probiotic strain NCC533 were targeted for analysis. We hypothesized that genes specific for this strain, which are expressed during passage of the bacterium through the gut, affect the phenotype. When the DNA of the type strain was hybridized against a microarray of the sequenced NCC533 strain, we identified 233 genes that were specific for the long-gut-persistence isolate. Whole-genome transcription analysis of the NCC533 strain using the microarray format identified 174 genes that were strongly and consistently expressed in the jejunum of mice monocolonized with this strain. Fusion of the two microarray data sets identified three gene loci that were both expressed in vivo and specific to the long-gut-persistence isolate. The identified genes included LJ1027 and LJ1028, two glycosyltransferase genes in the exopolysaccharide synthesis operon; LJ1654 to LJ1656, encoding a sugar phosphotransferase system (PTS) transporter annotated as mannose PTS; and LJ1680, whose product shares 30% amino acid identity with immunoglobulin A proteases from pathogenic bacteria. Knockout mutants were tested in vivo. The experiments revealed that deletion of LJ1654 to LJ1656 and LJ1680 decreased the gut residence time, while a mutant with a deleted exopolysaccharide biosynthesis cluster had a slightly increased residence time.

Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification.

PubMed

Wolf, Dieter A; Bähler, Jürg; Wise, Jo Ann

2017-04-03

Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis. © 2017 Cold Spring Harbor Laboratory Press.

21 CFR 864.7140 - Activated whole blood clotting time tests.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class II...

21 CFR 864.7140 - Activated whole blood clotting time tests.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class II...

21 CFR 864.7140 - Activated whole blood clotting time tests.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class II...

21 CFR 864.7140 - Activated whole blood clotting time tests.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class II...

21 CFR 864.7140 - Activated whole blood clotting time tests.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Activated whole blood clotting time tests. 864....7140 Activated whole blood clotting time tests. (a) Identification. An activated whole blood clotting... pulmonary embolism by measuring the coagulation time of whole blood. (b) Classification. Class II...

Combined analysis of whole human blood parameters by Raman spectroscopy and spectral-domain low-coherence interferometry

NASA Astrophysics Data System (ADS)

Gnyba, M.; Wróbel, M. S.; Karpienko, K.; Milewska, D.; Jedrzejewska-Szczerska, M.

2015-07-01

In this article the simultaneous investigation of blood parameters by complementary optical methods, Raman spectroscopy and spectral-domain low-coherence interferometry, is presented. Thus, the mutual relationship between chemical and physical properties may be investigated, because low-coherence interferometry measures optical properties of the investigated object, while Raman spectroscopy gives information about its molecular composition. A series of in-vitro measurements were carried out to assess sufficient accuracy for monitoring of blood parameters. A vast number of blood samples with various hematological parameters, collected from different donors, were measured in order to achieve a statistical significance of results and validation of the methods. Preliminary results indicate the benefits in combination of presented complementary methods and form the basis for development of a multimodal system for rapid and accurate optical determination of selected parameters in whole human blood. Future development of optical systems and multivariate calibration models are planned to extend the number of detected blood parameters and provide a robust quantitative multi-component analysis.

On-chip wavelength multiplexed detection of cancer DNA biomarkers in blood

PubMed Central

Cai, H.; Stott, M. A.; Ozcelik, D.; Parks, J. W.; Hawkins, A. R.; Schmidt, H.

2016-01-01

We have developed an optofluidic analysis system that processes biomolecular samples starting from whole blood and then analyzes and identifies multiple targets on a silicon-based molecular detection platform. We demonstrate blood filtration, sample extraction, target enrichment, and fluorescent labeling using programmable microfluidic circuits. We detect and identify multiple targets using a spectral multiplexing technique based on wavelength-dependent multi-spot excitation on an antiresonant reflecting optical waveguide chip. Specifically, we extract two types of melanoma biomarkers, mutated cell-free nucleic acids —BRAFV600E and NRAS, from whole blood. We detect and identify these two targets simultaneously using the spectral multiplexing approach with up to a 96% success rate. These results point the way toward a full front-to-back chip-based optofluidic compact system for high-performance analysis of complex biological samples. PMID:28058082

Plasma Homocysteine and Asymmetrical Dimethyl-l-Arginine (ADMA) and Whole Blood DNA Methylation in Early and Neovascular Age-Related Macular Degeneration: A Pilot Study.

PubMed

Pinna, Antonio; Zinellu, Angelo; Tendas, Donatella; Blasetti, Francesco; Carru, Ciriaco; Castiglia, Paolo

2016-01-01

To compare the plasma levels of homocysteine and asymmetrical dimethyl-l-arginine (ADMA) and the degree of whole blood DNA methylation in patients with early and neovascular age-related macular degeneration (AMD) and in controls without maculopathy of any sort. This observational case-control pilot study included 39 early AMD patients, 27 neovascular AMD patients and 132 sex- and age-matched controls without maculopathy. Plasma homocysteine and ADMA concentrations and the degree of whole blood DNA methylation were measured. Quantitative variables were compared by Student's t-test or Mann-Whitney test. Logistic regression models were used to investigate the significance of the association between early or wet AMD and some variables. There were no significant differences in mean plasma homocysteine and ADMA concentrations and in the degree of whole blood DNA methylation between patients with early or neovascular AMD and their controls. Similarly, logistic regression analysis disclosed that plasma homocysteine and ADMA levels were not associated with an increased risk for early or neovascular AMD. We failed to demonstrate an association between early or neovascular AMD and increased plasma homocysteine and/or ADMA. Results also suggest that the degree of whole blood DNA methylation is not a marker of AMD.

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia

PubMed Central

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-01-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre-processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)-gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein-DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid-repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF-pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF-gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA-box binding protein associated factor 1 and CCCTC-binding factor, which may be potential therapeutic targets of AML. PMID:28498449

Bioinformatic analysis of the effects and mechanisms of decitabine and cytarabine on acute myeloid leukemia.

PubMed

Zhou, Shiyong; Liu, Pengfei; Zhang, Huilai

2017-07-01

Acute myeloid leukemia (AML) is a frequently occurring malignant disease of the blood and may result from a variety of genetic disorders. The present study aimed to identify the underlying mechanisms associated with the therapeutic effects of decitabine and cytarabine on AML, using microarray analysis. The microarray datasets GSE40442 and GSE40870 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine via the Linear Models for Microarray Data package, following data pre‑processing. Gene Ontology (GO) analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Analysis Discovery. Genes corresponding to the differentially methylated sites were obtained using the annotation package of the methylation microarray platform. The overlapping genes were identified, which exhibited the opposite variation trend between gene expression and DNA methylation. Important transcription factor (TF)‑gene pairs were screened out, and a regulated network subsequently constructed. A total of 190 DEGs and 540 differentially methylated sites were identified in AML cells treated with decitabine compared with those treated with cytarabine. A total of 36 GO terms of DEGs were enriched, including nucleosomes, protein‑DNA complexes and the nucleosome assembly. The 540 differentially methylated sites were located on 240 genes, including the acid‑repeat containing protein (ACRC) gene that was additionally differentially expressed. In addition, 60 TF pairs and overlapped methylated sites, and 140 TF‑pairs and DEGs were screened out. The regulated network included 68 nodes and 140 TF‑gene pairs. The present study identified various genes including ACRC and proliferating cell nuclear antigen, in addition to various TFs, including TATA‑box binding protein associated factor 1 and CCCTC‑binding factor, which may be potential therapeutic targets of AML.

Cell cycle, apoptosis, cellular uptake and whole-transcriptome microarray gene expression analysis of HeLa cells treated with a ruthenium(II)-arene complex with an isoquinoline-3-carboxylic acid ligand.

PubMed

Jovanović, Katarina K; Tanić, Miljana; Ivanović, Ivanka; Gligorijević, Nevenka; Dojčinović, Biljana P; Radulović, Siniša

2016-10-01

Ruthenium(II)-arene complexes are promising drug candidates for the therapy of solid tumors. In previous work, seven new compounds of the general formula [Ru(η 6 -p-cymene)(L 1-7 )Cl] were synthesized and characterized, of which the complex with L=isoquinoline-3-carboxylic acid (RuT 7 ) was two times as active on HeLa cells compared to normal cell line MRC-5, as indicated by IC 50 values determined after 48h of incubation (45.4±3.0 vs. 84.2±5.7μM, respectively). In the present study, cell cycle analysis of HeLa cells treated with RuT 7 showed S phase arrest and an increase in sub-G1 population. The apoptotic potential of the title compound was confirmed with the Annexin V-FITC/PI assay together with a morphological evaluation of cells using fluorescent microscopy. Analysis of the intracellular accumulation of ruthenium showed 8.9ng Ru/10 6 cells after 6h of incubation. To gain further insight in the molecular mechanism of action of RuT 7 on HeLa cells, a whole-transcriptome microarray gene expression analysis was performed. Analysis of functional categories and signaling and biochemical pathways associated with the response of HeLa cells to treatment with RuT 7 showed that it leads the cells through the intrinsic (mitochondrial) apoptotic pathway, via indirect DNA damage due to the action of reactive oxygen species, and through direct DNA binding of RuT 7 . Statistical analysis for enrichment of gene sets associated with known drug-induced toxicities identified fewer associated toxicity profiles in RuT 7 -treated cells compared to cisplatin treatment. Altogether these results provide the basis for further development of RuT 7 in animal and pre-clinical studies as a potential drug candidate. Copyright © 2016 Elsevier Inc. All rights reserved.

Flow microfluorometric analysis of phagocyte degranulation in bacteria-infected whole human blood cell cultures

NASA Astrophysics Data System (ADS)

Kravtsov, Alexander L.; Bobyleva, Elena V.; Grebenyukova, Tatyana P.; Kuznetsov, Oleg S.; Kulyash, Youri V.

2002-07-01

A quantitative flow microfluorometric method was used to study the intensity of human blood phagocyte degranulation in response to viable staphylococcus aureus or Yersinia pestis cells. Microorganisms were added directly to defibrinated whole blood. Uninfected and infected blood samples were incubated at 37 degrees C to 8 h. The results were recorded in dynamics after the staining of whole blood with acridine orange solution. Lymphocytes with a low azurophilic granule per cell content were discriminated from phagocytes by the measurement of single cell red cytoplasmic granule fluorescence. 30,000 cells in each sample were examined. S. aureus cells caused a dose-dependent decrease in the number of phagocytes having a high red cytoplasmic fluorescence intensity and a corresponding increase in the weakly fluorescence cell population. In the presence of an initial S. aureus-to-phagocyte ratio more than 1:1, degranulation was measured after 3 h of incubation and to 8 h the percentage of degranulated phagocytes was at least 100 percent Y. pestis cells grown for 48 h at 28 degrees C caused at same condition as the degranulation only about 50 percent of cells. Y.pestis EV cells preincubated in broth for 12 h at 37 degrees C did no stimulate the phahocyte degranulation. The results of these studies suggest that analysis of cell populations via flow microfluorimeter technology may be a powerful tool in analysis bacterial infection.

Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

PubMed

Adan, Aysun; Baran, Yusuf

2016-05-01

Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

Unique Proteins Expressed by Blood Vessels in Skeletal Sites Colonized by Breast Cancer Cells

DTIC Science & Technology

2005-08-01

labeled acetylated LDL at an accelerated rate (3). After one week in culture BVECs and MVECs were harvested. Total RNA was extracted from both cell...bones where breast cancer cells tend to lodge, as compared to the vasculature of the central marrow cavity. We have found differences in RNA expression...by microarray analysis. The bone-derived vasculature expresses five RNA messages in greater abundance (2-fold or more) than the marrow-derived

A Transcriptional Signature of Fatigue Derived from Patients with Primary Sjögren’s Syndrome

PubMed Central

James, Katherine; Al-Ali, Shereen; Tarn, Jessica; Cockell, Simon J.; Gillespie, Colin S.; Hindmarsh, Victoria; Locke, James; Mitchell, Sheryl; Lendrem, Dennis; Bowman, Simon; Price, Elizabeth; Pease, Colin T.; Emery, Paul; Lanyon, Peter; Hunter, John A.; Gupta, Monica; Bombardieri, Michele; Sutcliffe, Nurhan; Pitzalis, Costantino; McLaren, John; Cooper, Annie; Regan, Marian; Giles, Ian; Isenberg, David; Saravanan, Vadivelu; Coady, David; Dasgupta, Bhaskar; McHugh, Neil; Young-Min, Steven; Moots, Robert; Gendi, Nagui; Akil, Mohammed; Griffiths, Bridget; Wipat, Anil; Newton, Julia; Jones, David E.; Isaacs, John; Hallinan, Jennifer; Ng, Wan-Fai

2015-01-01

Background Fatigue is a debilitating condition with a significant impact on patients’ quality of life. Fatigue is frequently reported by patients suffering from primary Sjögren’s Syndrome (pSS), a chronic autoimmune condition characterised by dryness of the eyes and the mouth. However, although fatigue is common in pSS, it does not manifest in all sufferers, providing an excellent model with which to explore the potential underpinning biological mechanisms. Methods Whole blood samples from 133 fully-phenotyped pSS patients stratified for the presence of fatigue, collected by the UK primary Sjögren’s Syndrome Registry, were used for whole genome microarray. The resulting data were analysed both on a gene by gene basis and using pre-defined groups of genes. Finally, gene set enrichment analysis (GSEA) was used as a feature selection technique for input into a support vector machine (SVM) classifier. Classification was assessed using area under curve (AUC) of receiver operator characteristic and standard error of Wilcoxon statistic, SE(W). Results Although no genes were individually found to be associated with fatigue, 19 metabolic pathways were enriched in the high fatigue patient group using GSEA. Analysis revealed that these enrichments arose from the presence of a subset of 55 genes. A radial kernel SVM classifier with this subset of genes as input displayed significantly improved performance over classifiers using all pathway genes as input. The classifiers had AUCs of 0.866 (SE(W) 0.002) and 0.525 (SE(W) 0.006), respectively. Conclusions Systematic analysis of gene expression data from pSS patients discordant for fatigue identified 55 genes which are predictive of fatigue level using SVM classification. This list represents the first step in understanding the underlying pathophysiological mechanisms of fatigue in patients with pSS. PMID:26694930

The Microarray Revolution: Perspectives from Educators

ERIC Educational Resources Information Center

Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

2004-01-01

In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research,…

Detection of AR-V7 mRNA in whole blood may not predict the effectiveness of novel endocrine drugs for castration-resistant prostate cancer.

PubMed

Takeuchi, Takumi; Okuno, Yumiko; Hattori-Kato, Mami; Zaitsu, Masayoshi; Mikami, Koji

2016-01-01

A splice variant of androgen receptor (AR), AR-V7, lacks in androgen-binding portion and leads to aggressive cancer characteristics. Reverse transcription-polymerase chain reactions (PCRs) and subsequent nested PCRs for the amplification of AR-V7 and prostate-specific antigen (PSA) transcripts were done for whole blood of patients with prostate cancer and male controls. With primary reverse transcription PCRs, AR-V7 and PSA were detected in 4.5% and 4.7% of prostate cancer, respectively. With nested PCRs, AR-V7 messenger RNA (mRNA) was positive in 43.8% of castration-sensitive prostate cancer and 48.1% of castration-resistant prostate cancer (CRPC), while PSA mRNA was positive in 6.3% of castration-sensitive prostate cancer and 18.5% of CRPC. Whole-blood samples of controls showed AR-V7 mRNA expression by nested PCR. Based on multivariate analysis, expression of AR-V7 mRNA in whole blood was not significantly correlated with clinical parameters and PSA mRNA in blood, while univariate analysis showed a correlation between AR-V7 mRNA and metastasis at initial diagnosis. Detection of AR-V7 mRNA did not predict the reduction of serum PSA in patients with CRPC following abiraterone and enzalutamide administration. In conclusion, AR-V7 mRNA expression in normal hematopoietic cells may have annihilated the manifestation of aggressiveness of prostate cancer and the prediction of the effectiveness of abiraterone and enzalutamide by the assessment of AR-V7 mRNA in blood.

Highly sensitive measurement of whole blood chromium by inductively coupled plasma mass spectrometry.

PubMed

Cieslak, Wendy; Pap, Kathleen; Bunch, Dustin R; Reineks, Edmunds; Jackson, Raymond; Steinle, Roxanne; Wang, Sihe

2013-02-01

Chromium (Cr), a trace metal element, is implicated in diabetes and cardiovascular disease. A hypochromic state has been associated with poor blood glucose control and unfavorable lipid metabolism. Sensitive and accurate measurement of blood chromium is very important to assess the chromium nutritional status. However, interferents in biological matrices and contamination make the sensitive analysis challenging. The primary goal of this study was to develop a highly sensitive method for quantification of total Cr in whole blood by inductively coupled plasma mass spectrometry (ICP-MS) and to validate the reference interval in a local healthy population. This method was developed on an ICP-MS with a collision/reaction cell. Interference was minimized using both kinetic energy discrimination between the quadrupole and hexapole and a selective collision gas (helium). Reference interval was validated in whole blood samples (n=51) collected in trace element free EDTA tubes from healthy adults (12 males, 39 females), aged 19-64 years (38.8±12.6), after a minimum of 8 h fasting. Blood samples were aliquoted into cryogenic vials and stored at -70 °C until analysis. The assay linearity was 3.42 to 1446.59 nmol/L with an accuracy of 87.7 to 99.8%. The high sensitivity was achieved by minimization of interference through selective kinetic energy discrimination and selective collision using helium. The reference interval for total Cr using a non-parametric method was verified to be 3.92 to 7.48 nmol/L. This validated ICP-MS methodology is highly sensitive and selective for measuring total Cr in whole blood. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Published by Elsevier Inc. All rights reserved.

PGD and aneuploidy screening for 24 chromosomes: advantages and disadvantages of competing platforms.

PubMed

Bisignano, A; Wells, D; Harton, G; Munné, S

2011-12-01

Diagnosis of embryos for chromosome abnormalities, i.e. aneuploidy screening, has been invigorated by the introduction of microarray-based testing methods allowing analysis of 24 chromosomes in one test. Recent data have been suggestive of increased implantation and pregnancy rates following microarray testing. Preimplantation genetic diagnosis for infertility aims to test for gross chromosome changes with the hope that identification and transfer of normal embryos will improve IVF outcomes. Testing by some methods, specifically single-nucleotide polymorphism (SNP) microarrays, allow for more information and potential insight into parental origin of aneuploidy and uniparental disomy. The usefulness and validity of reporting this information is flawed. Numerous papers have shown that the majority of meiotic errors occur in the egg, while mitotic errors in the embryo affect parental chromosomes at random. Potential mistakes made in assigning an error as meiotic or mitotic may lead to erroneous reporting of results with medical consequences. This study's data suggest that the bioinformatic cleaning used to 'fix' the miscalls that plague single-cell whole-genome amplification provides little improvement in the quality of useful data. Based on the information available, SNP-based aneuploidy screening suffers from a number of serious issues that must be resolved. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

PubMed

Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

2016-01-28

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function. © 2016 by The American Society of Hematology.

New approach to 'top-and-bottom' whole blood separation using the multiunit TACSI WB system: quality of blood components.

PubMed

Lotens, A; Najdovski, T; Cellier, N; Ernotte, B; Lambermont, M; Rapaille, A

2014-10-01

TACSI whole blood system is designed to combine primary and secondary processing of six whole blood bags into plasma units, buffy coat and red blood cell concentrates. The aim of this study was to investigate the specifications and in vitro storage parameters of blood components compared with standard centrifugation and separation processing. Whole blood bags, collected in CRC kits, were treated on a TACSI whole blood system. They were compared with whole blood bags collected in Composelect kits. In addition to routine quality control analyses, conservation studies were performed on red blood cell concentrates for 42 days and on plasma for 6 months. Platelets pools with five buffy coats were also created, and cellular contamination was evaluated. Red blood cell concentrates produced from TACSI whole blood met European quality requirements. For white blood cell count, one individual result exceeded 1 × 10(6) cells/unit. All plasma units fell within specifications for residual cellular contamination and storage parameters. The performances of the TACSI whole blood system allow for the preparation of low volume buffy coats with a recovery of 90% of whole blood platelets. Haemoglobin losses in TACSI BC are smaller, but this did not result in higher haemoglobin content of red cells. These BC are suitable for the production of platelet concentrates. From these in vitro data, red blood cell concentrates produced using TACSI whole blood are suitable for clinical use with a quality at least equivalent to the control group. © 2014 International Society of Blood Transfusion.

Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study

PubMed Central

Joehanes, Roby; Johnson, Andrew D.; Barb, Jennifer J.; Raghavachari, Nalini; Liu, Poching; Woodhouse, Kimberly A.; O'Donnell, Christopher J.; Munson, Peter J.

2012-01-01

Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL. PMID:22045913

EMAAS: An extensible grid-based Rich Internet Application for microarray data analysis and management

PubMed Central

Barton, G; Abbott, J; Chiba, N; Huang, DW; Huang, Y; Krznaric, M; Mack-Smith, J; Saleem, A; Sherman, BT; Tiwari, B; Tomlinson, C; Aitman, T; Darlington, J; Game, L; Sternberg, MJE; Butcher, SA

2008-01-01

Background Microarray experimentation requires the application of complex analysis methods as well as the use of non-trivial computer technologies to manage the resultant large data sets. This, together with the proliferation of tools and techniques for microarray data analysis, makes it very challenging for a laboratory scientist to keep up-to-date with the latest developments in this field. Our aim was to develop a distributed e-support system for microarray data analysis and management. Results EMAAS (Extensible MicroArray Analysis System) is a multi-user rich internet application (RIA) providing simple, robust access to up-to-date resources for microarray data storage and analysis, combined with integrated tools to optimise real time user support and training. The system leverages the power of distributed computing to perform microarray analyses, and provides seamless access to resources located at various remote facilities. The EMAAS framework allows users to import microarray data from several sources to an underlying database, to pre-process, quality assess and analyse the data, to perform functional analyses, and to track data analysis steps, all through a single easy to use web portal. This interface offers distance support to users both in the form of video tutorials and via live screen feeds using the web conferencing tool EVO. A number of analysis packages, including R-Bioconductor and Affymetrix Power Tools have been integrated on the server side and are available programmatically through the Postgres-PLR library or on grid compute clusters. Integrated distributed resources include the functional annotation tool DAVID, GeneCards and the microarray data repositories GEO, CELSIUS and MiMiR. EMAAS currently supports analysis of Affymetrix 3' and Exon expression arrays, and the system is extensible to cater for other microarray and transcriptomic platforms. Conclusion EMAAS enables users to track and perform microarray data management and analysis tasks through a single easy-to-use web application. The system architecture is flexible and scalable to allow new array types, analysis algorithms and tools to be added with relative ease and to cope with large increases in data volume. PMID:19032776

An Introduction to MAMA (Meta-Analysis of MicroArray data) System.

PubMed

Zhang, Zhe; Fenstermacher, David

2005-01-01

Analyzing microarray data across multiple experiments has been proven advantageous. To support this kind of analysis, we are developing a software system called MAMA (Meta-Analysis of MicroArray data). MAMA utilizes a client-server architecture with a relational database on the server-side for the storage of microarray datasets collected from various resources. The client-side is an application running on the end user's computer that allows the user to manipulate microarray data and analytical results locally. MAMA implementation will integrate several analytical methods, including meta-analysis within an open-source framework offering other developers the flexibility to plug in additional statistical algorithms.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

PubMed

Ji, Allena Ji; Wang, Haixing; Ziso-Qejvanaj, Enida; Zheng, Kefei; Chung, Lee Lee; Foley, Timothy; Chuang, Wei-Lien; Richards, Susan; Sung, Crystal

2015-01-01

Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. The new approach greatly improves assay precision and accuracy.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood.

PubMed

van der Maten, Erika; de Jonge, Marien I; de Groot, Ronald; van der Flier, Michiel; Langereis, Jeroen D

2017-02-08

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood

PubMed Central

van der Maten, Erika; de Jonge, Marien I.; de Groot, Ronald; van der Flier, Michiel; Langereis, Jeroen D.

2017-01-01

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood. PMID:28176849

Radiation Fibrosis of the Vocal Fold: From Man to Mouse

PubMed Central

Johns, Michael M.; Kolachala, Vasantha; Berg, Eric; Muller, Susan; Creighton, Frances X.; Branski, Ryan C.

2013-01-01

Objectives To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis to ultimately develop both treatment and prevention paradigms. Design Translational study using archived human and fresh murine irradiated vocal fold tissue. Methods 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis were identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray as well as real-time transcriptional analyses was performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis. Results Human irradiated vocal folds demonstrated increased collagen transcription with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-β increased in the lamina propria. Conclusion Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability that occurs in patients following laryngeal irradiation. Irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis. PMID:23242839

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients

PubMed Central

Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the “neurotrophin-MAPK signaling pathway” was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment. PMID:28900628

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients.

PubMed

Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Ji, Lin-Dan

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the "neurotrophin-MAPK signaling pathway" was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment.

Transcriptomic Studies of the Effect of nod Gene-Inducing Molecules in Rhizobia: Different Weapons, One Purpose

PubMed Central

Jiménez-Guerrero, Irene; Acosta-Jurado, Sebastián; Navarro-Gómez, Pilar; López-Baena, Francisco Javier; Ollero, Francisco Javier

2017-01-01

Simultaneous quantification of transcripts of the whole bacterial genome allows the analysis of the global transcriptional response under changing conditions. RNA-seq and microarrays are the most used techniques to measure these transcriptomic changes, and both complement each other in transcriptome profiling. In this review, we exhaustively compiled the symbiosis-related transcriptomic reports (microarrays and RNA sequencing) carried out hitherto in rhizobia. This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110, Rhizobium leguminosarum biovar viciae 3841, Rhizobium tropici CIAT 899, Sinorhizobium (=Ensifer) meliloti 1021 and S. fredii HH103, recognize inducing flavonoids, plant-exuded phenolic compounds that activate the biosynthesis and export of Nod factors (NF) in all analysed rhizobia. Interestingly, our global transcriptomic comparison also indicates that each rhizobial species possesses its own arsenal of molecular weapons accompanying the set of NF in order to establish a successful interaction with host legumes. PMID:29267254

MRI phenotypes with high neurodegeneration are associated with peripheral blood B-cell changes.

PubMed

Comabella, Manuel; Cantó, Ester; Nurtdinov, Ramil; Río, Jordi; Villar, Luisa M; Picón, Carmen; Castilló, Joaquín; Fissolo, Nicolás; Aymerich, Xavier; Auger, Cristina; Rovira, Alex; Montalban, Xavier

2016-01-15

Little is known about the mechanisms leading to neurodegeneration in multiple sclerosis (MS) and the role of peripheral blood cells in this neurodegenerative component. We aimed to correlate brain radiological phenotypes defined by high and low neurodegeneration with gene expression profiling of peripheral blood mononuclear cells (PBMC) from MS patients. Magnetic resonance imaging (MRI) scans from 64 patients with relapsing-remitting MS (RRMS) were classified into radiological phenotypes characterized by low (N = 27) and high (N = 37) neurodegeneration according to the number of contrast-enhancing lesions, the relative volume of non-enhancing black holes on T1-weighted images, and the brain parenchymal fraction. Gene expression profiling was determined in PBMC using microarrays, and validation of selected genes was performed by polymerase chain reaction (PCR). B-cell immunophenotyping was conducted by flow cytometry. Microarray analysis revealed the B-cell specific genes FCRL1, FCRL2, FCRL5 (Fc receptor-like 1, 2 and 5 respectively), and CD22 as the top differentially expressed genes between patients with high and low neurodegeneration. Levels for these genes were significantly down-regulated in PBMC from patients with MRI phenotypes characterized by high neurodegeneration and microarray findings were validated by PCR. In patients with high neurodegeneration, immunophenotyping showed a significant increase in the expression of the B-cell activation markers CD80 in naïve B cells (CD45+/CD19+/CD27-/IgD+), unswitched memory B cells (CD45+/CD19+/CD27+/IgD+), and switched memory B cells (CD45+/CD19+/CD27+/IgD-), and CD86 in naïve and switched memory B cells. These results suggest that RRMS patients with radiological phenotypes showing high neurodegeneration have changes in B cells characterized by down-regulation of B-cell-specific genes and increased activation status. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B cells and Idiotype Positive Marginal Zone B cells Before and After Immunization with Streptococcus pneumoniae 1

PubMed Central

Liu, Jiabin; Behrens, Timothy W.; Kearney, John F.

2014-01-01

Marginal Zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and Follicular (FO) B cells were sort-purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. 99 genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, idiotype positive and negative MZ B cells were sort-purified before (0 hour) or after (1 hour) i.v. immunization with heat killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up regulated or down regulated at 1 hour following immunization in the idiotype positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs. FO B cells and the specific regulation of gene expression in antigen-specific MZ B cells following interaction with antigen. PMID:18453586

Mapping autosomal recessive intellectual disability: combined microarray and exome sequencing identifies 26 novel candidate genes in 192 consanguineous families.

PubMed

Harripaul, R; Vasli, N; Mikhailov, A; Rafiq, M A; Mittal, K; Windpassinger, C; Sheikh, T I; Noor, A; Mahmood, H; Downey, S; Johnson, M; Vleuten, K; Bell, L; Ilyas, M; Khan, F S; Khan, V; Moradi, M; Ayaz, M; Naeem, F; Heidari, A; Ahmed, I; Ghadami, S; Agha, Z; Zeinali, S; Qamar, R; Mozhdehipanah, H; John, P; Mir, A; Ansar, M; French, L; Ayub, M; Vincent, J B

2018-04-01

Approximately 1% of the global population is affected by intellectual disability (ID), and the majority receive no molecular diagnosis. Previous studies have indicated high levels of genetic heterogeneity, with estimates of more than 2500 autosomal ID genes, the majority of which are autosomal recessive (AR). Here, we combined microarray genotyping, homozygosity-by-descent (HBD) mapping, copy number variation (CNV) analysis, and whole exome sequencing (WES) to identify disease genes/mutations in 192 multiplex Pakistani and Iranian consanguineous families with non-syndromic ID. We identified definite or candidate mutations (or CNVs) in 51% of families in 72 different genes, including 26 not previously reported for ARID. The new ARID genes include nine with loss-of-function mutations (ABI2, MAPK8, MPDZ, PIDD1, SLAIN1, TBC1D23, TRAPPC6B, UBA7 and USP44), and missense mutations include the first reports of variants in BDNF or TET1 associated with ID. The genes identified also showed overlap with de novo gene sets for other neuropsychiatric disorders. Transcriptional studies showed prominent expression in the prenatal brain. The high yield of AR mutations for ID indicated that this approach has excellent clinical potential and should inform clinical diagnostics, including clinical whole exome and genome sequencing, for populations in which consanguinity is common. As with other AR disorders, the relevance will also apply to outbred populations.

Whole-genome transcriptional analysis of heavy metal stresses inCaulobacter crescentus

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

Hu, Ping; Brodie, Eoin L.; Suzuki, Yohey

2005-09-21

The bacterium Caulobacter crescentus and related stalkbacterial species are known for their distinctive ability to live in lownutrient environments, a characteristic of most heavy metal contaminatedsites. Caulobacter crescentus is a model organism for studying cell cycleregulation with well developed genetics. We have identified the pathwaysresponding to heavy metal toxicity in C. crescentus to provide insightsfor possible application of Caulobacter to environmental restoration. Weexposed C. crescentus cells to four heavy metals (chromium, cadmium,selenium and uranium) and analyzed genome wide transcriptional activitiespost exposure using a Affymetrix GeneChip microarray. C. crescentusshowed surprisingly high tolerance to uranium, a possible mechanism forwhich may be formationmore » of extracellular calcium-uranium-phosphateprecipitates. The principal response to these metals was protectionagainst oxidative stress (up-regulation of manganese-dependent superoxidedismutase, sodA). Glutathione S-transferase, thioredoxin, glutaredoxinsand DNA repair enzymes responded most strongly to cadmium and chromate.The cadmium and chromium stress response also focused on reducing theintracellular metal concentration, with multiple efflux pumps employed toremove cadmium while a sulfate transporter was down-regulated to reducenon-specific uptake of chromium. Membrane proteins were also up-regulatedin response to most of the metals tested. A two-component signaltransduction system involved in the uranium response was identified.Several differentially regulated transcripts from regions previously notknown to encode proteins were identified, demonstrating the advantage ofevaluating the transcriptome using whole genome microarrays.« less

Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology

PubMed Central

Ding, Liang-Hao; Xie, Yang; Park, Seongmi; Xiao, Guanghua; Story, Michael D.

2008-01-01

Despite the tremendous growth of microarray usage in scientific studies, there is a lack of standards for background correction methodologies, especially in single-color microarray platforms. Traditional background subtraction methods often generate negative signals and thus cause large amounts of data loss. Hence, some researchers prefer to avoid background corrections, which typically result in the underestimation of differential expression. Here, by utilizing nonspecific negative control features integrated into Illumina whole genome expression arrays, we have developed a method of model-based background correction for BeadArrays (MBCB). We compared the MBCB with a method adapted from the Affymetrix robust multi-array analysis algorithm and with no background subtraction, using a mouse acute myeloid leukemia (AML) dataset. We demonstrated that differential expression ratios obtained by using the MBCB had the best correlation with quantitative RT–PCR. MBCB also achieved better sensitivity in detecting differentially expressed genes with biological significance. For example, we demonstrated that the differential regulation of Tnfr2, Ikk and NF-kappaB, the death receptor pathway, in the AML samples, could only be detected by using data after MBCB implementation. We conclude that MBCB is a robust background correction method that will lead to more precise determination of gene expression and better biological interpretation of Illumina BeadArray data. PMID:18450815

Microfluidic-Based Bacteria Isolation from Whole Blood for Diagnostics of Blood Stream Infection.

PubMed

Zelenin, Sergey; Ramachandraiah, Harisha; Faridi, Asim; Russom, Aman

2017-01-01

Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing.We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.

EFFECT OF A SHORT PERIOD WHOLE BODY VIBRATION WITH 10 HZ ON BLOOD BIOMARKERS IN WISTAR RATS.

PubMed

Monteiro, Milena de Oliveira Bravo; de Sá-Caputo, Danúbia da Cunha; Moreira-Marconi, Eloá; Frederico, Éric Heleno Freire Ferreira; de Sousa-Gonçalves, Cintia Renata; Bernardo, Luciana Camargo; Guimarães, Carlos Alberto Sampaio; Bernardo-Filho, Mario

2017-01-01

Exposure to whole body vibration exercises (WBVE), besides some biological effects, causes alterations in the concentration of some blood biomarkers. The aim of this study is to evaluate the action of vibration (10 Hz) of WBVE on the concentration of blood biomarkers in Wistar rats. Wistar rats were divided in 2 groups. The experimental group (EG) was subjected to vibrations of 10Hz (one min per day, one week, total time of seven min), while the control group (CG) has not experienced vibration. Samples of whole blood were drawn for biochemical analysis of the concentration of total cholesterol, triglycerides, HDL, LDL, VLDL, glucose, CPK, albumin, alkaline phosphates, TGP, TGO, γGT, lipase, amylase, urea and creatinine. White blood cell count and a platelet-hemogram were also performed. Significant (p<0.05) increase in TGP, TGO and white blood cells and decrease in LDL concentration was found after exposure of 10Hz mechanical vibration. Although these findings were obtained with rats, they might contribute to try to understand better these mechanisms that occur following exposure to a frequency of 10Hz.

Blood analysis by Raman spectroscopy.

PubMed

Enejder, Annika M K; Koo, Tae-Woong; Oh, Jeankun; Hunter, Martin; Sasic, Slobodan; Feld, Michael S; Horowitz, Gary L

2002-11-15

Concentrations of multiple analytes were simultaneously measured in whole blood with clinical accuracy, without sample processing, using near-infrared Raman spectroscopy. Spectra were acquired with an instrument employing nonimaging optics, designed using Monte Carlo simulations of the influence of light-scattering-absorbing blood cells on the excitation and emission of Raman light in turbid medium. Raman spectra were collected from whole blood drawn from 31 individuals. Quantitative predictions of glucose, urea, total protein, albumin, triglycerides, hematocrit, and hemoglobin were made by means of partial least-squares (PLS) analysis with clinically relevant precision (r(2) values >0.93). The similarity of the features of the PLS calibration spectra to those of the respective analyte spectra illustrates that the predictions are based on molecular information carried by the Raman light. This demonstrates the feasibility of using Raman spectroscopy for quantitative measurements of biomolecular contents in highly light-scattering and absorbing media.

21 CFR 864.7500 - Whole blood hemoglobin assays.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not include...

21 CFR 864.7500 - Whole blood hemoglobin assays.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not include...

21 CFR 864.7500 - Whole blood hemoglobin assays.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not include...

21 CFR 864.7500 - Whole blood hemoglobin assays.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not include...

21 CFR 864.7500 - Whole blood hemoglobin assays.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Whole blood hemoglobin assays. 864.7500 Section... blood hemoglobin assays. (a) Identification. A whole blood hemoglobin assay is a device consisting or... hemoglobin content of whole blood for the detection of anemia. This generic device category does not include...

Emergency Whole Blood Use in the Field: A Simplified Protocol for Collection and Transfusion

DTIC Science & Technology

2014-01-01

renewed interest in the reintroduction of fresh whole blood and cold- stored whole blood to patient care in austere environments. There is scant...blood use in the treat- ment of exsanguinating hemorrhage and have renewed interest in the reintroduction of warm whole blood (WWB) stored at 22-C for...data pro- vide a biological rationale for whole-blood use in the treatment of exsanguinating hemorrhage and have renewed interest in the reintroduction

Analysis of hydroxylated metabolites of PCBs (OH-PCBs) and other chlorinated phenolic compounds in whole blood from Canadian inuit.

PubMed Central

Sandau, C D; Ayotte, P; Dewailly, E; Duffe, J; Norstrom, R J

2000-01-01

In this study, we identified the main hydroxylated polychlorinated biphenyls (OH-PCBs) and other chlorinated phenolic compounds and we determined their relative concentrations in whole blood from 13 male and 17 female Inuit from northern Quebec, Canada, and from a pooled whole blood sample from southern Quebec. We also determined concentrations of polychlorinated biphenyls (PCBs). Total OH-PCB concentrations were variable among the Inuit samples, ranging over 2 orders of magnitude (0.117-11.6 ng/g whole blood wet weight). These concentrations were equal to and up to 70 times those found for the southern Quebec pooled whole blood sample. Geometric mean concentrations of total OH-PCBs were 1.73 and 1.01 ng/g whole blood for Inuit men and women, respectively, and 0.161 ng/g whole blood for the southern population pool. There are limited data available for comparison, but the levels of OH-PCBs in Inuit are higher than those previously reported in the literature for other populations. There was a significant correlation (p < 0.005) between OH-PCBs and PCBs (r = 0.84) and both correlated significantly (p < 0.005) with age (r = 0.68 and 0.78, respectively). The ratio of OH-PCBs to PCBs was lower in Inuit (0.11) than in the southern Quebec pool (0.33). There is no apparent explanation for the difference. There was considerable variability in the congener pattern of the identified OH-PCBs. The main metabolite, 4-OH-CB109 (4-OH-2,3,3',4', 5-pentachlorobiphenyl), constituted 12-62% of the total OH-PCBs in the samples. Pentachlorophenol (PCP) was the dominant phenolic compound in blood, constituting 46% (geometric mean) of the total quantitated chlorinated phenolic compounds. PCP concentrations in Inuit blood ranged from 0.558 to 7.77 ng/g on a wet weight basis. All but two Inuit samples had lower concentrations than the southern Quebec pool (6.29 ng/g). The possible role of OH-PCBs in mediating PCB-induced adverse effects needs to be investigated further. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10903613

Stability of blood potassium: effects of duration, temperature and transport during 10 hours storage of human whole blood in serum and plasma.

PubMed

Dromigny, Jacques-Albert; Robert, Emmanuel

2017-08-01

To comply with the pre-analytical requirements of ISO EN 15189, we investigated the stability of potassium, a very critical and sensitive analyte. We took into count effects of duration, temperature and transport after 10 hours storage of human whole blood in serum and plasma. Blood of 12 healthy subjects was analyzed after 4, 6, 8 and 10 hours of storage. Three study groups were designed: samples stored in laboratory at room temperature, transported by car during 4 hours at a temperature of 21±1̊C, with or without previous thermal shock (20 min at 4±1 ̊C) before transportation. Variations in concentration were expressed as mean bias from baseline using the analytical change limit (ACL) and the reference change value (RCV). Using RCV, we considered that potassium was biologically stable during 10 hours whatever our study groups. Considering ACL, potassium in serum was not stable after the thermal shock. We conclude that whole blood in lithium-heparin tubes may be used for routine potassium analysis even if long car transportation and previous thermal shock is involved. It confirms that potassium analysis can be still performed in locations distant from a medical laboratory.

Blood proteins analysis by Raman spectroscopy method

NASA Astrophysics Data System (ADS)

Artemyev, D. N.; Bratchenko, I. A.; Khristoforova, Yu. A.; Lykina, A. A.; Myakinin, O. O.; Kuzmina, T. P.; Davydkin, I. L.; Zakharov, V. P.

2016-04-01

This work is devoted to study the possibility of plasma proteins (albumin, globulins) concentration measurement using Raman spectroscopy setup. The blood plasma and whole blood were studied in this research. The obtained Raman spectra showed significant variation of intensities of certain spectral bands 940, 1005, 1330, 1450 and 1650 cm-1 for different protein fractions. Partial least squares regression analysis was used for determination of correlation coefficients. We have shown that the proposed method represents the structure and biochemical composition of major blood proteins.

Performance evaluation of the FDA-approved Determine™ HIV-1/2 Ag/Ab Combo assay using plasma and whole blood specimens.

PubMed

Masciotra, Silvina; Luo, Wei; Westheimer, Emily; Cohen, Stephanie E; Gay, Cynthia L; Hall, Laura; Pan, Yi; Peters, Philip J; Owen, S Michele

2017-06-01

The Determine™ HIV-1/2 Ag/Ab Combo (DC) rapid test can identify HIV-1 infection earlier than rapid antibody-only tests in plasma specimens. We compared the performance of DC with a laboratory-based antigen/antibody (Ag/Ab) combo assay in plasma and evaluated antigen reactivity in whole blood specimens. We tested by DC 508 plasma specimens collected in a prospective study and 107 sequential plasma and simulated whole blood specimens from 20 seroconversion panels. Previous results using the ARCHITECT (ARC) Ag/Ab combo assay were compared to DC results. In seroconversion panels, the days from the first HIV1 RNA-positive test to first DC-reactive in plasma and whole blood was compared. McNemar's and Wilcoxon signed rank tests were used for statistical analysis. Of 415 HIV-positive samples, ARC detected 396 (95.4%) and DC 337 (81.2%) (p<0.0001). DC was reactive in 50.0% of ARC-reactive/MS-negative, 78.6% of ARC-reactive/MS-indeterminate, and 99.6% of ARC-reactive/MS-HIV-1-positive or -undifferentiated specimens. DC antigen reactivity was higher among ARC-reactive/MS-negative than MS-indeterminate samples. In 20 HIV-1 seroconversion panels, there was a significant difference between DC reactivity in plasma (91.1%) and whole blood (56.4%) (p<0.0001). DC with whole blood showed a significant delay in reactivity compared to plasma (p=0.008). In plasma, DC was significantly less sensitive than an instrumented laboratory-based Ag/Ab combo assay. DC in plasma was significantly more sensitive compared to whole blood in early HIV-1 infections. With the U.S. laboratory-based diagnostic algorithm, DC as the first step would likely miss a high proportion of HIV-1 infections in early stages of seroconversion. Published by Elsevier B.V.

Correlation between viral load of cytomegalovirus and tacrolimus and sirolimus levels in transplanted pediatric patients.

PubMed

Reyes-Pérez, Herlinda; Sánchez-Huerta, José Luis; Varela-Fascinetto, Gustavo; Romo-Vázquez, José Carlos; Morales-Sánchez, Abigail; Fuentes-Pananá, Ezequiel M; Parra-Ortega, Israel; Ramírez-Ramírez, Graciela; López-Martínez, Briceida

Survival of transplant patients and grafts depends largely on the use of immunosuppressive drugs. However, a balance remains to be established among immunosuppression, transplant rejection and cytomegalovirus (CMV) infection, which results in a high rate of morbidity and mortality. The aim of this study was to define a better strategy for monitoring transplanted patients based on the analysis of the blood concentration of sirolimus and tacrolimus and the burden of CMV. Fifty five post-transplant (kidney and liver) pediatric patients, nine treated with sirolimus and 46 treated with tacrolimus, were included. A total of 541 measurements were obtained. In each measurement the concentration of immunosuppressant in whole blood and CMV viral load in plasma and whole blood was quantified by real-time PCR. Pearson correlation coefficient (r) was estimated. Values of r ≤0.0747 were found for the relationship between dose and concentration of immunosuppressant; r = 0.9406 for the relationship between viral load in whole blood and plasma, and r ≤0.4616 for the relationship between concentration of immunosuppressant and viral load. These data support that the doses of immunosuppressive drugs do not correlate with the levels of the same in whole blood. Therefore, systemic levels of immunosuppressant should be constantly monitored together with CMV load. Meanwhile, a high correlation between viral load measured in whole blood and plasma was found. Copyright © 2016 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Rotor for processing liquids using movable capillary tubes

DOEpatents

Johnson, Wayne F.; Burtis, Carl A.; Walker, William A.

1989-01-01

A rotor assembly for processing liquids, especially whole blood samples, is disclosed. The assembly includes apparatus for separating non-liquid components of whole blood samples from liquid components, apparatus for diluting the separated liquid component with a diluent and apparatus for transferring the diluted sample to an external apparatus for analysis. The rotor assembly employs several movable capillary tubes to handle the sample and diluents. A method for using the rotor assembly to process liquids is also described.

Rotor for processing liquids using movable capillary tubes

DOEpatents

Johnson, Wayne F [Loudon, TN; Burtis, Carl A [Oak Ridge, TN; Walker, William A [Knoxville, TN

1989-05-30

A rotor assembly for processing liquids, especially whole blood samples, is disclosed. The assembly includes apparatus for separating non-liquid components of whole blood samples from liquid components, apparatus for diluting the separated liquid component with a diluent and apparatus for transferring the diluted sample to an external apparatus for analysis. The rotor assembly employs several movable capillary tubes to handle the sample and diluents. A method for using the rotor assembly to process liquids is also described.

Decoding genes with coexpression networks and metabolomics - 'majority report by precogs'.

PubMed

Saito, Kazuki; Hirai, Masami Y; Yonekura-Sakakibara, Keiko

2008-01-01

Following the sequencing of whole genomes of model plants, high-throughput decoding of gene function is a major challenge in modern plant biology. In view of remarkable technical advances in transcriptomics and metabolomics, integrated analysis of these 'omics' by data-mining informatics is an excellent tool for prediction and identification of gene function, particularly for genes involved in complicated metabolic pathways. The availability of Arabidopsis public transcriptome datasets containing data of >1000 microarrays reinforces the potential for prediction of gene function by transcriptome coexpression analysis. Here, we review the strategy of combining transcriptome and metabolome as a powerful technology for studying the functional genomics of model plants and also crop and medicinal plants.

Murine Hyperglycemic Vasculopathy and Cardiomyopathy: Whole-Genome Gene Expression Analysis Predicts Cellular Targets and Regulatory Networks Influenced by Mannose Binding Lectin

PubMed Central

Zou, Chenhui; La Bonte, Laura R.; Pavlov, Vasile I.; Stahl, Gregory L.

2012-01-01

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies. PMID:22375142

Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

PubMed

Mendoza, A; Torrisi, D M; Sell, S; Cady, N C; Lawrence, D A

2016-01-21

Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice.

Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications.

PubMed

Baghbaderani, Behnam Ahmadian; Syama, Adhikarla; Sivapatham, Renuka; Pei, Ying; Mukherjee, Odity; Fellner, Thomas; Zeng, Xianmin; Rao, Mahendra S

2016-08-01

We have recently described manufacturing of human induced pluripotent stem cells (iPSC) master cell banks (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Reports, 5(4), 647-659, 2015). In this manuscript, we describe the detailed characterization of the two iPSC clones generated using this process, including whole genome sequencing (WGS), microarray, and comparative genomic hybridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibration material and with a reporter subclone and lines made by a similar process from different donors. We believe that iPSCs are likely to be used to make multiple clinical products. We further believe that the lines used as input material will be used at different sites and, given their immortal status, will be used for many years or even decades. Therefore, it will be important to develop assays to monitor the state of the cells and their drift in culture. We suggest that a detailed characterization of the initial status of the cells, a comparison with some calibration material and the development of reporter sublcones will help determine which set of tests will be most useful in monitoring the cells and establishing criteria for discarding a line.

Portable vibration-assisted filtration device for on-site isolation of blood cells or pathogenic bacteria from whole human blood.

PubMed

Kim, Yong Tae; Park, Kyun Joo; Kim, Seyl; Kim, Soon Ae; Lee, Seok Jae; Kim, Do Hyun; Lee, Tae Jae; Lee, Kyoung G

2018-03-01

Isolation of specific cells from whole blood is important to monitor disease prognosis and diagnosis. In this study, a vibration-assisted filtration (VF) device has been developed for isolation and recovery of specific cells such as leukocytes and pathogenic bacteria from human whole blood. The VF device is composed of three layers which was fabricated using injection molding with cyclic olefin copolymer (COC) pellets consisting of: a top layer with coin-type vibration motor (Ф = 10mm), a middle plate with a 1μm or 3μm-pore filter membrane to separate of Staphylococcus aureus (S. aureus) cells or leukocytes (i.e. white blood cells) respectively, and a bottom chamber with conical-shaped microstructure. One milliliter of human whole blood was injected into a sample loading chamber using a 3μm-pore filter equipped in the VF device and the coin-type vibration motor applied external vibration force by generating a rotational fluid which enhances the filtration velocity due to the prevention of the cell clogging on the filter membrane. The effluent blood such as erythrocytes, platelet, and plasma was collected at the bottom chamber while the leukocytes were sieved by the filter membrane. The vibration-assisted leukocyte separation was able to finish within 200s while leukocyte separation took 1200s without vibration. Moreover, we successfully separated S. aureus from human whole blood using a 1μm-pore filter equipped VF device and it was further confirmed by genetic analysis. The proposed VF device provides an advanced cell separation platform in terms of simplicity, fast separation, and portability in the fields of point-of-care diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique.

PubMed

Kawana, Shuichi; Nakagawa, Katsuhiro; Hasegawa, Yuki; Yamaguchi, Seiji

2010-11-15

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using ²H₈-Val, ²H₃-Leu, ²H₃-Met and ²H₅-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients=1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood. Copyright © 2010 Elsevier B.V. All rights reserved.

Changes in hematological indices and lymphocyte subsets in response to whole blood donation in healthy male donors.

PubMed

Borai, Anwar; Livingstone, Callum; Alsobhi, Enaam; Al Sofyani, Abeer; Balgoon, Dalal; Farzal, Anwar; Almohammadi, Mohammed; Al-Amri, Abdulafattah; Bahijri, Suhad; Alrowaili, Daad; Bassiuni, Wafaa; Saleh, Ayman; Alrowaili, Norah; Abdelaal, Mohamed

2017-04-01

Whole blood donation has immunomodulatory effects, and most of these have been observed at short intervals following blood donation. This study aimed to investigate the impact of whole blood donation on lymphocyte subsets over a typical inter-donation interval. Healthy male subjects were recruited to study changes in complete blood count (CBC) (n = 42) and lymphocyte subsets (n = 16) before and at four intervals up to 106 days following blood donation. Repeated measures ANOVA were used to compare quantitative variables between different visits. Following blood donation, changes in CBC and erythropoietin were as expected. The neutrophil count increased by 11.3% at 8 days (p < .001). Novel changes were observed in lymphocyte subsets as the CD4/CD8 ratio increased by 9.2% (p < .05) at 8 days and 13.7% (p < .05) at 22 days. CD16-56 cells decreased by 16.2% (p < .05) at 8 days. All the subsets had returned to baseline by 106 days. Regression analysis showed that the changes in CD16-56 cells and CD4/CD8 ratio were not significant (Wilk's lambda = 0.15 and 0.94, respectively) when adjusted for BMI. In conclusion, following whole blood donation, there are transient changes in lymphocyte subsets. The effect of BMI on lymphocyte subsets and the effect of this immunomodulation on the immune response merit further investigation.

Overexpression of GRß in colonic mucosal cell line partly reflects altered gene expression in colonic mucosa of patients with inflammatory bowel disease.

PubMed

Nagy, Zsolt; Acs, Bence; Butz, Henriett; Feldman, Karolina; Marta, Alexa; Szabo, Peter M; Baghy, Kornelia; Pazmany, Tamas; Racz, Karoly; Liko, Istvan; Patocs, Attila

2016-01-01

The glucocorticoid receptor (GR) plays a crucial role in inflammatory responses. GR has several isoforms, of which the most deeply studied are the GRα and GRß. Recently it has been suggested that in addition to its negative dominant effect on GRα, the GRß may have a GRα-independent transcriptional activity. The GRß isoform was found to be frequently overexpressed in various autoimmune diseases, including inflammatory bowel disease (IBD). In this study, we wished to test whether the gene expression profile found in a GRß overexpressing intestinal cell line (Caco-2GRß) might mimic the gene expression alterations found in patients with IBD. Whole genome microarray analysis was performed in both normal and GRß overexpressing Caco-2 cell lines with and without dexamethasone treatment. IBD-related genes were identified from a meta-analysis of 245 microarrays available in online microarray deposits performed on intestinal mucosa samples from patients with IBD and healthy individuals. The differentially expressed genes were further studied using in silico pathway analysis. Overexpression of GRß altered a large proportion of genes that were not regulated by dexamethasone suggesting that GRß may have a GRα-independent role in the regulation of gene expression. About 10% of genes differentially expressed in colonic mucosa samples from IBD patients compared to normal subjects were also detected in Caco-2 GRß intestinal cell line. Common genes are involved in cell adhesion and cell proliferation. Overexpression of GRß in intestinal cells may affect appropriate mucosal repair and intact barrier function. The proposed novel role of GRß in intestinal epithelium warrants further studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Changes of Gene Expression on Human Hair during Long-Spaceflight

NASA Astrophysics Data System (ADS)

Terada, Masahiro; Mukai, Chiaki; Ishioka, Noriaki; Majima, Hideyuki J.; Yamada, Shin; Seki, Masaya; Takahashi, Rika; Higashibata, Akira; Ohshima, Hiroshi; Sudoh, Masamichi; Minamisawa, Susumu

Hair has many advantages as the experimental sample. In a hair follicle, hair matrix cells actively divide and these active changes sensitively reflect physical condition on human body. The hair shaft records the metabolic conditions of mineral elements in our body. From human hairs, we can detect physiological informations about the human health. Therefore, we focused on using hair root analysis to understand the effects of spaceflight on astronauts. In 2009, we started a research program focusing on the analysis of astronauts’ hairs to examine the effects of long-term spaceflight on the gene expression in the human body. We want to get basic information to invent the effectivly diagnostic methods to detect the health situations of astronauts during space flight by analyzing human hair. We extracted RNA form the collected samples. Then, these extracted RNA was amplified. Amplified RNA was processed and hybridized to the Whole Human Genome (4×44K) Oligo Microarray (Agilent Technologies) according to the manufacturer’s protocol. Slide scanning was performed using the Agilent DNA Microarray Scanner. Scanning data were normalized with Agilent’s Feature Extraction software. Data preprocessing and analysis were performed using GeneSpring software 11.0.1. Next, Synthesis of cDNA (1 mg) was carried out using the PrimeScript RT reagent Kit (TaKaRa Bio) following the manufacturer’s instructions. The qRT-PCR experiment was performed with SYBR Premix Ex Taq (TaKaRa Bio) using the 7500 Real-Time PCR system (Applied Biosystems). We detected the changes of some gene expressions during spaceflight from both microarray and qRT-PCR data. These genes seems to be related with the hair proliferation. We believe that these results will lead to the discovery of the important factor effected during space flight on the hair.

Characterization of a novel Lactobacillus species closely related to Lactobacillus johnsonii using a combination of molecular and comparative genomics methods.

PubMed

Sarmiento-Rubiano, Luz-Adriana; Berger, Bernard; Moine, Déborah; Zúñiga, Manuel; Pérez-Martínez, Gaspar; Yebra, María J

2010-09-17

Comparative genomic hybridization (CGH) constitutes a powerful tool for identification and characterization of bacterial strains. In this study we have applied this technique for the characterization of a number of Lactobacillus strains isolated from the intestinal content of rats fed with a diet supplemented with sorbitol. Phylogenetic analysis based on 16S rRNA gene, recA, pheS, pyrG and tuf sequences identified five bacterial strains isolated from the intestinal content of rats as belonging to the recently described Lactobacillus taiwanensis species. DNA-DNA hybridization experiments confirmed that these five strains are distinct but closely related to Lactobacillus johnsonii and Lactobacillus gasseri. A whole genome DNA microarray designed for the probiotic L. johnsonii strain NCC533 was used for CGH analysis of L. johnsonii ATCC 33200T, L. johnsonii BL261, L. gasseri ATCC 33323T and L. taiwanensis BL263. In these experiments, the fluorescence ratio distributions obtained with L. taiwanensis and L. gasseri showed characteristic inter-species profiles. The percentage of conserved L. johnsonii NCC533 genes was about 83% in the L. johnsonii strains comparisons and decreased to 51% and 47% for L. taiwanensis and L. gasseri, respectively. These results confirmed the separate status of L. taiwanensis from L. johnsonii at the level of species, and also that L. taiwanensis is closer to L. johnsonii than L. gasseri is to L. johnsonii. Conventional taxonomic analyses and microarray-based CGH analysis have been used for the identification and characterization of the newly species L. taiwanensis. The microarray-based CGH technology has been shown as a remarkable tool for the identification and fine discrimination between phylogenetically close species, and additionally provided insight into the adaptation of the strain L. taiwanensis BL263 to its ecological niche.

The analysis of image feature robustness using cometcloud

PubMed Central

Qi, Xin; Kim, Hyunjoo; Xing, Fuyong; Parashar, Manish; Foran, David J.; Yang, Lin

2012-01-01

The robustness of image features is a very important consideration in quantitative image analysis. The objective of this paper is to investigate the robustness of a range of image texture features using hematoxylin stained breast tissue microarray slides which are assessed while simulating different imaging challenges including out of focus, changes in magnification and variations in illumination, noise, compression, distortion, and rotation. We employed five texture analysis methods and tested them while introducing all of the challenges listed above. The texture features that were evaluated include co-occurrence matrix, center-symmetric auto-correlation, texture feature coding method, local binary pattern, and texton. Due to the independence of each transformation and texture descriptor, a network structured combination was proposed and deployed on the Rutgers private cloud. The experiments utilized 20 randomly selected tissue microarray cores. All the combinations of the image transformations and deformations are calculated, and the whole feature extraction procedure was completed in 70 minutes using a cloud equipped with 20 nodes. Center-symmetric auto-correlation outperforms all the other four texture descriptors but also requires the longest computational time. It is roughly 10 times slower than local binary pattern and texton. From a speed perspective, both the local binary pattern and texton features provided excellent performance for classification and content-based image retrieval. PMID:23248759

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

PubMed

Jones, D L; Petty, J; Hoyle, D C; Hayes, A; Ragni, E; Popolo, L; Oliver, S G; Stateva, L I

2003-12-16

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Assessing differential expression in two-color microarrays: a resampling-based empirical Bayes approach.

PubMed

Li, Dongmei; Le Pape, Marc A; Parikh, Nisha I; Chen, Will X; Dye, Timothy D

2013-01-01

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth's ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth's parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth's parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.

Transfection microarray and the applications.

PubMed

Miyake, Masato; Yoshikawa, Tomohiro; Fujita, Satoshi; Miyake, Jun

2009-05-01

Microarray transfection has been extensively studied for high-throughput functional analysis of mammalian cells. However, control of efficiency and reproducibility are the critical issues for practical use. By using solid-phase transfection accelerators and nano-scaffold, we provide a highly efficient and reproducible microarray-transfection device, "transfection microarray". The device would be applied to the limited number of available primary cells and stem cells not only for large-scale functional analysis but also reporter-based time-lapse cellular event analysis.

Whole blood genome-wide gene expression profile in males after prolonged wakefulness and sleep recovery.

PubMed

Pellegrino, R; Sunaga, D Y; Guindalini, C; Martins, R C S; Mazzotti, D R; Wei, Z; Daye, Z J; Andersen, M L; Tufik, S

2012-11-01

Although the specific functions of sleep have not been completely elucidated, the literature has suggested that sleep is essential for proper homeostasis. Sleep loss is associated with changes in behavioral, neurochemical, cellular, and metabolic function as well as impaired immune response. Using high-resolution microarrays we evaluated the gene expression profiles of healthy male volunteers who underwent 60 h of prolonged wakefulness (PW) followed by 12 h of sleep recovery (SR). Peripheral whole blood was collected at 8 am in the morning before the initiation of PW (Baseline), after the second night of PW, and one night after SR. We identified over 500 genes that were differentially expressed. Notably, these genes were related to DNA damage and repair and stress response, as well as diverse immune system responses, such as natural killer pathways including killer cell lectin-like receptors family, as well as granzymes and T-cell receptors, which play important roles in host defense. These results support the idea that sleep loss can lead to alterations in molecular processes that result in perturbation of cellular immunity, induction of inflammatory responses, and homeostatic imbalance. Moreover, expression of multiple genes was downregulated following PW and upregulated after SR compared with PW, suggesting an attempt of the body to re-establish internal homeostasis. In silico validation of alterations in the expression of CETN3, DNAJC, and CEACAM genes confirmed previous findings related to the molecular effects of sleep deprivation. Thus, the present findings confirm that the effects of sleep loss are not restricted to the brain and can occur intensely in peripheral tissues.

The FDA's Experience with Emerging Genomics Technologies-Past, Present, and Future.

PubMed

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-07-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing.

The FDA’s Experience with Emerging Genomics Technologies—Past, Present, and Future

PubMed Central

Xu, Joshua; Thakkar, Shraddha; Gong, Binsheng; Tong, Weida

2016-01-01

The rapid advancement of emerging genomics technologies and their application for assessing safety and efficacy of FDA-regulated products require a high standard of reliability and robustness supporting regulatory decision-making in the FDA. To facilitate the regulatory application, the FDA implemented a novel data submission program, Voluntary Genomics Data Submission (VGDS), and also to engage the stakeholders. As part of the endeavor, for the past 10 years, the FDA has led an international consortium of regulatory agencies, academia, pharmaceutical companies, and genomics platform providers, which was named MicroArray Quality Control Consortium (MAQC), to address issues such as reproducibility, precision, specificity/sensitivity, and data interpretation. Three projects have been completed so far assessing these genomics technologies: gene expression microarrays, whole genome genotyping arrays, and whole transcriptome sequencing (i.e., RNA-seq). The resultant studies provide the basic parameters for fit-for-purpose application of these new data streams in regulatory environments, and the solutions have been made available to the public through peer-reviewed publications. The latest MAQC project is also called the SEquencing Quality Control (SEQC) project focused on next-generation sequencing. Using reference samples with built-in controls, SEQC studies have demonstrated that relative gene expression can be measured accurately and reliably across laboratories and RNA-seq platforms. Besides prediction performance comparable to microarrays in clinical settings and safety assessments, RNA-seq is shown to have better sensitivity for low expression and reveal novel transcriptomic features. Future effort of MAQC will be focused on quality control of whole genome sequencing and targeted sequencing. PMID:27116022

Hemorrhage and Subsequent Allogenic Red Blood Cell Transfusion are Associated With Characteristic Monocyte Messenger RNA Expression Patterns in Patients After Multiple Injury—A Genome Wide View

PubMed Central

Bogner, Viktoria; Baker, Henry V.; Kanz, Karl-Georg; Moldawer, L. L.; Mutschler, Wolf; Biberthaler, Peter

2014-01-01

Introduction As outcome to severe trauma is frequently affected by massive blood loss and consecutive hemorrhagic shock, replacement of red blood cell (RBC) units remains indispensable. Administration of RBC units is an independent risk factor for adverse outcome in patients with trauma. The impact of massive blood transfusion or uncrossmatched blood transfusion on the patients’ immune response in the early posttraumatic period remains unclear. Material Thirteen patients presenting with blunt multiple injuries (Injury Severity Score >16) were studied. Monocytes were obtained on admission and at 6, 12, 24, 48, and 72 hours after trauma. Biotinylated complementary RNA targets were hybridized to Affymetrix HG U 133A microarrays. The data were analyzed by a supervised analysis based on whether the patients received massive blood transfusions, and then subsequently, by hierarchical clustering, and by Ingenuity pathway analysis. Results Supervised analysis identified 224 probe sets to be differentially expressed (p < 0.001) in patients who received massive blood transfusion, when compared with those who did not. In addition, 331 probe sets were found differentially expressed (p < 0.001) in patients who received uncrossmatched RBC units in comparison with those who exclusively gained crossmatched ones. Functional pathway analysis of the respectively identified gene expression profiles suggests a contributory role by the AKT/PI3Kinase pathway, the mitogen-activated protein-kinase pathway, the Ubiquitin pathway, and the diverse inflammatory networks. Conclusion We exhibited for the first time a serial, sequential screening analysis of monocyte messenger RNA expression patterns in patients with multiple trauma indicating a strongly significant association between the patients’ genomic response in blood monocytes and massive or uncross-matched RBC substitution. PMID:19820587

Stability of 35 biochemical and immunological routine tests after 10 hours storage and transport of human whole blood at 21°C.

PubMed

Henriksen, Linda O; Faber, Nina R; Moller, Mette F; Nexo, Ebba; Hansen, Annebirthe B

2014-10-01

Suitable procedures for transport of blood samples from general practitioners to hospital laboratories are requested. Here we explore routine testing on samples stored and transported as whole blood in lithium-heparin or serum tubes. Blood samples were collected from 106 hospitalized patients, and analyzed on Architect c8000 or Advia Centaur XP for 35 analytes at base line, and after storage and transport of whole blood in lithium-heparin or serum tubes at 21 ± 1°C for 10 h. Bias and imprecision (representing variation from analysis and storage) were calculated from values at baseline and after storage, and differences tested by paired t-tests. Results were compared to goals set by the laboratory. We observed no statistically significant bias and results within the goal for imprecision between baseline samples and 10-h samples for albumin, alkaline phosphatase, antitrypsin, bilirubin, creatinine, free triiodothyronine, γ-glutamyl transferase, haptoglobin, immunoglobulin G, lactate dehydrogenase, prostate specific antigen, total carbon dioxide, and urea. Alanine aminotransferase, amylase, C-reactive protein, calcium, cholesterol, creatine kinase, ferritin, free thyroxine, immunoglobulin A, immunoglobulin M, orosomucoid, sodium, transferrin, and triglycerides met goals for imprecision, though they showed a minor, but statistically significant bias in results after storage. Cobalamin, folate, HDL-cholesterol, iron, phosphate, potassium, thyroid stimulating hormone and urate warranted concern, but only folate and phosphate showed deviations of clinical importance. We conclude that whole blood in lithium-heparin or serum tubes stored for 10 h at 21 ± 1°C, may be used for routine analysis without restrictions for all investigated analytes but folate and phosphate.

Microarray technology for major chemical contaminants analysis in food: current status and prospects.

PubMed

Zhang, Zhaowei; Li, Peiwu; Hu, Xiaofeng; Zhang, Qi; Ding, Xiaoxia; Zhang, Wen

2012-01-01

Chemical contaminants in food have caused serious health issues in both humans and animals. Microarray technology is an advanced technique suitable for the analysis of chemical contaminates. In particular, immuno-microarray approach is one of the most promising methods for chemical contaminants analysis. The use of microarrays for the analysis of chemical contaminants is the subject of this review. Fabrication strategies and detection methods for chemical contaminants are discussed in detail. Application to the analysis of mycotoxins, biotoxins, pesticide residues, and pharmaceutical residues is also described. Finally, future challenges and opportunities are discussed.

Rapid Separation of Bacteria from Blood—Review and Outlook

PubMed Central

Alizadeh, Mahsa; Husseini, Ghaleb A.; McClellan, Daniel S.; Buchanan, Clara M.; Bledsoe, Colin G.; Robison, Richard A.; Blanco, Rae; Roeder, Beverly L.; Melville, Madison; Hunter, Alex K.

2017-01-01

The high morbidity and mortality rate of bloodstream infections involving antibiotic-resistant bacteria necessitate a rapid identification of the infectious organism and its resistance profile. Traditional methods based on culturing the blood typically require at least 24 h, and genetic amplification by PCR in the presence of blood components has been problematic. The rapid separation of bacteria from blood would facilitate their genetic identification by PCR or other methods so that the proper antibiotic regimen can quickly be selected for the septic patient. Microfluidic systems that separate bacteria from whole blood have been developed, but these are designed to process only microliter quantities of whole blood or only highly diluted blood. However, symptoms of clinical blood infections can be manifest with bacterial burdens perhaps as low as 10 CFU/mL, and thus milliliter quantities of blood must be processed to collect enough bacteria for reliable genetic analysis. This review considers the advantages and shortcomings of various methods to separate bacteria from blood, with emphasis on techniques that can be done in less than 10 min on milliliter-quantities of whole blood. These techniques include filtration, screening, centrifugation, sedimentation, hydrodynamic focusing, chemical capture on surfaces or beads, field-flow fractionation, and dielectrophoresis. Techniques with the most promise include screening, sedimentation, and magnetic bead capture, as they allow large quantities of blood to be processed quickly. Some microfluidic techniques can be scaled up. PMID:27160415

Microarray profiling of human white adipose tissue after exogenous leptin injection.

PubMed

Taleb, S; Van Haaften, R; Henegar, C; Hukshorn, C; Cancello, R; Pelloux, V; Hanczar, B; Viguerie, N; Langin, D; Evelo, C; Zucker, J; Clément, K; Saris, W H M

2006-03-01

Leptin is a secreted adipocyte hormone that plays a key role in the regulation of body weight homeostasis. The leptin effect on human white adipose tissue (WAT) is still debated. The aim of this study was to assess whether the administration of polyethylene glycol-leptin (PEG-OB) in a single supraphysiological dose has transcriptional effects on genes of WAT and to identify its target genes and functional pathways in WAT. Blood samples and WAT biopsies were obtained from 10 healthy nonobese men before treatment and 72 h after the PEG-OB injection, leading to an approximate 809-fold increase in circulating leptin. The WAT gene expression profile before and after the PEG-OB injection was compared using pangenomic microarrays. Functional gene annotations based on the gene ontology of the PEG-OB regulated genes were performed using both an 'in house' automated procedure and GenMAPP (Gene Microarray Pathway Profiler), designed for viewing and analyzing gene expression data in the context of biological pathways. Statistical analysis of microarray data revealed that PEG-OB had a major down-regulated effect on WAT gene expression, as we obtained 1,822 and 100 down- and up-regulated genes, respectively. Microarray data were validated using reverse transcription quantitative PCR. Functional gene annotations of PEG-OB regulated genes revealed that the functional class related to immunity and inflammation was among the most mobilized PEG-OB pathway in WAT. These genes are mainly expressed in the cell of the stroma vascular fraction in comparison with adipocytes. Our observations support the hypothesis that leptin could act on WAT, particularly on genes related to inflammation and immunity, which may suggest a novel leptin target pathway in human WAT.

Comparison of Standardized Cytomegalovirus (CMV) Viral Load Thresholds in Whole Blood and Plasma of Solid Organ and Hematopoietic Stem Cell Transplant Recipients with CMV Infection and Disease.

PubMed

Dioverti, M Veronica; Lahr, Brian D; Germer, Jeffrey J; Yao, Joseph D; Gartner, Michelle L; Razonable, Raymund R

2017-01-01

Quantification of cytomegalovirus (CMV) deoxyribonucleic acid (DNA) has important diagnostic, prognostic, and therapeutic implications in the management of transplant recipients. We aimed to assess a viral load in plasma and whole blood that distinguishes CMV disease from asymptomatic infection in a cohort of solid organ and hematopoietic stem cell transplantation. We prospectively measured and compared CMV viral load in paired plasma and whole blood samples collected from transplant recipients with CMV infection and disease. Cytomegalovirus viral loads were determined by a commercially available US Food and Drug Administration-approved quantitative assay (COBAS AmpliPrep/COBAS TaqMan CMV Test [CAP/CTM CMV]) calibrated to the first World Health Organization International Standard for CMV DNA quantification. Moderate agreement of CMV viral load was observed between plasma and whole blood, with 31% of samples having discordant findings, particularly among samples with low DNA levels. Among the subset of samples where both paired samples had quantifiable levels, we observed a systematic bias that reflected higher viral load in whole blood compared with plasma. Based on receiver operating curve analysis, an initial plasma CMV viral load threshold of 1700 IU/mL in solid organ transplant recipients (sensitivity 80%, specificity 74%) and 1350 IU/mL in allogeneic hematopoietic stem cell transplant recipients (sensitivity 87%, specificity 87%) distinguished CMV disease and asymptomatic infection. This study identifies standardized viral load thresholds that distinguish CMV disease from asymptomatic infection using CAP/CTM CMV assay. We propose these thresholds as potential triggers to be evaluated in prospective studies of preemptive therapy. Plasma was better than whole blood for measuring viral load using the CAP/CTM CMV assay.

Hypoxia-Inducible Factor-1α (HIF-1α) Expression on Endothelial Cells in Juvenile Nasopharyngeal Angiofibroma: A Review of 70 cases and Tissue Microarray Analysis.

PubMed

Song, Xiaole; Yang, Chenhe; Zhang, Huankang; Wang, Jingjing; Sun, Xicai; Hu, Li; Liu, Zhuofu; Wang, Dehui

2018-06-01

To examine the expression of hypoxia-inducible factor-1α (HIF-1α) and its related molecules (cellular repressor of E1A-stimulated genes [CREG], osteopontin [OPN], proto-oncogene tyrosine-protein kinase Src [c-Src], and vascular endothelial growth factor [VEGF]) in juvenile nasopharyngeal angiofibroma (JNA) and explore the correlation between clinical prognosis and HIF-1α expression. The study performed a retrospective review of the clinical records of patients with JNA treated between 2003 and 2007. Specimens were analyzed by immunohistochemistry for HIF-1α, CREG, OPN, c-Src, and VEGF expression, and microvessel density (MVD) was assessed by tissue microarray. The correlation between expression levels and clinicopathological features including age, tumor stage, intraoperative blood loss, and recurrence was analyzed. HIF-1α, CREG, OPN, c-Src, and VEGF were upregulated in endothelial cells (ECs) of patients with JNA, and strong correlations in the expression of these molecules were observed. HIF-1α expression was higher in young patients ( P = .032) and in recurrent cases ( P = .01). Survival analysis showed that low HIF-1α levels in ECs predicted longer time to recurrence (log rank test P = .006). Receiver operating characteristic curve analysis showed that HIF-1α was a prognostic factor for recurrence (area under the curve = 0.690, P = .019). No correlation was found between the expression of molecules and Radkowski stage or intraoperative blood loss. In cases of JNA treated surgically, HIF-1α expression in ECs is a useful prognostic factor for tumor recurrence.

Translating plasma and whole blood fatty acid compositional data into the sum of eicosapentaenoic and docosahexaenoic acid in erythrocytes.

PubMed

Stark, Ken D; Aristizabal Henao, Juan J; Metherel, Adam H; Pilote, Louise

2016-01-01

Specific blood levels of eicosapentaenoic plus docosahexaenoic acid (EPA+DHA, wt% of total) in erythrocytes or "the omega-3 index" have been recommended for cardio-protection, but fatty acids are often measured in different blood fractions. The ability to estimate the % of EPA+DHA in erythrocytes from the fatty acid composition of other blood fractions would enable clinical assessments of omega-3 status when erythrocyte fractions are not available and increase the ability to compare blood levels of omega-3 fatty acids across clinical studies. The fatty acid composition of baseline plasma, erythrocytes and whole blood samples from participants (n=1104) in a prospective, multicenter study examining acute coronary syndrome were determined. The ability to predict the % of EPA+DHA in erythrocytes from other blood fractions were examined using bivariate and multiple linear regression modelling. Concordance analysis was also used to compare the actual erythrocytes EPA+DHA values to values estimated from other blood fractions. EPA+DHA in erythrocytes was significantly (p<0.001) correlated EPA+DHA in plasma (r(2)=0.54) and whole blood (r(2)=0.79). Using multiple linear regression to predict EPA+DHA in erythrocytes resulted in stronger coefficients of determination in both plasma (R(2)=0.70) and whole blood (R(2)=0.84). Concordance analyses indicated agreement between actual and estimated EPA+DHA in erythrocytes, although estimating from plasma fatty acids appears to require translation by categorization rather than by translation as continuous data. This study shows that the fatty acid composition of different blood fractions can be used to estimate erythrocyte EPA+DHA in a population with acute coronary syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of whole body heating on dynamic baroreflex regulation of heart rate in humans

NASA Technical Reports Server (NTRS)

Crandall, C. G.; Zhang, R.; Levine, B. D.

2000-01-01

The purpose of this project was to identify whether dynamic baroreflex regulation of heart rate (HR) is altered during whole body heating. In 14 subjects, dynamic baroreflex regulation of HR was assessed using transfer function analysis. In normothermic and heat-stressed conditions, each subject breathed at a fixed rate (0. 25 Hz) while beat-by-beat HR and systolic blood pressure (SBP) were obtained. Whole body heating significantly increased sublingual temperature, HR, and forearm skin blood flow. Spectral analysis of HR and SBP revealed that the heat stress significantly reduced HR and SBP variability within the high-frequency range (0.2-0.3 Hz), reduced SBP variability within the low-frequency range (0.03-0.15 Hz), and increased the ratio of low- to high-frequency HR variability (all P < 0.01). Transfer function gain analysis showed that the heat stress reduced dynamic baroreflex regulation of HR within the high-frequency range (from 1.04 +/- 0.06 to 0.54 +/- 0.6 beats. min(-1). mmHg(-1); P < 0.001) without significantly affecting the gain in the low-frequency range (P = 0.63). These data suggest that whole body heating reduced high-frequency dynamic baroreflex regulation of HR associated with spontaneous changes in blood pressure. Reduced vagal baroreflex regulation of HR may contribute to reduced orthostatic tolerance known to occur in humans during heat stress.

Fresh Whole Blood Transfusion: Military and Civilian Implications.

PubMed

Goforth, Carl W; Tranberg, John W; Boyer, Phillip; Silvestri, Peter J

2016-06-01

Uncontrolled hemorrhage and exsanguination are the leading cause of preventable death, and resuscitative therapy is a critical component for survival. In various combinations, fresh whole blood, blood components, colloids, and crystalloids have all been staples of trauma care. The use of fresh whole blood is a well-established military practice that has saved the lives of thousands of American and coalition military personnel. Civilian use of fresh whole blood is far less established owing to the wide availability of individual blood components. However, this highly tailored blood supply is vulnerable to both natural and man-made disasters. In the event of such disruption, such as a major hurricane, it may be necessary for civilian hospitals to rapidly enact a fresh whole blood program. Therefore, the aim of this article is to review the current use of blood therapy for trauma resuscitation, the US military's approach to fresh whole blood, and how maintaining a civilian capacity for fresh whole blood collection in the event of future man-made and natural disasters is key to promoting survival from trauma. ©2016 American Association of Critical-Care Nurses.

Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae.

PubMed

Zhang, Ke; Wu, Xue-Chang; Zheng, Dao-Qiong; Petes, Thomas D

2017-12-19

Although meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae , regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures. IMPORTANCE In the yeast Saccharomyces cerevisiae , recombination is usually studied in cells that undergo meiosis at 25°C or 30°C. In a genome-wide analysis, we showed that the locations of genomic regions with high and low levels of meiotic recombination (hot spots and cold spots, respectively) differed dramatically in cells sporulated at 14°C, 30°C, and 37°C. Thus, in yeast, and likely in other non-warm-blooded organisms, genetic maps are strongly affected by the environment. Copyright © 2017 Zhang et al.

Highly sensitive and multiplexed platforms for allergy diagnostics

NASA Astrophysics Data System (ADS)

Monroe, Margo R.

Allergy is a disorder of the immune system caused by an immune response to otherwise harmless environmental allergens. Currently 20% of the US population is allergic and 90% of pediatric patients and 60% of adult patients with asthma have allergies. These percentages have increased by 18.5% in the past decade, with predicted similar trends for the future. Here we design sensitive, multiplexed platforms to detect allergen-specific IgE using the Interferometric Reflectance Imaging Sensor (IRIS) for various clinical settings. A microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of patient blood sample. However, conventional fluorescent microarray technology is limited by i) the variation of probe immobilization, which hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics and ii) the use of fluorophore labels, which is not suitable for some clinical applications due to the tendency of fluorophores to stick to blood particulates and require daily calibration methods. This calibrated fluorescence enhancement (CaFE) method integrates the low magnification modality of IRIS with enhanced fluorescence sensing in order to directly correlate immobilized probe (major allergens) density to allergen-specific IgE in patient serum. However, this platform only operates in processed serum samples, which is not ideal for point of care testing. Thus, a high magnification modality of IRIS was adapted as an alternative allergy diagnostic platform to automatically discriminate and size single nanoparticles bound to specific IgE in unprocessed, characterized human blood and serum samples. These features make IRIS an ideal candidate for clinical and diagnostic applications, such a POC testing. The high magnification (nanoparticle counting) modality in conjunction with low magnification of IRIS in a combined instrument offers four significant advantages compared to existing sensing technologies: IRIS i) corrects for any variation in probe immobilization, ii) detects proteins from attomolar to nanomolar concentrations in unprocessed biological samples, iii) unambiguously discriminates nanoparticles tags on a robust and physically large sensor area, iv) detects protein targets with conjugated nanoparticle tags (~40nm diameter), which minimally affect assay kinetics compared to conventional microparticle tagging methods, and v) utilizes components that make the instrument inexpensive, robust, and portable. This platform was successfully validated on patient serum and whole blood samples with documented allergy profiles (ImmunoCAPRTM, ThermoFisher Scientific).

[Application of chromosomal microarray analysis for the diagnosis of children with intellectual disability/developmental delay and a normal karytype].

PubMed

Hu, Ting; Zhu, Hongmei; Zhang, Zhu; Wang, Jiamin; Liu, Hongqian; Zhang, Xuemei; Zhang, Haixia; Du, Ze; Li, Lingping; Wang, He; Liu, Shanling

2017-04-10

To assess the value of chromosomal microarray analysis (CMA) for the diagnosis of children with intellectual disability/developmental delay (ID/DD) but a normal karytype. Peripheral blood samples from 92 ID/DD patients were analyzed with CMA using Affymetrix CytoScan 750K arrays. The results were analyzed by ChAS v3.0 software. Eighteen cases (19.57%) were detected with abnormalities by CMA, among which 10 cases were diagnosed with microdeletion/microduplication syndromes. These included 2 Williams-Beuren syndromes, 2 Angelman syndromes, 2 Russell-Silver syndromes, 1 Smith-Magenis syndromes, 1 Wolf-Hirschhorn syndromes, 1 15q26 overgrowth syndrome and 1 Xq28 (MECP2) duplication syndrome. In addition, 8 cases were diagnosed with pathogenic copy number variations (pCNV). CMA can significantly improve the diagnostic rate for patients with ID/DD, which is of great value for the treatment of such children and guidance of reproduction for their parents. Therefore, CMA should become the first-line diagnostic test for patients with ID/DD.

Blood drop patterns: Formation and applications.

PubMed

Chen, Ruoyang; Zhang, Liyuan; Zang, Duyang; Shen, Wei

2016-05-01

The drying of a drop of blood or plasma on a solid substrate leads to the formation of interesting and complex patterns. Inter- and intra-cellular and macromolecular interactions in the drying plasma or blood drop are responsible for the final morphologies of the dried patterns. Changes in these cellular and macromolecular components in blood caused by diseases have been suspected to cause changes in the dried drop patterns of plasma and whole blood, which could be used as simple diagnostic tools to identify the health of humans and livestock. However, complex physicochemical driving forces involved in the pattern formation are not fully understood. This review focuses on the scientific development in microscopic observations and pattern interpretation of dried plasma and whole blood samples, as well as the diagnostic applications of pattern analysis. Dried drop patterns of plasma consist of intricate visible cracks in the outer region and fine structures in the central region, which are mainly influenced by the presence and concentration of inorganic salts and proteins during drying. The shrinkage of macromolecular gel and its adhesion to the substrate surface have been thought to be responsible for the formation of the cracks. Dried drop patterns of whole blood have three characteristic zones; their formation as functions of drying time has been reported in the literature. Some research works have applied engineering treatment to the evaporation process of whole blood samples. The sensitivities of the resultant patterns to the relative humidity of the environment, the wettability of the substrates, and the size of the drop have been reported. These research works shed light on the mechanisms of spreading, evaporation, gelation, and crack formation of the blood drops on solid substrates, as well as on the potential applications of dried drop patterns of plasma and whole blood in diagnosis. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Immunoassay and antibody microarray analysis of the HUPO Plasma Proteome Project reference specimens: Systematic variation between sample types and calibration of mass spectrometry data

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

Haab, Brian B.; Geierstanger, Bernhard H.; Michailidis, George

2005-08-01

Four different immunoassay and antibody microarray methods performed at four different sites were used to measure the levels of a broad range of proteins (N = 323 assays; 39, 88, 168, and 28 assays at the respective sites; 237 unique analytes) in the human serum and plasma reference specimens distributed by the Plasma Proteome Project (PPP) of the HUPO. The methods provided a means to (1) assess the level of systematic variation in protein abundances associated with blood preparation methods (serum, citrate-anticoagulated-plasma, EDTA-anticoagulated-plasma, or heparin-anticoagulated-plasma) and (2) evaluate the dependence on concentration of MS-based protein identifications from data sets usingmore » the HUPO specimens. Some proteins, particularly cytokines, had highly variable concentrations between the different sample preparations, suggesting specific effects of certain anticoagulants on the stability or availability of these proteins. The linkage of antibody-based measurements from 66 different analytes with the combined MS/MS data from 18 different laboratories showed that protein detection and the quality of MS data increased with analyte concentration. The conclusions from these initial analyses are that the optimal blood preparation method is variable between analytes and that the discovery of blood proteins by MS can be extended to concentrations below the ng/mL range under certain circumstances. Continued developments in antibody-based methods will further advance the scientific goals of the PPP.« less

Rotor for processing liquids using movable capillary tubes

DOEpatents

Johnson, W.F.; Burtis, C.A.; Walker, W.A.

1987-07-17

A rotor assembly for processing liquids, especially whole blood samples, is disclosed. The assembly includes apparatus for separating non-liquid components of whole blood samples from liquid components, apparatus for diluting the separated liquid component with a diluent and apparatus for transferring the diluted sample to an external apparatus for analysis. The rotor assembly employs several movable capillary tubes to handle the sample and diluents. A method for using the rotor assembly to process liquids is also described. 5 figs.

Strategy for the hemocompatibility testing of microparticles.

PubMed

Braune, S; Basu, S; Kratz, K; Johansson, J Bäckemo; Reinthaler, M; Lendlein, A; Jung, F

2016-01-01

Polymer-based microparticles are applied as non-thrombogenic or thrombogenic materials in a wide variety of intra- or extra-corporeal medical devices. As demanded by the regulatory agencies, the hemocompatibility of these blood contacting biomaterials has to be evaluated in vitro to ensure that the particle systems appropriately fulfill the envisioned function without causing undesired events such as thrombosis or inflammation. Currently described in vitro assays for hemocompatibility testing of particles comprise tests with different single cell types (e.g. erythrocytes or leukocytes), varying concentrations/dilutions of the used blood cells or whole blood, which are not standardized.Here, we report about an in vitro dynamic test system for studying the hemocompatibility of polymeric microparticles utilizing fresh human whole blood from apparently healthy subjects, collected and processed under standardized conditions. Spherical poly(ether imide) microparticles with an average diameter of 140±30 μm were utilized as model systems. Reported as candidate materials for the removal of uremic toxins, these microparticles are anticipated to facilitate optimal flow conditions in a dialyzer with minimal backflow and blood cell damage. Pristine (PEI) and potassium hydroxide (PEI-KOH) functionalized microparticles exhibited similarly nanoporous surfaces (PEI: ØExternal pore = 90±60 nm; PEI-KOH ØExternal pore = 150±130 nm) but varying water wettabilities (PEI: θadv = 112±10° PEI-KOH θadv = 60±2°). The nanoporosity of the microparticle surfaces allows the exchange of toxic solutes from blood towards the interconnective pores in the particle core, while an immigration of the substantially larger blood cells is inhibited.Sterilized PEI microparticles were incorporated -air-free -in a syringe-based test system and exposed to whole blood for 60 minutes under gentle agitation. Thereafter, thrombi formation on the particles surfaces were analyzed microscopically. In the collected whole blood the non-adherent/circulating single blood cells were quantified via a differentiated complete blood cell count and the activation of platelets (P-Selectin expression, secretion and release), platelet function (PFA100 closure time) as well as thrombin formation (thrombin-antithrombin-complex) was analyzed. Free hemoglobin (HGB) levels were quantified as a measure of hemolysis.Microscopic evaluation revealed thrombi formation and particle aggregates for all tested microparticles. Reduction of circulating blood cells differed significantly between the particle types. Particularly, platelet and monocyte counts decreased up to 50% compared to the control (syringe filled with whole blood but without microparticles). In accordance, platelet activation, thrombin levels and degrees of hemolysis were clearly elevated in the particle loaded test systems and allowed a differentiation between the particle types. Increased PFA100 closure times (as activating agent a combination of collagen/ADP was used) indicated a similarly reduced ability of platelets to adhere and form stable aggregates independent from the particle type tested. This observation is most probably a consequence of the strong thrombus formation in the test system, which is associated with a reduction of the circulating blood cells.The reported in vitro dynamic whole blood test system allowed the sensitive analysis of the hemocompatibility of polymer-based microparticles and was successfully validated for porous PEI microparticles with different water wettabilities. Beyond the qualitative and quantitative analysis of cell-material interactions, the test also allowed the functional evaluation of platelets in whole blood.

Contributions to Statistical Problems Related to Microarray Data

ERIC Educational Resources Information Center

Hong, Feng

2009-01-01

Microarray is a high throughput technology to measure the gene expression. Analysis of microarray data brings many interesting and challenging problems. This thesis consists three studies related to microarray data. First, we propose a Bayesian model for microarray data and use Bayes Factors to identify differentially expressed genes. Second, we…

[Identification of Animal Whole Blood Based on Near Infrared Transmission Spectroscopy].

PubMed

Wan, Xiong; Wang, Jian; Liu, Peng-xi; Zhang, Ting-ting

2016-01-01

The inspection and classification for blood products are important but complicated in import-export ports or inspection and quarantine departments. For the inspection of whole blood products, open sampling can cause pollution and virulence factors in bloods samples may even endanger inspectors. Thus non-contact classification and identification methods for whole bloods of animals are needed. Spectroscopic techniques adopted in the flowcytometry need sampling blood cells during the detection; therefore they can not meet the demand of non-contact identification and classification for whole bloods of animals. Infrared absorption spectroscopy is a technique that can be used to analyze the molecular structure and chemical bonds of detected samples under the condition of non-contact. To find a feasible spectroscopic approach of non-contact detection for the species variation in whole blood samples, a near infrared transmitted spectra (NITS, 4 497.669 - 7 506.4 cm(-1)) experiment of whole blood samples of three common animals including chickens, dogs and cats has been conducted. During the experiment, the spectroscopic resolution is 5 cm(-1), and each spectrogram is an average of 5 measured spectral data. Experimental results show that all samples have a sharp absorption peak between 5 184 and 5 215 cm(-1), and a gentle absorption peak near 7 000 cm(-1). Besides, the NITS curves of different samples of same animals are similar, and only have slight differences in the whole transmittance. A correlation coefficient (CC) is induced to distinguish the differences of the three animals' whole bloods in NITS curves, and the computed CCs between NITS curves of different samples of the same animals, are greater than 0.99, whereas CCs between NITS curves of the whole bloods of different animals are from 0.509 48 to 0.916 13. Among which CCs between NITS curves of the whole bloods of chickens and cats are from 0.857 23 to 0.912 44, CCs between NITS curves of the whole bloods of chickens and dogs are from 0.509 48 to 0.664 82, and CCs between NITS curves of the whole bloods of cats and dogs are from 0.872 75 to 0.916 13. The cat and the dog belong to the class of mammal, and the CCs between their whole bloods NITS curves are greater than those between chickens and cats, or chickens and dogs, which are hetero-class animals. Namely, the whole bloods NITS curves of the cat and the dog have higher similarity. These results of NITS provide a feasible method of non-contact identification of animal whole bloods.

Effects of subchronic benzo(a)pyrene exposure on neurotransmitter receptor gene expression in the rat hippocampus related with spatial learning and memory change.

PubMed

Qiu, Chongying; Cheng, Shuqun; Xia, Yinyin; Peng, Bin; Tang, Qian; Tu, Baijie

2011-11-18

Exposure of laboratory rats to Benzo(a)pyrene (BaP), an environmental contaminant with its high lipophilicify which is widely dispersed in the environment and can easily cross the blood brain barrier presenting in the central nervous system, is associated with impaired learning and memory. The purpose of the research was to examine whether subchronic exposure to BaP affects spatial learning and memory, and how it alters normal gene expression in hippocampus, as well as selection of candidate genes involving neurotransmitter receptor attributed to learning and memory. Morris water maze (MWM) was used to evaluate behavioral differences between BaP-treated and vehicle-treated groups. To gain a better insight into the mechanism of BaP-induced neurotoxicity on learning and memory, we used whole genome oligo microarrays as well as Polymerase Chain Reaction (PCR) to assess the global impact of gene expression. Male Sprague-Dawley rats were intraperitoneally injected with 6.25mg/kg of BaP or vehicle for 14 weeks. The results from the Morris water maze (MWM) test showed that rats treated with BaP exhibited significantly higher mean latencies as compared to vehicle controls. BaP exposure significantly decreased the number of crossing the platform and the time spent in the target area. After the hippocampus was collected from each rat, total RNA was isolated. Microarray and PCR revealed that exposure to BaP affected mRNA expression of neurotransmitter receptors. The web tool DAVID was used to analyze the significantly enriched gene ontology (GO) and KEGG pathways in the differentially expressed genes. Analysis showed that the most significantly affected gene ontology category was behavior. Furthermore, the fourth highest significantly affected gene ontology category was learning and memory. KEGG molecular pathway analysis showed that "neuroactive ligand-receptor interaction" was affected by BaP with highest statistical significance, and 9 candidate neurotransmitter receptor genes involving learning and memory were selected out. Our results revealed a close link between behavioral changes and altered neurotransmitter receptor gene expression in BaP-treated rats. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Creation of antifouling microarrays by photopolymerization of zwitterionic compounds for protein assay and cell patterning.

PubMed

Sun, Xiuhua; Wang, Huaixin; Wang, Yuanyuan; Gui, Taijiang; Wang, Ke; Gao, Changlu

2018-04-15

Nonspecific binding or adsorption of biomolecules presents as a major obstacle to higher sensitivity, specificity and reproducibility in microarray technology. We report herein a method to fabricate antifouling microarray via photopolymerization of biomimetic betaine compounds. In brief, carboxybetaine methacrylate was polymerized as arrays for protein sensing, while sulfobetaine methacrylate was polymerized as background. With the abundant carboxyl groups on array surfaces and zwitterionic polymers on the entire surfaces, this microarray allows biomolecular immobilization and recognition with low nonspecific interactions due to its antifouling property. Therefore, low concentration of target molecules can be captured and detected by this microarray. It was proved that a concentration of 10ngmL -1 bovine serum albumin in the sample matrix of bovine serum can be detected by the microarray derivatized with anti-bovine serum albumin. Moreover, with proper hydrophilic-hydrophobic designs, this approach can be applied to fabricate surface-tension droplet arrays, which allows surface-directed cell adhesion and growth. These light controllable approaches constitute a clear improvement in the design of antifouling interfaces, which may lead to greater flexibility in the development of interfacial architectures and wider application in blood contact microdevices. Copyright © 2017 Elsevier B.V. All rights reserved.

Microarray platform for omics analysis

NASA Astrophysics Data System (ADS)

Mecklenburg, Michael; Xie, Bin

2001-09-01

Microarray technology has revolutionized genetic analysis. However, limitations in genome analysis has lead to renewed interest in establishing 'omic' strategies. As we enter the post-genomic era, new microarray technologies are needed to address these new classes of 'omic' targets, such as proteins, as well as lipids and carbohydrates. We have developed a microarray platform that combines self- assembling monolayers with the biotin-streptavidin system to provide a robust, versatile immobilization scheme. A hydrophobic film is patterned on the surface creating an array of tension wells that eliminates evaporation effects thereby reducing the shear stress to which biomolecules are exposed to during immobilization. The streptavidin linker layer makes it possible to adapt and/or develop microarray based assays using virtually any class of biomolecules including: carbohydrates, peptides, antibodies, receptors, as well as them ore traditional DNA based arrays. Our microarray technology is designed to furnish seamless compatibility across the various 'omic' platforms by providing a common blueprint for fabricating and analyzing arrays. The prototype microarray uses a microscope slide footprint patterned with 2 by 96 flat wells. Data on the microarray platform will be presented.

Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease

PubMed Central

Eshoo, Mark W.; Crowder, Christopher C.; Rebman, Alison W.; Rounds, Megan A.; Matthews, Heather E.; Picuri, John M.; Soloski, Mark J.; Ecker, David J.; Schutzer, Steven E.; Aucott, John N.

2012-01-01

Direct molecular tests in blood for early Lyme disease can be insensitive due to low amount of circulating Borrelia burgdorferi DNA. To address this challenge, we have developed a sensitive strategy to both detect and genotype B. burgdorferi directly from whole blood collected during the initial patient visit. This strategy improved sensitivity by employing 1.25 mL of whole blood, a novel pre-enrichment of the entire specimen extract for Borrelia DNA prior to a multi-locus PCR and electrospray ionization mass spectrometry detection assay. We evaluated the assay on blood collected at the initial presentation from 21 endemic area patients who had both physician-diagnosed erythema migrans (EM) and positive two-tiered serology either at the initial visit or at a follow-up visit after three weeks of antibiotic therapy. Results of this DNA analysis showed detection of B. burgdorferi in 13 of 21 patients (62%). In most cases the new assay also provided the B. burgdorferi genotype. The combined results of our direct detection assay with initial physician visit serology resulted in the detection of early Lyme disease in 19 of 21 (90%) of patients at the initial visit. In 5 of 21 cases we demonstrate the ability to detect B. burgdorferi in early Lyme disease directly from whole blood specimens prior to seroconversion. PMID:22590620

Expression Profile of Long Noncoding RNAs in Peripheral Blood Mononuclear Cells from Multiple Sclerosis Patients.

PubMed

Zhang, Fang; Gao, Chao; Ma, Xiao-Feng; Peng, Xiao-Lin; Zhang, Rong-Xin; Kong, De-Xin; Simard, Alain R; Hao, Jun-Wei

2016-04-01

Long noncoding RNAs (lncRNAs) play a key role in regulating immunological functions. Their impact on the chronic inflammatory disease multiple sclerosis (MS), however, remains unknown. We investigated the expression of lncRNAs in peripheral blood mononuclear cells (PBMCs) of patients with MS and attempt to explain their possible role in the process of MS. For this study, we recruited 26 patients with MS according to the revised McDonald criteria. Then, we randomly chose 6 patients for microarray analysis. Microarray assays identified outstanding differences in lncRNA expression, which were verified through real-time PCR. LncRNA functions were annotated for target genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and regulatory relationships between lncRNAs and target genes were analyzed using the "cis" and "trans" model. There were 2353 upregulated lncRNAs, 389 downregulated lncRNAs, 1037 upregulated mRNAs, and 279 downregulated mRNAs in patients with MS compared to healthy control subjects (fold change >2.0). Real-time PCR results of six aberrant lncRNAs were consistent with the microarray data. The coexpression network comprised 864 lncRNAs and 628 mRNAs. Among differentially expressed lncRNAs, 10 lncRNAs were predicted to have 10 cis-regulated target genes, and 33 lncRNAs might regulate their trans target genes. We identified a subset of dysregulated lncRNAs and mRNAs. The differentially expressed lncRNAs may be important in the process of MS. However, the specific molecular mechanisms and biological functions of these lncRNAs in the pathogenesis of MS need further study. © 2016 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.

Microarrays in brain research: the good, the bad and the ugly.

PubMed

Mirnics, K

2001-06-01

Making sense of microarray data is a complex process, in which the interpretation of findings will depend on the overall experimental design and judgement of the investigator performing the analysis. As a result, differences in tissue harvesting, microarray types, sample labelling and data analysis procedures make post hoc sharing of microarray data a great challenge. To ensure rapid and meaningful data exchange, we need to create some order out of the existing chaos. In these ground-breaking microarray standardization and data sharing efforts, NIH agencies should take a leading role

The application of silicon sol-gel technology to forensic blood substitute development: Mimicking aspects of whole human blood rheology.

PubMed

Stotesbury, Theresa; Illes, Mike; Wilson, Paul; Vreugdenhil, Andrew J

2017-01-01

Solution-gelation chemistry has promising applications in forensic synthetic blood substitute development. This research offers a silicon-based sol-gel approach to creating stable materials that share similar rheological properties to that of whole human blood samples. Room temperature, high water content, silicon sol-gels were created using the organosilane precursors 3-glycidoxypropyltrimethoxysilane and tetraethylorthosilicate along with various concentrations of filler and pigment. Shear-thinning non-Newtonian properties were observed within most formulations of the presented materials. The effects of colloidal concentration, temperature, age and filler addition on the viscosity of the sol-gels were investigated. SEM-EDS analysis was used to identify the behavior of the fillers within the film and support their inclusion for basic bloodstain pattern simulation. A final proposed candidate sol-gel was assessed using a previously reported passive drip simulation test on a hard, dry surface and passed. This works represents encouraging development in providing safe material alternatives to using whole human blood for forensic training and research. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Rapid killing of Capnocytophaga canimorsus and Capnocytophaga cynodegmi by human whole blood and serum is mediated via the complement system.

PubMed

Zangenah, Salah; Bergman, Peter

2015-01-01

Capnocytophaga canimorsus (Cani) and Capnocytophaga cynodegmi (Cyno) are found in the oral cavities of dogs and cats. They can be transmitted to humans via licks or bites and cause wound infections as well as severe systemic infections. Cani is considered to be more pathogenic than Cyno, but the pathophysiological mechanisms are not elucidated. Cani has been suggested to be resistant to serum bactericidal effects. Thus, we hypothesized that the more invasive Cani would exhibit a higher degree of serum-resistance than the less pathogenic Cyno. Whole blood and serum bactericidal assays were performed against Cani- (n = 8) and Cyno-strains (n = 15) isolated from blood and wound-specimens, respectively. Analysis of complement-function was performed by heat-inactivation, EGTA-treatment and by using C1q-depleted serum. Serum and whole blood were collected from healthy individuals and from patients (n = 3) with a history of sepsis caused by Cani. Both Cani and Cyno were equally susceptible to human whole blood and serum. Cani was preferentially killed by the classical pathway of the complement-system whereas Cyno was killed by a partly different mechanism. Serum from 2/3 Cani-infected patients were deficient in MBL-activity but still exhibited the same killing effect as control sera. Both Cani and Cyno were readily killed by human whole blood and serum in a complement-dependent way. Thus, it is not likely that serum bactericidal capacity is the key determinant for the clinical outcome in Cani or Cyno-infections.

West Nile virus nucleic acid persistence in whole blood months after clearance in plasma: implication for transfusion and transplantation safety

PubMed Central

Lanteri, Marion C.; Lee, Tzong-Hae; Wen, Li; Kaidarova, Zhanna; Bravo, Marjorie D.; Kiely, Nancy E.; Kamel, Hany T.; Tobler, Leslie H.; Norris, Philip J.; Busch, Michael P.

2014-01-01

BACKGROUND Previous reports of WNV RNA persistence in blood compartments have raised concerns around the remaining risk of WNV transfusion-transmission. This study characterized the dynamics of WNV viremia in blood compartments in a longitudinal cohort of 54 WNV-infected blood donors. STUDY DESIGN AND METHODS Blood samples were collected throughout the year after WNV RNA+ blood donation (index) and characterized for anti-WNV IgM and IgG antibodies and for WNV RNA by real-time reverse-transcription polymerase chain reaction. WNV viral loads were compared in plasma and whole blood samples and correlated with blood groups and clinical outcomes. RESULTS WNV RNA persisted in the red blood cell (RBC) compartment up to three months post-index in 42% of the donors. Donors with the highest WNV RNA levels in plasma at index maintained the highest WNV RNA levels in whole blood over the three months post-index. Blood group A donors maintained higher post-index WNV viral load in whole blood than blood group O individuals (P=0.027). Despite a trend for WNV RNA to persist longer in whole blood from symptomatic subjects, no significant association was found between WNV RNA levels in whole blood and disease outcome. CONCLUSION This study confirmed that WNV RNA persists in the RBC fraction in whole blood and further suggested that the level of persistence in whole blood may be a reflection of initial viral burden in plasma. The association with blood groups suggests that WNV adherence to RBCs may be mediated by molecules overrepresented at the surface of blood group A RBCs. PMID:24965017

Negative Enrichment and Isolation of Circulating Tumor Cells for Whole Genome Amplification.

PubMed

Kanwar, Nisha; Done, Susan J

2017-01-01

Circulating tumor cells (CTCs) are a rare population of cells found in the peripheral blood of patients with many types of cancer such as breast, prostate, colon, and lung cancers. Higher numbers of these cells in blood are associated with a poorer prognosis of patients. Genomic profiling of CTCs would help characterize markers specific for the identification of these cells in blood, and also define genomic alterations that give these cells a metastatic advantage over other cells in the primary tumor. Here, we describe an immunomagnetic method to enrich CTCs from the blood of patients with breast cancer, followed by single-cell laser capture microdissection to isolate single CTCs. Whole genome amplification of isolated CTCs allows for many downstream applications to be performed to aide in their characterization, such as whole genome or exome sequencing, Single Nucleotide Polymorphism (SNP) and copy number analysis, and targeted sequencing or quantitative Polymerase Chain Reaction (qPCR) for genomic analyses.

Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity

PubMed Central

2010-01-01

Background The European sea bass (Dicentrarchus labrax) is a marine fish of great importance for fisheries and aquaculture. Functional genomics offers the possibility to discover the molecular mechanisms underlying productive traits in farmed fish, and a step towards the application of marker assisted selection methods in this species. To this end, we report here on the development of an oligo DNA microarray for D. labrax. Results A database consisting of 19,048 unique transcripts was constructed, of which 12,008 (63%) could be annotated by similarity and 4,692 received a GO functional annotation. Two non-overlapping 60mer probes were designed for each unique transcript and in-situ synthesized on glass slides using Agilent SurePrint™ technology. Probe design was positively completed for 19,035 target clusters; the oligo microarray was then applied to profile gene expression in mandibles and whole-heads of fish affected by prognathism, a skeletal malformation that strongly affects sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish, with a significant enrichment in genes related to nervous system development and functioning. A set of genes spanning a wide dynamic range in gene expression level were selected for quantitative RT-PCR validation. Fold change correlation between microarray and qPCR data was always significant. Conclusions The microarray platform developed for the European sea bass has a high level of flexibility, reliability, and reproducibility. Despite the well known limitations in achieving a proper functional annotation in non-model species, sufficient information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on putative mechanisms involved on mandibular prognathism, suggesting that bone/nervous system development might play a role in this phenomenon. PMID:20525278

HDBStat!: a platform-independent software suite for statistical analysis of high dimensional biology data.

PubMed

Trivedi, Prinal; Edwards, Jode W; Wang, Jelai; Gadbury, Gary L; Srinivasasainagendra, Vinodh; Zakharkin, Stanislav O; Kim, Kyoungmi; Mehta, Tapan; Brand, Jacob P L; Patki, Amit; Page, Grier P; Allison, David B

2005-04-06

Many efforts in microarray data analysis are focused on providing tools and methods for the qualitative analysis of microarray data. HDBStat! (High-Dimensional Biology-Statistics) is a software package designed for analysis of high dimensional biology data such as microarray data. It was initially developed for the analysis of microarray gene expression data, but it can also be used for some applications in proteomics and other aspects of genomics. HDBStat! provides statisticians and biologists a flexible and easy-to-use interface to analyze complex microarray data using a variety of methods for data preprocessing, quality control analysis and hypothesis testing. Results generated from data preprocessing methods, quality control analysis and hypothesis testing methods are output in the form of Excel CSV tables, graphs and an Html report summarizing data analysis. HDBStat! is a platform-independent software that is freely available to academic institutions and non-profit organizations. It can be downloaded from our website http://www.soph.uab.edu/ssg_content.asp?id=1164.

Transcriptional signature associated with early rheumatoid arthritis and healthy individuals at high risk to develop the disease

PubMed Central

Macías-Segura, N.; Bastian, Y.; Santiago-Algarra, D.; Castillo-Ortiz, J. D.; Alemán-Navarro, A. L.; Jaime-Sánchez, E.; Gomez-Moreno, M.; Saucedo-Toral, C. A.; Lara-Ramírez, Edgar E.; Zapata-Zuñiga, M.; Enciso-Moreno, L.; González-Amaro, R.; Ramos-Remus, C.; Enciso-Moreno, J. A.

2018-01-01

Background Little is known regarding the mechanisms underlying the loss of tolerance in the early and preclinical stages of autoimmune diseases. The aim of this work was to identify the transcriptional profile and signaling pathways associated to non-treated early rheumatoid arthritis (RA) and subjects at high risk. Several biomarker candidates for early RA are proposed. Methods Whole blood total RNA was obtained from non-treated early RA patients with <1 year of evolution as well as from healthy first-degree relatives of patients with RA (FDR) classified as ACCP+ and ACCP- according to their antibodies serum levels against cyclic citrullinated peptides. Complementary RNA (cRNA) was synthetized and hybridized to high-density microarrays. Data was analyzed in Genespring Software and functional categories were assigned to a specific transcriptome identified in subjects with RA and FDR ACCP positive. Specific signaling pathways for genes associated to RA were identified. Gene expression was evaluated by qPCR. Receiver operating characteristic (ROC) analysis was used to evaluate these genes as biomarkers. Results A characteristic transcriptome of 551 induced genes and 4,402 repressed genes were identified in early RA patients. Bioinformatics analysis of the data identified a specific transcriptome in RA patients. Moreover, some overlapped transcriptional profiles between patients with RA and ACCP+ were identified, suggesting an up-regulated distinctive transcriptome from the preclinical stages up to progression to an early RA state. A total of 203 pathways have up-regulated genes that are shared between RA and ACCP+. Some of these genes show potential to be used as progression biomarkers for early RA with area under the curve of ROC > 0.92. These genes come from several functional categories associated to inflammation, Wnt signaling and type I interferon pathways. Conclusion The presence of a specific transcriptome in whole blood of RA patients suggests the activation of a specific inflammatory transcriptional signature in early RA development. The set of overexpressed genes in early RA patients that are shared with ACCP+ subjects but not with ACCP- subjects, can represent a transcriptional signature involved with the transition of a preclinical to a clinical RA stage. Some of these particular up-regulated and down-regulated genes are related to inflammatory processes and could be considered as biomarker candidates for disease progression in subjects at risk to develop RA. PMID:29584756

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

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

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

DOE PAGES

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

2016-04-07

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

Global transcriptional responses of Bacillus subtilis to xenocoumacin 1.

PubMed

Zhou, T; Zeng, H; Qiu, D; Yang, X; Wang, B; Chen, M; Guo, L; Wang, S

2011-09-01

To determine the global transcriptional response of Bacillus subtilis to an antimicrobial agent, xenocoumacin 1 (Xcn1). Subinhibitory concentration of Xcn1 applied to B. subtilis was measured according to Hutter's method for determining optimal concentrations. cDNA microarray technology was used to study the global transcriptional response of B. subtilis to Xcn1. Real-time RT-PCR was employed to verify alterations in the transcript levels of six genes. The subinhibitory concentration was determined to be 1 μg ml(-1). The microarray data demonstrated that Xcn1 treatment of B. subtilis led to more than a 2.0-fold up-regulation of 480 genes and more than a 2.0-fold down-regulation of 479 genes (q ≤ 0.05). The transcriptional responses of B. subtilis to Xcn1 were determined, and several processes were affected by Xcn1. Additionally, cluster analysis of gene expression profiles after treatment with Xcn1 or 37 previously studied antibiotics indicated that Xcn1 has similar mechanisms of action to protein synthesis inhibitors. These microarray data showed alterations of gene expression in B. subtilis after exposure to Xcn1. From the results, we identified various processes affected by Xcn1. This study provides a whole-genome perspective to elucidate the action of Xcn1 as a potential antimicrobial agent. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Gene-body hypermethylation of ATM in peripheral blood DNA of bilateral breast cancer patients

PubMed Central

Flanagan, James M.; Munoz-Alegre, Marta; Henderson, Stephen; Tang, Thomas; Sun, Ping; Johnson, Nichola; Fletcher, Olivia; dos Santos Silva, Isabel; Peto, Julian; Boshoff, Chris; Narod, Steven; Petronis, Arturas

2009-01-01

Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility; however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer-related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared with 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulphite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared with 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared with controls (P = 0.0017), with the highest quartile of methylation associated with a 3-fold increased risk of breast cancer (OR 3.20, 95% CI 1.78–5.86, P = 0.000083). Increased methylation of this locus is associated with lower steady-state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age–phenotype-related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer-related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk. Accession numbers: The microarray data and associated .BED and .WIG files can be accessed through Gene Expression Omnibus accession number: GSE14603. PMID:19153073

EFFECT OF A SHORT PERIOD WHOLE BODY VIBRATION WITH 10 HZ ON BLOOD BIOMARKERS IN WISTAR RATS

PubMed Central

Monteiro, Milena de Oliveira Bravo; de Sá-Caputo, Danúbia da Cunha; Moreira-Marconi, Eloá; Frederico, Éric Heleno Freire Ferreira; de Sousa-Gonçalves, Cintia Renata; Bernardo, Luciana Camargo; Guimarães, Carlos Alberto Sampaio; Bernardo-Filho, Mario

2017-01-01

Background: Exposure to whole body vibration exercises (WBVE), besides some biological effects, causes alterations in the concentration of some blood biomarkers. The aim of this study is to evaluate the action of vibration (10 Hz) of WBVE on the concentration of blood biomarkers in Wistar rats. Materials and Methods: Wistar rats were divided in 2 groups. The experimental group (EG) was subjected to vibrations of 10Hz (one min per day, one week, total time of seven min), while the control group (CG) has not experienced vibration. Samples of whole blood were drawn for biochemical analysis of the concentration of total cholesterol, triglycerides, HDL, LDL, VLDL, glucose, CPK, albumin, alkaline phosphates, TGP, TGO, γGT, lipase, amylase, urea and creatinine. Results: White blood cell count and a platelet-hemogram were also performed. Significant (p<0.05) increase in TGP, TGO and white blood cells and decrease in LDL concentration was found after exposure of 10Hz mechanical vibration. Conclusion: Although these findings were obtained with rats, they might contribute to try to understand better these mechanisms that occur following exposure to a frequency of 10Hz. PMID:28740939

Elasto-inertial microfluidics for bacteria separation from whole blood for sepsis diagnostics.

PubMed

Faridi, Muhammad Asim; Ramachandraiah, Harisha; Banerjee, Indradumna; Ardabili, Sahar; Zelenin, Sergey; Russom, Aman

2017-01-04

Bloodstream infections (BSI) remain a major challenge with high mortality rate, with an incidence that is increasing worldwide. Early treatment with appropriate therapy can reduce BSI-related morbidity and mortality. However, despite recent progress in molecular based assays, complex sample preparation steps have become critical roadblock for a greater expansion of molecular assays. Here, we report a size based, label-free, bacteria separation from whole blood using elasto-inertial microfluidics. In elasto-inertial microfluidics, the viscoelastic flow enables size based migration of blood cells into a non-Newtonian solution, while smaller bacteria remain in the streamline of the blood sample entrance and can be separated. We first optimized the flow conditions using particles, and show continuous separation of 5 μm particles from 2 μm at a yield of 95% for 5 µm particle and 93% for 2 µm particles at respective outlets. Next, bacteria were continuously separated at an efficiency of 76% from undiluted whole blood sample. We demonstrate separation of bacteria from undiluted while blood using elasto-inertial microfluidics. The label-free, passive bacteria preparation method has a great potential for downstream phenotypic and molecular analysis of bacteria.

Gene expression profiling in whole blood of patients with coronary artery disease

PubMed Central

Taurino, Chiara; Miller, William H.; McBride, Martin W.; McClure, John D.; Khanin, Raya; Moreno, María U.; Dymott, Jane A.; Delles, Christian; Dominiczak, Anna F.

2010-01-01

Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease. PMID:20528768

Use of diagnostic accuracy as a metric for evaluating laboratory proficiency with microarray assays using mixed-tissue RNA reference samples.

PubMed

Pine, P S; Boedigheimer, M; Rosenzweig, B A; Turpaz, Y; He, Y D; Delenstarr, G; Ganter, B; Jarnagin, K; Jones, W D; Reid, L H; Thompson, K L

2008-11-01

Effective use of microarray technology in clinical and regulatory settings is contingent on the adoption of standard methods for assessing performance. The MicroArray Quality Control project evaluated the repeatability and comparability of microarray data on the major commercial platforms and laid the groundwork for the application of microarray technology to regulatory assessments. However, methods for assessing performance that are commonly applied to diagnostic assays used in laboratory medicine remain to be developed for microarray assays. A reference system for microarray performance evaluation and process improvement was developed that includes reference samples, metrics and reference datasets. The reference material is composed of two mixes of four different rat tissue RNAs that allow defined target ratios to be assayed using a set of tissue-selective analytes that are distributed along the dynamic range of measurement. The diagnostic accuracy of detected changes in expression ratios, measured as the area under the curve from receiver operating characteristic plots, provides a single commutable value for comparing assay specificity and sensitivity. The utility of this system for assessing overall performance was evaluated for relevant applications like multi-laboratory proficiency testing programs and single-laboratory process drift monitoring. The diagnostic accuracy of detection of a 1.5-fold change in signal level was found to be a sensitive metric for comparing overall performance. This test approaches the technical limit for reliable discrimination of differences between two samples using this technology. We describe a reference system that provides a mechanism for internal and external assessment of laboratory proficiency with microarray technology and is translatable to performance assessments on other whole-genome expression arrays used for basic and clinical research.

Whole mitochondrial genome screening in maternally inherited non-syndromic hearing impairment using a microarray resequencing mitochondrial DNA chip.

PubMed

Lévêque, Marianne; Marlin, Sandrine; Jonard, Laurence; Procaccio, Vincent; Reynier, Pascal; Amati-Bonneau, Patrizia; Baulande, Sylvain; Pierron, Denis; Lacombe, Didier; Duriez, Françoise; Francannet, Christine; Mom, Thierry; Journel, Hubert; Catros, Hélène; Drouin-Garraud, Valérie; Obstoy, Marie-Françoise; Dollfus, Hélène; Eliot, Marie-Madeleine; Faivre, Laurence; Duvillard, Christian; Couderc, Remy; Garabedian, Eréa-Noël; Petit, Christine; Feldmann, Delphine; Denoyelle, Françoise

2007-11-01

Mitochondrial DNA (mtDNA) mutations have been implicated in non-syndromic hearing loss either as primary or as predisposing factors. As only a part of the mitochondrial genome is usually explored in deafness, its prevalence is probably under-estimated. Among 1350 families with non-syndromic sensorineural hearing loss collected through a French collaborative network, we selected 29 large families with a clear maternal lineage and screened them for known mtDNA mutations in 12S rRNA, tRNASer(UCN) and tRNALeu(UUR) genes. When no mutation could be identified, a whole mitochondrial genome screening was performed, using a microarray resequencing chip: the MitoChip version 2.0 developed by Affymetrix Inc. Known mtDNA mutations was found in nine of the 29 families, which are described in the article: five with A1555G, two with the T7511C, one with 7472insC and one with A3243G mutation. In the remaining 20 families, the resequencing Mitochip detected 258 mitochondrial homoplasmic variants and 107 potentially heteroplasmic variants. Controls were made by direct sequencing on selected fragments and showed a high sensibility of the MitoChip but a low specificity, especially for heteroplasmic variations. An original analysis on the basis of species conservation, frequency and phylogenetic investigation was performed to select the more probably pathogenic variants. The entire genome analysis allowed us to identify five additional families with a putatively pathogenic mitochondrial variant: T669C, C1537T, G8078A, G12236A and G15077A. These results indicate that the new MitoChip platform is a rapid and valuable tool for identification of new mtDNA mutations in deafness.

Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

PubMed Central

Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

2009-01-01

Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks. PMID:19298644

Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum.

PubMed

Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

2009-03-19

Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

Differential loss of natural killer cell activity in patients with acute myocardial infarction and stable angina pectoris.

PubMed

Yan, Wenwen; Zhou, Lin; Wen, Siwan; Duan, Qianglin; Huang, Feifei; Tang, Yu; Liu, Xiaohong; Chai, Yongyan; Wang, Lemin

2015-01-01

To evaluate the activity of natural killer cells through their inhibitory and activating receptors and quantity in peripheral blood mononuclear cells extracted from patients with acute myocardial infarction, stable angina pectoris and the controls. 100 patients with myocardial infarction, 100 with stable angina, and 20 healthy volunteers were recruited into the study. 20 randomly chosen people per group were examined for the whole human genome microarray analysis to detect the gene expressions of all 40 inhibitory and activating natural killer cell receptors. Flow cytometry analysis was applied to all 200 patients to measure the quantity of natural killer cells. In myocardial infarction group, the mRNA expressions of six inhibitory receptors KIR2DL2, KIR3DL3, CD94, NKG2A, KLRB1, KLRG1, and eight activating receptors KIR2DS3, KIR2DS5, NKp30, NTB-A, CRACC, CD2, CD7 and CD96 were significantly down-regulated (P<0.05) compared with both angina patients and the controls. There was no statistical difference in receptor expressions between angina patients and control group. The quantity of natural killer cells was significantly decreased in both infarction and angina patients compared with normal range (P<0.001). The significant mRNAs down-regulation of several receptors in myocardial infarction group and reduction in the quantity of natural killer cells in both myocardial infarction and angina patients showed a quantitative loss and dysfunction of natural killer cells in myocardial infarction patients.

Identification of side- and shear-dependent microRNAs regulating porcine aortic valve pathogenesis

NASA Astrophysics Data System (ADS)

Rathan, Swetha; Ankeny, Casey J.; Arjunon, Sivakkumar; Ferdous, Zannatul; Kumar, Sandeep; Fernandez Esmerats, Joan; Heath, Jack M.; Nerem, Robert M.; Yoganathan, Ajit P.; Jo, Hanjoong

2016-05-01

Aortic valve (AV) calcification is an inflammation driven process that occurs preferentially in the fibrosa. To explore the underlying mechanisms, we investigated if key microRNAs (miRNA) in the AV are differentially expressed due to disturbed blood flow (oscillatory shear (OS)) experienced by the fibrosa compared to the ventricularis. To identify the miRNAs involved, endothelial-enriched RNA was isolated from either side of healthy porcine AVs for microarray analysis. Validation using qPCR confirmed significantly higher expression of 7 miRNAs (miR-100, -130a, -181a/b, -199a-3p, -199a-5p, and -214) in the fibrosa versus the ventricularis. Upon bioinformatics analysis, miR-214 was selected for further investigation using porcine AV leaflets in an ex vivo shear system. Fibrosa and ventricularis sides were exposed to either oscillatory or unidirectional pulsatile shear for 2 days and 3 & 7 days in regular and osteogenic media, respectively. Higher expression of miR-214, increased thickness of the fibrosa, and calcification was observed when the fibrosa was exposed to OS compared to the ventricularis. Silencing of miR-214 by anti-miR-214 in whole AV leaflets with the fibrosa exposed to OS significantly increased the protein expression of TGFβ1 and moderately increased collagen content but did not affect AV calcification. Thus, miR-214 is identified as a side- and shear-dependent miRNA that regulates key mechanosensitive gene in AV such as TGFβ1.

Coagulopathy in patients with acute pulmonary embolism: a pilot study of whole blood coagulation and markers of endothelial damage.

PubMed

Lehnert, Per; Johansson, Pär I; Ostrowski, Sisse R; Møller, Christian H; Bang, Lia E; Olsen, Peter Skov; Carlsen, Jørn

2017-02-01

Whole blood coagulation and markers of endothelial damage were studied in patients with acute pulmonary embolism (PE), and evaluated in relation to PE severity. Twenty-five patients were enrolled prospectively each having viscoelastical analysis of whole blood done using thrombelastography (TEG) and Multiplate aggregometry. Fourteen of these patients were investigated for endothelial damage by ELISA measurements of Syndecan-1 (endothelial glycocalyx degradation), soluble endothelial Selectin (endothelial cell activation), soluble Thrombomodulin (endothelial cell injury) and Histone Complexed DNA fragments (endothelial cytotoxic histones). The mean values of TEG and Multiplate parameters were all within the reference levels, but a significant difference between patients with high and intermediate risk PE was observed for Ly30 (lytic activity) 1.5% [0-10] vs. 0.2% [0-2.2] p = .04, and ADP (platelet reactivity) 92 U [20-145] vs. 59 U [20-111] p = .03. A similar difference was indicated for functional fibrinogen 21 mm [17-29] vs. 18 mm [3-23] p = .05. Analysis of endothelial markers identified a significant difference in circulating levels between high and intermediate risk PE patients for Syndecan-1 118.6 ng/mL [76-133] vs. 36.3 ng/mL [11.8-102.9] p = .008. In conclusion, patients with acute PE had normal whole blood coagulation, but high risk PE patients had signs of increased activity of the haemostatic system and significantly increased level of endothelial glycocalyx degradation.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives.

PubMed

Zhao, Min; Wang, Qingguo; Wang, Quan; Jia, Peilin; Zhao, Zhongming

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives

PubMed Central

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development. PMID:24564169

Evaluation of the testicular toxicity of prenatal exposure to bisphenol A based on microarray analysis combined with MeSH annotation.

PubMed

Tainaka, Hitoshi; Takahashi, Hikari; Umezawa, Masakazu; Tanaka, Hiromitsu; Nishimune, Yoshitake; Oshio, Shigeru; Takeda, Ken

2012-01-01

Bisphenol A (BPA) is known to be an endocrine disruptor that affects the development of reproductive system. The aim of the present study was to investigate a group of testicular genes dysregulated by prenatal exposure to BPA. Pregnant ICR mice were treated with BPA by subcutaneous administration on days 7 and 14 of pregnancy. Tissue and blood samples were collected from 6-week-old male offspring. Testes were subjected to gene expression analysis using a testis-specific microarray (Testis2), consisting of 2,482 mouse cDNA clones annotated with Medical Subject Headings (MeSH) terms indicative of testicular components and functions. To interpret the microarray data, we used the MeSH terms significantly associated with the altered genes. As a result, MeSH terms related to androgens and Sertoli cells were extracted in BPA-treated groups. Among the genes related to Sertoli cells, downregulation of Msi1h, Ncoa1, Nid1, Hspb2, and Gata6 were detected in the testis of mice treated with BPA (twice administered 50 mg/kg). The MeSH terms associated with this group of genes may provide useful means to interpret the testicular toxicity of BPA. This article concludes that prenatal BPA exposure downregulates expression of genes associated with Sertoli cell function and affects the reproductive function of male offspring. Additionally, a method using MeSH to extract a group of genes was useful for predicting the testicular and reproductive toxicity of prenatal BPA exposure.

Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma

PubMed Central

Konishi, H; Ichikawa, D; Komatsu, S; Shiozaki, A; Tsujiura, M; Takeshita, H; Morimura, R; Nagata, H; Arita, T; Kawaguchi, T; Hirashima, S; Fujiwara, H; Okamoto, K; Otsuji, E

2012-01-01

Background: Recently, it was reported that plasma microRNAs (miRNAs) are low-invasive useful biomarkers for cancer. We attempted to isolate gastric cancer (GC)-associated miRNAs comparing pre- and post-operative paired plasma, thereby excluding the possible effects of individual variability. Methods: This study was divided into four steps: (1) microarray analysis comparing pre- and post-operative plasma; (2) validation of candidate miRNAs by quantitative RT–PCR; (3) validation study of selected miRNAs using paired plasma; and (4) comparison of the levels of selected miRNAs in plasma between healthy controls and patients. Results: From the results of microarray analysis, nine candidate miRNAs the levels of which were markedly decreased in post-operative plasma were selected for further studies. After confirmation of their post-operative marked reduction, two candidate miRNAs, miR-451 and miR-486, were selected as plasma biomarkers, considering the abundance in plasma, and marked decrease in post-operative samples. In validation, the two miRNAs were found to decrease in post-operative plasma in 90 and 93% of patients (both P<0.01). In comparison with healthy controls, the levels of both miRNAs were found to be significantly higher in patients, and the area under the curve values were high at 0.96 and 0.92. Conclusion: Plasma miR-451 and miR-486 could be useful blood-based biomarkers for screening GC. PMID:22262318

Epigenetics of prostate cancer.

PubMed

McKee, Tawnya C; Tricoli, James V

2015-01-01

The introduction of novel technologies that can be applied to the investigation of the molecular underpinnings of human cancer has allowed for new insights into the mechanisms associated with tumor development and progression. They have also advanced the diagnosis, prognosis and treatment of cancer. These technologies include microarray and other analysis methods for the generation of large-scale gene expression data on both mRNA and miRNA, next-generation DNA sequencing technologies utilizing a number of platforms to perform whole genome, whole exome, or targeted DNA sequencing to determine somatic mutational differences and gene rearrangements, and a variety of proteomic analysis platforms including liquid chromatography/mass spectrometry (LC/MS) analysis to survey alterations in protein profiles in tumors. One other important advancement has been our current ability to survey the methylome of human tumors in a comprehensive fashion through the use of sequence-based and array-based methylation analysis (Bock et al., Nat Biotechnol 28:1106-1114, 2010; Harris et al., Nat Biotechnol 28:1097-1105, 2010). The focus of this chapter is to present and discuss the evidence for key genes involved in prostate tumor development, progression, or resistance to therapy that are regulated by methylation-induced silencing.

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity

PubMed Central

Pantazatos, Spiro P.; Huang, Yung-yu; Rosoklija, Gorazd B.; Dwork, Andrew J.; Arango, Victoria; Mann, J. John

2016-01-01

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing (NGS) approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden-death medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA expression (9 samples per group). DeSeq2 identified thirty-five genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted p<0.1). In depression, altered genes include humanin like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted p<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted p<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders. PMID:27528462

Whole-transcriptome brain expression and exon-usage profiling in major depression and suicide: evidence for altered glial, endothelial and ATPase activity.

PubMed

Pantazatos, S P; Huang, Y-Y; Rosoklija, G B; Dwork, A J; Arango, V; Mann, J J

2017-05-01

Brain gene expression profiling studies of suicide and depression using oligonucleotide microarrays have often failed to distinguish these two phenotypes. Moreover, next generation sequencing approaches are more accurate in quantifying gene expression and can detect alternative splicing. Using RNA-seq, we examined whole-exome gene and exon expression in non-psychiatric controls (CON, N=29), DSM-IV major depressive disorder suicides (MDD-S, N=21) and MDD non-suicides (MDD, N=9) in the dorsal lateral prefrontal cortex (Brodmann Area 9) of sudden death medication-free individuals post mortem. Using small RNA-seq, we also examined miRNA expression (nine samples per group). DeSeq2 identified 35 genes differentially expressed between groups and surviving adjustment for false discovery rate (adjusted P<0.1). In depression, altered genes include humanin-like-8 (MTRNRL8), interleukin-8 (IL8), and serpin peptidase inhibitor, clade H (SERPINH1) and chemokine ligand 4 (CCL4), while exploratory gene ontology (GO) analyses revealed lower expression of immune-related pathways such as chemokine receptor activity, chemotaxis and cytokine biosynthesis, and angiogenesis and vascular development in (adjusted P<0.1). Hypothesis-driven GO analysis suggests lower expression of genes involved in oligodendrocyte differentiation, regulation of glutamatergic neurotransmission, and oxytocin receptor expression in both suicide and depression, and provisional evidence for altered DNA-dependent ATPase expression in suicide only. DEXSEq analysis identified differential exon usage in ATPase, class II, type 9B (adjusted P<0.1) in depression. Differences in miRNA expression or structural gene variants were not detected. Results lend further support for models in which deficits in microglial, endothelial (blood-brain barrier), ATPase activity and astrocytic cell functions contribute to MDD and suicide, and identify putative pathways and mechanisms for further study in these disorders.

Warm fresh whole blood and thoracic traumain iraq and afghanistan.

PubMed

Keneally, Ryan J; Parsons, Andrew M; Willett, Peter B

2015-01-01

Thoracic trauma occurred in 10% of the patients seen at US military treatment facilities in Iraq and Afghanistan and 52% of those patients were transfused. Among those transfused, 281 patients received warm fresh whole blood. A previous report documented improved survival with warm fresh whole blood in patients injured in combat without stratification by injury pattern. A later report described an increase in acute lung injuries after its administration. Survivorship and warm fresh whole blood have never been analyzed in a subpopulation at highest risk for lung injuries, such as patients with thoracic trauma. There may be a heterogeneous relationship between whole blood and survival based on likelihood of a concomitant pulmonary injury. In this report, the relationship between warm fresh whole blood and survivorship was analyzed among patients at highest risk for concomitant pulmonary injuries. Patients with thoracic trauma who received a transfusion were identified in the Joint Theater Trauma Registry. Gross mortality rates were compared between whole blood recipients and patients transfused with component therapy only. The association between each blood component and mortality was determined in a regression model. The overall mortality risk was compared between warm fresh whole blood recipients and non-recipients. Patients transfused with warm fresh whole blood in addition to component therapy had a higher mortality rate than patients transfused only separated blood components (21.3% vs. 12.8%, P < 0.001). When controlling for covariates, transfusion of warm fresh whole blood in addition to component therapy was not associated with increased mortality risk compared with the transfusion of component therapy only (OR 1.247 [95% CI 0.760-2.048], P = 0.382). Patients with combat related thoracic trauma transfused with warm fresh whole blood were not at increased risk for mortality compared to those who received component therapy alone when controlling for covariates.

A Diagnosis of Insomnia Is Associated With Differential Expression of Sleep-Regulating Genes in Military Personnel.

PubMed

Gill, Jessica M; Lee, Hyunhwa; Baxter, Tristin; Reddy, Swarnalatha Y; Barr, Taura; Kim, Hyung-Suk; Wang, Dan; Mysliwiec, Vincent

2015-07-01

Sleep disturbance is a common and disturbing symptom in military personnel, with many individuals progressing to the development of insomnia, which is characterized by increased arousals, wakefulness after sleep onset, and distorted sleep architecture. The molecular mechanisms underlying insomnia remain elusive, limiting future therapeutic development to address this critical issue. We examined whole gene expression profiles associated with insomnia. We compared subjects with insomnia (n = 25) to controls (n = 13) without insomnia using microarray gene expression profiles obtained from peripheral samples of whole blood obtained from military personnel. Compared to controls, participants with insomnia had differential expression of 44 transcripts from 43 identified genes. Among the identified genes, urotensin 2 was downregulated by more than 6 times in insomnia participants, and the fold-change remained significant after controlling for depression, posttraumatic stress disorder, and medication use. Urotensin 2 is involved in regulation of orexin A and B activity and rapid eye movement during sleep. These findings suggest that differential expression of these sleep-regulating genes contributes to symptoms of insomnia and, specifically, that switching between rapid eye movement and nonrapid eye movement sleep stages underlies insomnia symptoms. Future work to identify therapeutic agents that are able to regulate these pathways may provide novel treatments for insomnia. © The Author(s) 2015.

Whole Blood Gene Expression Profiling Predicts Severe Morbidity and Mortality in Cystic Fibrosis: A 5-Year Follow-Up Study.

PubMed

Saavedra, Milene T; Quon, Bradley S; Faino, Anna; Caceres, Silvia M; Poch, Katie R; Sanders, Linda A; Malcolm, Kenneth C; Nichols, David P; Sagel, Scott D; Taylor-Cousar, Jennifer L; Leach, Sonia M; Strand, Matthew; Nick, Jerry A

2018-05-01

Cystic fibrosis pulmonary exacerbations accelerate pulmonary decline and increase mortality. Previously, we identified a 10-gene leukocyte panel measured directly from whole blood, which indicates response to exacerbation treatment. We hypothesized that molecular characteristics of exacerbations could also predict future disease severity. We tested whether a 10-gene panel measured from whole blood could identify patient cohorts at increased risk for severe morbidity and mortality, beyond standard clinical measures. Transcript abundance for the 10-gene panel was measured from whole blood at the beginning of exacerbation treatment (n = 57). A hierarchical cluster analysis of subjects based on their gene expression was performed, yielding four molecular clusters. An analysis of cluster membership and outcomes incorporating an independent cohort (n = 21) was completed to evaluate robustness of cluster partitioning of genes to predict severe morbidity and mortality. The four molecular clusters were analyzed for differences in forced expiratory volume in 1 second, C-reactive protein, return to baseline forced expiratory volume in 1 second after treatment, time to next exacerbation, and time to morbidity or mortality events (defined as lung transplant referral, lung transplant, intensive care unit admission for respiratory insufficiency, or death). Clustering based on gene expression discriminated between patient groups with significant differences in forced expiratory volume in 1 second, admission frequency, and overall morbidity and mortality. At 5 years, all subjects in cluster 1 (very low risk) were alive and well, whereas 90% of subjects in cluster 4 (high risk) had suffered a major event (P = 0.0001). In multivariable analysis, the ability of gene expression to predict clinical outcomes remained significant, despite adjustment for forced expiratory volume in 1 second, sex, and admission frequency. The robustness of gene clustering to categorize patients appropriately in terms of clinical characteristics, and short- and long-term clinical outcomes, remained consistent, even when adding in a secondary population with significantly different clinical outcomes. Whole blood gene expression profiling allows molecular classification of acute pulmonary exacerbations, beyond standard clinical measures, providing a predictive tool for identifying subjects at increased risk for mortality and disease progression.

DNA methylation analysis of phenotype specific stratified Indian population.

PubMed

Rotti, Harish; Mallya, Sandeep; Kabekkodu, Shama Prasada; Chakrabarty, Sanjiban; Bhale, Sameer; Bharadwaj, Ramachandra; Bhat, Balakrishna K; Dedge, Amrish P; Dhumal, Vikram Ram; Gangadharan, G G; Gopinath, Puthiya M; Govindaraj, Periyasamy; Joshi, Kalpana S; Kondaiah, Paturu; Nair, Sreekumaran; Nair, S N Venugopalan; Nayak, Jayakrishna; Prasanna, B V; Shintre, Pooja; Sule, Mayura; Thangaraj, Kumarasamy; Patwardhan, Bhushan; Valiathan, Marthanda Varma Sankaran; Satyamoorthy, Kapaettu

2015-05-08

DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

An Integrated Lab-on-Chip for Rapid Identification and Simultaneous Differentiation of Tropical Pathogens

PubMed Central

Sato, Mitsuharu; Watthanaworawit, Wanitda; Ling, Clare L.; Mauduit, Marjorie; Malleret, Benoît; Grüner, Anne-Charlotte; Tan, Rosemary; Nosten, François H.; Snounou, Georges; Rénia, Laurent; Ng, Lisa F. P.

2014-01-01

Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens. PMID:25078474

MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation

PubMed Central

Zubakov, Dmitry; Boersma, Anton W. M.; Choi, Ying; van Kuijk, Patricia F.; Wiemer, Erik A. C.

2010-01-01

MicroRNAs (miRNAs) are non-protein coding molecules with important regulatory functions; many have tissue-specific expression patterns. Their very small size in principle makes them less prone to degradation processes, unlike messenger RNAs (mRNAs), which were previously proposed as molecular tools for forensic body fluid identification. To identify suitable miRNA markers for forensic body fluid identification, we first screened total RNA samples derived from saliva, semen, vaginal secretion, and venous and menstrual blood for the expression of 718 human miRNAs using a microarray platform. All body fluids could be easily distinguished from each other on the basis of complete array-based miRNA expression profiles. Results from quantitative reverse transcription PCR (RT-PCR; TaqMan) assays for microarray candidate markers confirmed strong over-expression in the targeting body fluid of several miRNAs for venous blood and several others for semen. However, no candidate markers from array experiments for other body fluids such as saliva, vaginal secretion, or menstrual blood could be confirmed by RT-PCR. Time-wise degradation of venous blood and semen stains for at least 1 year under lab conditions did not significantly affect the detection sensitivity of the identified miRNA markers. The detection limit of the TaqMan assays tested for selected venous blood and semen miRNA markers required only subpicogram amounts of total RNA per single RT-PCR test, which is considerably less than usually needed for reliable mRNA RT-PCR detection. We therefore propose the application of several stable miRNA markers for the forensic identification of blood stains and several others for semen stain identification, using commercially available TaqMan assays. Additional work remains necessary in search for suitable miRNA markers for other forensically relevant body fluids. Electronic supplementary material The online version of this article (doi:10.1007/s00414-009-0402-3) contains supplementary material, which is available to authorized users. PMID:20145944

ArrayNinja: An Open Source Platform for Unified Planning and Analysis of Microarray Experiments.

PubMed

Dickson, B M; Cornett, E M; Ramjan, Z; Rothbart, S B

2016-01-01

Microarray-based proteomic platforms have emerged as valuable tools for studying various aspects of protein function, particularly in the field of chromatin biochemistry. Microarray technology itself is largely unrestricted in regard to printable material and platform design, and efficient multidimensional optimization of assay parameters requires fluidity in the design and analysis of custom print layouts. This motivates the need for streamlined software infrastructure that facilitates the combined planning and analysis of custom microarray experiments. To this end, we have developed ArrayNinja as a portable, open source, and interactive application that unifies the planning and visualization of microarray experiments and provides maximum flexibility to end users. Array experiments can be planned, stored to a private database, and merged with the imaged results for a level of data interaction and centralization that is not currently attainable with available microarray informatics tools. © 2016 Elsevier Inc. All rights reserved.

Distribution of Parvovirus B19 DNA in Blood Compartments and Persistence of Virus in Blood Donors

PubMed Central

Lee, Tzong-Hae; Kleinman, Steven H.; Wen, Li; Montalvo, Lani; Todd, Deborah S.; Wright, David J.; Tobler, Leslie H.; Busch, Michael P.

2013-01-01

Introduction Because the receptor for Parvovirus B19 (B19V) is on erythrocytes, we investigated B19V distribution in blood by in-vitro spiking experiments and evaluated viral compartmentalization and persistence in natural infection. Methods Two whole blood protocols (ultracentrifugation and a rapid RBC lysis/removal protocol) were evaluated using quantitative real-time PCR. Whole blood (WB) was spiked with known concentrations of B19V and recovery in various blood fractions was determined. The rapid RBC lysis/removal protocol was then used to compare B19V concentrations in 104 paired whole blood and plasma samples collected longitudinally from 43 B19V infected donors with frozen specimens in the REDS Allogeneic Donor and Recipient Repository (RADAR). Results In B19V spiking experiments, ~one-third of viral DNA was recovered in plasma and two-thirds was loosely bound to erythrocytes. In the IgM positive stage of infection in blood donors when plasma B19V DNA concentrations were > 100 IU/mL, median DNA concentrations were ~30-fold higher in WB than in plasma. In contrast, when IgM was absent and when the B19V DNA concentration was lower, the median whole blood to plasma ratio was ~1. Analysis of longitudinal samples demonstrated persistent detection of B19V in WB but declining ratios of WB/plasma B19V with declining plasma VL levels and loss of IgM-reactivity. Conclusions The WB/plasma B19V DNA ratio varies by stage of infection. Further study is required to determine if this is related to the presence of circulating DNA-positive erythrocytes derived from B19V infected erythroblasts, B19V-specific IgM mediated binding of virus to cells, or other factors. PMID:21303368

Smoking induces transcription of the heat shock protein system in the joints.

PubMed

Ospelt, Caroline; Camici, Giovanni G; Engler, Anna; Kolling, Christoph; Vogetseder, Alexander; Gay, Renate E; Michel, Beat A; Gay, Steffen

2014-07-01

Smoking increases the risk of developing rheumatoid arthritis (RA) and worsens the course of the disease. In the current study we analysed whether smoking can affect gene expression directly in the joints. Synovial fibroblasts were incubated with 5% cigarette smoke extract and changes in gene expression were detected using whole genome microarrays and verified with real-time PCR. Synovial tissues were obtained from smoking and non-smoking patients with RA undergoing joint replacement surgery and from mice exposed to cigarette smoke or ambient air in a whole body exposure chamber for 3 weeks. Microarray and real-time PCR analysis showed a significant upregulation of the heat shock proteins DnaJA4, DnaJB4, DnaJC6, HspB8 and Hsp70 after stimulation of synovial fibroblasts with 5% cigarette smoke extract. Similarly, in synovial tissues of smokers with RA the expression of DnaJB4, DnaJC6, HspB8 and Hsp70 was significantly higher compared with non-smokers with RA. Upregulation of DnaJB4 and DnaJC6 in joints by smoking was also confirmed in mice exposed to cigarette smoke. Our data clearly show that smoking can change gene expression in the joints, which can lead to the activation of signalling pathways that promote development of autoimmunity and chronic joint inflammation. 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.

Moving Toward Integrating Gene Expression Profiling Into High-Throughput Testing: A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium

PubMed Central

Ryan, Natalia; Chorley, Brian; Tice, Raymond R.; Judson, Richard; Corton, J. Christopher

2016-01-01

Microarray profiling of chemical-induced effects is being increasingly used in medium- and high-throughput formats. Computational methods are described here to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through chromatin immunoprecipitation coupled with DNA sequencing analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression datasets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including “very weak” agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals, the balanced accuracies were 95% and 98% for activation or suppression, respectively. These results demonstrate that the ERα gene expression biomarker can accurately identify ERα modulators in large collections of microarray data derived from MCF-7 cells. PMID:26865669

Harnessing pain heterogeneity and RNA transcriptome to identify blood-based pain biomarkers: a novel correlational study design and bioinformatics approach in a graded chronic constriction injury model.

PubMed

Grace, Peter M; Hurley, Daniel; Barratt, Daniel T; Tsykin, Anna; Watkins, Linda R; Rolan, Paul E; Hutchinson, Mark R

2012-09-01

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fcε signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Increasing hemoglobin oxygen saturation levels in sickle trait donor whole blood prevents hemoglobin S polymerization and allows effective white blood cell reduction by filtration.

PubMed

Stroncek, David F; Byrne, Karen M; Noguchi, Constance T; Schechter, Alan N; Leitman, Susan F

2004-09-01

BACKGROUND Red blood cell (RBC) components from donors with sickle cell trait (Hb AS) often occlude white blood cell (WBC) reduction filters. Techniques were investigated to successfully filter Hb AS donor blood by increasing the Hb oxygen saturation with storage bags and conditions suitable for transfusion products. Oxygenation kinetics were measured over 3 days in whole-blood units stored in standard-sized 600-mL polyvinylchloride (PVC) bags and whole-blood units divided into three equal parts and stored in standard-sized blood bags made from PVC, tri-2-(ethylhexyl)trimellitate (CLX) plastic, or Teflon. The filterability of Hb AS blood stored for 3 days was tested with whole-blood filters. Oxygen saturation levels did not increase in full whole-blood units from donors without sickle cell trait during 3 days of storage in 600-mL PVC bags. In divided Hb AS whole-blood units stored for 3 days, oxygen saturation levels increased from baseline levels of 45 to 56, 66, and 94 percent after storage in 600-mL PVC, CLX, and Teflon bags, respectively (n = 5, p < 0.02), and all components filtered completely. When full Hb AS whole-blood units from eight donors were stored for 3 days in 1.5-L CLX bags, all units filtered completely, but one had a high residual WBC count. Storage of Hb AS whole blood in large-capacity oxygen-permeable bags increases oxygen tension and allows more effective WBC reduction by filtration.

Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry

PubMed Central

Fung, Erik; Esposito, Laura; Todd, John A.; Wicker, Linda S.

2010-01-01

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (dendritic cells, monocytes, B lymphocytes) in minimally manipulated whole blood. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, to demonstrate the quantification of surface expression levels of molecules involved in chemotaxis (e.g. CX3CR1, CCR2), adhesion (e.g. CD11b, CD62L), antigen presentation (e.g. CD83, CD86, CD209) and immune regulation (e.g. CD101) on circulating antigen-presenting cells. Each immunostaining reaction requires as little as 50–100 μl of peripheral whole blood, no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h. PMID:20134434

Parameter uncertainty analysis of a biokinetic model of caesium

DOE PAGES

Li, W. B.; Klein, W.; Blanchardon, Eric; ...

2014-04-17

Parameter uncertainties for the biokinetic model of caesium (Cs) developed by Leggett et al. were inventoried and evaluated. The methods of parameter uncertainty analysis were used to assess the uncertainties of model predictions with the assumptions of model parameter uncertainties and distributions. Furthermore, the importance of individual model parameters was assessed by means of sensitivity analysis. The calculated uncertainties of model predictions were compared with human data of Cs measured in blood and in the whole body. It was found that propagating the derived uncertainties in model parameter values reproduced the range of bioassay data observed in human subjects atmore » different times after intake. The maximum ranges, expressed as uncertainty factors (UFs) (defined as a square root of ratio between 97.5th and 2.5th percentiles) of blood clearance, whole-body retention and urinary excretion of Cs predicted at earlier time after intake were, respectively: 1.5, 1.0 and 2.5 at the first day; 1.8, 1.1 and 2.4 at Day 10 and 1.8, 2.0 and 1.8 at Day 100; for the late times (1000 d) after intake, the UFs were increased to 43, 24 and 31, respectively. The model parameters of transfer rates between kidneys and blood, muscle and blood and the rate of transfer from kidneys to urinary bladder content are most influential to the blood clearance and to the whole-body retention of Cs. For the urinary excretion, the parameters of transfer rates from urinary bladder content to urine and from kidneys to urinary bladder content impact mostly. The implication and effect on the estimated equivalent and effective doses of the larger uncertainty of 43 in whole-body retention in the later time, say, after Day 500 will be explored in a successive work in the framework of EURADOS.« less

WebArray: an online platform for microarray data analysis

PubMed Central

Xia, Xiaoqin; McClelland, Michael; Wang, Yipeng

2005-01-01

Background Many cutting-edge microarray analysis tools and algorithms, including commonly used limma and affy packages in Bioconductor, need sophisticated knowledge of mathematics, statistics and computer skills for implementation. Commercially available software can provide a user-friendly interface at considerable cost. To facilitate the use of these tools for microarray data analysis on an open platform we developed an online microarray data analysis platform, WebArray, for bench biologists to utilize these tools to explore data from single/dual color microarray experiments. Results The currently implemented functions were based on limma and affy package from Bioconductor, the spacings LOESS histogram (SPLOSH) method, PCA-assisted normalization method and genome mapping method. WebArray incorporates these packages and provides a user-friendly interface for accessing a wide range of key functions of limma and others, such as spot quality weight, background correction, graphical plotting, normalization, linear modeling, empirical bayes statistical analysis, false discovery rate (FDR) estimation, chromosomal mapping for genome comparison. Conclusion WebArray offers a convenient platform for bench biologists to access several cutting-edge microarray data analysis tools. The website is freely available at . It runs on a Linux server with Apache and MySQL. PMID:16371165

Comparison of whole blood and plasma colloid osmotic pressure in healthy cats.

PubMed

Jackson, Mary L; Kerl, Marie E; Tynan, Beth; Mann, F A

2014-01-01

To establish reference intervals for whole blood and plasma colloid osmotic pressure (COP) in healthy cats between the ages of 1 and 10 years using a cage-side colloid osmometer. Prospective, observational study. University veterinary teaching hospital. Sixty-three healthy cats. Phlebotomy. Whole blood COP mean was 24.4 (±2.78) mmHg and plasma COP mean was 24.3 (±2.59) mmHg. Reference interval for our study population of feline whole blood COP was 18.9 to 30.4 mmHg, and for our study population of feline plasma COP was 18.3 to 30.8 mmHg. Difference of paired whole blood COP and plasma COP was +0.23 ± 1.68 mmHg (P = 0.32). There was no significant difference when comparing COP from neutered male and neutered female cats. Total protein and albumin were significantly correlated with whole blood COP (total protein to whole blood COP P < 0.0001, r = 0.53; albumin to whole blood COP P <0.0001, r = 0.68) and plasma COP (total protein to plasma COP P = 0.0025, r = 0.41; albumin to plasma COP P < 0.0001, r = 0.66). No significant difference was found between mean whole blood and plasma COP in this study population of cats. Even though not statistically significant, evaluation of paired whole blood COP and plasma COP did reveal a slight difference; therefore, it seems prudent to maintain sample consistency for serial evaluations in cats. © Veterinary Emergency and Critical Care Society 2014.

Microfluidic immunomagnetic cell separation from whole blood.

PubMed

Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

2016-02-01

Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Longitudinal peripheral blood transcriptional analysis of a patient with severe Ebola virus disease

PubMed Central

Kash, John C.; Walters, Kathie-Anne; Kindrachuk, Jason; Baxter, David; Scherler, Kelsey; Janosko, Krisztina B.; Adams, Rick D.; Herbert, Andrew S.; James, Rebekah M.; Stonier, Spencer W.; Memoli, Matthew J.; Dye, John M.; Davey, Richard T.; Chertow, Daniel S.; Taubenberger, Jeffery K.

2017-01-01

The 2013–2015 outbreak of Ebola virus disease in Guinea, Liberia, and Sierra Leone was unprecedented in the number of documented cases, but there have been few published reports on immune responses in clinical cases and their relationships with the course of illness and severity of Ebola virus disease. Symptoms of Ebola virus disease can include severe headache, myalgia, asthenia, fever, fatigue, diarrhea, vomiting, abdominal pain, and hemorrhage. Although experimental treatments are in development, there are no current U.S. Food and Drug Administration–approved vaccines or therapies. We report a detailed study of host gene expression as measured by microarray in daily peripheral blood samples collected from a patient with severe Ebola virus disease. This individual was provided with supportive care without experimental therapies at the National Institutes of Health Clinical Center from before onset of critical illness to recovery. Pearson analysis of daily gene expression signatures revealed marked gene expression changes in peripheral blood leukocytes that correlated with changes in serum and peripheral blood leukocytes, viral load, antibody responses, coagulopathy, multiple organ dysfunction, and then recovery. This study revealed marked shifts in immune and antiviral responses that preceded changes in medical condition, indicating that clearance of replicating Ebola virus from peripheral blood leukocytes is likely important for systemic viral clearance. PMID:28404864

Phenotypic Profiling of Scedosporium aurantiacum, an Opportunistic Pathogen Colonizing Human Lungs

PubMed Central

Kaur, Jashanpreet; Duan, Shu Yao; Vaas, Lea A. I.; Penesyan, Anahit; Meyer, Wieland; Paulsen, Ian T.; Nevalainen, Helena

2015-01-01

Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains. PMID:25811884

Phenotypic profiling of Scedosporium aurantiacum, an opportunistic pathogen colonizing human lungs.

PubMed

Kaur, Jashanpreet; Duan, Shu Yao; Vaas, Lea A I; Penesyan, Anahit; Meyer, Wieland; Paulsen, Ian T; Nevalainen, Helena

2015-01-01

Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.

A curated collection of tissue microarray images and clinical outcome data of prostate cancer patients

PubMed Central

Zhong, Qing; Guo, Tiannan; Rechsteiner, Markus; Rüschoff, Jan H.; Rupp, Niels; Fankhauser, Christian; Saba, Karim; Mortezavi, Ashkan; Poyet, Cédric; Hermanns, Thomas; Zhu, Yi; Moch, Holger; Aebersold, Ruedi; Wild, Peter J.

2017-01-01

Microscopy image data of human cancers provide detailed phenotypes of spatially and morphologically intact tissues at single-cell resolution, thus complementing large-scale molecular analyses, e.g., next generation sequencing or proteomic profiling. Here we describe a high-resolution tissue microarray (TMA) image dataset from a cohort of 71 prostate tissue samples, which was hybridized with bright-field dual colour chromogenic and silver in situ hybridization probes for the tumour suppressor gene PTEN. These tissue samples were digitized and supplemented with expert annotations, clinical information, statistical models of PTEN genetic status, and computer source codes. For validation, we constructed an additional TMA dataset for 424 prostate tissues, hybridized with FISH probes for PTEN, and performed survival analysis on a subset of 339 radical prostatectomy specimens with overall, disease-specific and recurrence-free survival (maximum 167 months). For application, we further produced 6,036 image patches derived from two whole slides. Our curated collection of prostate cancer data sets provides reuse potential for both biomedical and computational studies. PMID:28291248

THE EFFECT OF PROPRANOLOL ON GENE EXPRESSION DURING THE BLOOD ALCOHOL CYCLE OF RATS FED ETHANOL INTRAGASTRICALLY

PubMed Central

Li, Jun; Bardag-Gorce, F; Joan, Oliva; French, BA; Dedes, J; French, SW

2010-01-01

Propranolol, a beta adrenergic blocker prevents the blood alcohol (BAL) cycle in rats fed ethanol intragastrically at a constant rate by preventing the cyclic changes in the metabolic rate caused by fluctuating levels of norepinephrine released into the blood. The change in the rate of metabolism changes the rate of alcohol elimination in the blood which causes the BAL to cycle. Microarray analysis of the livers from the rats fed ethanol and propranolol showed similar changes in clusters of functionally related gene expressions. The controls and the trough of the cycle differed dramatically from the cluster pattern seen in the rats at the peaks of the blood alcohol cycle. The changes in gene expression induced by ethanol were similar when propranolol was fed without ethanol especially with the changes in the kinases and phosphatases, Toll-like receptor signaling and cytokine-cytokine receptor interaction were also changed. The changes in gene expression caused by ethanol and propranolol feeding are alike probably because both drugs induce β adrenergic receptor desensitization. PMID:19925788

A reverse engineering approach to optimize experiments for the construction of biological regulatory networks.

PubMed

Zhang, Xiaomeng; Shao, Bin; Wu, Yangle; Qi, Ouyang

2013-01-01

One of the major objectives in systems biology is to understand the relation between the topological structures and the dynamics of biological regulatory networks. In this context, various mathematical tools have been developed to deduct structures of regulatory networks from microarray expression data. In general, from a single data set, one cannot deduct the whole network structure; additional expression data are usually needed. Thus how to design a microarray expression experiment in order to get the most information is a practical problem in systems biology. Here we propose three methods, namely, maximum distance method, trajectory entropy method, and sampling method, to derive the optimal initial conditions for experiments. The performance of these methods is tested and evaluated in three well-known regulatory networks (budding yeast cell cycle, fission yeast cell cycle, and E. coli. SOS network). Based on the evaluation, we propose an efficient strategy for the design of microarray expression experiments.

Real-Time Electrical Impedimetric Monitoring of Blood Coagulation Process under Temperature and Hematocrit Variations Conducted in a Microfluidic Chip

PubMed Central

Lei, Kin Fong; Chen, Kuan-Hao; Tsui, Po-Hsiang; Tsang, Ngan-Ming

2013-01-01

Blood coagulation is an extremely complicated and dynamic physiological process. Monitoring of blood coagulation is essential to predict the risk of hemorrhage and thrombosis during cardiac surgical procedures. In this study, a high throughput microfluidic chip has been developed for the investigation of the blood coagulation process under temperature and hematocrit variations. Electrical impedance of the whole blood was continuously recorded by on-chip electrodes in contact with the blood sample during coagulation. Analysis of the impedance change of the blood was conducted to investigate the characteristics of blood coagulation process and the starting time of blood coagulation was defined. The study of blood coagulation time under temperature and hematocrit variations was shown a good agreement with results in the previous clinical reports. The electrical impedance measurement for the definition of blood coagulation process provides a fast and easy measurement technique. The microfluidic chip was shown to be a sensitive and promising device for monitoring blood coagulation process even in a variety of conditions. It is found valuable for the development of point-of-care coagulation testing devices that utilizes whole blood sample in microliter quantity. PMID:24116099

A microfluidic approach for hemoglobin detection in whole blood

NASA Astrophysics Data System (ADS)

Taparia, Nikita; Platten, Kimsey C.; Anderson, Kristin B.; Sniadecki, Nathan J.

2017-10-01

Diagnosis of anemia relies on the detection of hemoglobin levels in a blood sample. Conventional blood analyzers are not readily available in most low-resource regions where anemia is prevalent, so detection methods that are low-cost and point-of-care are needed. Here, we present a microfluidic approach to measure hemoglobin concentration in a sample of whole blood. Unlike conventional approaches, our microfluidic approach does not require hemolysis. We detect the level of hemoglobin in a blood sample optically by illuminating the blood in a microfluidic channel at a peak wavelength of 540 nm and measuring its absorbance using a CMOS sensor coupled with a lens to magnify the image onto the detector. We compare measurements in microchannels with channel heights of 50 and 115 μm and found the channel with the 50 μm height provided a better range of detection. Since we use whole blood and not lysed blood, we fit our data to an absorption model that includes optical scattering in order to obtain a calibration curve for our system. Based on this calibration curve and data collected, we can measure hemoglobin concentration within 1 g/dL for severe cases of anemia. In addition, we measured optical density for blood flowing at a shear rate of 500 s-1 and observed it did not affect the nonlinear model. With this method, we provide an approach that uses microfluidic detection of hemoglobin levels that can be integrated with other microfluidic approaches for blood analysis.

Fast imputation using medium or low-coverage sequence data

USDA-ARS?s Scientific Manuscript database

Accurate genotype imputation can greatly reduce costs and increase benefits by combining whole-genome sequence data of varying read depth and microarray genotypes of varying densities. For large populations, an efficient strategy chooses the two haplotypes most likely to form each genotype and updat...

Insights into the fluoride-resistant regulation mechanism of Acidithiobacillus ferrooxidans ATCC 23270 based on whole genome microarrays.

PubMed

Ma, Liyuan; Li, Qian; Shen, Li; Feng, Xue; Xiao, Yunhua; Tao, Jiemeng; Liang, Yili; Yin, Huaqun; Liu, Xueduan

2016-10-01

Acidophilic microorganisms involved in uranium bioleaching are usually suppressed by dissolved fluoride ions, eventually leading to reduced leaching efficiency. However, little is known about the regulation mechanisms of microbial resistance to fluoride. In this study, the resistance of Acidithiobacillus ferrooxidans ATCC 23270 to fluoride was investigated by detecting bacterial growth fluctuations and ferrous or sulfur oxidation. To explore the regulation mechanism, a whole genome microarray was used to profile the genome-wide expression. The fluoride tolerance of A. ferrooxidans cultured in the presence of FeSO4 was better than that cultured with the S(0) substrate. The differentially expressed gene categories closely related to fluoride tolerance included those involved in energy metabolism, cellular processes, protein synthesis, transport, the cell envelope, and binding proteins. This study highlights that the cellular ferrous oxidation ability was enhanced at the lower fluoride concentrations. An overview of the cellular regulation mechanisms of extremophiles to fluoride resistance is discussed.

Assessment of DNA extracted from FTA® cards for use on the Illumina iSelect BeadChip

PubMed Central

McClure, Matthew C; McKay, Stephanie D; Schnabel, Robert D; Taylor, Jeremy F

2009-01-01

Background As FTA® cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes ≥ 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. Findings An ANOVA analysis indicated no significant difference (P > 0.72) in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. Conclusion We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform. PMID:19531223

Assessment of DNA extracted from FTA cards for use on the Illumina iSelect BeadChip.

PubMed

McClure, Matthew C; McKay, Stephanie D; Schnabel, Robert D; Taylor, Jeremy F

2009-06-16

As FTA cards provide an ideal medium for the field collection of DNA we sought to assess the quality of genomic DNA extracted from this source for use on the Illumina BovineSNP50 iSelect BeadChip which requires unbound, relatively intact (fragment sizes >or= 2 kb), and high-quality DNA. Bovine blood and nasal swab samples collected on FTA cards were extracted using the commercially available GenSolve kit with a minor modification. The call rate and concordance of genotypes from each sample were compared to those obtained from whole blood samples extracted by standard PCI extraction. An ANOVA analysis indicated no significant difference (P > 0.72) in BovineSNP50 genotype call rate between DNA extracted from FTA cards by the GenSolve kit or extracted from whole blood by PCI. Two sample t-tests demonstrated that the DNA extracted from the FTA cards produced genotype call and concordance rates that were not different to those produced by assaying DNA samples extracted by PCI from whole blood. We conclude that DNA extracted from FTA cards by the GenSolve kit is of sufficiently high quality to produce results comparable to those obtained from DNA extracted from whole blood when assayed by the Illumina iSelect technology. Additionally, we validate the use of nasal swabs as an alternative to venous blood or buccal samples from animal subjects for reliably producing high quality genotypes on this platform.

Direct and quantitative analysis of underivatized acylcarnitines in serum and whole blood using paper spray mass spectrometry

PubMed Central

Yang, Qian; Manicke, Nicholas E.; Wang, He; Petucci, Christopher; Cooks, R. Graham

2013-01-01

A simple protocol for rapid quantitation of acylcarnitines in serum and whole blood has been developed using paper spray mass spectrometry. Dried serum and whole blood containing a mixture of ten acylcarnitines at various concentrations were analyzed as spots from paper directly without any sample pretreatment, separation, or derivatization. The composition of the spray solvent was found to be a critical factor: for serum samples, spray solvent of methanol/water/formic acid (80:20:0.1) gave the best signal intensity while for blood samples which contain more matrix components, acetonitrile/water (90:10) was a much more suitable spray solvent. For the paper type and size used, 0.5 μL of sample provided an optimal signal for both serum and whole blood samples. For quantitative profiling, the limits of quantitation obtained from both serum and blood were much lower than the clinically validated cutoff values for diagnosis of fatty acid oxidation disorders in newborn screening. Linearity (R2>0.95) and reproducibility (RSD ~10 %) were achieved in the concentration ranges from 100 nM to 5 μM for the C2 acylcarnitine, and for other acylcarnitines, these values were from 10 to 500 nM. Acylcarnitine profiles offer an effective demonstration of the fact that paper spray mass spectrometry is an appropriate, simple, rapid method with high sensitivity and high reproducibility applicable to newborn screening tests. PMID:22760507

Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping

NASA Technical Reports Server (NTRS)

Royce, Thomas E.; Rozowsky, Joel S.; Bertone, Paul; Samanta, Manoj; Stolc, Viktor; Weissman, Sherman; Snyder, Michael; Gerstein, Mark

2005-01-01

Traditional microarrays use probes complementary to known genes to quantitate the differential gene expression between two or more conditions. Genomic tiling microarray experiments differ in that probes that span a genomic region at regular intervals are used to detect the presence or absence of transcription. This difference means the same sets of biases and the methods for addressing them are unlikely to be relevant to both types of experiment. We introduce the informatics challenges arising in the analysis of tiling microarray experiments as open problems to the scientific community and present initial approaches for the analysis of this nascent technology.

Practical considerations of image analysis and quantification of signal transduction IHC staining.

PubMed

Grunkin, Michael; Raundahl, Jakob; Foged, Niels T

2011-01-01

The dramatic increase in computer processing power in combination with the availability of high-quality digital cameras during the last 10 years has fertilized the grounds for quantitative microscopy based on digital image analysis. With the present introduction of robust scanners for whole slide imaging in both research and routine, the benefits of automation and objectivity in the analysis of tissue sections will be even more obvious. For in situ studies of signal transduction, the combination of tissue microarrays, immunohistochemistry, digital imaging, and quantitative image analysis will be central operations. However, immunohistochemistry is a multistep procedure including a lot of technical pitfalls leading to intra- and interlaboratory variability of its outcome. The resulting variations in staining intensity and disruption of original morphology are an extra challenge for the image analysis software, which therefore preferably should be dedicated to the detection and quantification of histomorphometrical end points.

Genome-wide Integration Study of Circulating miRNAs and Peripheral Whole-Blood mRNAs of Male Acute Ischemic Stroke Patients.

PubMed

Xue, Yang; Yin, Pengqi; Li, Guozhong; Zhong, Di

2018-06-01

Several circulating microRNAs (miRNAs) have been proved to serve as stable biomarkers in blood for acute ischemic stroke (AIS). However, the functions of these biomarkers remain elusive. By conducting the integration analysis of circulating miRNAs and peripheral whole-blood mRNAs using bioinformatics methods, we explored the biological role of these circulating markers in peripheral whole blood at the genome-wide level. Stroke-related circulating miRNA profile data (GSE86291) and peripheral whole-blood mRNA expression data (GSE16561) were collected from the Gene Expression Omnibus (GEO) datasets. We selected male patients to avoid any gender differences in stroke pathology. Male stroke-related miRNAs (M-miRNAs) and mRNAs (M-mRNAs) were detected using GEO2R. Nine M-miRNAs (five up- and four down-regulated) were applied to TargetScan to predict the possible target mRNAs. Next, we intersected these targets with the M-mRNAs (38 up- and three down-regulated) to obtain the male stroke-related overlapped mRNAs (Mo-mRNAs). Finally, we analyzed biological functions of Mo-mRNAs using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and constructed networks among the Mo-mRNAs, overlapped M-miRNAs (Mo-miRNAs), and their functions. The Mo-mRNAs were enriched in functions such as platelet degranulation, immune response, and pathways associated with phagosome biology and Staphylococcus aureus infection. This study provides an integrated view of interactions among circulating miRNAs and peripheral whole-blood mRNAs involved in the pathophysiological processes of male AIS. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Microwave Energy Increases Fatty Acid Methyl Ester Yield in Human Whole Blood Due to Increased Sphingomyelin Transesterification.

PubMed

Metherel, Adam H; Aristizabal Henao, Juan J; Ciobanu, Flaviu; Taha, Ameer Y; Stark, Ken D

2015-09-01

Dried blood spots (DBS) by fingertip prick collection for fatty acid profiling are becoming increasingly popular due to ease of collection, minimal invasiveness and its amenability to high-throughput analyses. Herein, we assess a microwave-assisted direct transesterification method for the production of fatty acid methyl esters (FAME) from DBS. Technical replicates of human whole blood were collected and 25-μL aliquots were applied to chromatography strips prior to analysis by a standard 3-h transesterification method or microwave-assisted direct transesterification method under various power (variable vs constant), time (1-5 min) and reagent (1-10% H2SO4 in methanol) conditions. In addition, a standard method was compared to a 5-min, 30-W power microwave in 1% H2SO4 method for FAME yield from whole blood sphingomyelin, and sphingomyelin standards alone and spiked in whole blood. Microwave-assisted direct transesterification yielded no significant differences in both quantitative (nmol/100 µL) and qualitative (mol%) fatty acid assessments after as little as 1.5- and 1-min reaction times, respectively, using the variable power method and 5% H2SO4 in methanol. However, 30-W power for 5 min increased total FAME yield of the technical replicates by 14%. This increase appears largely due to higher sphingomyelin-derived FAME yield of up to 109 and 399% compared to the standard method when determined from whole blood or pure standards, respectively. In conclusion, microwave-assisted direct transesterification of DBS achieved in as little as 1-min, and 5-min reaction times increase total fatty acids primarily by significantly improving sphingomyelin-derived fatty acid yield.

Erythrocyte sedimentation rate and fibrinogen concentration of whole blood influences the cellular composition of platelet-rich plasma obtained from centrifugation methods.

PubMed

Yin, Wenjing; Xu, Zhengliang; Sheng, Jiagen; Xie, Xuetao; Zhang, Changqing

2017-09-01

Erythrocyte sedimentation rate (ESR), which reflects the sedimentation rate of platelets, leukocytes and erythrocytes in response to centrifugal force, may influence the cellular composition of platelet-rich plasma (PRP) obtained via centrifugation methods. However, no relevant studies have substantiated this. In the present study, blood was collected from 40 healthy volunteers and used to prepare PRP with two plasma-based preparation systems [YinPRP and Plasma Rich in Growth Factor (PRGF) systems] and two buffy coat-based systems (RegenPRP and WEGOPRP systems) in a single-donor model. Volumes of PRP and platelet-poor plasma (PPP) that were removed in the preparation process were recorded. Analyses of ESR, haematocrit, C-reaction protein, coagulation, serum glucose and serum lipid of the whole blood used for PRP preparation were performed to evaluate the levels of ESR and the factors known to influence it. Whole blood analysis was performed to evaluate the cellular composition of PRP. Results demonstrated that there were marked positive correlations between the ESR of the whole blood used for PRP preparation and PPP removal efficiencies, platelet concentrations, platelet capture efficiencies and platelet enrichment factors of PRP formulations obtained from plasma-based systems, and PRP yield efficiency of RegenPRP and PPP removal efficiency of WEGOPRP. Furthermore, there were marked negative correlations between ESR and concentrations and enrichment factors of platelets, leukocytes and erythrocytes of RegenPRP. Fibrinogen concentration of the whole blood, which had a marked positive correlation with ESR, also influenced the cellular composition of PRP. These findings may increase the understanding of PRP preparation and provide substantial evidence for the individualised optimisation of PRP preparation systems used in clinical practice.

Liquid chromatography-electrospray mass spectrometry determination of ibogaine and noribogaine in human plasma and whole blood. Application to a poisoning involving Tabernanthe iboga root.

PubMed

Kontrimaviciūte, Violeta; Breton, Hélène; Mathieu, Olivier; Mathieu-Daudé, Jean-Claude; Bressolle, Françoise M M

2006-11-07

A liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the first time for the determination of ibogaine and noribogaine in human plasma and whole blood. The method involved solid phase extraction of the compounds and the internal standard (fluorescein) from the two matrices using OasisHLB columns. LC separation was performed on a Zorbax eclipse XD8 C8 column (5 microm) with a mobile phase of acetonitrile containing 0.02% (v/v) trimethylamine and 2mM ammonium formate buffer. MS data were acquired in single ion monitoring mode at m/z 311.2, 297.2 and 332.5 for ibogaine, noribogaine and fluorescein, respectively. The drug/internal standard peak area ratios were linked via a quadratic relationship to plasma (0.89-179 microg/l for ibogaine; 1-200 microg/l for noribogaine) and to whole blood concentrations (1.78-358 microg/kg for ibogaine; 2-400 microg/kg for noribogaine). Precision ranged from 4.5 to 13% and accuracy was 89-102%. Dilution of the samples had no influence on the performance of the method. Extraction recoveries were > or =94% in plasma and > or =57% in whole blood. The lower limits of quantitation were 0.89 microg/l for ibogaine and 1 microg/l for noribogaine in plasma, and 1.78 microg/kg for ibogaine and 2 microg/kg for noribogaine in whole blood. In frozen plasma samples, the two drugs were stable for at least 1 year. In blood, ibogaine and noribogaine were stable for 4h at 4 degrees C and 20 degrees C and 2 months at -20 degrees C. The method was successfully used for the analysis of a poisoning involving Tabernanthe iboga root.

Specific Transcriptome Changes Associated with Blood Pressure Reduction in Hypertensive Patients After Relaxation Response Training.

PubMed

Bhasin, Manoj K; Denninger, John W; Huffman, Jeff C; Joseph, Marie G; Niles, Halsey; Chad-Friedman, Emma; Goldman, Roberta; Buczynski-Kelley, Beverly; Mahoney, Barbara A; Fricchione, Gregory L; Dusek, Jeffery A; Benson, Herbert; Zusman, Randall M; Libermann, Towia A

2018-05-01

Mind-body practices that elicit the relaxation response (RR) have been demonstrated to reduce blood pressure (BP) in essential hypertension (HTN) and may be an adjunct to antihypertensive drug therapy. However, the molecular mechanisms by which the RR reduces BP remain undefined. Genomic determinants associated with responsiveness to an 8-week RR-based mind-body intervention for lowering HTN in 13 stage 1 hypertensive patients classified as BP responders and 11 as nonresponders were identified. Transcriptome analysis in peripheral blood mononuclear cells identified 1771 genes regulated by the RR in responders. Biological process- and pathway-based analysis of transcriptome data demonstrated enrichment in the following gene categories: immune regulatory pathways and metabolism (among downregulated genes); glucose metabolism, cardiovascular system development, and circadian rhythm (among upregulated genes). Further in silico estimation of cell abundance from the microarray data showed enrichment of the anti-inflammatory M2 subtype of macrophages in BP responders. Nuclear factor-κB, vascular endothelial growth factor, and insulin were critical molecules emerging from interactive network analysis. These findings provide the first insights into the molecular mechanisms that are associated with the beneficial effects of the RR on HTN.

Specific Transcriptome Changes Associated with Blood Pressure Reduction in Hypertensive Patients After Relaxation Response Training

PubMed Central

Bhasin, Manoj K.; Denninger, John W.; Huffman, Jeff C.; Joseph, Marie G.; Niles, Halsey; Chad-Friedman, Emma; Goldman, Roberta; Buczynski-Kelley, Beverly; Mahoney, Barbara A.; Fricchione, Gregory L.; Dusek, Jeffery A.; Benson, Herbert; Zusman, Randall M.

2018-01-01

Abstract Objective: Mind–body practices that elicit the relaxation response (RR) have been demonstrated to reduce blood pressure (BP) in essential hypertension (HTN) and may be an adjunct to antihypertensive drug therapy. However, the molecular mechanisms by which the RR reduces BP remain undefined. Design: Genomic determinants associated with responsiveness to an 8-week RR-based mind–body intervention for lowering HTN in 13 stage 1 hypertensive patients classified as BP responders and 11 as nonresponders were identified. Results: Transcriptome analysis in peripheral blood mononuclear cells identified 1771 genes regulated by the RR in responders. Biological process- and pathway-based analysis of transcriptome data demonstrated enrichment in the following gene categories: immune regulatory pathways and metabolism (among downregulated genes); glucose metabolism, cardiovascular system development, and circadian rhythm (among upregulated genes). Further in silico estimation of cell abundance from the microarray data showed enrichment of the anti-inflammatory M2 subtype of macrophages in BP responders. Nuclear factor-κB, vascular endothelial growth factor, and insulin were critical molecules emerging from interactive network analysis. Conclusions: These findings provide the first insights into the molecular mechanisms that are associated with the beneficial effects of the RR on HTN. PMID:29616846

[Demography and donation frequencies of blood and plasma donor populations in Germany].

PubMed

Ritter, Sabine; Willand, L; Reinhard, B; Offergeld, R; Hamouda, O

2008-08-01

According to Article 22 of the Transfusion Act, the Robert Koch Institute collects and evaluates nationwide data on the prevalence and incidence of transfusion-relevant infections among blood and plasma donors in Germany. Due to revision of the Transfusion Act in 2005 not only the number of donations but also the number of donors has become available for analysis. Here we give a detailed account on the demographic profile and donation frequencies of German whole blood, plasma and platelet donors in 2006. Overall, 4 % of the German population eligible to donate were active as repeat whole blood donors in 2006; 0.3 % repeatedly donated plasma or platelets. Irrespective of the type of donation, the percentage of donors among the general population was highest among the youngest age group (18 to 24 years). While the age distribution of whole blood repeat donors roughly resembled that of the general population, with the greatest number among those aged 35 to 44, younger age groups were overrepresented among repeat plasma donors. Donation frequency varied depending on donor age and sex, with an average of 1.9 per year for whole blood donations, 11.9 for plasmapheresis and 4.0 for plateletpheresis. With the exception of the latter, men donated more frequently than women. For both sexes, donation frequency increased with age. Detailed knowledge of the demographic profile and changes in the composition of donor populations are essential for planning adequate blood supply. The data presented may serve as reference for assessing the consequences of measures that affect the number of donors and/or donations (for example changing deferral criteria) in Germany.

Effect of renal replacement therapy on viscosity in end-stage renal disease patients.

PubMed

Feriani, M; Kimmel, P L; Kurantsin-Mills, J; Bosch, J P

1992-02-01

Viscosity, an important determinant of microcirculatory hemodynamics, is related to hematocrit (HCT), and may be altered by renal failure or its treatment. To assess these factors, we studied the effect of dialysis on the viscosity of whole blood, plasma, and reconstituted 70% HCT blood of eight continuous ambulatory peritoneal dialysis (CAPD) and nine hemodialysis (HD) patients under steady shear flow conditions at different shear rates, before and after dialysis, compared with nine normal subjects. The density of the red blood cells (RBCs), a marker of cell hydration, was measured in HD patients by a nonaqueous differential floatation technique. Whole blood viscosity was higher in controls than patients, and correlated with HCT before treatment (P less than 0.05) at shear rates of 11.5 to 230 s-1) in HD patients, and 23 to 230 s-1 in all end-stage renal disease (ESRD) patients. In contrast, whole blood viscosity correlated with HCT in CAPD patients only at the lowest shear rates (2.3 and 5.75 s-1, P less than 0.05). Plasma viscosity was higher in CAPD patients than both HD patients before treatment and controls (P less than 0.05, analysis of variance [ANOVA]), despite lower plasma total protein, albumin, and similar fibrinogen concentration compared with HD patients. When all samples were reconstituted to 70% HCT, CAPD patients had higher whole blood viscosity than control subjects'. The high HCT blood viscosity of the ESRD patients was higher than control subjects' at capillary shear rates, suggesting increased RBC aggregation and decreased RBC deformability in patients with renal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic Dissection of Learning and Memory in Mice

PubMed Central

Mineur, Yann S.; Crusio, Wim E.; Sluyter, Frans

2004-01-01

In this minireview, we discuss different strategies to dissect genetically the keystones of learning and memory. First, we broadly sketch the neurogenetic analysis of complex traits in mice. We then discuss two general strategies to find genes affecting learning and memory: candidate gene studies and whole genome searches. Next, we briefly review more recently developed techniques, such as microarrays and RNA interference. In addition, we focus on gene-environment interactions and endophenotypes. All sections are illustrated with examples from the learning and memory field, including a table summarizing the latest information about genes that have been shown to have effects on learning and memory. PMID:15656270

Toward a patient-based paradigm for blood transfusion.

PubMed

Farrugia, Albert; Vamvakas, Eleftherios

2014-01-01

The current "manufacturing paradigm" of transfusion practice has detached transfusion from the clinical environment. As an example, fresh whole blood in large-volume hemorrhage may be superior to whole blood reconstituted from multiple components. Multicomponent apheresis can overcome logistical difficulties in matching patient needs with fresh component availability and can deliver the benefits of fresh whole blood. Because of the different transfusion needs of patients in emerging economies and the vulnerability of these blood systems to emerging infections, fresh whole blood and multicomponent apheresis can better meet patient needs when compared with transplants of the "manufacturing paradigm". We propose that patient blood management, along with panels of repeat, paid, accredited apheresis and fresh whole-blood donors can be used in emerging economies to support decentralized blood services. This alternative transfusion-medicine paradigm could eventually also be adopted by established economies to focus transfusion medicine on local patient needs and to alleviate the problem of the aging volunteer donor base.

Identification of a neutrophil-related gene expression signature that is enriched in adult systemic lupus erythematosus patients with active nephritis: Clinical/pathologic associations and etiologic mechanisms

PubMed Central

Prokopec, Stephenie D.; Noamani, Babak; Chang, Nan-Hua; Bonilla, Dennisse; Touma, Zahi; Avila-Casado, Carmen; Reich, Heather N.; Scholey, James; Fortin, Paul R.; Boutros, Paul C.; Landolt-Marticorena, Carolina

2018-01-01

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited. PMID:29742110

hemaClass.org: Online One-By-One Microarray Normalization and Classification of Hematological Cancers for Precision Medicine.

PubMed

Falgreen, Steffen; Ellern Bilgrau, Anders; Brøndum, Rasmus Froberg; Hjort Jakobsen, Lasse; Have, Jonas; Lindblad Nielsen, Kasper; El-Galaly, Tarec Christoffer; Bødker, Julie Støve; Schmitz, Alexander; H Young, Ken; Johnsen, Hans Erik; Dybkær, Karen; Bøgsted, Martin

2016-01-01

Dozens of omics based cancer classification systems have been introduced with prognostic, diagnostic, and predictive capabilities. However, they often employ complex algorithms and are only applicable on whole cohorts of patients, making them difficult to apply in a personalized clinical setting. This prompted us to create hemaClass.org, an online web application providing an easy interface to one-by-one RMA normalization of microarrays and subsequent risk classifications of diffuse large B-cell lymphoma (DLBCL) into cell-of-origin and chemotherapeutic sensitivity classes. Classification results for one-by-one array pre-processing with and without a laboratory specific RMA reference dataset were compared to cohort based classifiers in 4 publicly available datasets. Classifications showed high agreement between one-by-one and whole cohort pre-processsed data when a laboratory specific reference set was supplied. The website is essentially the R-package hemaClass accompanied by a Shiny web application. The well-documented package can be used to run the website locally or to use the developed methods programmatically. The website and R-package is relevant for biological and clinical lymphoma researchers using affymetrix U-133 Plus 2 arrays, as it provides reliable and swift methods for calculation of disease subclasses. The proposed one-by-one pre-processing method is relevant for all researchers using microarrays.

Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

PubMed

Cox, Brian; Sharma, Parveen; Evangelou, Andreas I; Whiteley, Kathie; Ignatchenko, Vladimir; Ignatchenko, Alex; Baczyk, Dora; Czikk, Marie; Kingdom, John; Rossant, Janet; Gramolini, Anthony O; Adamson, S Lee; Kislinger, Thomas

2011-12-01

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti‐citrulline autoreactivity is associated with up regulation of proinflammatory cytokines

PubMed Central

Hueber, Wolfgang; Tomooka, Beren H; Zhao, Xiaoyan; Kidd, Brian A; Drijfhout, Jan W; Fries, James F; van Venrooij, Walther J; Metzger, Allan L; Genovese, Mark C; Robinson, William H

2007-01-01

Objectives To identify peripheral blood autoantibody and cytokine profiles that characterise clinically relevant subgroups of patients with early rheumatoid arthritis using arthritis antigen microarrays and a multiplex cytokine assay. Methods Serum samples from 56 patients with a diagnosis of rheumatoid arthritis of <6 months' duration were tested. Cytokine profiles were also determined in samples from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (n = 21), and from healthy individuals (n = 19). Data were analysed using Kruskal–Wallis test with Dunn's adjustment for multiple comparisons, linear correlation tests, significance analysis of microarrays (SAM) and hierarchical clustering software. Results Distinct antibody profiles were associated with subgroups of patients who exhibited high serum levels of tumour necrosis factor (TNF)α, interleukin (IL)1β, IL6, IL13, IL15 and granulocyte macrophage colony‐stimulating factor. Significantly increased autoantibody reactivity against citrullinated epitopes was observed in patients within the cytokine “high” subgroup. Increased levels of TNFα, IL1α, IL12p40 and IL13, and the chemokines eotaxin/CCL11, monocyte chemoattractant protein‐1 and interferon‐inducible protein 10, were present in early rheumatoid arthritis as compared with controls (p<0.001). Chemokines showed some of the most impressive differences. Only IL8/CXCL8 concentrations were higher in patients with PsA/ankylosing spondylitis (p = 0.02). Conclusions Increased blood levels of proinflammatory cytokines are associated with autoantibody targeting of citrullinated antigens and surrogate markers of disease activity in patients with early rheumatoid arthritis. Proteomic analysis of serum autoantibodies, cytokines and chemokines enables stratification of patients with early rheumatoid arthritis into molecular subgroups. PMID:16901957

Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression.

PubMed

Tsunoda, Fumiyoshi; Lamon-Fava, Stefania; Asztalos, Bela F; Iyer, Lakshmanan K; Richardson, Kris; Schaefer, Ernst J

2015-08-01

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (PBMC). Subjects were sampled at baseline and six weeks after receiving either: olive oil 6.0 g/day (n = 16), EPA 1.8 g/day (n = 16), or DHA 1.8 g/day (n = 18). PBMC were subjected to gene expression analysis by microarray with key findings confirmed by quantitative real-time polymerase chain reaction (Q-PCR). Plasma phospholipid EPA increased 3 fold in the EPA group, and DHA increased 63% in the DHA group (both p < 0.01), while no effects were observed in the olive oil group. Microarray analysis indicated that EPA but not DHA or olive oil significantly affected the gene expression in the following pathways: 1) interferon signaling, 2) receptor recognition of bacteria and viruses, 3) G protein signaling, glycolysis and glycolytic shunting, 4) S-adenosyl-l-methionine biosynthesis, and 5) cAMP-mediated signaling including cAMP responsive element protein 1 (CREB1), as well as many other individual genes including hypoxia inducible factor 1, α subunit (HIF1A). The findings for CREB1 and HIF1A were confirmed by Q-PCR analysis. Our data indicate that EPA supplementation was associated with significant effects on gene expression involving the interferon pathway as well as down-regulation of CREB1 and HIF1A, which may relate to its beneficial effect on CVD risk reduction. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Evidence of inflammatory immune signaling in chronic fatigue syndrome: A pilot study of gene expression in peripheral blood.

PubMed

Aspler, Anne L; Bolshin, Carly; Vernon, Suzanne D; Broderick, Gordon

2008-09-26

Genomic profiling of peripheral blood reveals altered immunity in chronic fatigue syndrome (CFS) however interpretation remains challenging without immune demographic context. The object of this work is to identify modulation of specific immune functional components and restructuring of co-expression networks characteristic of CFS using the quantitative genomics of peripheral blood. Gene sets were constructed a priori for CD4+ T cells, CD8+ T cells, CD19+ B cells, CD14+ monocytes and CD16+ neutrophils from published data. A group of 111 women were classified using empiric case definition (U.S. Centers for Disease Control and Prevention) and unsupervised latent cluster analysis (LCA). Microarray profiles of peripheral blood were analyzed for expression of leukocyte-specific gene sets and characteristic changes in co-expression identified from topological evaluation of linear correlation networks. Median expression for a set of 6 genes preferentially up-regulated in CD19+ B cells was significantly lower in CFS (p = 0.01) due mainly to PTPRK and TSPAN3 expression. Although no other gene set was differentially expressed at p < 0.05, patterns of co-expression in each group differed markedly. Significant co-expression of CD14+ monocyte with CD16+ neutrophil (p = 0.01) and CD19+ B cell sets (p = 0.00) characterized CFS and fatigue phenotype groups. Also in CFS was a significant negative correlation between CD8+ and both CD19+ up-regulated (p = 0.02) and NK gene sets (p = 0.08). These patterns were absent in controls. Dissection of blood microarray profiles points to B cell dysfunction with coordinated immune activation supporting persistent inflammation and antibody-mediated NK cell modulation of T cell activity. This has clinical implications as the CD19+ genes identified could provide robust and biologically meaningful basis for the early detection and unambiguous phenotyping of CFS.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood.

PubMed

Toepfner, Nicole; Herold, Christoph; Otto, Oliver; Rosendahl, Philipp; Jacobi, Angela; Kräter, Martin; Stächele, Julia; Menschner, Leonhard; Herbig, Maik; Ciuffreda, Laura; Ranford-Cartwright, Lisa; Grzybek, Michal; Coskun, Ünal; Reithuber, Elisabeth; Garriss, Geneviève; Mellroth, Peter; Henriques-Normark, Birgitta; Tregay, Nicola; Suttorp, Meinolf; Bornhäuser, Martin; Chilvers, Edwin R; Berner, Reinhard; Guck, Jochen

2018-01-13

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis. © 2018, Toepfner et al.

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood

PubMed Central

Toepfner, Nicole; Herold, Christoph; Otto, Oliver; Rosendahl, Philipp; Jacobi, Angela; Kräter, Martin; Stächele, Julia; Menschner, Leonhard; Herbig, Maik; Ciuffreda, Laura; Ranford-Cartwright, Lisa; Grzybek, Michal; Coskun, Ünal; Reithuber, Elisabeth; Garriss, Geneviève; Mellroth, Peter; Henriques-Normark, Birgitta; Tregay, Nicola; Suttorp, Meinolf; Bornhäuser, Martin; Chilvers, Edwin R; Berner, Reinhard

2018-01-01

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis. PMID:29331015

Karyotype versus microarray testing for genetic abnormalities after stillbirth.

PubMed

Reddy, Uma M; Page, Grier P; Saade, George R; Silver, Robert M; Thorsten, Vanessa R; Parker, Corette B; Pinar, Halit; Willinger, Marian; Stoll, Barbara J; Heim-Hall, Josefine; Varner, Michael W; Goldenberg, Robert L; Bukowski, Radek; Wapner, Ronald J; Drews-Botsch, Carolyn D; O'Brien, Barbara M; Dudley, Donald J; Levy, Brynn

2012-12-06

Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).

GPFrontend and GPGraphics: graphical analysis tools for genetic association studies.

PubMed

Uebe, Steffen; Pasutto, Francesca; Krumbiegel, Mandy; Schanze, Denny; Ekici, Arif B; Reis, André

2010-09-21

Most software packages for whole genome association studies are non-graphical, purely text based programs originally designed to run with UNIX-like operating systems. Graphical output is often not intended or supposed to be performed with other command line tools, e.g. gnuplot. Using the Microsoft .NET 2.0 platform and Visual Studio 2005, we have created a graphical software package to analyze data from microarray whole genome association studies, both for a DNA-pooling based approach as well as regular single sample data. Part of this package was made to integrate with GenePool 0.8.2, a previously existing software suite for GNU/Linux systems, which we have modified to run in a Microsoft Windows environment. Further modifications cause it to generate some additional data. This enables GenePool to interact with the .NET parts created by us. The programs we developed are GPFrontend, a graphical user interface and frontend to use GenePool and create metadata files for it, and GPGraphics, a program to further analyze and graphically evaluate output of different WGA analysis programs, among them also GenePool. Our programs enable regular MS Windows users without much experience in bioinformatics to easily visualize whole genome data from a variety of sources.

Whole transcriptome RNA-Seq analysis of breast cancer recurrence risk using formalin-fixed paraffin-embedded tumor tissue.

PubMed

Sinicropi, Dominick; Qu, Kunbin; Collin, Francois; Crager, Michael; Liu, Mei-Lan; Pelham, Robert J; Pho, Mylan; Dei Rossi, Andrew; Jeong, Jennie; Scott, Aaron; Ambannavar, Ranjana; Zheng, Christina; Mena, Raul; Esteban, Jose; Stephans, James; Morlan, John; Baker, Joffre

2012-01-01

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.

Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue

PubMed Central

Sinicropi, Dominick; Qu, Kunbin; Collin, Francois; Crager, Michael; Liu, Mei-Lan; Pelham, Robert J.; Pho, Mylan; Rossi, Andrew Dei; Jeong, Jennie; Scott, Aaron; Ambannavar, Ranjana; Zheng, Christina; Mena, Raul; Esteban, Jose; Stephans, James; Morlan, John; Baker, Joffre

2012-01-01

RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts. PMID:22808097

Systems biology of cancer biomarker detection.

PubMed

Mitra, Sanga; Das, Smarajit; Chakrabarti, Jayprokas

2013-01-01

Cancer systems-biology is an ever-growing area of research due to explosion of data; how to mine these data and extract useful information is the problem. To have an insight on carcinogenesis one need to systematically mine several resources, such as databases, microarray and next-generation sequences. This review encompasses management and analysis of cancer data, databases construction and data deposition, whole transcriptome and genome comparison, analysing results from high throughput experiments to uncover cellular pathways and molecular interactions, and the design of effective algorithms to identify potential biomarkers. Recent technical advances such as ChIP-on-chip, ChIP-seq and RNA-seq can be applied to get epigenetic information transformed into a high-throughput endeavour to which systems biology and bioinformatics are making significant inroads. The data from ENCODE and GENCODE projects available through UCSC genome browser can be considered as benchmark for comparison and meta-analysis. A pipeline for integrating next generation sequencing data, microarray data, and putting them together with the existing database is discussed. The understanding of cancer genomics is changing the way we approach cancer diagnosis and treatment. To give a better understanding of utilizing available resources' we have chosen oral cancer to show how and what kind of analysis can be done. This review is a computational genomic primer that provides a bird's eye view of computational and bioinformatics' tools currently available to perform integrated genomic and system biology analyses of several carcinoma.

Emerging Use of Gene Expression Microarrays in Plant Physiology

DOE PAGES

Wullschleger, Stan D.; Difazio, Stephen P.

2003-01-01

Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology weremore » selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.« less

A genome-wide 20 K citrus microarray for gene expression analysis

PubMed Central

Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

2008-01-01

Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. PMID:18598343

Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column-switching high-performance liquid chromatography and mass spectrometry detection.

PubMed

Paniagua, Ricardo; Campbell, Andrew; Changelian, Paul S; Reitz, Bruce A; Prakash, Chandra; Borie, Dominic C

2005-10-01

A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.

Surface-enhanced Raman scattering of whole human blood, blood plasma, and red blood cells: cellular processes and bioanalytical sensing.

PubMed

Premasiri, W R; Lee, J C; Ziegler, L D

2012-08-09

SERS spectra of whole human blood, blood plasma, and red blood cells on Au nanoparticle SiO(2) substrates excited at 785 nm have been observed. For the sample preparation procedure employed here, the SERS spectrum of whole blood arises from the blood plasma component only. This is in contrast to the normal Raman spectrum of whole blood excited at 785 nm and open to ambient air, which is exclusively due to the scattering of oxyhemoglobin. The SERS spectrum of whole blood shows a storage time dependence that is not evident in the non-SERS Raman spectrum of whole blood. Hypoxanthine, a product of purine degradation, dominates the SERS spectrum of blood after ~10-20 h of storage at 8 °C. The corresponding SERS spectrum of plasma isolated from the stored blood shows the same temporal release of hypoxanthine. Thus, blood cellular components (red blood cells, white blood cells, and/or platelets) are releasing hypoxanthine into the plasma over this time interval. The SERS spectrum of red blood cells (RBCs) excited at 785 nm is reported for the first time and exhibits well-known heme group marker bands as well as other bands that may be attributed to cell membrane components or protein denaturation contributions. SERS, as well as normal Raman spectra, of oxy- and met-RBCs are reported and compared. These SERS results can have significant impact in the area of clinical diagnostics, blood supply management, and forensics.

Surface Enhanced Raman Scattering of Whole Human Blood, Blood Plasma and Red Blood Cells: Cellular Processes and Bioanalytical Sensing

PubMed Central

Premasiri, W. R.; Lee, J. C.; Ziegler, L. D.

2013-01-01

SERS spectra of whole human blood, blood plasma and red blood cells on Au nanoparticle SiO2 substrates excited at 785 nm have been observed. For the sample preparation procedure employed here, the SERS spectrum of whole blood arises from the blood plasma component only. This is in contrast to the normal Raman spectrum of whole blood excited at 785 nm and open to ambient air, which is exclusively due to the scattering of oxyhemoglobin. The SERS spectrum of whole blood shows a storage time dependence that is not evident in the non-SERS Raman spectrum of whole blood. Hypoxanthine, a product of purine degradation, dominates the SERS spectrum of blood after ~10 – 20 hours of storage at 8 °C. The corresponding SERS spectrum of plasma isolated from the stored blood shows the same temporal release of hypoxanthine. Thus, blood cellular components (red blood cells, white blood cells and/or platelets) are releasing hypoxanthine into the plasma over this time interval. The SERS spectrum of red blood cells (RBCs) excited at 785 nm is reported for the first time and exhibits well known heme group marker bands, as well as other bands that may be attributed to cell membrane components or protein denaturation contributions. SERS, as well as normal Raman spectra, of oxy- and met-RBCs are reported and compared. These SERS results can have significant impact in the area of clinical diagnostics, blood supply management and forensics. PMID:22780445

A Java-based tool for the design of classification microarrays.

PubMed

Meng, Da; Broschat, Shira L; Call, Douglas R

2008-08-04

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for analysis of subsequent experimental data. Additionally, PLASMID can be used to construct virtual microarrays with genomes from public databases, which can then be used to identify an optimal set of probes.

Enhancing interdisciplinary mathematics and biology education: a microarray data analysis course bridging these disciplines.

PubMed

Tra, Yolande V; Evans, Irene M

2010-01-01

BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course.

Enhancing Interdisciplinary Mathematics and Biology Education: A Microarray Data Analysis Course Bridging These Disciplines

PubMed Central

Evans, Irene M.

2010-01-01

BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course. PMID:20810954

The Bioinformatic Analysis of the Dysregulated Genes and MicroRNAs in Entorhinal Cortex, Hippocampus, and Blood for Alzheimer's Disease

PubMed Central

Pang, Xiaocong; Zhao, Ying; Wang, Jinhua; Zhou, Qimeng; Xu, Lvjie; Kang, De

2017-01-01

Aim The incidence of Alzheimer's disease (AD) has been increasing in recent years, but there exists no cure and the pathological mechanisms are not fully understood. This study aimed to find out the pathogenesis of learning and memory impairment, new biomarkers, potential therapeutic targets, and drugs for AD. Methods We downloaded the microarray data of entorhinal cortex (EC) and hippocampus (HIP) of AD and controls from Gene Expression Omnibus (GEO) database, and then the differentially expressed genes (DEGs) in EC and HIP regions were analyzed for functional and pathway enrichment. Furthermore, we utilized the DEGs to construct coexpression networks to identify hub genes and discover the small molecules which were capable of reversing the gene expression profile of AD. Finally, we also analyzed microarray and RNA-seq dataset of blood samples to find the biomarkers related to gene expression in brain. Results We found some functional hub genes, such as ErbB2, ErbB4, OCT3, MIF, CDK13, and GPI. According to GO and KEGG pathway enrichment, several pathways were significantly dysregulated in EC and HIP. CTSD and VCAM1 were dysregulated significantly in blood, EC, and HIP, which were potential biomarkers for AD. Target genes of four microRNAs had similar GO_terms distribution with DEGs in EC and HIP. In addtion, small molecules were screened out for AD treatment. Conclusion These biological pathways and DEGs or hub genes will be useful to elucidate AD pathogenesis and identify novel biomarkers or drug targets for developing improved diagnostics and therapeutics against AD. PMID:29359159

Differential gene expression in notochord and nerve cord fate segregation in the Ciona intestinalis embryo.

PubMed

Kobayashi, Kenji; Yamada, Lixy; Satou, Yutaka; Satoh, Nori

2013-09-01

During early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanisms underlying developmental fate restriction are one of the major research subjects within developmental biology. In this article, this subject was addressed by combining blastomere isolation with microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord precursor cells and the other to nerve cord precursors. Approximately 2,200 each of notochord and nerve cord precursor cells were isolated, and their mRNA expression profiles were compared by microarray. This analysis identified 106 and 68 genes, respectively, that are differentially expressed in notochord and nerve cord precursor cells. These included not only genes for transcription factors and signaling molecules but also those with generalized functions observed in many types of cells. In addition, whole-mount in situ hybridization showed dynamic spatial expression profiles of these genes during segregation of the two fates: partitioning of transcripts present in the mother cells into either type of daughter cells, and initiation of preferential gene expression in either type of cells. Copyright © 2013 Wiley Periodicals, Inc.

Global gene expression profiling of oral cavity cancers suggests molecular heterogeneity within anatomic subsites

PubMed Central

Severino, Patricia; Alvares, Adriana M; Michaluart, Pedro; Okamoto, Oswaldo K; Nunes, Fabio D; Moreira-Filho, Carlos A; Tajara, Eloiza H

2008-01-01

Background Oral squamous cell carcinoma (OSCC) is a frequent neoplasm, which is usually aggressive and has unpredictable biological behavior and unfavorable prognosis. The comprehension of the molecular basis of this variability should lead to the development of targeted therapies as well as to improvements in specificity and sensitivity of diagnosis. Results Samples of primary OSCCs and their corresponding surgical margins were obtained from male patients during surgery and their gene expression profiles were screened using whole-genome microarray technology. Hierarchical clustering and Principal Components Analysis were used for data visualization and One-way Analysis of Variance was used to identify differentially expressed genes. Samples clustered mostly according to disease subsite, suggesting molecular heterogeneity within tumor stages. In order to corroborate our results, two publicly available datasets of microarray experiments were assessed. We found significant molecular differences between OSCC anatomic subsites concerning groups of genes presently or potentially important for drug development, including mRNA processing, cytoskeleton organization and biogenesis, metabolic process, cell cycle and apoptosis. Conclusion Our results corroborate literature data on molecular heterogeneity of OSCCs. Differences between disease subsites and among samples belonging to the same TNM class highlight the importance of gene expression-based classification and challenge the development of targeted therapies. PMID:19014556

Application of Whole Genome Expression Analysis to Assess Bacterial Responses to Environmental Conditions

NASA Astrophysics Data System (ADS)

Vukanti, R. V.; Mintz, E. M.; Leff, L. G.

2005-05-01

Bacterial responses to environmental signals are multifactorial and are coupled to changes in gene expression. An understanding of bacterial responses to environmental conditions is possible using microarray expression analysis. In this study, the utility of microarrays for examining changes in gene expression in Escherichia coli under different environmental conditions was assessed. RNA was isolated, hybridized to Affymetrix E. coli Genome 2.0 chips and analyzed using Affymetrix GCOS and Genespring software. Major limiting factors were obtaining enough quality RNA (107-108 cells to get 10μg RNA)and accounting for differences in growth rates under different conditions. Stabilization of RNA prior to isolation and taking extreme precautions while handling RNA were crucial. In addition, use of this method in ecological studies is limited by availability and cost of commercial arrays; choice of primers for cDNA synthesis, reproducibility, complexity of results generated and need to validate findings. This method may be more widely applicable with the development of better approaches for RNA recovery from environmental samples and increased number of available strain-specific arrays. Diligent experimental design and verification of results with real-time PCR or northern blots is needed. Overall, there is a great potential for use of this technology to discover mechanisms underlying organisms' responses to environmental conditions.

De Novo Deep Transcriptome Analysis of Medicinal Plants for Gene Discovery in Biosynthesis of Plant Natural Products.

PubMed

Han, R; Rai, A; Nakamura, M; Suzuki, H; Takahashi, H; Yamazaki, M; Saito, K

2016-01-01

Study on transcriptome, the entire pool of transcripts in an organism or single cells at certain physiological or pathological stage, is indispensable in unraveling the connection and regulation between DNA and protein. Before the advent of deep sequencing, microarray was the main approach to handle transcripts. Despite obvious shortcomings, including limited dynamic range and difficulties to compare the results from distinct experiments, microarray was widely applied. During the past decade, next-generation sequencing (NGS) has revolutionized our understanding of genomics in a fast, high-throughput, cost-effective, and tractable manner. By adopting NGS, efficiency and fruitful outcomes concerning the efforts to elucidate genes responsible for producing active compounds in medicinal plants were profoundly enhanced. The whole process involves steps, from the plant material sampling, to cDNA library preparation, to deep sequencing, and then bioinformatics takes over to assemble enormous-yet fragmentary-data from which to comb and extract information. The unprecedentedly rapid development of such technologies provides so many choices to facilitate the task, which can cause confusion when choosing the suitable methodology for specific purposes. Here, we review the general approaches for deep transcriptome analysis and then focus on their application in discovering biosynthetic pathways of medicinal plants that produce important secondary metabolites. © 2016 Elsevier Inc. All rights reserved.

Energy dispersive X-ray fluorescence spectrometry for the direct multi-element analysis of dried blood spots

NASA Astrophysics Data System (ADS)

Marguí, E.; Queralt, I.; García-Ruiz, E.; García-González, E.; Rello, L.; Resano, M.

2018-01-01

Home-based collection protocols for clinical specimens are actively pursued as a means of improving life quality of patients. In this sense, dried blood spots (DBS) are proposed as a non-invasive and even self-administered alternative to sampling whole venous blood. This contribution explores the potential of energy dispersive X-ray fluorescence spectrometry for the simultaneous and direct determination of some major (S, Cl, K, Na), minor (P, Fe) and trace (Ca, Cu, Zn) elements in blood, after its deposition onto clinical filter papers, thus giving rise to DBS. For quantification purposes the best strategy was to use matrix-matched blood samples of known analyte concentrations. The accuracy and precision of the method were evaluated by analysis of a blood reference material (Seronorm™ trace elements whole blood L3). Quantitative results were obtained for the determination of P, S, Cl, K and Fe, and limits of detection for these elements were adequate, taking into account their typical concentrations in real blood samples. Determination of Na, Ca, Cu and Zn was hampered by the occurrence of high sample support (Na, Ca) and instrumental blanks (Cu, Zn). Therefore, the quantitative determination of these elements at the levels expected in blood samples was not feasible. The methodology developed was applied to the analysis of several blood samples and the results obtained were compared with those reported by standard techniques. Overall, the performance of the method developed is promising and it could be used to determine the aforementioned elements in blood samples in a simple, fast and economic way. Furthermore, its non-destructive nature enables further analyses by means of complementary techniques to be carried out.

Endothelial Inflammatory Transcriptional Responses Induced by Plasma Following Inhalation of Diesel Emissions

PubMed Central

Schisler, Jonathan C.; Ronnebaum, Sarah M.; Madden, Michael; Channell, Meghan M.; Campen, Matthew J.; Willis, Monte S.

2016-01-01

Background Air pollution, especially emissions derived from traffic sources, is associated with adverse cardiovascular outcomes. However, it remains unclear how inhaled factors drive extrapulmonary pathology. Objectives Previously, we found that canonical inflammatory response transcripts were elevated in cultured endothelial cells treated with plasma obtained after exposure compared with pre-exposure samples or filtered air (sham) exposures. While the findings confirmed the presence of bioactive factor(s) in the plasma after diesel inhalation, we wanted to better examine the complete genomic response to investigate 1) major responsive transcripts and 2) collected response pathways and ontogeny that may help to refine this method and inform the pathogenesis. Methods We assayed endothelial RNA with gene expression microarrays, examining the responses of cultured endothelial cells to plasma obtained from 6 healthy human subjects exposed to 100 μg/m3 diesel exhaust or filtered air for 2 h on separate occasions. In addition to pre-exposure baseline samples, we investigated samples obtained immediately-post and 24h-post exposure. Results Microarray analysis of the coronary artery endothelial cells challenged with plasma identified 855 probes that changed over time following diesel exhaust exposure. Over-representation analysis identified inflammatory cytokine pathways were upregulated both at the 2 and 24 h condition. Novel pathways related to FOX transcription factors and secreted extracellular factors were also identified in the microarray analysis. Conclusions These outcomes are consistent with our recent findings that plasma contains bioactive and inflammatory factors following pollutant inhalation. The specific study design implicates a novel pathway related to inflammatory blood borne components that may drive the extrapulmonary toxicity of ambient air pollutants. PMID:25942053

Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis

PubMed Central

Wapner, Ronald J.; Martin, Christa Lese; Levy, Brynn; Ballif, Blake C.; Eng, Christine M.; Zachary, Julia M.; Savage, Melissa; Platt, Lawrence D.; Saltzman, Daniel; Grobman, William A.; Klugman, Susan; Scholl, Thomas; Simpson, Joe Leigh; McCall, Kimberly; Aggarwal, Vimla S.; Bunke, Brian; Nahum, Odelia; Patel, Ankita; Lamb, Allen N.; Thom, Elizabeth A.; Beaudet, Arthur L.; Ledbetter, David H.; Shaffer, Lisa G.; Jackson, Laird

2013-01-01

Background Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Methods Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. Results We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down’s syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. Conclusions In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.) PMID:23215555

Molecular classification of benign prostatic hyperplasia: A gene expression profiling study in a rat model.

PubMed

Hata, Junya; Satoh, Yuichi; Akaihata, Hidenori; Hiraki, Hiroyuki; Ogawa, Soichiro; Haga, Nobuhiro; Ishibashi, Kei; Aikawa, Ken; Kojima, Yoshiyuki

2016-07-01

To characterize the molecular features of benign prostatic hyperplasia by carrying out a gene expression profiling analysis in a rat model. Fetal urogenital sinus isolated from 20-day-old male rat embryo was implanted into a pubertal male rat ventral prostate. The implanted urogenital sinus grew time-dependently, and the pathological findings at 3 weeks after implantation showed epithelial hyperplasia as well as stromal hyperplasia. Whole-genome oligonucleotide microarray analysis utilizing approximately 30 000 oligonucleotide probes was carried out using prostate specimens during the prostate growth process (3 weeks after implantation). Microarray analyses showed 926 upregulated (>2-fold change, P < 0.01) and 3217 downregulated genes (<0.5-fold change, P < 0.01) in benign prostatic hyperplasia specimens compared with normal prostate. Gene ontology analyses of upregulated genes showed predominant genetic themes of involvement in development (162 genes, P = 2.01 × 10(-4) ), response to stimulus (163 genes, P = 7.37 × 10(-13) ) and growth (32 genes, P = 1.93 × 10(-5) ). When we used both normal prostate and non-transplanted urogenital sinuses as controls to identify benign prostatic hyperplasia-specific genes, 507 and 406 genes were upregulated and downregulated, respectively. Functional network and pathway analyses showed that genes associated with apoptosis modulation by heat shock protein 70, interleukin-1, interleukin-2 and interleukin-5 signaling pathways, KIT signaling pathway, and secretin-like G-protein-coupled receptors, class B, were relatively activated during the growth process in the benign prostatic hyperplasia specimens. In contrast, genes associated with cholesterol biosynthesis were relatively inactivated. Our microarray analyses of the benign prostatic hyperplasia model rat might aid in clarifying the molecular mechanism of benign prostatic hyperplasia progression, and identifying molecular targets for benign prostatic hyperplasia treatment. © 2016 The Japanese Urological Association.

Correcting for intra-experiment variation in Illumina BeadChip data is necessary to generate robust gene-expression profiles.

PubMed

Kitchen, Robert R; Sabine, Vicky S; Sims, Andrew H; Macaskill, E Jane; Renshaw, Lorna; Thomas, Jeremy S; van Hemert, Jano I; Dixon, J Michael; Bartlett, John M S

2010-02-24

Microarray technology is a popular means of producing whole genome transcriptional profiles, however high cost and scarcity of mRNA has led many studies to be conducted based on the analysis of single samples. We exploit the design of the Illumina platform, specifically multiple arrays on each chip, to evaluate intra-experiment technical variation using repeated hybridisations of universal human reference RNA (UHRR) and duplicate hybridisations of primary breast tumour samples from a clinical study. A clear batch-specific bias was detected in the measured expressions of both the UHRR and clinical samples. This bias was found to persist following standard microarray normalisation techniques. However, when mean-centering or empirical Bayes batch-correction methods (ComBat) were applied to the data, inter-batch variation in the UHRR and clinical samples were greatly reduced. Correlation between replicate UHRR samples improved by two orders of magnitude following batch-correction using ComBat (ranging from 0.9833-0.9991 to 0.9997-0.9999) and increased the consistency of the gene-lists from the duplicate clinical samples, from 11.6% in quantile normalised data to 66.4% in batch-corrected data. The use of UHRR as an inter-batch calibrator provided a small additional benefit when used in conjunction with ComBat, further increasing the agreement between the two gene-lists, up to 74.1%. In the interests of practicalities and cost, these results suggest that single samples can generate reliable data, but only after careful compensation for technical bias in the experiment. We recommend that investigators appreciate the propensity for such variation in the design stages of a microarray experiment and that the use of suitable correction methods become routine during the statistical analysis of the data.

Correcting for intra-experiment variation in Illumina BeadChip data is necessary to generate robust gene-expression profiles

PubMed Central

2010-01-01

Background Microarray technology is a popular means of producing whole genome transcriptional profiles, however high cost and scarcity of mRNA has led many studies to be conducted based on the analysis of single samples. We exploit the design of the Illumina platform, specifically multiple arrays on each chip, to evaluate intra-experiment technical variation using repeated hybridisations of universal human reference RNA (UHRR) and duplicate hybridisations of primary breast tumour samples from a clinical study. Results A clear batch-specific bias was detected in the measured expressions of both the UHRR and clinical samples. This bias was found to persist following standard microarray normalisation techniques. However, when mean-centering or empirical Bayes batch-correction methods (ComBat) were applied to the data, inter-batch variation in the UHRR and clinical samples were greatly reduced. Correlation between replicate UHRR samples improved by two orders of magnitude following batch-correction using ComBat (ranging from 0.9833-0.9991 to 0.9997-0.9999) and increased the consistency of the gene-lists from the duplicate clinical samples, from 11.6% in quantile normalised data to 66.4% in batch-corrected data. The use of UHRR as an inter-batch calibrator provided a small additional benefit when used in conjunction with ComBat, further increasing the agreement between the two gene-lists, up to 74.1%. Conclusion In the interests of practicalities and cost, these results suggest that single samples can generate reliable data, but only after careful compensation for technical bias in the experiment. We recommend that investigators appreciate the propensity for such variation in the design stages of a microarray experiment and that the use of suitable correction methods become routine during the statistical analysis of the data. PMID:20181233

Analysis of microarray leukemia data using an efficient MapReduce-based K-nearest-neighbor classifier.

PubMed

Kumar, Mukesh; Rath, Nitish Kumar; Rath, Santanu Kumar

2016-04-01

Microarray-based gene expression profiling has emerged as an efficient technique for classification, prognosis, diagnosis, and treatment of cancer. Frequent changes in the behavior of this disease generates an enormous volume of data. Microarray data satisfies both the veracity and velocity properties of big data, as it keeps changing with time. Therefore, the analysis of microarray datasets in a small amount of time is essential. They often contain a large amount of expression, but only a fraction of it comprises genes that are significantly expressed. The precise identification of genes of interest that are responsible for causing cancer are imperative in microarray data analysis. Most existing schemes employ a two-phase process such as feature selection/extraction followed by classification. In this paper, various statistical methods (tests) based on MapReduce are proposed for selecting relevant features. After feature selection, a MapReduce-based K-nearest neighbor (mrKNN) classifier is also employed to classify microarray data. These algorithms are successfully implemented in a Hadoop framework. A comparative analysis is done on these MapReduce-based models using microarray datasets of various dimensions. From the obtained results, it is observed that these models consume much less execution time than conventional models in processing big data. Copyright © 2016 Elsevier Inc. All rights reserved.

Whole genome sequencing reveals mycobacterial microevolution among concurrent isolates from sputum and blood in HIV infected TB patients.

PubMed

Ssengooba, Willy; de Jong, Bouke C; Joloba, Moses L; Cobelens, Frank G; Meehan, Conor J

2016-08-05

In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments. From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis. Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16-101)/mm(3) and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical. Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly.

Determining the effect of different environmental conditions on Ebola virus viability in clinically relevant specimens.

PubMed

Palyi, Bernadett; Magyar, Nora; Henczko, Judit; Szalai, Balint; Farkas, Agnes; Strecker, Thomas; Takacs, Maria; Kis, Zoltan

2018-03-29

In 2013-2016, West Africa experienced the largest and longest Ebola virus disease outbreak ever documented. The wide geographic spread and magnitude of the outbreak often limited the timely and rapid testing of diagnostic samples from patients with suspected Ebola virus disease, raising questions regarding the optimal storage and shipping conditions of clinically relevant specimens, including EDTA-whole blood, plasma, capillary blood, urine and seminal fluid (associated with sexual transmission of the Ebola virus after recovery from the disease). Therefore, the aim of our study was to identify the extent to which storage temperature and clinical specimen type influence Ebola virus viability. Virus infectivity was determined using a fluorescent focus-forming assay. In our study, we show that Ebola virus was the most stable in EDTA-whole blood and plasma samples, whereas rapid decay of infectivity was observed in simulated capillary blood, urine and semen samples, especially when these samples were stored at higher temperatures. The analysis of variance results demonstrated that both temperature and clinical specimen type have significant effects on virus viability, whereas donor differences were not observed. Repeated freeze and thaw cycles of the samples also had a notable impact on virus viability in EDTA-whole blood and urine. Due to the rapid temperature- and specimen-dependent degradation of the virus observed here, our study highlights the importance of proper clinical sample storage at low temperatures during transportation and laboratory analysis.

In Vivo and In Vitro Nitinol Corrosion Properties

NASA Astrophysics Data System (ADS)

Lonn, Melissa K.; Metcalf, Justin M.; Choules, Brian D.

2015-09-01

Regulatory authorities often require in vitro testing on medical devices prior to approval. Current standardized corrosion testing methods (ASTM F2129) require testing in a non-physiologic, de-oxygenated solution for a pre-exposure time of ≤1 h; however, no correlations between the prescribed simulated environment and whole blood conditions have been elucidated. This study compared open circuit potential (OCP), breakdown potentials (Eb), Eb - OCP, and cyclic polarization curves tested in vivo (OCP only) and in vitro in whole blood to those tested in phosphate-buffered saline (PBS). Two oxide thicknesses of Nitinol, two solution oxygen contents (deaerated and aerated solutions), and two pre-exposure durations (acute and chronic) were investigated. The in vitro OCP in whole blood was not significantly different than the in vivo OCP, suggesting that whole blood in vitro can be used to determine baseline corrosion behavior of medical implants. Eb - OCP tested per ASTM F2129 was comparable to acute whole blood and was conservative compared to chronic whole blood for both oxide thicknesses. However, OCP, Eb, and cyclic polarization curves were not always comparable to whole blood. Testing in aerated PBS achieved Eb, Eb - OCP, and cyclic polarization curves that were comparable to or more conservative than whole blood testing, regardless of pre-exposure duration and oxide thickness.