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Sample records for cell-free plasma dna

  1. Plasma cell-free DNA in patients needing mechanical ventilation

    PubMed Central

    2011-01-01

    Introduction Concentrations of plasma cell-free DNA are increased in various diseases and have shown some prognostic value in many patient groups, including critically ill patients. Pathophysiological processes behind the need for mechanical ventilation and the treatment itself could raise plasma levels of cell-free DNA. We evaluated levels of plasma cell-free DNA and their prognostic value in patients needing mechanical ventilation. Methods We studied prospectively 580 mechanically ventilated critically ill patients. Blood samples were taken at study admission (Day 0) and on Day 2. Plasma cell-free DNA concentrations were measured by real-time quantitative PCR assay for the β-globin gene and are expressed as genome equivalents (GE)/ml. Results Median (interquartile range, IQR) plasma cell-free DNA concentration was 11,853 GE/ml (5,304 to 24,620 GE/mL) at study admission, and 11,610 GE/mL (6,411 to 21,558 GE/mL) on Day 2. Concentrations at admission were significantly higher in 90-day non-survivors than survivors, 16,936 GE/mL (7,262 to 46,866 GE/mL) versus 10,026 GE/mL (4,870 to 19,820 GE/mL), P < 0.001. In a multivariate logistic regression analysis plasma cell-free DNA concentration over 16,000 GE/ml remained an independent predictor of 90-day mortality (adjusted odds ratio 2.16, 95% confidence interval CI 1.37 to 3.40). Positive likelihood ratio of plasma cell-free DNA at admission for the prediction of 90-day mortality was 1.72 (95% CI 1.40 to 2.11). Conclusions Plasma levels of cell-free DNA were significantly higher in non-survivors than survivors. Plasma DNA level at baseline was an independent predictor of 90-day mortality. However, its clinical benefit as a prognostic marker seems to be limited. PMID:21838858

  2. Kinetics of Circulating Plasma Cell-Free DNA in Paediatric Classical Hodgkin Lymphoma

    PubMed Central

    Primerano, Simona; Burnelli, Roberta; Carraro, Elisa; Pillon, Marta; Elia, Caterina; Farruggia, Piero; Sala, Alessandra; Vinti, Luciana; Buffardi, Salvatore; Basso, Giuseppe; Mascarin, Maurizio; Mussolin, Lara

    2016-01-01

    Levels of plasma cell-free DNA (cfDNA) of a large series of children with classical Hodgkin lymphoma (cHL) were evaluated and analyzed at diagnosis and during chemotherapy treatment in relation with clinical characteristics. CfDNA levels in cHL patients were significantly higher compared with controls (p=0.002). CfDNA at diagnosis was correlated with presence of B symptoms (p=0.027) and high erythrocyte sedimentation rate (p=0.049). We found that the increasing of plasma cfDNA after first chemotherapy cycle seems to be associated with a worse prognosis (p=0.049). Levels of plasma cfDNA might constitute an interesting non-invasive tool in cHL patients' management. PMID:26918050

  3. An Advanced Model to Precisely Estimate the Cell-Free Fetal DNA Concentration in Maternal Plasma

    PubMed Central

    Xu, Huixin; Jiang, Haojun; Xie, Weiwei; Chen, Fang; Zeng, Peng; Li, Xuchao; Xie, Yifan; Liu, Hongtai; Huang, Guodong; Chen, Dayang; Liu, Ping; Jiang, Hui; Zhang, Xiuqing

    2016-01-01

    Background With the speedy development of sequencing technologies, noninvasive prenatal testing (NIPT) has been widely applied in clinical practice for testing for fetal aneuploidy. The cell-free fetal DNA (cffDNA) concentration in maternal plasma is the most critical parameter for this technology because it affects the accuracy of NIPT-based sequencing for fetal trisomies 21, 18 and 13. Several approaches have been developed to calculate the cffDNA fraction of the total cell-free DNA in the maternal plasma. However, most approaches depend on specific single nucleotide polymorphism (SNP) allele information or are restricted to male fetuses. Methods In this study, we present an innovative method to accurately deduce the concentration of the cffDNA fraction using only maternal plasma DNA. SNPs were classified into four maternal-fetal genotype combinations and three boundaries were added to capture effective SNP loci in which the mother was homozygous and the fetus was heterozygous. The median value of the concentration of the fetal DNA fraction was estimated using the effective SNPs. A depth-bias correction was performed using simulated data and corresponding regression equations for adjustments when the depth of the sequencing data was below 100-fold or the cffDNA fraction is less than 10%. Results Using our approach, the median of the relative bias was 0.4% in 18 maternal plasma samples with a median sequencing depth of 125-fold. There was a significant association (r = 0.935) between our estimations and the estimations inferred from the Y chromosome. Furthermore, this approach could precisely estimate a cffDNA fraction as low as 3%, using only maternal plasma DNA at the targeted region with a sequencing depth of 65-fold. We also used PCR instead of parallel sequencing to calculate the cffDNA fraction. There was a significant association (r = 98.2%) between our estimations and those inferred from the Y chromosome. PMID:27662469

  4. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    PubMed Central

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis. PMID:27141267

  5. Plasma circulating cell-free mitochondrial DNA in the assessment of Friedreich's ataxia.

    PubMed

    Dantham, Subrahamanyam; Srivastava, Achal K; Gulati, Sheffali; Rajeswari, Moganty R

    2016-06-15

    Friedreich's ataxia (FRDA) is one of the most devastating childhood onset neurodegenerative disease affecting multiple organs in the course of progression. FRDA is associated with mitochondrial dysfunction due to deficit in a nuclear encoded mitochondrial protein, frataxin. Identification of disease-specific biomarker for monitoring the severity remains to be a challenging topic. This study was aimed to identify whether circulating cell-free nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in blood plasma can be a potential biomarker for FRDA. Clinical information was assessed using International Cooperative Ataxia Rating Scale and the disease was confirmed using Long-range PCR for GAA repeat expansion within the gene encoding frataxin. The frataxin expression was measured using Western blot. Plasma nDNA and mtDNA levels were quantified by Multiplex real-time PCR. The major observation is that the levels of nDNA found to be increased, whereas mtDNA levels were reduced significantly in the plasma of FRDA patients (n=21) as compared to healthy controls (n=21). Further, plasma mtDNA levels showed high sensitivity (90%) and specificity (76%) in distinguishing from healthy controls with optimal cutoff indicated at 4.1×10(5)GE/mL. Interestingly, a small group of follow-up patients (n=9) on intervention with, a nutrient supplement, omega-3 fatty acid (a known enhancer of mitochondrial metabolism) displayed a significant improvement in the levels of plasma mtDNA, supporting our hypothesis that plasma mtDNA can be a potential monitoring or prognosis biomarker for FRDA.

  6. Plasma circulating cell-free mitochondrial DNA in the assessment of Friedreich's ataxia.

    PubMed

    Dantham, Subrahamanyam; Srivastava, Achal K; Gulati, Sheffali; Rajeswari, Moganty R

    2016-06-15

    Friedreich's ataxia (FRDA) is one of the most devastating childhood onset neurodegenerative disease affecting multiple organs in the course of progression. FRDA is associated with mitochondrial dysfunction due to deficit in a nuclear encoded mitochondrial protein, frataxin. Identification of disease-specific biomarker for monitoring the severity remains to be a challenging topic. This study was aimed to identify whether circulating cell-free nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in blood plasma can be a potential biomarker for FRDA. Clinical information was assessed using International Cooperative Ataxia Rating Scale and the disease was confirmed using Long-range PCR for GAA repeat expansion within the gene encoding frataxin. The frataxin expression was measured using Western blot. Plasma nDNA and mtDNA levels were quantified by Multiplex real-time PCR. The major observation is that the levels of nDNA found to be increased, whereas mtDNA levels were reduced significantly in the plasma of FRDA patients (n=21) as compared to healthy controls (n=21). Further, plasma mtDNA levels showed high sensitivity (90%) and specificity (76%) in distinguishing from healthy controls with optimal cutoff indicated at 4.1×10(5)GE/mL. Interestingly, a small group of follow-up patients (n=9) on intervention with, a nutrient supplement, omega-3 fatty acid (a known enhancer of mitochondrial metabolism) displayed a significant improvement in the levels of plasma mtDNA, supporting our hypothesis that plasma mtDNA can be a potential monitoring or prognosis biomarker for FRDA. PMID:27206881

  7. Direct measurement of cell-free DNA from serially collected capillary plasma during incremental exercise.

    PubMed

    Breitbach, Sarah; Sterzing, Björn; Magallanes, Carlos; Tug, Suzan; Simon, Perikles

    2014-07-15

    To investigate the kinetics of cell-free DNA (cfDNA) due to exercise, we established a direct real-time PCR for the quantification of cfDNA from unpurified capillary plasma by amplification of a 90- and a 222-bp multilocus L1PA2 sequence. Twenty-six male athletes performed an incremental treadmill test. For cfDNA measurement, capillary samples were collected serially from the fingertip preexercise, during, and several times postexercise. Venous blood was drawn before and immediately after exercise to compare capillary and venous cfDNA values. To elucidate the strongest association of cfDNA accumulations with either cardiorespiratory or metabolic function during exercise, capillary cfDNA values were correlated with standard measures like heart rate, oxygen consumption, or lactate concentrations. The venous cfDNA concentrations were significantly higher compared with the capillary plasma, but in both fractions cfDNA increased 9.8-fold and the values correlated significantly (r = 0.796). During incremental treadmill running, the capillary cfDNA concentrations increased nearly parallel to the lactate values. The values correlated best with heart rate and energy expenditure, followed by oxygen consumption, Borg values, and lactate levels (0.710 ≤ r ≥ 0.808). With this article, we present a sensitive procedure for the direct quantification of cfDNA in unpurified capillary plasma instead of purified venous plasma. Further studies should investigate the differences between capillary and venous cfDNA that might mirror different physiological mechanisms. Enhanced cardiorespiratory function during exercise might lead to the accumulation of cfDNA via the release of stress hormones that already increase at intensities below the anaerobic threshold. Furthermore, cfDNA might be released by neutrophil extracellular traps.

  8. Direct Quantification of Cell-Free, Circulating DNA from Unpurified Plasma

    PubMed Central

    Breitbach, Sarah; Tug, Suzan; Helmig, Susanne; Zahn, Daniela; Kubiak, Thomas; Michal, Matthias; Gori, Tommaso; Ehlert, Tobias; Beiter, Thomas; Simon, Perikles

    2014-01-01

    Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1–183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6–23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions. PMID:24595313

  9. Plasma cell-free mitochondrial DNA declines in response to prolonged moderate aerobic exercise.

    PubMed

    Shockett, Penny E; Khanal, Januka; Sitaula, Alina; Oglesby, Christopher; Meachum, William A; Castracane, V Daniel; Kraemer, Robert R

    2016-01-01

    Increased plasma cell-free mitochondrial DNA (cf-mDNA), a damage-associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients and arises in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf-mDNA response to prolonged moderate aerobic exercise. Seven healthy moderately trained young men (age = 22.4 ± 1.2) completed a treadmill exercise trial for 90 min at 60% VO2 max and a resting control trial. Blood was sampled immediately prior to exercise (0 min = baseline), during (+18, +54 min), immediately after (+90 min), and after recovery (R40). Plasma was analyzed for cf-mDNA, IL-6, and lactate. A significant difference in cf-mDNA response was observed between exercise and control trials, with cf-mDNA levels reduced during exercise at +54 and +90 (with or without plasma volume shift correction). Declines in cf-mDNA were accompanied by increased lactate and followed by an increase in IL-6, suggesting a temporal association with muscle stress and inflammatory processes. Our novel finding of cf-mDNA decline with prolonged moderate treadmill exercise provides evidence for increased clearance from or reduced release of cf-mDNA into the blood with prolonged exercise. These studies contrast with previous investigations involving exhaustive short-term treadmill exercise, in which no change in cf-mDNA levels were reported, and contribute to our understanding of differences between exercise- and trauma-induced inflammation. We propose that transient declines in cf-mDNA may induce health benefits, by reducing systemic inflammation. PMID:26755735

  10. Plasma cell-free mitochondrial DNA declines in response to prolonged moderate aerobic exercise.

    PubMed

    Shockett, Penny E; Khanal, Januka; Sitaula, Alina; Oglesby, Christopher; Meachum, William A; Castracane, V Daniel; Kraemer, Robert R

    2016-01-01

    Increased plasma cell-free mitochondrial DNA (cf-mDNA), a damage-associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients and arises in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf-mDNA response to prolonged moderate aerobic exercise. Seven healthy moderately trained young men (age = 22.4 ± 1.2) completed a treadmill exercise trial for 90 min at 60% VO2 max and a resting control trial. Blood was sampled immediately prior to exercise (0 min = baseline), during (+18, +54 min), immediately after (+90 min), and after recovery (R40). Plasma was analyzed for cf-mDNA, IL-6, and lactate. A significant difference in cf-mDNA response was observed between exercise and control trials, with cf-mDNA levels reduced during exercise at +54 and +90 (with or without plasma volume shift correction). Declines in cf-mDNA were accompanied by increased lactate and followed by an increase in IL-6, suggesting a temporal association with muscle stress and inflammatory processes. Our novel finding of cf-mDNA decline with prolonged moderate treadmill exercise provides evidence for increased clearance from or reduced release of cf-mDNA into the blood with prolonged exercise. These studies contrast with previous investigations involving exhaustive short-term treadmill exercise, in which no change in cf-mDNA levels were reported, and contribute to our understanding of differences between exercise- and trauma-induced inflammation. We propose that transient declines in cf-mDNA may induce health benefits, by reducing systemic inflammation.

  11. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    PubMed Central

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A.; Khush, Kiran K.; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10−5, Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10−5) and microbial cfDNA (71.3x, p 10−5). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  12. Genome aberrations in canine mammary carcinomas and their detection in cell-free plasma DNA.

    PubMed

    Beck, Julia; Hennecke, Silvia; Bornemann-Kolatzki, Kirsten; Urnovitz, Howard B; Neumann, Stephan; Ströbel, Philipp; Kaup, Franz-Josef; Brenig, Bertram; Schütz, Ekkehard

    2013-01-01

    Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences. There is lack of knowledge about the genomes of these common dog tumors. Five tumors of three different histological subtypes were evaluated. Massive parallel sequencing (MPS) was performed in comparison to the respective somatic genome of each animal. Copy number and structural aberrations were validated using droplet digital PCR (ddPCR). Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas one tumor was almost normal. These aberrations affect several known cancer associated genes such as cMYC, and KIT. One common deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N = 20). Breakpoint specific dPCRs were established for four tumors and tumor specific cell-free DNA (cfDNA) was detected in the plasma. In one animal tumor-specific cfDNA was found >1 year after surgery, attributable to a lung metastasis. Paired-end sequencing proved that copy-number imbalances of the tumor are reflected by the cfDNA. This report on chromosomal instability of canine mammary cancers reveals similarities to human breast cancers as well as special canine alterations. This animal model provides a framework for using MPS for screening for individual cancer biomarkers with cost effective confirmation and monitoring using ddPCR. The possibility exists that ddPCR can be expanded to screening for common cancer related variants. PMID:24098698

  13. Increased plasma cell-free DNA is associated with low pregnancy rates among women undergoing IVF-embryo transfer.

    PubMed

    Czamanski-Cohen, Johanna; Sarid, Orly; Cwikel, Julie; Lunenfeld, Eitan; Douvdevani, Amos; Levitas, Eliahu; Har-Vardi, Iris

    2013-01-01

    This prospective repeated measures study was designed to examine the cell-free DNA (cfDNA) concentrations during ovarian stimulation and the relationship between cfDNA concentration and pregnancy rates in women undergoing IVF-embryo transfer. The study examined 37 women undergoing IVF treatment in an IVF unit in a university medical centre in southern Israel. cfDNA concentrations were measured by a direct fluorescence assay, pregnancy rates were identified by plasma β human chorionic gonadotrophin (HCG) concentrations and verified by vaginal ultrasound to determine gestational sac and fetal heart beats. Throughout the IVF cycle, at the three time points measured, the mean concentration of plasma cfDNA among all participants did not statistically significantly change. However, on the day of βHCG test in patients undergoing IVF-embryo transfer, plasma cfDNA concentrations were statistically significantly higher among women who did not conceive in comparison to those who conceived. Plasma cfDNA may reflect the presence of factors which interfere with embryo implantation. Further research is required to determine the usefulness of cfDNA as a biomarker of IVF outcome and to examine the underlying pathologies as potential sources for increased plasma cfDNA concentrations. Cell-free DNA (cfDNA) is particles of DNA which are released from the cell nucleus and are found in high concentrations during a variety of illnesses and injuries. This study was designed to examine the cfDNA concentrations during IVF treatment and the relationship between cfDNA concentration in the bloodstream and pregnancy rates in women undergoing IVF. This study examined 37 women in treatment at the IVF unit of the University Medical Centre in southern Israel. cfDNA concentrations in the bloodstream were measured at three time points by a direct test. Pregnancy rates were identified by pregnancy hormone concentrations in the bloodstream and verified by vaginal ultrasound to determine a pregnancy

  14. Elevated Cell-Free Plasma DNA Level as an Independent Predictor of Mortality in Patients with Severe Traumatic Brain Injury

    PubMed Central

    Rodrigues Filho, Edison Moraes; Simon, Daniel; Ikuta, Nilo; Klovan, Caroline; Dannebrock, Fernando Augusto; Oliveira de Oliveira, Carla

    2014-01-01

    Abstract Trauma is the leading cause of death in individuals less than 45 years old worldwide, and up to 50% of trauma fatalities are because of brain injury. Prediction of outcome is one of the major problems associated with severe traumatic brain injury (TBI), and research efforts have focused on the investigation of biomarkers with prognostic value after TBI. Therefore, our aim was to investigate whether cell-free DNA concentrations correlated to short-term primary outcome (survival or death) and Glasgow Coma Scale (GCS) scores after severe TBI. A total of 188 patients with severe TBI were enrolled in this prospective study; outcome variables comprised survival and neurological assessment using the GCS at intensive care unit (ICU) discharge. Control blood samples were obtained from 25 healthy volunteers. Peripheral venous blood was collected at admission to the ICU. Plasma DNA was measured using a real-time quantitative polymerase chain reaction (PCR) assay for the β-globin gene. There was correlation between higher DNA levels and both fatal outcome and lower hospital admission GCS scores. Plasma DNA concentrations at the chosen cutoff point (≥171,381 kilogenomes-equivalents/L) predicted mortality with a specificity of 90% and a sensitivity of 43%. Logistic regression analysis showed that elevated plasma DNA levels were independently associated with death (p<0.001). In conclusion, high cell-free DNA concentration was a predictor of short-term mortality after severe TBI. PMID:24827371

  15. Elevated cell-free plasma DNA level as an independent predictor of mortality in patients with severe traumatic brain injury.

    PubMed

    Rodrigues Filho, Edison Moraes; Simon, Daniel; Ikuta, Nilo; Klovan, Caroline; Dannebrock, Fernando Augusto; Oliveira de Oliveira, Carla; Regner, Andrea

    2014-10-01

    Trauma is the leading cause of death in individuals less than 45 years old worldwide, and up to 50% of trauma fatalities are because of brain injury. Prediction of outcome is one of the major problems associated with severe traumatic brain injury (TBI), and research efforts have focused on the investigation of biomarkers with prognostic value after TBI. Therefore, our aim was to investigate whether cell-free DNA concentrations correlated to short-term primary outcome (survival or death) and Glasgow Coma Scale (GCS) scores after severe TBI. A total of 188 patients with severe TBI were enrolled in this prospective study; outcome variables comprised survival and neurological assessment using the GCS at intensive care unit (ICU) discharge. Control blood samples were obtained from 25 healthy volunteers. Peripheral venous blood was collected at admission to the ICU. Plasma DNA was measured using a real-time quantitative polymerase chain reaction (PCR) assay for the β-globin gene. There was correlation between higher DNA levels and both fatal outcome and lower hospital admission GCS scores. Plasma DNA concentrations at the chosen cutoff point (≥171,381 kilogenomes-equivalents/L) predicted mortality with a specificity of 90% and a sensitivity of 43%. Logistic regression analysis showed that elevated plasma DNA levels were independently associated with death (p<0.001). In conclusion, high cell-free DNA concentration was a predictor of short-term mortality after severe TBI.

  16. Plasma cell-free DNA levels and integrity in patients with chest radiological findings: NSCLC versus benign lung nodules.

    PubMed

    Szpechcinski, Adam; Rudzinski, Piotr; Kupis, Wlodzimierz; Langfort, Renata; Orlowski, Tadeusz; Chorostowska-Wynimko, Joanna

    2016-05-01

    Effective discrimination between lung cancer and benign tumours is a common clinical problem in the differential diagnosis of solitary pulmonary nodules. The analysis of cell-free DNA (cfDNA) in blood may greatly aid the early detection of lung cancer by evaluating cancer-related alterations. The plasma cfDNA levels and integrity were analysed in 65 non-small cell lung cancer (NSCLC) patients, 28 subjects with benign lung tumours, and 16 healthy controls using real-time PCR. The NSCLC patients demonstrated significantly higher mean plasma cfDNA levels compared with those with benign tumours (P = 0.0009) and healthy controls (P < 0.0001). The plasma cfDNA integrity in healthy individuals was significantly different than that found in patients with NSCLC or benign lung tumours (P < 0.0003). In ROC curve analysis, plasma cfDNA levels >2.8 ng/ml provided 86.4% sensitivity and 61.4% specificity in discriminating NSCLC from benign lung pathologies and healthy controls. cfDNA integrity showed better discriminatory power (91% sensitivity, 68.2% specificity). These data demonstrate that plasma cfDNA concentration and integrity analyses can significantly differentiate between NSCLC and benign lung tumours. The diagnostic capacity of the quantitative cfDNA assay is comparable to the values presented by conventional imaging modalities used in clinical practice.

  17. Ultradeep sequencing detects GNAQ and GNA11 mutations in cell-free DNA from plasma of patients with uveal melanoma.

    PubMed

    Metz, Claudia Hd; Scheulen, Max; Bornfeld, Norbert; Lohmann, Dietmar; Zeschnigk, Michael

    2013-04-01

    Elevated levels of cell-free DNA (cfDNA) are frequently observed in tumor patients. Activating mutations in exon 4 (R183) and exon 5 (Q209) of GNAQ and GNA 11 are almost exclusively found in uveal melanoma, thus providing a highly specific marker for the presence of circulating tumor DNA (ctDNA). To establish a reliable, noninvasive assay that might allow early detection and monitoring of metastatic disease, we determined the proportion of GNAQ or GNA 11 mutant reads in cfDNA of uveal melanoma patients by ultradeep sequencing. Cell-free DNA from 28 uveal melanoma patients with metastases or extraocular growth was isolated and quantified by real-time polymerase chain reaction (PCR) (7-1550 ng DNA/mL plasma). GNAQ and GNA 11 regions of interest were amplified in 22 of 28 patients and ultradeep sequencing of amplicons was performed to detect even low proportions of mutant reads. We detected Q209 mutations (2-38% mutant reads) in either GNAQ or GNA 11 in the plasma of 9 of 22 metastasized patients. No correlation between the proportion of mutant reads and the concentration of cfDNA could be detected. Among the nine ctDNA-positive patients, four had metastases in bone, whereas no metastases were detected in the 13 ctDNA-negative patients at this location (P = 0.025). Furthermore, ctDNA-positive patients tended to be younger at initial diagnosis and show larger metastases. The results show that ultradeep amplicon sequencing can be used to detect tumor DNA in plasma of metastasized uveal melanoma patients. It remains to be shown if this approach can be used for early detection of disseminated tumor disease.

  18. Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer

    PubMed Central

    Tsyba, Liudmyla; Onyshchenko, Kateryna; Kashparova, Olena; Nikolaienko, Oleksii; Panasenko, Grigory; Vozianov, Sergii; Romanenko, Alina; Rynditch, Alla

    2016-01-01

    The critical point for successful treatment of cancer is diagnosis at early stages of tumor development. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA (cfDNA) circulating in the blood is a convenient tumor-associated DNA marker. Therefore methylated cfDNA can be used as a minimally invasive diagnostic marker. We analysed the concentration of plasma cfDNA and methylation of six tumor suppressor genes in samples of 27 patients with renal cancer and 15 healthy donors as controls. The cfDNA concentrations in samples from cancer patients and healthy donors was measured using two different methods, the SYBR Green I fluorescence test and quantitative real-time PCR. Both methods revealed a statistically significant increase of cfDNA concentrations in cancer patients. Hypermethylation on cfDNA was detected for the LRRC3B (74.1%), APC (51.9%), FHIT (55.6%), and RASSF1 (62.9%) genes in patients with renal cancer. Promoter methylation of VHL and ITGA9 genes was not found on cfDNA. Our results confirmed that the cfDNA level and methylation of CpG islands of RASSF1A, FHIT, and APC genes in blood plasma can be used as noninvasive diagnostic markers of cancer. PMID:27725787

  19. Aberrant reduction of telomere repetitive sequences in plasma cell-free DNA for early breast cancer detection

    PubMed Central

    Wu, Xi; Tanaka, Hiromi

    2015-01-01

    Excessive telomere shortening is observed in breast cancer lesions when compared to adjacent non-cancerous tissues, suggesting that telomere length may represent a key biomarker for early cancer detection. Because tumor-derived, cell-free DNA (cfDNA) is often released from cancer cells and circulates in the bloodstream, we hypothesized that breast cancer development is associated with changes in the amount of telomeric cfDNA that can be detected in the plasma. To test this hypothesis, we devised a novel, highly sensitive and specific quantitative PCR (qPCR) assay, termed telomeric cfDNA qPCR, to quantify plasma telomeric cfDNA levels. Indeed, the internal reference primers of our design correctly reflected input cfDNA amount (R2 = 0.910, P = 7.82 × 10−52), implying accuracy of this assay. We found that plasma telomeric cfDNA levels decreased with age in healthy individuals (n = 42, R2 = 0.094, P = 0.048), suggesting that cfDNA is likely derived from somatic cells in which telomere length shortens with increasing age. Our results also showed a significant decrease in telomeric cfDNA level from breast cancer patients with no prior treatment (n = 47), compared to control individuals (n = 42) (P = 4.06 × 10−8). The sensitivity and specificity for the telomeric cfDNA qPCR assay was 91.49% and 76.19%, respectively. Furthermore, the telomeric cfDNA level distinguished even the Ductal Carcinoma In Situ (DCIS) group (n = 7) from the healthy group (n = 42) (P = 1.51 × 10−3). Taken together, decreasing plasma telomeric cfDNA levels could be an informative genetic biomarker for early breast cancer detection. PMID:26356673

  20. Prognostic value of cell-free plasma DNA in patients with cardiac arrest outside the hospital: an observational cohort study

    PubMed Central

    2010-01-01

    Introduction Many approaches have been examined to try to predict patient outcome after cardiopulmonary resuscitation. It has been shown that plasma DNA could predict mortality in critically ill patients but no data are available regarding its clinical value in patients after out-of-hospital cardiac arrest. In this study we investigated whether plasma DNA on arrival at the emergency room may be useful in predicting the outcome of these patients. Methods We performed a prospective study of out-of-hospital patients with cardiac arrest who achieved return of spontaneous circulation after successful resuscitation. Cardiovascular co-morbidities and resuscitation history were recorded according to the Utstein Style. The outcome measures were 24 h and overall in-hospital mortality. Cell-free plasma DNA was measured by real-time quantitative PCR assay for the β-globin gene in blood samples drawn within two hours after the arrest. Descriptive statistics, multiple logistic regression analysis, and receiver operator characteristic (ROC) curves were calculated. Results Eighty-five consecutive patients were analyzed with a median time to return of spontaneous circulation of 27 minutes (interquartile range (IQR) 18 to 35). Thirty patients died within 24 h and 58 died during the hospital course. Plasma DNA concentrations at admission were higher in non-survivors at 24 h than in survivors (median 5,520 genome equivalents (GE)/ml, vs 2810 GE/ml, P < 0.01), and were also higher in patients who died in the hospital than in survivors to discharge (median 4,150 GE/ml vs 2,460 GE/ml, P < 0.01). Lactate clearance at six hours was significantly higher in 24 h survivors (P < 0.05). The area under the ROC curves for plasma DNA to predict 24-hour mortality and in-hospital mortality were 0.796 (95% confidence interval (CI) 0.701 to 0.890) and 0.652 (95% CI 0.533 to 0.770). The best cut-off value of plasma DNA for 24-h mortality was 4,340 GE/ml (sensitivity 76%, specificity 83%), and for in

  1. Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

    PubMed Central

    Lim, Ji Hyae; Kim, Min Hyoung; Han, You Jung; Lee, Da Eun; Park, So Yeon; Han, Jung Yeol; Kim, Moon Young; Ryu, Hyun Mee

    2013-01-01

    Background Cell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy. Methodology/Principal Findings A nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC) curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both). The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001) than in SA women with normal karyotype (r = 0.465, P = 0.002) and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037). The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852–0.945) and 0.939 (95% CI, 0.903–0.975), respectively. Conclusions Significantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA with fetal

  2. Non-Invasive Prenatal Testing Using Cell Free DNA in Maternal Plasma: Recent Developments and Future Prospects.

    PubMed

    Benn, Peter

    2014-05-21

    Recent advances in molecular genetic technologies have facilitated non-invasive prenatal testing (NIPT) through the analysis of cell-free fetal DNA in maternal plasma. NIPT can be used to identify monogenic disorders including the identification of autosomal recessive disorders where the maternally inherited mutation needs to be identified in the presence of an excess of maternal DNA that contains the same mutation. In the future, simultaneous screening for multiple monogenic disorders is anticipated. Several NIPT methods have been developed to screen for trisomy. These have been shown to be effective for fetal trisomy 21, 18 and 13. Although the testing has been extended to sex chromosome aneuploidy, robust estimates of the efficacy are not yet available and maternal mosaicism for gain or loss of an X-chromosome needs to be considered. Using methods based on the analysis of single nucleotide polymorphisms, diandric triploidy can be identified. NIPT is being developed to identify a number of microdeletion syndromes including α-globin gene deletion. NIPT is a profoundly important development in prenatal care that is substantially advancing the individual patient and public health benefits achieved through conventional prenatal screening and diagnosis.

  3. Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

    PubMed

    Suzumori, Nobuhiro; Ebara, Takeshi; Yamada, Takahiro; Samura, Osamu; Yotsumoto, Junko; Nishiyama, Miyuki; Miura, Kiyonori; Sawai, Hideaki; Murotsuki, Jun; Kitagawa, Michihiro; Kamei, Yoshimasa; Masuzaki, Hideaki; Hirahara, Fumiki; Saldivar, Juan-Sebastian; Dharajiya, Nilesh; Sago, Haruhiko; Sekizawa, Akihiko

    2016-07-01

    The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging. PMID:26984559

  4. Plasma Circulating Cell-free DNA Integrity as a Promising Biomarker for Diagnosis and Surveillance in Patients with Hepatocellular Carcinoma

    PubMed Central

    Huang, Ao; Zhang, Xin; Zhou, Shao-Lai; Cao, Ya; Huang, Xiao-Wu; Fan, Jia; Yang, Xin-Rong; Zhou, Jian

    2016-01-01

    The clinical significance of circulating cell-free DNA (cfDNA) integrity as diagnostic and surveillance biomarker in hepatocellular carcinoma (HCC) was investigated and compared to that of alpha fetoprotein (AFP). Liver cancer patients had lower cfDNA integrity than those with benign diseases (P = 0.0167) and healthy individuals (P = 0.0025). Patients with HCC and non-HCC liver cancers (P = 0.7356), and patients with benign diseases and healthy individuals (P = 0.9138) had comparable cfDNA integrity respectively. cfDNA integrity increased after hepatectomy in cancer patients (P = 0.0003). The AUCs for detecting HCC by cfDNA integrity and AFP were 0.705 (P = 0.005) and 0.605 (P = 0.156), respectively. We found cfDNA integrity decreased in HCC patients and has the potential as promising biomarker for HCC diagnosis and treatment surveillance.

  5. Bioinformatics analysis of circulating cell-free DNA sequencing data.

    PubMed

    Chan, Landon L; Jiang, Peiyong

    2015-10-01

    The discovery of cell-free DNA molecules in plasma has opened up numerous opportunities in noninvasive diagnosis. Cell-free DNA molecules have become increasingly recognized as promising biomarkers for detection and management of many diseases. The advent of next generation sequencing has provided unprecedented opportunities to scrutinize the characteristics of cell-free DNA molecules in plasma in a genome-wide fashion and at single-base resolution. Consequently, clinical applications of circulating cell-free DNA analysis have not only revolutionized noninvasive prenatal diagnosis but also facilitated cancer detection and monitoring toward an era of blood-based personalized medicine. With the remarkably increasing throughput and lowering cost of next generation sequencing, bioinformatics analysis becomes increasingly demanding to understand the large amount of data generated by these sequencing platforms. In this Review, we highlight the major bioinformatics algorithms involved in the analysis of cell-free DNA sequencing data. Firstly, we briefly describe the biological properties of these molecules and provide an overview of the general bioinformatics approach for the analysis of cell-free DNA. Then, we discuss the specific upstream bioinformatics considerations concerning the analysis of sequencing data of circulating cell-free DNA, followed by further detailed elaboration on each key clinical situation in noninvasive prenatal diagnosis and cancer management where downstream bioinformatics analysis is heavily involved. We also discuss bioinformatics analysis as well as clinical applications of the newly developed massively parallel bisulfite sequencing of cell-free DNA. Finally, we offer our perspectives on the future development of bioinformatics in noninvasive diagnosis.

  6. A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

    PubMed Central

    Xu, Xu-Ping; Gan, Hai-Yan; Li, Fen-Xia; Tian, Qi; Zhang, Jun; Liang, Rong-Liang; Li, Ming

    2016-01-01

    Objective The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure. Methods Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction. Results A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B. Conclusion A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability. PMID:26765738

  7. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    PubMed

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P < .05). Branched DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  8. BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

    PubMed

    Janku, Filip; Huang, Helen J; Claes, Bart; Falchook, Gerald S; Fu, Siqing; Hong, David; Ramzanali, Nishma M; Nitti, Giovanni; Cabrilo, Goran; Tsimberidou, Apostolia M; Naing, Aung; Piha-Paul, Sarina A; Wheler, Jennifer J; Karp, Daniel D; Holley, Veronica R; Zinner, Ralph G; Subbiah, Vivek; Luthra, Rajyalakshmi; Kopetz, Scott; Overman, Michael J; Kee, Bryan K; Patel, Sapna; Devogelaere, Benoit; Sablon, Erwin; Maertens, Geert; Mills, Gordon B; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-06-01

    Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR.

  9. An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping

    PubMed Central

    Gordon, Paul M. K.; Khan, Aneal; Sajid, Umair; Chang, Nicholas; Suresh, Varun; Dimnik, Leo; Lamont, Ryan E.; Parboosingh, Jillian S.; Martin, Steven R.; Pon, Richard T.; Weatherhead, Jene; Wegener, Shelly; Isaac, Debra; Greenway, Steven C.

    2016-01-01

    Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detect in vivo engraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation. PMID:27713880

  10. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA

    PubMed Central

    Kuang, Yanan; Mach, Stacy L.; O'Connell, Allison; Messineo, Melissa M.; Luke, Jason J.; Butaney, Mohit; Kirschmeier, Paul; Jackman, David M.; Jänne, Pasi A.

    2014-01-01

    Purpose Tumor genotyping using cell free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype. Experimental Design ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with non-overlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in EGFR-mutant lung cancer patients on a prospective trial of erlotinib. Results We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic (ROC) curve analysis of 4 assays demonstrated an area under the curve in the range of 0.80-0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging prior to objective progression. Conclusions Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks prior to radiographic progression. PMID:24429876

  11. Sensitivity of plasma BRAFmutant and NRASmutant cell-free DNA assays to detect metastatic melanoma in patients with low RECIST scores and non-RECIST disease progression.

    PubMed

    Chang, Gregory A; Tadepalli, Jyothirmayee S; Shao, Yongzhao; Zhang, Yilong; Weiss, Sarah; Robinson, Eric; Spittle, Cindy; Furtado, Manohar; Shelton, Dawne N; Karlin-Neumann, George; Pavlick, Anna; Osman, Iman; Polsky, David

    2016-01-01

    Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.

  12. Circulating Cell Free DNA in the Diagnosis of Trophoblastic Tumors

    PubMed Central

    Openshaw, Mark R.; Harvey, Richard A.; Sebire, Neil J.; Kaur, Baljeet; Sarwar, Naveed; Seckl, Michael J.; Fisher, Rosemary A.

    2015-01-01

    Gestational trophoblastic neoplasia (GTN) represents a group of diseases characterized by production of human chorionic gonadotropin (hCG). Since non-gestational tumors may occasionally secrete hCG, histopathological diagnosis is important for appropriate clinical management. However, a histopathological diagnosis is not always available. We therefore investigated the feasibility of extracting cell free DNA (cfDNA) from the plasma of women with GTN for use as a “liquid biopsy” in patients without histopathological diagnosis. cfDNA was prepared from the plasma of 20 women with a diagnosis of GTN and five with hCG-secreting tumors of unknown origin. Genotyping of cfDNA from the patient, genomic DNA from her and her partner and DNA from the tumor tissue identified circulating tumor DNA (ctDNA) (from 9% to 53% of total cfDNA) in 12 of 20 patients with GTN. In one case without a tissue diagnosis, ctDNA enabled a diagnosis of GTN originating in a non-molar conception and in another a diagnosis of non-gestational tumor, based on the high degree of allelic instability and loss of heterozygosity in the ctDNA. In summary ctDNA can be detected in the plasma of women with GTN and can facilitate the diagnosis of both gestational and non-gestational trophoblastic tumors in cases without histopathological diagnosis. PMID:26981554

  13. Fetal Sex Determination using Non-Invasive Method of Cell-free Fetal DNA in Maternal Plasma of Pregnant Women During 6(th)- 10(th) Weeks of Gestation.

    PubMed

    Zargari, Maryam; Sadeghi, Mohammad Reza; Shahhosseiny, Mohammad Hassan; Kamali, Koroush; Saliminejad, Kyomars; Esmaeilzadeh, Ali; Khorshid, Hamid Reza Khorram

    2011-10-01

    In previous years, identification of fetal cells in maternal blood circulation has caused a new revolution in non-invasive method of prenatal diagnosis. Low number of fetal cells in maternal blood and long-term survival after pregnancy limited the use of fetal cells in diagnostic and clinical applications. With the discovery of cell-free fetal DNA (cffDNA) in plasma of pregnant women, access to genetic material of the fetus had become possible to determine early gender of a fetus in pregnancies at the risk of X-linked genetic conditions instead of applying invasive methods. Therefore in this study, the probability of detecting sequences on the Y chromosome in pregnant women has been evaluated to identify the gender of fetuses. Peripheral blood samples were obtained from 80 pregnant women at 6(th) to 10(th) weeks of gestation and then the fetal DNA was extracted from the plasma. Nested PCR was applied to detect the sequences of single copy SRY gene and multi copy DYS14 & DAZ genes on the Y chromosome of the male fetuses. At the end, all the obtained results were compared with the actual gender of the newborns. In 40 out of 42 born baby boys, the relevant gene sequences were identified and 95.2% sensitivity was obtained. Non-invasive early determination of fetal gender using cffDNA could be employed as a pre-test in the shortest possible time and with a high reliability to avoid applying invasive methods in cases where a fetus is at the risk of genetic diseases.

  14. Cell-free DNA: Comparison of Technologies.

    PubMed

    Dar, Pe'er; Shani, Hagit; Evans, Mark I

    2016-06-01

    Cell-free fetal DNA screening for Down syndrome has gained rapid acceptance over the past few years with increasing market penetration. Three main laboratory methodologies are currently used: a massive parallel shotgun sequencing (MPSS), a targeted massive parallel sequencing (t-MPS) and a single nucleotide polymorphism (SNP) based approach. Although each of these technologies has its own advantages and disadvantages, the performance of all was shown to be comparable and superior to that of traditional first-trimester screening for the detection of trisomy 21 in a routine prenatal population. Differences in performance were predominantly shown for chromosomal anomalies other than trisomy 21. Understanding the limitations and benefits of each technology is essential for proper counseling to patients. These technologies, as well as few investigational technologies described in this review, carry a great potential beyond screening for the common aneuploidies. PMID:27235906

  15. Israeli Society of Medical Genetics NIPT Committee Opinion 072013: Non-invasive prenatal testing of cell-free DNA in maternal plasma for detection of fetal aneuploidy.

    PubMed

    Michaelson-Cohen, Rachel; Gershoni-Baruch, Ruth; Sharoni, Reuven; Shochat, Mordechai; Yaron, Yuval; Singer, Amihood

    2014-01-01

    Non-invasive prenatal testing (NIPT) of cell-free fetal DNA in maternal plasma is a novel approach, designed for detecting common aneuploidies in the fetus. The Israeli Society of Medical Geneticists (ISMG) supports its use according to the guidelines stated herein. The clinical data collected thus far indicate that NIPT is highly sensitive in detecting trisomies 21 and 18, and fairly sensitive in detecting trisomy 13 and sex chromosome aneuploidies. Because false-positive results may occur, an abnormal result must be validated by invasive prenatal testing. At this juncture, NIPT does not replace existing prenatal screening tests for Down syndrome, as these are relatively inexpensive and cost-effective. Nonetheless, NIPT may be offered to women considered to be at high risk for fetal chromosomal abnormalities as early as 10 weeks of gestation. The ISMG states that NIPT should be an informed patient choice, and that pretest counseling regarding the limitations of NIPT is warranted. Women at high risk for genetic disorders not detected by NIPT should be referred for genetic counseling. A normal test result may be conveyed by a relevant healthcare provider, while an abnormal result should be discussed during a formal genetic consultation session.

  16. Cell-Free Fetal DNA Testing for Prenatal Diagnosis.

    PubMed

    Drury, S; Hill, M; Chitty, L S

    2016-01-01

    Prenatal diagnosis and screening have undergone rapid development in recent years, with advances in molecular technology driving the change. Noninvasive prenatal testing (NIPT) for Down syndrome as a highly sensitive screening test is now available worldwide through the commercial sector with many countries moving toward implementation into their publically funded maternity systems. Noninvasive prenatal diagnosis (NIPD) can now be performed for definitive diagnosis of some recessive and X-linked conditions, rather than just paternally inherited dominant and de novo conditions. NIPD/T offers pregnant couples greater choice during their pregnancy as these safer methods avoid the risk of miscarriage associated with invasive testing. As the cost of sequencing falls and technology develops further, there may well be potential for whole exome and whole genome sequencing of the unborn fetus using cell-free DNA in the maternal plasma. How such assays can or should be implemented into the clinical setting remain an area of significant debate, but it is clear that the progress made to date for safer prenatal testing has been welcomed by expectant couples and their healthcare professionals. PMID:27645814

  17. Cell-free DNA screening and sex chromosome aneuploidies.

    PubMed

    Mennuti, Michael T; Chandrasekaran, Suchitra; Khalek, Nahla; Dugoff, Lorraine

    2015-10-01

    Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed.

  18. Monitoring of organ transplants through genomic analyses of circulating cell-free DNA

    NASA Astrophysics Data System (ADS)

    de Vlaminck, Iwijn

    Solid-organ transplantation is the preferred treatment for patients with end-stage organ diseases, but complications due to infection and acute rejection undermine its long-term benefits. While clinicians strive to carefully monitor transplant patients, diagnostic options are currently limited. My colleagues and I in the lab of Stephen Quake have found that a combination of next-generation sequencing with a phenomenon called circulating cell-free DNA enables non-invasive diagnosis of both infection and rejection in transplantation. A substantial amount of small fragments of cell-free DNA circulate in blood that are the debris of dead cells. We discovered that donor specific DNA is released in circulation during injury to the transplant organ and we show that the proportion of donor DNA in plasma is predictive of acute rejection in heart and lung transplantation. We profiled viral and bacterial DNA sequences in plasma of transplant patients and discovered that the relative representation of different viruses and bacteria is informative of immunosuppression. This discovery suggested a novel biological measure of a person's immune strength, a finding that we have more recently confirmed via B-cell repertoire sequencing. Lastly, our studies highlight applications of shotgun sequencing of cell-free DNA in the broad, hypothesis free diagnosis of infection.

  19. #36: Prenatal aneuploidy screening using cell-free DNA.

    PubMed

    2015-06-01

    Recent advances in technology have created exciting opportunities to expand and improve genetic testing options that are available to women during pregnancy. However, the novelty and complexity of these technologies, combined with the commercial interest to implement these tests rapidly into routine clinical care, have created challenges for physicians and patients and potentially will lead to misunderstanding, misuse, and unintended consequences. The purpose of this document was to aid clinicians in their day-to-day practice of counseling patients regarding prenatal aneuploidy testing options with cell-free DNA screening, which includes how it compares to current testing methods, potential benefits and harms, and its limitations and caveats.

  20. Quantitative analysis of cell-free DNA in ovarian cancer

    PubMed Central

    SHAO, XUEFENG; He, YAN; JI, MIN; CHEN, XIAOFANG; QI, JING; SHI, WEI; HAO, TIANBO; JU, SHAOQING

    2015-01-01

    The aim of the present study was to investigate the association between cell-free DNA (cf-DNA) levels and clinicopathological characteristics of patients with ovarian cancer using a branched DNA (bDNA) technique, and to determine the value of quantitative cf-DNA detection in assisting with the diagnosis of ovarian cancer. Serum specimens were collected from 36 patients with ovarian cancer on days 1, 3 and 7 following surgery, and additional serum samples were also collected from 22 benign ovarian tumor cases, and 19 healthy, non-cancerous ovaries. bDNA techniques were used to detect serum cf-DNA concentrations. All data were analyzed using SPSS version 18.0. The cf-DNA levels were significantly increased in the ovarian cancer group compared with those of the benign ovarian tumor group and healthy ovarian group (P<0.01). Furthermore, cf-DNA levels were significantly increased in stage III and IV ovarian cancer compared with those of stages I and II (P<0.01). In addition, cf-DNA levels were significantly increased on the first day post-surgery (P<0.01), and subsequently demonstrated a gradual decrease. In the ovarian cancer group, the area under the receiver operating characteristic curve of cf-DNA and the sensitivity were 0.917 and 88.9%, respectively, which was higher than those of cancer antigen 125 (0.724, 75%) and human epididymis protein 4 (0.743, 80.6%). There was a correlation between the levels of serum cf-DNA and the occurrence and development of ovarian cancer in the patients evaluated. bDNA techniques possessed higher sensitivity and specificity than other methods for the detection of serum cf-DNA in patients exhibiting ovarian cancer, and bDNA techniques are more useful for detecting cf-DNA than other factors. Thus, the present study demonstrated the potential value for the use of bDNA as an adjuvant diagnostic method for ovarian cancer. PMID:26788153

  1. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    PubMed Central

    Cao, Yang; Hoppman, Nicole L.; Kerr, Sarah E.; Sattler, Christopher A.; Borowski, Kristi S.; Wick, Myra J.; Highsmith, W. Edward; Aypar, Umut

    2016-01-01

    Background. Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA) screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information. PMID:26998368

  2. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13.

    PubMed

    Cao, Yang; Hoppman, Nicole L; Kerr, Sarah E; Sattler, Christopher A; Borowski, Kristi S; Wick, Myra J; Highsmith, W Edward; Aypar, Umut

    2016-01-01

    Background. Noninvasive prenatal screening (NIPS) is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA) circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA) screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information. PMID:26998368

  3. The Long and Short of Circulating Cell-Free DNA and the Ins and Outs of Molecular Diagnostics.

    PubMed

    Jiang, Peiyong; Lo, Y M Dennis

    2016-06-01

    The discovery of cell-free tumor and fetal DNA molecules in the plasma of cancer patients and pregnant women, respectively, has opened up exciting opportunities in molecular diagnosis. The understanding of the biological properties of circulating cell-free DNA (cfDNA) molecules would be essential for us to make the best use of such molecules in different clinical settings. In this review we start by exploring the technologies that have been used for analyzing the size profiles of cfDNA in plasma. We then review the size profiles of cfDNA in different clinical scenarios, including cancer, pregnancy, transplantation, and autoimmune diseases. Finally, we discuss the potential diagnostic applications of plasma DNA size profiling.

  4. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    PubMed Central

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Results In this study, we investigated the association between epigenetic landscapes of human tissues evident in the patterns of cfDNA in plasma by deep sequencing of human cfDNA samples. We have demonstrated that baseline characteristics of cfDNA fragmentation pattern are in concordance with the ones corresponding to cell lines-derived. To identify the loci differentially represented in cfDNA fragment, we mapped the transcription start sites within the sequenced cfDNA fragments and tested for association of these genomic coordinates with the relative strength and the patterns of gene expressions. Preselected sets of house-keeping and tissue specific genes were used as models for actively expressed and silenced genes. Developed measure of gene regulation was able to differentiate these two sets based on sequencing coverage near gene transcription start site. Conclusion Experimental outcomes suggest that cfDNA retains characteristics previously noted in genome-wide analysis of chromatin structure, in particular, in MNase-seq assays. Thus far the analysis of the DNA fragmentation pattern may aid further developing of cfDNA based biomarkers for a variety of human conditions. PMID:26693644

  5. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer.

    PubMed

    Yamada, Takeshi; Iwai, Takuma; Takahashi, Goro; Kan, Hayato; Koizumi, Michihiro; Matsuda, Akihisa; Shinji, Seiichi; Yamagishi, Aya; Yokoyama, Yasuyuki; Tatsuguchi, Atsushi; Kawagoe, Tatsuro; Kitano, Shiro; Nakayama, Masato; Matsumoto, Satoshi; Uchida, Eiji

    2016-07-01

    In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. PMID:27116474

  6. Noninvasive determination of fetal rh blood group, D antigen status by cell-free DNA analysis in maternal plasma: experience in a Brazilian population.

    PubMed

    Chinen, Paulo Alexandre; Nardozza, Luciano Marcondes Machado; Martinhago, Ciro Dresch; Camano, Luiz; Daher, Silvia; Pares, David Baptista da Silva; Minett, Thais; Araujo Júnior, Edward; Moron, Antonio Fernandes

    2010-11-01

    We evaluated the diagnostic accuracy of Rh blood group, D antigen (RHD) fetal genotyping, using real-time polymerase chain reaction in maternal blood samples, in a racially mixed population. We performed a prospective study conducted between January 2006 and December 2007, analyzing fetal RHD genotype in the plasma of 102 D- pregnant women by real-time polymerase chain reaction, targeting exons 7 and 10 of the RHD gene. Genotype results were compared with cord blood phenotype obtained after delivery or before the first intrauterine transfusion when necessary. Most of the participants (75.5%) were under 28 weeks of pregnancy, and 87.5% had at least one relative of black ancestry. By combining amplification of two exons, the accuracy of genotyping was 98%, sensitivity was 100%, and specificity was 92%. The positive likelihood ratio was 12.5, and the negative likelihood ratio was 0. The two false-positive cases were confirmed to be pseudogene RHD by real-time polymerase chain reaction. There were no differences between the patients with positive or negative Coombs test ( P = 0.479). Determination of fetal RHD status in maternal peripheral blood was highly sensitive in this racially mixed population and was not influenced by the presence of antierythrocyte antibodies.

  7. Isolation and analysis of cell-free fetal DNA from maternal peripheral blood in Chinese women.

    PubMed

    Yang, W C; Zhu, L; Qiu, Y M; Zhou, B X; Cheng, J L; Wei, C L; Chen, H C; Li, L Y; Fu, X D; Fu, J J

    2015-12-22

    Non-invasive prenatal diagnosis is used to detect the genetic material of the fetus by isolating the cell-free fetal DNA (cffDNA) from maternal peripheral blood. In order to establish an isolation method for cffDNA from maternal peripheral blood in Chinese women, the cffDNA was acquired with a two-step centrifugation using a QlAamp DNA Blood mini kit. The SRY gene of plasma DNA was amplified by polymerase chain reaction (PCR). Real-time quantitative PCR was used to measure the concentration of cffDNA in maternal peripheral blood in different pregnant women. The results of the SRY gene amplification of plasma DNA from pregnant women was the same as that of the amniocyte DNA. The average concentration of cffDNA in maternal peripheral blood of pregnant women in different gestational stages was 0.98 ng/mL (0.26-1.49 ng/mL), 1.43 ng/mL (0.46- 2.34 ng/mL), and 1.95 ng/mL (0.65-6.81 ng/mL) from early, middle, and late gestational stages, respectively. The mean of cffDNA from total DNA in plasma in different stages of gestation was 22.28% (9.86-27.81%). The lowest concentration of DNA amplified by nested-PCR in our research was 10-4-10-3 ng/μL. The isolation method for cffDNA from maternal peripheral blood was successfully established and further research into its applications will be conducted.

  8. Circulating Cell-Free Tumour DNA in the Management of Cancer

    PubMed Central

    Francis, Glenn; Stein, Sandra

    2015-01-01

    With the development of new sensitive molecular techniques, circulating cell-free tumour DNA containing mutations can be identified in the plasma of cancer patients. The applications of this technology may result in significant changes to the care and management of cancer patients. Whilst, currently, these “liquid biopsies” are used to supplement the histological diagnosis of cancer and metastatic disease, in the future these assays may replace the need for invasive procedures. Applications include the monitoring of tumour burden, the monitoring of minimal residual disease, monitoring of tumour heterogeneity, monitoring of molecular resistance and early diagnosis of tumours and metastatic disease. PMID:26101870

  9. Diagnostic potential of circulating cell-free DNA in patients needing mechanical ventilation: promises and challenges

    PubMed Central

    2011-01-01

    Circulating cell-free DNA (cf-DNA) mainly comes from apoptotic cells and can reflect the extent of cellular damage. Increased plasma levels of cf-DNA have been found in many acute disorders, including septic and clinically ill patients, and usually correlate well with clinical outcome. Acute respiratory failure, the most frequent organ failure in ICU patients, can be related to various acute diseases that may cause cell death and release of DNA into the bloodstream. In a recent issue of Critical Care, Okkonen and colleagues evaluate levels of cf-DNA in plasma as a prognostic marker in patients needing mechanical ventilation. They report that plasma cf-DNA was higher than normal in patients with mechanical ventilation, and even higher in patients who eventually died compared to survivors. However, its usefulness as a death predictor may be limited in the heterogeneous group of mechanically ventilated patients, probably due to confounding effects of co-morbidities, among other factors. PMID:21978490

  10. Modifying Risk of Aneuploidy with a Positive Cell-Free Fetal DNA Result.

    PubMed

    Long, A Ashleigh; Abuhamad, Alfred Z; Warsof, Steven L

    2016-06-01

    Noninvasive genomic assessments of the fetus while in utero have been made possible by the analysis of cell-free fetal DNA fragments from the serum of pregnant women, as part of a noninvasive prenatal testing screening strategy. Between 7% and 10% of total cell-free DNA in the maternal blood comes from placental trophoblasts, allowing for identification of the DNA associated with the fetal component of the placenta. Using simple venipuncture in the outpatient setting, this cell-free, extracellular fetal DNA can be isolated in the maternal serum from a single blood draw as early as the seventh week of gestation. PMID:27235910

  11. Quantification of Somatic Chromosomal Rearrangements in Circulating Cell-Free DNA from Ovarian Cancers

    PubMed Central

    Harris, Faye R.; Kovtun, Irina V.; Smadbeck, James; Multinu, Francesco; Jatoi, Aminah; Kosari, Farhad; Kalli, Kimberly R.; Murphy, Stephen J.; Halling, Geoffrey C.; Johnson, Sarah H.; Liu, Minetta C.; Mariani, Andrea; Vasmatzis, George

    2016-01-01

    Recently, the use of a liquid biopsy has shown promise in monitoring tumor burden. While point mutations have been extensively studied, chromosomal rearrangements have demonstrated greater tumor specificity. Such rearrangements can be identified in the tumor and subsequently detected in the plasma of patients using quantitative PCR (qPCR). In this study we used a whole-genome mate-pair protocol to characterize a landscape of genomic rearrangements in the primary tumors of ten ovarian cancer patients. Individualized tumor-specific primer panels of aberrant chromosomal junctions were identified for each case and detected by qPCR within the cell-free DNA. Selected chromosomal junctions were detected in pre-surgically drawn blood in eight of the ten patients. Of these eight, three demonstrated the continued presence of circulating tumor DNA (ctDNA) post-surgery, consistent with their documented presence of disease, and in five ctDNA was undetectable in the post-surgical blood collection, consistent with their lack of detectable disease. The ctDNA fraction was calculated using a novel algorithm designed for the unique challenges of quantifying ctDNA using qPCR to allow observations of real-time tumor dynamics. In summary, a panel of individualized junctions derived from tumor DNA could be an effective way to monitor cancer patients for relapse and therapeutic efficacy using cfDNA. PMID:27436510

  12. Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma

    PubMed Central

    Salvianti, Francesca; Orlando, Claudio; Massi, Daniela; De Giorgi, Vincenzo; Grazzini, Marta; Pazzagli, Mario; Pinzani, Pamela

    2016-01-01

    Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and

  13. Cell-free DNA testing: an aid to prenatal sonographic diagnosis.

    PubMed

    Chitty, Lyn S

    2014-04-01

    Sonographic diagnosis of fetal abnormalities is based on the recognition of sonographic patterns associated with structural abnormalities. Although diagnosis in some situations, such as neural tube defects, gastroschisis, and omphalocoele, can be straightforward, in many situations, the constellation of fetal abnormalities suggest an underlying chromosomal or genetic cause. In these situations, invasive testing is needed to provide the information required to make a definitive diagnosis, and thus accurately counsel parents. Since the identification of cell-free fetal DNA in maternal plasma, the potential for non-invasive prenatal diagnosis is increasingly becoming possible. In this chapter, the current role and future potential of non-invasive prenatal diagnosis, combined with new molecular techniques as an aid to sonographic diagnosis, will be discussed.

  14. Fetal DNA in maternal plasma.

    PubMed

    Lo, Y M

    2000-04-01

    Recently, cell-free fetal DNA has been found in maternal plasma and serum. This discovery opens up a new field of investigation and provides an easily accessible source of fetal genetic material for prenatal diagnosis. Prenatal diagnostic applications of fetal DNA in maternal plasma include the investigation of sex-linked disorders and fetal rhesus D status determination. Cell-free fetal DNA has been found to be present in much higher fractional concentrations than fetal nucleated cells in maternal blood. The concentration of fetal DNA increases throughout pregnancy, with a sharp rise towards the end of gestation. Abnormally high levels of cell-free DNA have been found in pregnancies complicated by preeclampsia and preterm labor, an observation that has potential diagnostic and pathophysiologic implications. Much remains to be learned regarding the mechanisms of production and clearance of maternal plasma fetal DNA. It is hoped that the eagerly awaited answers to these and other questions may ultimately enhance our understanding of the fetomaternal relationship.

  15. Cell-free DNA for diagnosing myocardial infarction: not ready for prime time.

    PubMed

    Lippi, Giuseppe; Sanchis-Gomar, Fabian; Cervellin, Gianfranco

    2015-11-01

    A modest amount of cell-free DNA is constantly present in human blood, originating from programmed cell death, apoptosis and rupture of blood cells or pathogens. Acute or chronic cell injury contributes to enhance the pool of circulating nucleic acids, so that their assessment may be regarded as an appealing perspective for diagnosing myocardial ischemia. We performed a search in Medline, Web of Science and Scopus to identify clinical studies that investigated the concentration of cell-free DNA in patients with myocardial ischemia. Overall, eight case-control studies could be detected and reviewed. Although the concentration of cell-free DNA was found to be higher in the diseased than in the healthy population, the scenario was inconclusive due to the fact that the overall number of subjects studied was modest, the populations were unclearly defined, cases and controls were not adequately matched, the methodology for measuring the reference cardiac biomarkers was inadequately described, and the diagnostic performance of cell-free DNA was not benchmarked against highly sensitive troponin immunoassays. Several biological and technical hurdles were also identified in cell-free DNA testing, including the lack of specificity and unsuitable kinetics for early diagnosis of myocardial ischemia, the long turnaround time and low throughput, the need for specialized instrumentation and dedicated personnel, the lack of standardization or harmonization of analytical techniques, the incremental costs and the high vulnerability to preanalytical variables. Hence it seems reasonable to conclude that the analysis of cell-free DNA is not ready for prime time in diagnostics of myocardial ischemia.

  16. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    PubMed

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-01-01

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis. PMID:27188728

  17. Cell-free fetal DNA in amniotic fluid supernatant for prenatal diagnosis.

    PubMed

    Soltani, M; Nemati, M; Maralani, M; Estiar, M A; Andalib, S; Fardiazar, Z; Sakhinia, E

    2016-04-30

    In widespread conviction, amniotic fluid is utilized for prenatal diagnosis. Amniotic fluid supernatant is usually discarded, notwithstanding being a good source of fetal DNA. The aim of the present study was to assess cell-free fetal DNA extracted from amniotic fluid supernatant for application in prenatal diagnosis such as gender determination and early diagnosis of β-thalassemia. Samples of amniotic fluid of 70 pregnant women were collected and went through routine tests along with tests for cell-free fetal DNA from amniotic fluid supernatant. The DNA in the amniotic fluid supernatant was extracted and analyzed for gender determination by PCR and Real-time PCR. ARMS-PCR was applied to test early diagnosis of IVS II-I mutation (common β-thalassemia mutation) and E7V mutation for sickle cell anemia using DNA extracted from the amniotic fluid supernatant. Using the cell-free fetal DNA extracted from the amniotic fluid supernatant, the sensitivity of PCR and Real-time PCR for gender detection was compared with the routine cytogenetic method. The fetus tested for sickle cell anemia and β-thalassemia was observed to be healthy but heterozygous for IVS II-I mutation. The findings indicated that cell-free fetal DNA from amniotic fluid supernatant can be a good source of fetal DNA and be used in early prenatal diagnosis since because of its fast and accurate application. Therefore, it would be suggested that the amniotic fluid supernatant's disposal is prevented because if the tests needs to be repeated, cell-free fetal DNA extracted from the amniotic fluid supernatant can be used as an alternative source for prenatal diagnosis.

  18. SMFM Statement: clarification of recommendations regarding cell-free DNA aneuploidy screening.

    PubMed

    2015-12-01

    The purpose of this statement is to clarify that the Society for Maternal-Fetal Medicine (SMFM) does not recommend that cell-free DNA aneuploidy screening be offered to all pregnant women, nor does it suggest a requirement for insurance coverage for cell-free DNA screening in women at low risk of aneuploidy. However, SMFM believes, due to the ethics of patient autonomy, that the option should be available to women who request additional testing beyond what is currently recommended by professional societies.

  19. Understanding the Limitations of Circulating Cell Free Fetal DNA: An Example of Two Unique Cases.

    PubMed

    Clark-Ganheart, Cecily A; Iqbal, Sara N; Brown, Donna L; Black, Susan; Fries, Melissa H

    2014-05-01

    Circulating cell free fetal DNA (cffDNA) is an effective screening modality for fetal aneuploidy. We report two cases of false positive results. The first case involves a female, with self-reported Down syndrome. CffDNA returned positive for trisomy 18 leading to a maternal diagnosis of mosaicism chromosome 18 with normal fetal karyotype. The second case involves a patient with an anomalous fetal ultrasound and cffDNA positive for trisomy 13. Amniocentesis demonstrated a chromosome 8p duplication/deletion. False positive cffDNA may arise in clinical scenarios where diagnostic testing is clearly indicated. Practitioners should recognize the limitations of cffDNA. PMID:25298847

  20. CpG methylation as a tool to characterize cell-free Kaposi sarcoma herpesvirus DNA.

    PubMed

    Shamay, Meir; Hand, Nicholas; Lemas, M Victor; Koon, Henry B; Krown, Susan E; Wrangle, John; Desai, Prashant; Ramos, Juan Carlos; Ambinder, Richard F

    2012-04-01

    We studied the presence of Kaposi sarcoma herpesvirus sequences in cell-free DNA (cfDNA) isolated from the blood of patients with AIDS-related Kaposi sarcoma (KS) and primary effusion lymphoma (PEL). The use of paramagnetic beads linked to methyl-CpG binding domain protein allowed separation of virion and cell-derived DNA. Only virion DNA was detected in the blood of KS patients, whereas cell-derived DNA was detected in a patient with AIDS-related PEL. The difference in the origins of cfDNA in these settings may in part reflect very different proliferative indices in KS and PEL tumor tissue. PMID:22357696

  1. Cell free fetal DNA testing in maternal blood of Romanian pregnant women

    PubMed Central

    Radoi, Viorica E; Bohiltea, Camil L; Bohiltea, Roxana E; Albu, Dragos N

    2015-01-01

    Background: The discovery of circulating fetal DNA in maternal blood led to the discovery of new strategies to perform noninvasive testing for prenatal diagnosis. Objective: The purpose of the study was to detect fetal aneuploidy at chromosomes 13, 18, 21, X, and Y by analysis of fetal cell-free DNA from maternal blood, without endangering pregnancy. Materials and Methods: This retrospective study has been performed in Bucharest at Medlife Maternal and Fetal Medicine Department between 2013-2014. In total 201 women were offered noninvasive prenatal test. Maternal plasma samples were collected from women at greater than 9 weeks of gestation after informed consent and genetics counseling. Results: From 201 patients; 28 (13.93%) had screening test with high risk for trisomy 21, 116 (57.71%) had advanced maternal age, 1 (0.49%) had second trimester ultrasound markers and the remaining 56 patients (27.86%) performed the test on request. Of those patients, 189 (94.02%) had a “low risk” result (<1/10,000). Of those who had a low risk result, 2 continued on to have amniocentesis with normal results.Five patients (2.48%) received “high risk” results (>99% risk) all for trisomy 21 (T21). T21 was confirmed by amniocentesis in 1 patient and the other 4 patients declined confirmation. The 7 remaining patients (3.48%) had a low fetal fraction of DNA. Conclusion: It is probably that prenatal diagnosis using fetal DNA in maternal blood would play an increasingly role in the future practice of prenatal testing because of high accuracy. PMID:26644790

  2. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  3. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

  4. Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients

    PubMed Central

    Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

    2016-01-01

    Background The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. Results ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). Methods A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). Conclusions We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information. PMID:27102299

  5. Vanished Twins and Misdiagnosed Sex: A Case Report with Implications in Prenatal Counseling Using Noninvasive Cell-Free DNA Screening.

    PubMed

    Kelley, James F; Henning, George; Ambrose, Anthony; Adelman, Alan

    2016-01-01

    Cell-free DNA testing is a recently introduced method for screening pregnant women for fetal trisomy, which is associated with some common significant genetic diseases, as well as the sex of the fetus. The case described here demonstrates the connection between the ultrasound "vanishing twin" phenomenon and the misdiagnosis of prenatal sex using cell-free DNA testing. PMID:27170800

  6. Circulating DNA in plasma or serum.

    PubMed

    Anker, P; Stroun, M

    2000-01-01

    Small amounts of DNA circulate in both healthy and diseased human plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreased strand stability, the presence of specific oncogene or tumor suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. Following the discovery of tumor derived DNA in plasma or serum, cell-free fetal DNA has also been found in maternal plasma and serum. This discovery provides an easily accessible source of fetal genetic material for prenatal diagnosis.

  7. Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients

    PubMed Central

    Wen, Lu; Li, Jingyi; Guo, Huahu; Liu, Xiaomeng; Zheng, Shengmin; Zhang, Dafang; Zhu, Weihua; Qu, Jianhui; Guo, Limin; Du, Dexiao; Jin, Xiao; Zhang, Yuhao; Gao, Yun; Shen, Jie; Ge, Hao; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

    2015-01-01

    Despite advances in DNA methylome analyses of cells and tissues, current techniques for genome-scale profiling of DNA methylation in circulating cell-free DNA (ccfDNA) remain limited. Here we describe a methylated CpG tandems amplification and sequencing (MCTA-Seq) method that can detect thousands of hypermethylated CpG islands simultaneously in ccfDNA. This highly sensitive technique can work with genomic DNA as little as 7.5 pg, which is equivalent to 2.5 copies of the haploid genome. We have analyzed a cohort of tissue and plasma samples (n = 151) of hepatocellular carcinoma (HCC) patients and control subjects, identifying dozens of high-performance markers in blood for detecting small HCC (≤ 3 cm). Among these markers, 4 (RGS10, ST8SIA6, RUNX2 and VIM) are mostly specific for cancer detection, while the other 15, classified as a novel set, are already hypermethylated in the normal liver tissues. Two corresponding classifiers have been established, combination of which achieves a sensitivity of 94% with a specificity of 89% for the plasma samples from HCC patients (n = 36) and control subjects including cirrhosis patients (n = 17) and normal individuals (n = 38). Notably, all 15 alpha-fetoprotein-negative HCC patients were successfully identified. Comparison between matched plasma and tissue samples indicates that both the cancer and noncancerous tissues contribute to elevation of the methylation markers in plasma. MCTA-Seq will facilitate the development of ccfDNA methylation biomarkers and contribute to the improvement of cancer detection in a clinical setting. PMID:26516143

  8. Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients.

    PubMed

    Wen, Lu; Li, Jingyi; Guo, Huahu; Liu, Xiaomeng; Zheng, Shengmin; Zhang, Dafang; Zhu, Weihua; Qu, Jianhui; Guo, Limin; Du, Dexiao; Jin, Xiao; Zhang, Yuhao; Gao, Yun; Shen, Jie; Ge, Hao; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

    2015-11-01

    Despite advances in DNA methylome analyses of cells and tissues, current techniques for genome-scale profiling of DNA methylation in circulating cell-free DNA (ccfDNA) remain limited. Here we describe a methylated CpG tandems amplification and sequencing (MCTA-Seq) method that can detect thousands of hypermethylated CpG islands simultaneously in ccfDNA. This highly sensitive technique can work with genomic DNA as little as 7.5 pg, which is equivalent to 2.5 copies of the haploid genome. We have analyzed a cohort of tissue and plasma samples (n = 151) of hepatocellular carcinoma (HCC) patients and control subjects, identifying dozens of high-performance markers in blood for detecting small HCC (≤ 3 cm). Among these markers, 4 (RGS10, ST8SIA6, RUNX2 and VIM) are mostly specific for cancer detection, while the other 15, classified as a novel set, are already hypermethylated in the normal liver tissues. Two corresponding classifiers have been established, combination of which achieves a sensitivity of 94% with a specificity of 89% for the plasma samples from HCC patients (n = 36) and control subjects including cirrhosis patients (n = 17) and normal individuals (n = 38). Notably, all 15 alpha-fetoprotein-negative HCC patients were successfully identified. Comparison between matched plasma and tissue samples indicates that both the cancer and noncancerous tissues contribute to elevation of the methylation markers in plasma. MCTA-Seq will facilitate the development of ccfDNA methylation biomarkers and contribute to the improvement of cancer detection in a clinical setting.

  9. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    PubMed Central

    Aljurf, Mahmoud; Abalkhail, Hala; Alseraihy, Amal; Mohamed, Said Y.; Ayas, Mouhab; Alsharif, Fahad; Alzahrani, Hazza; Al-Jefri, Abdullah; Aldawsari, Ghuzayel; Al-Ahmari, Ali; Belgaumi, Asim F.; Walter, Claudia Ulrike; El-Solh, Hassan; Rasheed, Walid; Albitar, Maher

    2016-01-01

    Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126) showed that, of 84 patients with 100% donor DNA in PMN, 16 (19%) had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19%) showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41%) showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse. PMID:27006832

  10. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    PubMed Central

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients. PMID:26562020

  11. Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas

    PubMed Central

    Zill, Oliver A.; Greene, Claire; Sebisanovic, Dragan; Siew, LaiMun; Leng, Jim; Vu, Mary; Hendifar, Andrew E.; Wang, Zhen; Atreya, Chloe E.; Kelley, Robin K.; Van Loon, Katherine; Ko, Andrew H.; Tempero, Margaret A.; Bivona, Trever G.; Munster, Pamela N.; Talasaz, AmirAli; Collisson, Eric A.

    2015-01-01

    Patients with pancreatic and biliary carcinomas lack personalized treatment options, in part because biopsies are often inadequate for molecular characterization. Cell-free DNA (cfDNA) sequencing may enable a precision oncology approach in this setting. We attempted to prospectively analyze 54 genes in tumor and cfDNA for 26 patients. Tumor sequencing failed in nine patients (35%). In the remaining 17, 90.3% (95% CI: 73.1–97.5%) of mutations detected in tumor biopsies were also detected in cfDNA. The diagnostic accuracy of cfDNA sequencing was 97.7%, with 92.3% average sensitivity and 100% specificity across five informative genes. Changes in cfDNA correlated well with tumor marker dynamics in serial sampling (r=0.93). We demonstrate that cfDNA sequencing is feasible, accurate, and sensitive in identifying tumor-derived mutations without prior knowledge of tumor genotype or the abundance of circulating tumor DNA. cfDNA sequencing should be considered in pancreatobiliary cancer trials where tissue sampling is unsafe, infeasible, or otherwise unsuccessful. PMID:26109333

  12. A novel cell-free system reveals a mechanism of circular DNA formation from tandem repeats.

    PubMed

    Cohen, S; Mechali, M

    2001-06-15

    One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). High levels of eccDNA are associated with genomic instability, exposure to carcinogens and aging. We have recently reported developmentally regulated formation of eccDNA that occurs preferentially in pre-blastula Xenopus laevis embryos. Multimers of tandemly repeated sequences were over-represented in the circle population while dispersed sequences were not detected, indicating that circles were not formed at random from any chromosomal sequence. Here we present detailed mechanistic studies of eccDNA formation in a cell-free system derived from Xenopus egg extracts. We show that naked chromosomal DNA from sperm or somatic tissues serves as a substrate for direct tandem repeat circle formation. Moreover, a recombinant bacterial tandem repeat can generate eccDNA in the extract through a de novo mechanism which is independent of DNA replication. These data suggest that the presence of a high level of any direct tandem repeat can confer on DNA the ability to be converted into circular multimers in the early embryo irrespective of its sequence and that homologous recombination is involved in this process. PMID:11410662

  13. Cell-free DNA as a molecular tool for monitoring disease progression and response to therapy in breast cancer patients.

    PubMed

    Liang, Diana H; Ensor, Joe E; Liu, Zhe-Bin; Patel, Asmita; Patel, Tejal A; Chang, Jenny C; Rodriguez, Angel A

    2016-01-01

    Due to the spatial and temporal genomic heterogeneity of breast cancer, genomic sequencing obtained from a single biopsy may not capture the complete genomic profile of tumors. Thus, we propose that cell-free DNA (cfDNA) in plasma may be an alternate source of genomic information to provide comprehensive data throughout a patient's clinical course. We performed a retrospective chart review of 100 patients with stage 4 or high-risk stage 3 breast cancer. The degree of agreement between genomic alterations found in tumor DNA (tDNA) and cfDNA was determined by Cohen's Kappa. Clinical disease progression was compared to mutant allele frequency using a two-sided Fisher's exact test. The presence of mutations and mutant allele frequency was correlated with progression-free survival (PFS) using a Cox proportional hazards model and a log-rank test. The most commonly found genomic alterations were mutations in TP53 and PIK3CA, and amplification of EGFR and ERBB2. PIK3CA mutation and ERBB2 amplification demonstrated robust agreement between tDNA and cfDNA (Cohen's kappa = 0.64 and 0.77, respectively). TP53 mutation and EGFR amplification demonstrated poor agreement between tDNA and cfDNA (Cohen's kappa = 0.18 and 0.33, respectively). The directional changes of TP53 and PIK3CA mutant allele frequency were closely associated with response to therapy (p = 0.002). The presence of TP53 mutation (p = 0.0004) and PIK3CA mutant allele frequency [p = 0.01, HR 1.074 (95 % CI 1.018-1.134)] was excellent predictors of PFS. Identification of selected cancer-specific genomic alterations from cfDNA may be a noninvasive way to monitor disease progression, predict PFS, and offer targeted therapy.

  14. Detection and prognostic value of recurrent exportin 1 mutations in tumor and cell-free circulating DNA of patients with classical Hodgkin lymphoma

    PubMed Central

    Camus, Vincent; Stamatoullas, Aspasia; Mareschal, Sylvain; Viailly, Pierre-Julien; Sarafan-Vasseur, Nasrin; Bohers, Elodie; Dubois, Sydney; Picquenot, Jean Michel; Ruminy, Philippe; Maingonnat, Catherine; Bertrand, Philippe; Cornic, Marie; Tallon-Simon, Valérie; Becker, Stéphanie; Veresezan, Liana; Frebourg, Thierry; Vera, Pierre; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

    2016-01-01

    Classical Hodgkin lymphoma is one of the most common lymphomas and shares clinical and genetic features with primary mediastinal B-cell lymphoma. In this retrospective study, we analyzed the recurrent hotspot mutation of the exportin 1 (XPO1, p.E571K) gene, previously identified in primary mediastinal B-cell lymphoma, in biopsies and plasma circulating cell-free DNA from patients with classical Hodgkin lymphoma using a highly sensitive digital PCR technique. A total of 94 patients were included in the present study. This widely expressed XPO1 E571K mutation is present in one quarter of classical Hodgkin lymphoma patients (24.2%). Mutated and wild-type classical Hodgkin lymphomas were similar regarding the main clinical features. Patients with a detectable XPO1 mutation at the end of treatment displayed a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation in plasma cell-free DNA (2-year progression-free survival: 57.1%, 95% confidence interval: 30.1–100% versus 2-year progression-free survival: 90.5%, 95% confidence interval: 78.8–100%, respectively, P=0.0601). To conclude, the detection of the XPO1 E571K mutation in biopsy and plasma cell-free DNA by digital PCR may be used as a novel biomarker in classical Hodgkin lymphoma for both diagnosis and minimal residual disease, and pinpoints a crucial role of XPO1 in classical Hodgkin lymphoma pathogenesis. The detection of somatic mutation in the plasma cell-free DNA of patients represents a major technological advance in the context of liquid biopsies and noninvasive management of classical Hodgkin lymphoma. PMID:27479820

  15. Detection and prognostic value of recurrent exportin 1 mutations in tumor and cell-free circulating DNA of patients with classical Hodgkin lymphoma.

    PubMed

    Camus, Vincent; Stamatoullas, Aspasia; Mareschal, Sylvain; Viailly, Pierre-Julien; Sarafan-Vasseur, Nasrin; Bohers, Elodie; Dubois, Sydney; Picquenot, Jean Michel; Ruminy, Philippe; Maingonnat, Catherine; Bertrand, Philippe; Cornic, Marie; Tallon-Simon, Valérie; Becker, Stéphanie; Veresezan, Liana; Frebourg, Thierry; Vera, Pierre; Bastard, Christian; Tilly, Hervé; Jardin, Fabrice

    2016-09-01

    Classical Hodgkin lymphoma is one of the most common lymphomas and shares clinical and genetic features with primary mediastinal B-cell lymphoma. In this retrospective study, we analyzed the recurrent hotspot mutation of the exportin 1 (XPO1, p.E571K) gene, previously identified in primary mediastinal B-cell lymphoma, in biopsies and plasma circulating cell-free DNA from patients with classical Hodgkin lymphoma using a highly sensitive digital PCR technique. A total of 94 patients were included in the present study. This widely expressed XPO1 E571K mutation is present in one quarter of classical Hodgkin lymphoma patients (24.2%). Mutated and wild-type classical Hodgkin lymphomas were similar regarding the main clinical features. Patients with a detectable XPO1 mutation at the end of treatment displayed a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation in plasma cell-free DNA (2-year progression-free survival: 57.1%, 95% confidence interval: 30.1-100% versus 2-year progression-free survival: 90.5%, 95% confidence interval: 78.8-100%, respectively, P=0.0601). To conclude, the detection of the XPO1 E571K mutation in biopsy and plasma cell-free DNA by digital PCR may be used as a novel biomarker in classical Hodgkin lymphoma for both diagnosis and minimal residual disease, and pinpoints a crucial role of XPO1 in classical Hodgkin lymphoma pathogenesis. The detection of somatic mutation in the plasma cell-free DNA of patients represents a major technological advance in the context of liquid biopsies and noninvasive management of classical Hodgkin lymphoma. PMID:27479820

  16. Feasibility of cell-free circulating tumor DNA testing for lung cancer.

    PubMed

    Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael

    2016-01-01

    Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications.

  17. The Prognostic Value of Circulating Cell-Free DNA in Colorectal Cancer: A Meta-Analysis

    PubMed Central

    Basnet, Shiva; Zhang, Zhen-yu; Liao, Wen-qiang; Li, Shu-heng; Li, Ping-shu; Ge, Hai-yan

    2016-01-01

    Background: Circulating cell-free DNA (cfDNA) is a promising candidate biomarker for detection, monitoring and survival prediction of colorectal cancer (CRC). However, its prognostic significance for patients with CRC remains controversial. To derive a precise estimation of the prognostic significance of cfDNA, a meta-analysis was performed. Methods: We made a systematic search in data base of the Science Citation Index Embase and Pubmed for studies reporting prognostic data of cfDNA in CRC patients. The data of cfDNA on recurrences-free survival (RFS) and overall survival (OS) were extracted and measured in hazard rates (HRs) and 95% confident intervals (CIs). Subgroup analyses were carried out as well. Finally, the meta-analysis is accompanied with nine studies including 19 subunits. Results: The pooled HRs with 95% CIs revealed strong associations between cfDNA and RFS (HR [95%CI]=2.78[2.08-3.72], I2=32.23%, n=7) along with OS (HR [95%CI]=3.03[2.51-3.66], I2=29.24%, n=12) in patients with CRC. Entire subgroup analyses indicated strong prognostic value of cfDNA irrespective tumor stage, study size, tumor markers, detection methods and marker origin. Conclusions: All the results exhibits that appearance of cfDNA in blood is an indicator for adverse RFS and OS in CRC patients. PMID:27326254

  18. Cell-Free DNA Screening: Complexities and Challenges of Clinical Implementation.

    PubMed

    Grace, Matthew R; Hardisty, Emily; Dotters-Katz, Sarah K; Vora, Neeta L; Kuller, Jeffrey A

    2016-08-01

    Screening for fetal aneuploidy in pregnant women using cell-free DNA has increased dramatically since the technology became commercially available in 2011. Since that time, numerous trials have demonstrated high sensitivity and specificity to screen for common aneuploidies in high-risk populations. Studies assessing the performance of these tests in low-risk populations have also demonstrated improved detection rates compared with traditional, serum-based screening strategies. Concurrent with the increased use of this technology has been a decrease in invasive procedures (amniocentesis and chorionic villus sampling). As the technology becomes more widely understood, available, and utilized, challenges regarding its clinical implementation have become apparent. Some of these challenges include test failures, false-positive and false-negative results, limitations in positive predictive value in low-prevalence populations, and potential maternal health implications of abnormal results. In addition, commercial laboratories are expanding screening beyond common aneuploidies to include microdeletion screening and whole genome screening. This review article is intended to provide the practicing obstetrician with a summary of the complexities of cell-free DNA screening and the challenges of implementing it in the clinical setting. PMID:27526871

  19. Cell-free DNA screening for fetal aneuploidy as a clinical service.

    PubMed

    Cuckle, Howard; Benn, Peter; Pergament, Eugene

    2015-10-01

    Non-invasive prenatal testing (NIPT) through the analysis of cell free (cf)DNA is revolutionizing prenatal screening for fetal aneuploidy. Current methods used in clinical practice include shotgun massively parallel sequencing (s-MPS); targeted (t-MPS); and an approach that takes advantage of single nucleotide polymorphism (SNP) differences between mother and fetus. Efficacy of cfDNA testing for the common autosomal trisomies far exceeds that of conventional screening. Depending on the methodology used, reasons for discordancy between cfDNA results and fetal karyotype can include true fetal mosaicism, confined placental mosaicism, presence of a maternal karyotype abnormality, insufficient counting due to low fetal fraction, and a vanishing twin. Among the possible cfDNA strategies a Primary test has the highest performance but is expensive, while a Contingent cfDNA test can achieve high performance at a relatively low cost. Practicalities to be considered in the provision of testing include pretest counseling about the scope and accuracy of the testing, the interpretation of results when there is a low fetal fraction and follow-up studies for positive test results. The role of first trimester nuchal translucency measurement and conventional biochemical testing needs to be reassessed in the context of the use of cfDNA. PMID:25732593

  20. Cell-free Xenopus egg extracts for studying DNA damage response pathways

    PubMed Central

    CUPELLO, STEVEN; RICHARDSON, CHRISTINE; YAN, SHAN

    2016-01-01

    In response to a variety of DNA replication stress or DNA damaging agents, the DNA damage response (DDR) pathways are triggered for cells to coordinate DNA repair, cell cycle checkpoints, apoptosis, and senescence. Cell-free Xenopus egg extracts, derived from the eggs of African clawed frogs (Xenopus laevis), have been widely used for studies concerning DDR pathways. In this review we focus on how different experimental systems have been established using Xenopus egg extracts to investigate the DDR pathways that are activated in response to DNA replication stress, double-strand breaks (DSBs), inter-strand crosslinks (ICLs), and oxidative stress. We summarize how molecular details of DDR pathways are dissected by the mechanistic studies with Xenopus egg extracts. We also provide an update on the regulation of translesion DNA synthesis (TLS) polymerases (Pol κ and REV1) in the DDR pathways. A better understanding of DDR pathways using Xenopus egg extracts has opened new avenues for future cancer therapeutics. Finally, we offer our perspectives of future directions for studies of DDR pathways with Xenopus egg extracts. PMID:27160070

  1. Efficient Capture and Isolation of Tumor-Related Circulating Cell-Free DNA from Cancer Patients Using Electroactive Conducting Polymer Nanowire Platforms

    PubMed Central

    Jeon, SeungHyun; Lee, HyungJae; Bae, Kieun; Yoon, Kyong-Ah; Lee, Eun Sook; Cho, Youngnam

    2016-01-01

    Circulating cell-free DNA (cfDNA) is currently recognized as a key non-invasive biomarker for cancer diagnosis and progression and therapeutic efficacy monitoring. Because cfDNA has been detected in patients with diverse types of cancers, the use of efficient strategies to isolate cfDNA not only provides valuable insights into tumour biology, but also offers the potential for developing new cancer-specific targets. However, the challenges associated with conventional cfDNA extraction methods prevent their further clinical applications. Here, we developed a nanostructured conductive polymer platform for the efficient capture and release of circulating cfDNA and demonstrated its potential clinical utility using unprocessed plasma samples from patients with breast and lung cancers. Our results confirmed that the platform's enhanced efficiency allows tumor-specific circulating cfDNA to be recovered at high yield and purity. PMID:27162553

  2. Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

    PubMed Central

    Yu, Hong; Shen, Yanting; Ge, Qinyu; He, Youji; Qiao, Dongyan; Ren, Mulan; Zhang, Jianqiong

    2013-01-01

    The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA) level of gravidas developed into early-onset preeclampsia (EOPE) subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged) cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68) was higher than controls (median, 1.79; interquartile range, 1.46–2.53). The increased level of (logged) cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG) level (r = 0.628, p < 0.001). Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively). The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta. PMID:23567271

  3. Demographic and experiential correlates of public attitudes towards cell-free fetal DNA screening

    PubMed Central

    Sayres, Lauren C.; Allyse, Megan; Goodspeed, Taylor A.; Cho, Mildred K.

    2014-01-01

    This study seeks to inform clinical application of cell-free fetal DNA (cffDNA) screening as a novel method for prenatal trisomy detection by investigating public attitudes towards this technology and demographic and experiential characteristics related to these attitudes. Two versions of a 25-item survey assessing interest in cffDNA and existing first-trimester combined screening for either trisomy 13 and 18 or trisomy 21 were distributed among 3,164 members of the United States public. Logistic regression was performed to determine variables predictive of interest in screening options. Approximately 47% of respondents expressed an interest in cffDNA screening for trisomy 13, 18, and 21, with a majority interested in cffDNA screening as a stand-alone technique. A significantly greater percent would consider termination of pregnancy following a diagnosis of trisomy 13 or 18 (52%) over one of trisomy 21 (44%). Willingness to consider abortion of an affected pregnancy was the strongest correlate to interest in both cffDNA and first-trimester combined screening, although markedly more respondents expressed an interest in some form of screening (69% and 71%, respectively) than would consider termination. Greater educational attainment, higher income, and insurance coverage predicted interest in cffDNA screening; stronger religious identification also corresponded to decreased interest. Prior experience with disability and genetic testing was associated with increased interest in cffDNA screening. Several of these factors, in addition to advanced age and Asian race, were, in turn, predictive of respondents’ increased willingness to consider post-diagnosis termination of pregnancy. In conclusion, divergent attitudes towards cffDNA screening - and prenatal options more generally – appear correlated with individual socioeconomic and religious backgrounds and experiences with disability and genetic testing. Clinical implementation and counseling for novel prenatal

  4. Cell-free plasma hemoglobin removal by dialyzers with various permeability profiles.

    PubMed

    Hulko, Michael; Kunz, Melanie; Yildirim, Mehmet; Homeyer, Sandra; Amon, Oliver; Krause, Bernd

    2015-11-10

    The release of hemoglobin from mechanically stressed erythrocytes into plasma is a general side effect of extracorporeal therapies, such as extracorporeal membrane oxygenation or hemodialysis. In many reported cases dialysis patients showed elevated cell-free plasma hemoglobin (CPH) levels which are associated with pathophysiological effects. In this in vitro study, the CPH clearance capacity of various filters with different permeability profiles was measured. Simulated dialysis treatments were conducted and clearance was calculated from variations in CPH concentrations over time by measuring plasma absorbance at 405 nm. Conventional high-flux filters exhibited no detectable clearance of CPH. High-flux filters with extended permeability exhibited clearances between 5.8 ± 1.2 and 12.7 ± 1.7 ml/min when tested with plasma and between 5.8 ± 1.2 and 11.3 ± 1.6 ml/min when tested with whole blood. septeX high-cutoff filters had clearances between 13.8 ± 1.8 and 15.5 ± 1.7 ml/min when tested with plasma and of 22.6 ± 2.9 ml/min when tested with whole blood. This study demonstrated that filters with extended permeability and the septeX filter enable CPH removal when used as in chronic and acute settings.

  5. Distinct subclonal tumour responses to therapy revealed by circulating cell-free DNA

    PubMed Central

    Gremel, G.; Lee, R. J.; Girotti, M. R.; Mandal, A. K.; Valpione, S.; Garner, G.; Ayub, M.; Wood, S.; Rothwell, D. G.; Fusi, A.; Wallace, A.; Brady, G.; Dive, C.; Dhomen, N.; Lorigan, P.; Marais, R.

    2016-01-01

    Background The application of precision medicine in oncology requires in-depth characterisation of a patient's tumours and the dynamics of their responses to treatment. Patients and methods We used next-generation sequencing of circulating cell-free DNA (cfDNA) to monitor the response of a KIT p.L576P-mutant metastatic vaginal mucosal melanoma to sequential targeted, immuno- and chemotherapy. Results Despite a KIT mutation, the response to imatinib was mixed. Unfortunately, tumours were not accessible for molecular analysis. To study the mechanism underlying the mixed clinical response, we carried out whole-exome sequencing and targeted longitudinal analysis of cfDNA. This revealed two tumour subclones; one with a KIT mutation that responded to imatinib and a second KIT-wild-type subclone that did not respond to imatinib. Notably, the subclones also responded differently to immunotherapy. However, both subclones responded to carboplatin/paclitaxel, and although the KIT-wild-type subclone progressed after chemotherapy, it responded to subsequent re-administration of paclitaxel. Conclusion We show that cfDNA can reveal tumour evolution and subclonal responses to therapy even when biopsies are not available. PMID:27502704

  6. Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

    PubMed

    Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

    2000-04-14

    There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

  7. Unfair discrimination in prenatal aneuploidy screening using cell-free DNA?

    PubMed

    Rolfes, Vasilija; Schmitz, Dagmar

    2016-03-01

    Non-invasive prenatal testing on the basis of cell-free DNA of placental origin (NIPT) changed the landscape of prenatal care and is seen as superior to all other up to now implemented prenatal screening procedures - at least in the high-risk population. NIPT has spread almost worldwide commercially, but only in a few countries the costs of NIPT are covered by insurance companies. Such financial barriers in prenatal testing can lead to significant restrictions to the average range of opportunities of pregnant women and couples, which on an intersubjective level can be defined as unfair discrimination and on an individual level weakens reproductive autonomy. Given that enabling reproductive autonomy is the main ethical justification for offering prenatal (genetic) testing, these barriers are not only an issue of justice in health care, but are potentially counteracting the primary purpose of these testing procedures. PMID:26773245

  8. Unfair discrimination in prenatal aneuploidy screening using cell-free DNA?

    PubMed

    Rolfes, Vasilija; Schmitz, Dagmar

    2016-03-01

    Non-invasive prenatal testing on the basis of cell-free DNA of placental origin (NIPT) changed the landscape of prenatal care and is seen as superior to all other up to now implemented prenatal screening procedures - at least in the high-risk population. NIPT has spread almost worldwide commercially, but only in a few countries the costs of NIPT are covered by insurance companies. Such financial barriers in prenatal testing can lead to significant restrictions to the average range of opportunities of pregnant women and couples, which on an intersubjective level can be defined as unfair discrimination and on an individual level weakens reproductive autonomy. Given that enabling reproductive autonomy is the main ethical justification for offering prenatal (genetic) testing, these barriers are not only an issue of justice in health care, but are potentially counteracting the primary purpose of these testing procedures.

  9. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients.

    PubMed

    Kim, Seung Tae; Lee, Won-Suk; Lanman, Richard B; Mortimer, Stefanie; Zill, Oliver A; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-11-24

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

  10. Cell free DNA testing-interpretation of results using an online calculator.

    PubMed

    Grace, Matthew R; Hardisty, Emily; Green, Noah S; Davidson, Emily; Stuebe, Alison M; Vora, Neeta L

    2015-07-01

    All pregnant women, regardless of age, should be offered screening or invasive testing for chromosomal abnormalities at <20 weeks' gestation. Noninvasive prenatal screening for fetal aneuploidy with the use of cell-free DNA (cfDNA) is a screening method that offers high sensitivity and specificity in validation studies and has reduced the need for unnecessary invasive procedures. Laboratories often advertise and report a test's sensitivity and specificity as a means to describe the test's accuracy. The positive predictive value (PPV) of a screening test (the proportion of positive results that are truly positive) is a function of the prevalence of the condition in a population and often is not reported in direct-to-patient advertising. False-positive cfDNA screening tests have been reported, and there is evidence that some women are deciding to terminate their pregnancy without confirmatory testing. We believe that laboratories should disclose the patient-specific PPV of cfDNA screening for aneuploidy on result reports. To assist with counseling patients about the benefits, risks, and limitations of aneuploidy screening with the use of cfDNA and to demonstrate the relationship between an a priori risk and PPV, we developed a web-based calculator to estimate the PPV of the 4 commercially available cfDNA testing platforms for which data have been published. Estimates are made with the use of a patient's age and gestational age-related risk of trisomy 21, 18 and 13 or an a priori risk that is based on other findings. This web-based calculator is an aid for providers and genetic counselors to illustrate the relationship between disease prevalence and a test's PPV. It has enhanced our counseling of patients both before they elect noninvasive prenatal screening and after they receive a positive result.

  11. Cell free DNA testing-interpretation of results using an online calculator.

    PubMed

    Grace, Matthew R; Hardisty, Emily; Green, Noah S; Davidson, Emily; Stuebe, Alison M; Vora, Neeta L

    2015-07-01

    All pregnant women, regardless of age, should be offered screening or invasive testing for chromosomal abnormalities at <20 weeks' gestation. Noninvasive prenatal screening for fetal aneuploidy with the use of cell-free DNA (cfDNA) is a screening method that offers high sensitivity and specificity in validation studies and has reduced the need for unnecessary invasive procedures. Laboratories often advertise and report a test's sensitivity and specificity as a means to describe the test's accuracy. The positive predictive value (PPV) of a screening test (the proportion of positive results that are truly positive) is a function of the prevalence of the condition in a population and often is not reported in direct-to-patient advertising. False-positive cfDNA screening tests have been reported, and there is evidence that some women are deciding to terminate their pregnancy without confirmatory testing. We believe that laboratories should disclose the patient-specific PPV of cfDNA screening for aneuploidy on result reports. To assist with counseling patients about the benefits, risks, and limitations of aneuploidy screening with the use of cfDNA and to demonstrate the relationship between an a priori risk and PPV, we developed a web-based calculator to estimate the PPV of the 4 commercially available cfDNA testing platforms for which data have been published. Estimates are made with the use of a patient's age and gestational age-related risk of trisomy 21, 18 and 13 or an a priori risk that is based on other findings. This web-based calculator is an aid for providers and genetic counselors to illustrate the relationship between disease prevalence and a test's PPV. It has enhanced our counseling of patients both before they elect noninvasive prenatal screening and after they receive a positive result. PMID:25957020

  12. Cell-Free Fetal DNA, Telomeres, and the Spontaneous Onset of Parturition.

    PubMed

    Phillippe, Mark

    2015-10-01

    Multiple previous reports have provided compelling support for the premise that spontaneous parturition is mediated by activation of inflammation-related signaling pathways leading to increased secretion of cytokines and chemokines, the influx of neutrophils and macrophages into the pregnant uterus, increased production of uterine activation proteins (eg, connexin-43, cyclo-oxygenase-2, oxytocin receptors, etc), activation of matrix metalloproteinases, and the release of uterotonins leading to cervical ripening, membrane rupture, and myometrial contractions. The missing link has been the fetal/placental signal that triggers these proinflammatory events in the absence of microbial invasion and intrauterine infection. This article reviews the biomedical literature regarding the increase in cell-free fetal DNA (cffDNA), which is released during apoptosis in the placenta and fetal membranes at term, the ability of apoptosis modified vertebrate DNA to stimulate toll-like receptor-9 (TLR9) leading to increased release of cytokines and chemokines, and the potential "fail-safe" role for the anti-inflammatory cytokine IL-10. This article also reviews the literature supporting the key role that telomere loss plays in regard to increasing the ability of vertebrate (including placental) DNA to stimulate TLR9, and in regard to signaling the onset of apoptosis in the placenta and fetal membranes, thereby providing a biologic clock that determines the length of gestation and the timing for the onset of parturition. In summary, this literature review provides a strong rationale for future research to test the hypothesis that telomere loss and increased cffDNA levels trigger the proinflammatory events leading to the spontaneous onset of parturition in mammals: the "cffDNA/telomere hypothesis."

  13. Cell-free coupled transcription-translation system for investigation of linear DNA segments.

    PubMed Central

    Yang, H L; Ivashkiv, L; Chen, H Z; Zubay, G; Cashel, M

    1980-01-01

    Heretofore the DNA-directed coupled transcription-translation system, most useful in gene expression analysis, has been limited to the use of circular or long linear DNAs. Linear DNAs are degraded in this system by an exonucleolytic activity that can be eliminated by making the synthetic extracts from a suitable recB mutant of Escherichia coli. Using these extracts, we have examined the gene expression of a variety of linear DNAs. In particular, the complex pattern of expression of ribosomal protein genes and RNA polymerase genes in the rpoBC-rplLJ region has been analyzed by comparing the protein products obtained when using lambda rifd18 DNA with the product obtained when using the same DNA segmented with various restriction enzymes. The results obtained confirm the conclusions of others obtained by much more elaborate in vivo techniques. It seems highly likely that this cell-free system will have extensive applications in the area of analysis of gene expression. Images PMID:6261235

  14. Graft-Derived Cell-Free DNA as a Marker of Transplant Graft Injury.

    PubMed

    Oellerich, Michael; Walson, Philip D; Beck, Julia; Schmitz, Jessica; Kollmar, Otto; Schütz, Ekkehard

    2016-04-01

    Although short-term success after solid organ transplantation is good, long-term graft and recipient survival are both not satisfactory. Despite therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs), both excessive and insufficient immunosuppression still do occur. There is a need for new biomarkers that, when combined with TDM, can be used to provide more effective and less toxic, personalized immunosuppression to improve long-term survival. Currently used methods are insufficient to rapidly, cost-effectively, and directly interrogate graft integrity after solid organ transplantation. However, because organ transplants are also genome transplants, measurement of graft-derived circulating cell-free DNA (GcfDNA) has shown promise as a way to improve both graft and recipient outcomes after solid organ transplantation through the early detection of severe graft injury, enabling an early intervention. A newly developed droplet digital polymerase chain reaction (ddPCR) method has advantages over expensive high-throughput sequencing methods to rapidly quantify GcfDNA percentages and absolute amounts. This procedure does not require donor DNA and therefore can be applied to any organ donor/recipient pair. The droplet digital polymerase chain reaction method allows for the early, sensitive, specific, and cost-effective direct assessment of graft integrity and can be used to define individual responses to ISDs including the minimal ISD exposures necessary to prevent rejection. This is especially important in patients undergoing ISD switches due to ISD toxicity, infections, or malignancies. Although prospective, multicenter clinical trials in liver, heart, and kidney transplantation have not been completed, early results suggest that GcfDNA can be combined with TDM to guide changes in immunosuppression to provide more effective, and less toxic treatment. Personalized immunosuppression will shift emphasis in transplantation from reaction to prevention and could

  15. Evaluation of patient education materials: the example of circulating cell free DNA testing for aneuploidy.

    PubMed

    Kloza, Edward M; Haddow, Paula K; Halliday, Jacquelyn V; O'Brien, Barbara M; Lambert-Messerlian, Geralyn M; Palomaki, Glenn E

    2015-04-01

    Informed consent is the process by which the treating health care provider discloses appropriate information to a competent patient so that the patient may make a voluntary choice to accept or refuse treatment. When the analysis of circulating cell free DNA (ccfDNA) became commercially available in 2011 through the Prenatal Diagnostic Laboratory at Women & Infants Hospital of Providence, Rhode Island to "high-risk" women, it provided an opportunity to examine how commercial laboratories informed potential consumers. We identified, via an internet search, four laboratories offering such testing in the United States and one in Europe. We evaluated patient educational materials (PEMs) from each using the Flesch Reading Ease method and a modified version of the Suitability Assessment of Materials (SAM) criteria. Pamphlets were also reviewed for their inclusion of content recommendations from the International Society for Prenatal Diagnosis, the National Society of Genetic Counselors, the American College of Obstetricians and Gynecologists jointly with the Society of Maternal Fetal Medicine, and the American College of Genetics and Genomics. Reading levels were typically high (10th-12th grade). None of the pamphlets met all SAM criteria evaluated nor did any pamphlet include all recommended content items. To comply with readability and content recommendations more closely, Women & Infants Hospital created a new pamphlet to which it applied the same criteria, and also subjected it to focus group assessment. These types of analyses can serve as a model for future evaluations of similar patient educational materials.

  16. Circulating Tumor Cell and Cell-free Circulating Tumor DNA in Lung Cancer.

    PubMed

    Nurwidya, Fariz; Zaini, Jamal; Putra, Andika Chandra; Andarini, Sita; Hudoyo, Achmad; Syahruddin, Elisna; Yunus, Faisal

    2016-09-01

    Circulating tumor cells (CTCs) are tumor cells that are separated from the primary site or metastatic lesion and disseminate in blood circulation. CTCs are considered to be part of the long process of cancer metastasis. As a 'liquid biopsy', CTC molecular examination and investigation of single cancer cells create an important opportunity for providing an understanding of cancer biology and the process of metastasis. In the last decade, we have seen dramatic development in defining the role of CTCs in lung cancer in terms of diagnosis, genomic alteration determination, treatment response and, finally, prognosis prediction. The aims of this review are to understand the basic biology and to review methods of detection of CTCs that apply to the various types of solid tumor. Furthermore, we explored clinical applications, including treatment monitoring to anticipate therapy resistance as well as biomarker analysis, in the context of lung cancer. We also explored the potential use of cell-free circulating tumor DNA (ctDNA) in the genomic alteration analysis of lung cancer. PMID:27689025

  17. Circulating Tumor Cell and Cell-free Circulating Tumor DNA in Lung Cancer

    PubMed Central

    Zaini, Jamal; Putra, Andika Chandra; Andarini, Sita; Hudoyo, Achmad; Syahruddin, Elisna; Yunus, Faisal

    2016-01-01

    Circulating tumor cells (CTCs) are tumor cells that are separated from the primary site or metastatic lesion and disseminate in blood circulation. CTCs are considered to be part of the long process of cancer metastasis. As a 'liquid biopsy', CTC molecular examination and investigation of single cancer cells create an important opportunity for providing an understanding of cancer biology and the process of metastasis. In the last decade, we have seen dramatic development in defining the role of CTCs in lung cancer in terms of diagnosis, genomic alteration determination, treatment response and, finally, prognosis prediction. The aims of this review are to understand the basic biology and to review methods of detection of CTCs that apply to the various types of solid tumor. Furthermore, we explored clinical applications, including treatment monitoring to anticipate therapy resistance as well as biomarker analysis, in the context of lung cancer. We also explored the potential use of cell-free circulating tumor DNA (ctDNA) in the genomic alteration analysis of lung cancer. PMID:27689025

  18. Circulating Tumor Cell and Cell-free Circulating Tumor DNA in Lung Cancer

    PubMed Central

    Zaini, Jamal; Putra, Andika Chandra; Andarini, Sita; Hudoyo, Achmad; Syahruddin, Elisna; Yunus, Faisal

    2016-01-01

    Circulating tumor cells (CTCs) are tumor cells that are separated from the primary site or metastatic lesion and disseminate in blood circulation. CTCs are considered to be part of the long process of cancer metastasis. As a 'liquid biopsy', CTC molecular examination and investigation of single cancer cells create an important opportunity for providing an understanding of cancer biology and the process of metastasis. In the last decade, we have seen dramatic development in defining the role of CTCs in lung cancer in terms of diagnosis, genomic alteration determination, treatment response and, finally, prognosis prediction. The aims of this review are to understand the basic biology and to review methods of detection of CTCs that apply to the various types of solid tumor. Furthermore, we explored clinical applications, including treatment monitoring to anticipate therapy resistance as well as biomarker analysis, in the context of lung cancer. We also explored the potential use of cell-free circulating tumor DNA (ctDNA) in the genomic alteration analysis of lung cancer.

  19. Cell-free fetal DNA in the maternal circulation originates from the cytotrophoblast: proof from an unique case

    PubMed Central

    Hochstenbach, Ron; Nikkels, Peter G J; Elferink, Martin G; Oudijk, Martijn A; van Oppen, Carla; van Zon, Patrick; van Harssel, Jeske; Schuring-Blom, Heleen; Page-Christiaens, Godelieve C M L

    2015-01-01

    Key Clinical Message Noninvasive prenatal testing (NIPT) and direct karyotyping of cytotrophoblast were normal for a male fetus, but cultured chorionic villus mesenchymal cells and umbilical cord fibroblasts showed nonmosaic trisomy 18. This observation provides direct evidence for the cytotrophoblastic origin of cell-free fetal DNA and yields a biological explanation for falsely reassuring NIPT results. PMID:26185654

  20. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

    PubMed

    Butler, Timothy M; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J; Macey, Tara A; Korkola, James E; Koppie, Theresa M; Corless, Christopher L; Gray, Joe W; Spellman, Paul T

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

  1. [Molecular Karyotyping of Cell-Free DNA from Blastocoele Fluid as a Basis for Noninvasive Preimplantation Genetic Screening of Aneuploidy].

    PubMed

    Skryabin, N A; Lebedev, I N; Artukhova, V G; Zhigalina, D I; Stepanov, I A; Krivoschekova, G V; Svetlakov, A V

    2015-11-01

    The discovery of DNA fragments in the blastocoele fluid is promising for the development of new noninvasive methods for the preimplantation genetic diagnosis of chromosomal diseases. However, to date there are no data confirming the concordance between the molecular karyotype of cell-free DNA from blastocoele fluid and the blastocyst cells per se. This paper reports on this concordance according to the results of molecular-cytogenetic analysis of the chromosomal set with the use of comparative genomic hybridization. PMID:26845860

  2. Non-invasive prenatal diagnosis of β-thalassemia by detection of the cell-free fetal DNA in maternal circulation: a systematic review and meta-analysis.

    PubMed

    Zafari, Mandana; Kosaryan, Mehrnoush; Gill, Pooria; Alipour, Abbass; Shiran, Mohammadreza; Jalalli, Hossein; Banihashemi, Ali; Fatahi, Fatemeh

    2016-08-01

    The discovery of fetal DNA (f-DNA) opens the possibility of early non-invasive procedure for detection of paternally inherited mutation of beta-thalassemia. Since 2002, some studies have examined the sensitivity and specificity of this method for detection of paternally inherited mutation of thalassemia in pregnant women at risk of having affected babies. We conducted a systematic review of published articles that evaluated using this method for early detection of paternally inherited mutation in maternal plasma. A sensitive search of multiple databases was done in which nine studies met our inclusion criteria. The sensitivity and specificity was 99 and 99 %, respectively. The current study found that detection of paternally inherited mutation of thalassemia using analysis of cell-free fetal DNA is highly accurate. This method could replace conventional and invasive methods. PMID:26968552

  3. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

    PubMed

    Lanman, Richard B; Mortimer, Stefanie A; Zill, Oliver A; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A; Divers, Stephen G; Hoon, Dave S B; Kopetz, E Scott; Lee, Jeeyun; Nikolinakos, Petros G; Baca, Arthur M; Kermani, Bahram G; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

  4. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

    PubMed Central

    Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

  5. Prenatal assessment of fetal chromosomal and genetic disorders through maternal plasma DNA analysis.

    PubMed

    Liao, Gary J W; Chiu, Rossa W K; Lo, Y M Dennis

    2012-02-01

    The existence of cell free DNA derived from the fetus in the plasma of pregnant women was first demonstrated in 1997. This discovery offered the possibility of non-invasive sampling of fetal genetic material simply through the collection of a maternal blood sample. Such cell free fetal DNA molecules in the maternal circulation have subsequently been shown to originate from the placenta and could be detected from about 7 weeks of gestation. It has been shown that cell free fetal DNA analysis could offer highly accurate assessment of fetal genotype and chromosomal makeup for some applications. Thus, cell free fetal DNA analysis has been incorporated as a part of prenatal screening programs for the prenatal management of sex-linked and sex-associated diseases, rhesus D incompatibility as well as the prenatal detection of Down's syndrome.Cell free fetal DNA analysis may lead to a change in the way prenatal assessments are made.

  6. Current Status of Testing for Microdeletion Syndromes and Rare Autosomal Trisomies Using Cell-Free DNA Technology.

    PubMed

    Yaron, Yuval; Jani, Jacques; Schmid, Maximilian; Oepkes, Dick

    2015-11-01

    Noninvasive prenatal testing using cell-free DNA in maternal blood for trisomy 21 was introduced in 2011. This technology has continuously evolved with the addition of screening for trisomy 18 and trisomy 13 followed by the inclusion of sex chromosome aneuploidies. Expanded noninvasive prenatal test panels have recently become available, which enable screening for microdeletion syndromes such as the 22q11.2 deletion (associated with the velocardiofacial syndrome) and others. However, the performance data for these microdeletion syndromes are derived from a small number of samples, mostly generated in vitro. Rigorous performance evaluation, as was done at least for trisomy 21 testing using cell-free DNA analysis, is difficult to perform given the rarity of each condition. In addition, detection rates may vary considerably depending on deletion size. Importantly, positive predictive values (PPVs), strongly influenced by the low prevalence, are expected to be significantly lower than 10% for most conditions. Thus, screening in an average-risk population is likely to have many more false-positives than affected cases detected. Conversely, testing in a high-risk population such as fetuses with cardiac anomalies may have higher PPVs, but a negative result needs to be considered carefully as a result of uncertain information about detection rates and a significant residual risk for other copy number variants and single gene disorders. This article integrates current knowledge on cell-free DNA testing for microdeletions with the aim to assist clinicians and policymakers in designing optimal programs for screening in pregnancy.

  7. Quantification of Cell-Free DNA in Red Blood Cell Units in Different Whole Blood Processing Methods

    PubMed Central

    Bhagirath, Vinai C.; Heddle, Nancy M.; Liu, Yang; Eikelboom, John W.; Liaw, Patricia C.

    2016-01-01

    Background. Whole blood donations in Canada are processed by either the red cell filtration (RCF) or whole blood filtration (WBF) methods, where leukoreduction is potentially delayed in WBF. Fresh WBF red blood cells (RBCs) have been associated with increased in-hospital mortality after transfusion. Cell-free DNA (cfDNA) is released by neutrophils prior to leukoreduction, degraded during RBC storage, and is associated with adverse patient outcomes. We explored cfDNA levels in RBCs prepared by RCF and WBF and different storage durations. Methods. Equal numbers of fresh (stored ≤14 days) and older RBCs were sampled. cfDNA was quantified by spectrophotometry and PicoGreen. Separate regression models determined the association with processing method and storage duration and their interaction on cfDNA. Results. cfDNA in 120 RBC units (73 RCF, 47 WBF) were measured. Using PicoGreen, WBF units overall had higher cfDNA than RCF units (p = 0.0010); fresh WBF units had higher cfDNA than fresh RCF units (p = 0.0093). Using spectrophotometry, fresh RBC units overall had higher cfDNA than older units (p = 0.0031); fresh WBF RBCs had higher cfDNA than older RCF RBCs (p = 0.024). Conclusion. Higher cfDNA in fresh WBF was observed compared to older RCF blood. Further study is required for association with patient outcomes. PMID:27774338

  8. Recommended pre-test counseling points for noninvasive prenatal testing using cell-free DNA: a 2015 perspective.

    PubMed

    Sachs, Amy; Blanchard, Leah; Buchanan, Amanda; Norwitz, Errol; Bianchi, Diana W

    2015-10-01

    Noninvasive prenatal testing (NIPT) using cell-free DNA is being offered to an increasing number of women. Comprehensive pre-test counseling is complicated by emerging information about the benefits and limitations of testing, as well as the potential to detect incidental findings. Genetic counselors are trained to facilitate informed decision-making; however, not all centers have access to these professionals. To aid in the informed consent process, we have summarized key points to be included in discussions with patients who are considering NIPT.

  9. DASAF: An R Package for Deep Sequencing-Based Detection of Fetal Autosomal Abnormalities from Maternal Cell-Free DNA.

    PubMed

    Liu, Baohong; Tang, Xiaoyan; Qiu, Feng; Tao, Chunmei; Gao, Junhui; Ma, Mengmeng; Zhong, Tingyan; Cai, JianPing; Li, Yixue; Ding, Guohui

    2016-01-01

    Background. With the development of massively parallel sequencing (MPS), noninvasive prenatal diagnosis using maternal cell-free DNA is fast becoming the preferred method of fetal chromosomal abnormality detection, due to its inherent high accuracy and low risk. Typically, MPS data is parsed to calculate a risk score, which is used to predict whether a fetal chromosome is normal or not. Although there are several highly sensitive and specific MPS data-parsing algorithms, there are currently no tools that implement these methods. Results. We developed an R package, detection of autosomal abnormalities for fetus (DASAF), that implements the three most popular trisomy detection methods-the standard Z-score (STDZ) method, the GC correction Z-score (GCCZ) method, and the internal reference Z-score (IRZ) method-together with one subchromosome abnormality identification method (SCAZ). Conclusions. With the cost of DNA sequencing declining and with advances in personalized medicine, the demand for noninvasive prenatal testing will undoubtedly increase, which will in turn trigger an increase in the tools available for subsequent analysis. DASAF is a user-friendly tool, implemented in R, that supports identification of whole-chromosome as well as subchromosome abnormalities, based on maternal cell-free DNA sequencing data after genome mapping. PMID:27437397

  10. DASAF: An R Package for Deep Sequencing-Based Detection of Fetal Autosomal Abnormalities from Maternal Cell-Free DNA

    PubMed Central

    Tang, Xiaoyan; Qiu, Feng; Tao, Chunmei; Gao, Junhui; Ma, Mengmeng; Zhong, Tingyan; Cai, JianPing; Li, Yixue

    2016-01-01

    Background. With the development of massively parallel sequencing (MPS), noninvasive prenatal diagnosis using maternal cell-free DNA is fast becoming the preferred method of fetal chromosomal abnormality detection, due to its inherent high accuracy and low risk. Typically, MPS data is parsed to calculate a risk score, which is used to predict whether a fetal chromosome is normal or not. Although there are several highly sensitive and specific MPS data-parsing algorithms, there are currently no tools that implement these methods. Results. We developed an R package, detection of autosomal abnormalities for fetus (DASAF), that implements the three most popular trisomy detection methods—the standard Z-score (STDZ) method, the GC correction Z-score (GCCZ) method, and the internal reference Z-score (IRZ) method—together with one subchromosome abnormality identification method (SCAZ). Conclusions. With the cost of DNA sequencing declining and with advances in personalized medicine, the demand for noninvasive prenatal testing will undoubtedly increase, which will in turn trigger an increase in the tools available for subsequent analysis. DASAF is a user-friendly tool, implemented in R, that supports identification of whole-chromosome as well as subchromosome abnormalities, based on maternal cell-free DNA sequencing data after genome mapping. PMID:27437397

  11. Cell-free fetal DNA testing for fetal aneuploidy and beyond: clinical integration challenges in the US context.

    PubMed

    Allyse, Megan; Sayres, Lauren C; King, Jaime S; Norton, Mary E; Cho, Mildred K

    2012-11-01

    The recent release of new, non-invasive prenatal tests for fetal aneuploidy using cell-free fetal DNA (cffDNA) has been hailed as a revolution in prenatal testing and has triggered significant commercial interest in the field. Ongoing research portends the arrival of a wide range of cffDNA tests. However, it is not yet clear how these tests will be integrated into well-established prenatal testing strategies in the USA, as the timing of such testing and the degree to which new non-invasive tests will supplement or replace existing screening and diagnostic tools remain uncertain. We argue that there is an urgent need for policy-makers, regulators and professional societies to provide guidance on the most efficient and ethical manner for such tests to be introduced into clinical practice in the USA.

  12. Cell-free fetal DNA testing for fetal aneuploidy and beyond: clinical integration challenges in the US context

    PubMed Central

    Allyse, Megan; Sayres, Lauren C.; King, Jaime S.; Norton, Mary E.; Cho, Mildred K.

    2012-01-01

    The recent release of new, non-invasive prenatal tests for fetal aneuploidy using cell-free fetal DNA (cffDNA) has been hailed as a revolution in prenatal testing and has triggered significant commercial interest in the field. Ongoing research portends the arrival of a wide range of cffDNA tests. However, it is not yet clear how these tests will be integrated into well-established prenatal testing strategies in the USA, as the timing of such testing and the degree to which new non-invasive tests will supplement or replace existing screening and diagnostic tools remain uncertain. We argue that there is an urgent need for policy-makers, regulators and professional societies to provide guidance on the most efficient and ethical manner for such tests to be introduced into clinical practice in the USA. PMID:22863603

  13. Circulating levels of maternal plasma cell-free pregnancy-associated placenta-specific microRNAs are associated with placental weight.

    PubMed

    Miura, K; Morisaki, S; Abe, S; Higashijima, A; Hasegawa, Y; Miura, S; Tateishi, S; Mishima, H; Yoshiura, K; Masuzaki, H

    2014-10-01

    The aim of this study was to investigate the relationship between plasma concentration of cell-free pregnancy-associated placenta-specific microRNAs and clinical variables (placental weight, maternal body mass index, and neonatal birth weight). Circulating levels of cell-free pregnancy-associated placenta-specific microRNAs (miR-515-3p, miR-517a, miR-517c and miR-518b) in maternal plasma were measured by quantitative real-time RT-PCR in sixty-two pregnant women. The levels of cell-free pregnancy-associated placenta-specific microRNAs were significantly associated with placental weight, but not associated with body mass index or birth weight. Therefore, the measurement of cell-free pregnancy-associated placenta-specific miRNAs levels in maternal plasma may reflect the pregnancy status related to placenta volume.

  14. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

    SciTech Connect

    Jones, E.V.; Moss, B.

    1984-01-01

    The previous demonstration that a phosphonoacetate (PAA)-resistant (PAA/sup r/) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAA/sup s/) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAA/sup r/ mutant virus. Formation of PAA/sup r/ recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAA/sup r/ phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAA/sup r/ vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.

  15. Protein synthesis directly from PCR: progress and applications of cell-free protein synthesis with linear DNA.

    PubMed

    Schinn, Song-Min; Broadbent, Andrew; Bradley, William T; Bundy, Bradley C

    2016-06-25

    A rapid, versatile method of protein expression and screening can greatly facilitate the future development of therapeutic biologics, proteomic drug targets and biocatalysts. An attractive candidate is cell-free protein synthesis (CFPS), a cell-lysate-based in vitro expression system, which can utilize linear DNA as expression templates, bypassing time-consuming cloning steps of plasmid-based methods. Traditionally, such linear DNA expression templates (LET) have been vulnerable to degradation by nucleases present in the cell lysate, leading to lower yields. This challenge has been significantly addressed in the recent past, propelling LET-based CFPS as a useful tool for studying, screening and engineering proteins in a high-throughput manner. Currently, LET-based CFPS has promise in fields such as functional proteomics, protein microarrays, and the optimization of complex biological systems. PMID:27085957

  16. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology.

    PubMed

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E; Demko, Zachary; Pelham, Robert J; Kareht, Stephanie; Simon, Alexander L; Jinnett, Kristine N; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew

    2015-10-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. PMID:26500031

  17. Genetic counselors' experience with cell-free fetal DNA testing as a prenatal screening option for aneuploidy.

    PubMed

    Horsting, Julie M H; Dlouhy, Stephen R; Hanson, Katelyn; Quaid, Kimberly; Bai, Shaochun; Hines, Karrie A

    2014-06-01

    First identified in 1997, cell-free fetal DNA (cffDNA) has just recently been used to detect fetal aneuploidy of chromosomes 13, 18, and 21, showing its potential to revolutionize prenatal genetic testing as a non-invasive screening tool. Although this technological advancement is exciting and has certain medical applications, it has been unclear how it will be implemented in a clinical setting. Genetic counselors will likely be instrumental in answering that question, but to date, there is no published research regarding prenatal counselors' implementation of and experiences with cffDNA testing. We developed a 67 question survey to gather descriptive information from counselors regarding their personal opinions, experiences, thoughts, and concerns regarding the validity, usefulness, and implementation of this new technology. A total of 236 individuals completed a portion of the survey; not all respondents answered all questions. Qualitative questions complemented quantitative survey items, allowing respondents to voice their thoughts directly. Results indicate that counselors value cffDNA testing as a screening option but are concerned regarding how some obstetricians and patients make use of this testing. Further results, discussion, and practice implications are presented.

  18. Fetal DNA in maternal plasma in preeclamptic pregnancies.

    PubMed

    Vlková, Barbora; Turňa, Ján; Celec, Peter

    2015-02-01

    Cell-free fetal DNA present in maternal circulation has revolutionized non-invasive prenatal diagnosis of genetic diseases. In preeclampsia, the quantity of fetal DNA in maternal plasma has been studied and found to be higher in comparison to healthy pregnant women. Whether the quantity of fetal DNA can be used as a reliable predictive biomarker of preeclampsia is currently uncertain. This is a systematic review on studies quantifying fetal DNA in preeclamptic pregnancies. Using a PubMed search 22 studies were identified. In all of them, elevated levels of fetal DNA in maternal plasma in preeclampsia were found. In some of the studies, the higher concentration of fetal DNA was observed before the onset of clinical symptoms. This shows that fetal DNA levels might have a potential informative value as an early diagnostic biomarker of preeclampsia. However, in most of the studies important data are missing and there is an enormous variability in the reported results between the studies. From the available data it is currently not possible to perform a meta-analysis due to the variation between studies. If once fetal DNA should be used as a marker for determining preeclampsia at early stage, it is necessary to reduce these variations via standardized protocols for the quantification of cell-free fetal DNA as well as its reporting in the publications.

  19. Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy?

    PubMed

    Mennuti, Michael T; Cherry, Athena M; Morrissette, Jennifer J D; Dugoff, Lorraine

    2013-11-01

    Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making.

  20. Is it time to sound an alarm about false-positive cell-free DNA testing for fetal aneuploidy?

    PubMed

    Mennuti, Michael T; Cherry, Athena M; Morrissette, Jennifer J D; Dugoff, Lorraine

    2013-11-01

    Testing cell-free DNA (cfDNA) in maternal blood samples has been shown to have very high sensitivity for the detection of fetal aneuploidy with very low false-positive results in high-risk patients who undergo invasive prenatal diagnosis. Recent observation in clinical practice of several cases of positive cfDNA tests for trisomy 18 and trisomy 13, which were not confirmed by cytogenetic testing of the pregnancy, may reflect a limitation of the positive predictive value of this quantitative testing, particularly when it is used to detect rare aneuploidies. Analysis of a larger number of false-positive cases is needed to evaluate whether these observations reflect the positive predictive value that should be expected. Infrequently, mechanisms (such as low percentage mosaicism or confined placental mosaicism) might also lead to positive cfDNA testing that is not concordant with standard prenatal cytogenetic diagnosis. The need to explore these and other possible causes of false-positive cfDNA testing is exemplified by 2 of these cases. Additional evaluation of cfDNA testing in clinical practice and a mechanism for the systematic reporting of false-positive and false-negative cases will be important before this test is offered widely to the general population of low-risk obstetric patients. In the meantime, incorporating information about the positive predictive value in pretest counseling and in clinical laboratory reports is recommended. These experiences reinforce the importance of offering invasive testing to confirm cfDNA results before parental decision-making. PMID:23529082

  1. Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study.

    PubMed

    Eraky, Maysa Ahmad; Aly, Nagwa Shaban Mohamed

    2016-09-01

    Searching for a more sensitive and accurate marker for schistosomiasis diagnosis and treatment follow up is a potential necessity. Hereby, we evaluated usefulness of circulating free DNA as a marker for schistosomiasis diagnosis, assessing drug efficacy and monitoring the control interventions impact using SYBR green real-time PCR. A batch of mice were infected by 90 ± 10 Schistosoma mansoni cercariae. Starting from the 2nd day post infection (p.i.), groups of 2 or 3 mice were sacrificed every 3 days until 30 days p.i. The remaining animals were treated by a single dose of 400 mg/kg mefloquine and sacrificed in group at 5, 10, 21 days post treatment (35, 40, 51 days p.i.). Using SYBR green real time qPCR, pooled sera DNA were extracted and amplified. The results showed that, circulating free S. mansoni DNA was detected from the 2nd day post infection (p.i.) onwards with gradual decrease in the cycle threshold value Ct which indicates the gradual elevation of the DNA level (Log quantity was 2.6-3.1 IU/ml), As the infection progressed, DNA quantity was increased(Log quantity was 6.29 IU/ml). Initial increase of circulating free DNA was observed 10 days post treatment (40 days p.i.) (Log quantity was 7.38 IU/ml). That was followed by a progressive decrease in DNA level by the end of 21st day, post treatment (51 p.i.) (Log quantity 4.35 IU/ml). In conclusion, circulating free S. mansoni DNA is a reliable marker in the diagnosis of schistosomiasis and for assessing drug efficacy and monitoring the impact of control interventions. PMID:27605830

  2. Cell Free DNA of Tumor Origin Induces a ‘Metastatic’ Expression Profile in HT-29 Cancer Cell Line

    PubMed Central

    Fűri, István; Kalmár, Alexandra; Wichmann, Barnabás; Spisák, Sándor; Schöller, Andrea; Barták, Barbara; Tulassay, Zsolt; Molnár, Béla

    2015-01-01

    Background Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions. Aims To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts. Materials and Methods DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin), DNA methyltransferase 3a (DNMT3a) and NFκB (for treated HDFα cells). Results Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05) mRNA level alteration in 118 genes (logFc≥1, p≤0.05), including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A), metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1), tumor biomarker (CEACAM5), metabolic genes (i.e. INSIG1, LIPG), messenger molecule genes (i.e. DAPP, CREB3L2). Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05), including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β), STING pathway (ADAR, IRF7, CXCL10, CASP1) and the FGF2 gene. Conclusions DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from

  3. Molecular Monitoring of Cell-Free Circulating Tumor DNA in Non-Hodgkin Lymphoma.

    PubMed

    Melani, Christopher; Roschewski, Mark

    2016-08-01

    The ability to precisely monitor the effectiveness of therapy for non-Hodgkin lymphoma has important clinical implications. In patients with curable lymphomas, such as diffuse large B-cell lymphoma, the eradication of all disease is necessary for cure. In patients with incurable lymphomas, such as follicular lymphoma and mantle cell lymphoma, deep and durable remissions are associated with improvements in survival. Radiographic imaging modalities such as computed tomography and positron emission tomography are the current gold standard for monitoring therapy, but they are fundamentally limited by radiation risks, costs, lack of tumor specificity, and inability to detect disease at the molecular level. Novel sequencing-based methods can detect circulating tumor DNA (ctDNA) in the peripheral blood with great sensitivity, which opens new opportunities for molecular monitoring before, during, and after therapy. Beyond monitoring, ctDNA can also be used as a "liquid biopsy" to assess for molecular changes after therapy that may identify treatment-resistant clones. ctDNA is an emerging tool that may transform our ability to offer precision therapy in non-Hodgkin lymphoma. PMID:27539624

  4. Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes.

    PubMed

    Traver, Sabine; Scalici, Elodie; Mullet, Tiffany; Molinari, Nicolas; Vincens, Claire; Anahory, Tal; Hamamah, Samir

    2015-01-01

    Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7-10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66-0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management. PMID:26288130

  5. A cell-free assay to determine the stoichiometry of plasma membrane proteins.

    PubMed

    Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

    2013-04-01

    Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

  6. Prenatal screening for fetal aneuploidies with cell-free DNA in the general pregnancy population: a cost-effectiveness analysis

    PubMed Central

    Fairbrother, Genevieve; Burigo, John; Sharon, Thomas; Song, Ken

    2016-01-01

    Abstract Objective: To estimate the cost-effectiveness of fetal aneuploidy screening in the general pregnancy population using non-invasive prenatal testing (NIPT) as compared to first trimester combined screening (FTS) with serum markers and NT ultrasound. Methods: Using a decision-analytic model, we estimated the number of fetal T21, T18, and T13 cases identified prenatally, the number of invasive procedures performed, corresponding normal fetus losses, and costs of screening using FTS or NIPT with cell-free DNA (cfDNA). Modeling was based on a 4 million pregnant women cohort, which represents annual births in the U.S. Results: For the general pregnancy population, NIPT identified 15% more trisomy cases, reduced invasive procedures by 88%, and reduced iatrogenic fetal loss by 94% as compared to FTS. The cost per trisomy case identified with FTS was $497 909. At a NIPT unit, cost of $453 and below, there were cost savings as compared to FTS. Accounting for additional trisomy cases identified by NIPT, a NIPT unit cost of $665 provided the same per trisomy cost as that of FTS. Conclusions: NIPT in the general pregnancy population leads to more prenatal identification of fetal trisomy cases as compared to FTS and is more economical at a NIPT unit cost of $453. PMID:26000626

  7. Silver Nanoscale Hexagonal Column Chips for Detecting Cell-free DNA and Circulating Nucleosomes in Cancer Patients

    PubMed Central

    Ito, Hiroaki; Hasegawa, Katsuyuki; Hasegawa, Yuuki; Nishimaki, Tadashi; Hosomichi, Kazuyoshi; Kimura, Satoshi; Ohba, Motoi; Yao, Hiroshi; Onimaru, Manabu; Inoue, Ituro; Inoue, Haruhiro

    2015-01-01

    Blood tests, which are commonly used for cancer screening, generally have low sensitivity. Here, we developed a novel rapid and simple method to generate silver nanoscale hexagonal columns (NHCs) for use in surface-enhanced Raman scattering (SERS). We reported that the intensity of SERS spectra of clinical serum samples obtained from gastrointestinal cancer patients is was significantly higher than that of SERS spectra of clinical serum samples obtained from non-cancer patients. We estimated the combined constituents on silver NHCs by using a field emission-type scanning electron microscope, Raman microscopes, and a 3D laser scanning confocal microscope. We obtained the Raman scattering spectra of samples of physically fractured cells and clinical serum. No spectra were obtained for chemically lysed cultured cells and DNA, RNA, and protein extracted from cultured cells. We believe that our method, which uses SERS with silver NHCs to detect circulating nucleosomes bound by methylated cell-free DNA, may be successfully implemented in blood tests for cancer screening. PMID:25994878

  8. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer

    PubMed Central

    MATTHAIOS, DIMITRIOS; BALGKOURANIDOU, IOANNA; KARAYIANNAKIS, ANASTASIOS; BOLANAKI, HELEN; XENIDIS, NIKOLAOS; AMARANTIDIS, KYRIAKOS; CHELIS, LEONIDAS; ROMANIDIS, KONSTANTINOS; CHATZAKI, AIKATERINI; LIANIDOU, EVI; TRYPSIANIS, GRIGORIOS; KAKOLYRIS, STYLIANOS

    2016-01-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with

  9. Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma

    PubMed Central

    Kirk, Jason L.; Merwat, Shehzad N.; Ju, Hyunsu; Soloway, Roger D.; Wieck, Lucas R.; Li, Albert; Okorodudu, Anthony O.; Petersen, John R.; Abdulla, Nihal E.; Duchini, Andrea; Cicalese, Luca; Rastellini, Cristiana; Hu, Peter C.; Dong, Jianli

    2015-01-01

    Background Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%–80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. Methods We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection. PMID:24406287

  10. Serial Next Generation Sequencing of Circulating Cell Free DNA Evaluating Tumour Clone Response To Molecularly Targeted Drug Administration

    PubMed Central

    Frenel, Jean Sebastien; Carreira, Suzanne; Goodall, Jane; Roda, Desam; Perez-Lopez, Raquel; Tunariu, Nina; Riisnaes, Ruth; Miranda, Susana; Figueiredo, Ines; NavaRodrigues, Daniel; Smith, Alan; Leux, Christophe; Garcia-Murillas, Isaac; Ferraldeschi, Roberta; Lorente, David; Mateo, Joaquin; Ong, Michael; Yap, Timothy A; Banerji, Udai; Tandefelt, Delila Gasi; Turner, Nick; Attard, Gerhardt; de Bono, Johann S

    2015-01-01

    Background We evaluated whether next generation sequencing (NGS) of cfDNA could be used for patient selection and as a tumor clone response biomarker in patients with advanced cancers participating in early phase clinical trials of targeted drugs. Methods Plasma samples from patients with known tumor mutations who completed at least 2 courses of investigational targeted therapy were collected monthly, until disease progression. NGS was performed sequentially on the Ion Torrent PGM platform. Results cfDNA was extracted from 39 patients with various tumor types. Treatments administered targeted mailnly the PI3K-AKT-mTOR pathway (n=28) or MEK (n=7). Overall 159 plasma samples were sequenced with a mean sequencing coverage achieved of 1,685X across experiments. At trial initiation (C1D1), 23 of 39 (59%) patients had at least one mutation identified in cfDNA (mean 2, range 1-5). TP53, PIK3CA and KRAS were the top 3 mutated genes identified, with 16 (39%), 9 (22%) and 8 (17%) different mutations, respectively. Out of these 23 patients, 13 received a targeted drug matching their tumor profile. For the 23 patients with cfDNA mutation at C1D1, the monitoring of mutation allele frequency (AF) in consecutive plasma samples during treatment with targeted drugs demonstrated potential treatment associated clonal responses. Longitudinal monitoring of cfDNA samples with multiple mutations indicated the presence of separate clones behaving discordantly. Molecular changes at cfDNA mutation level were associated with time to disease progression by RECIST criteria. Conclusion Targeted NGS of cfDNA has potential clinical utility to monitor the delivery of targeted therapies. PMID:26085511

  11. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation.

    PubMed

    Lowes, Lori E; Bratman, Scott V; Dittamore, Ryan; Done, Susan; Kelley, Shana O; Mai, Sabine; Morin, Ryan D; Wyatt, Alexander W; Allan, Alison L

    2016-01-01

    Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes. PMID:27618023

  12. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation

    PubMed Central

    Lowes, Lori E.; Bratman, Scott V.; Dittamore, Ryan; Done, Susan; Kelley, Shana O.; Mai, Sabine; Morin, Ryan D.; Wyatt, Alexander W.; Allan, Alison L.

    2016-01-01

    Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes. PMID:27618023

  13. Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation.

    PubMed

    Lowes, Lori E; Bratman, Scott V; Dittamore, Ryan; Done, Susan; Kelley, Shana O; Mai, Sabine; Morin, Ryan D; Wyatt, Alexander W; Allan, Alison L

    2016-09-08

    Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes.

  14. Single Nucleotide Polymorphism-Based Analysis of Cell-Free Fetal DNA in 3000 Cases from Germany and Austria

    PubMed Central

    Eiben, B.; Krapp, M.; Borth, H.; Kutur, N.; Kreiselmaier, P.; Glaubitz, R.; Deutinger, J.; Merz, E.

    2015-01-01

    Background & Patient: Data from 3 008 patients, who underwent single-nucleotide-polymorphism (SNP)-based noninvasive prenatal testing (NIPT) are presented. Method: The PanoramaTM test (Natera, San Carlos, CA) was used to analyze cell-free fetal DNA from maternal blood for trisomies 21, 18, and 13, triploidy and sex-chromosome aneuploidies. Result: In 2 942 (97.8%) cases, a result was obtained. The average fetal fraction was 10.2%. A high-risk result for fetal aneuploidy was made for 65 (2.2%) cases. In 59 (90.8%) of these cases, invasive testing confirmed the aneuploidy. There were 6 false-positive cases. In the false-positive group, the fetal fraction was significantly lower. The overall positive predictive value was 90.8%. No false-negative cases were reported but many patients in this study have not delivered yet. Therefore, exact data cannot be given for potential false-negative cases. Conclusion: SNP-based NIPT is a reliable screening method for evaluating the risk of aneuploidies of chromosomes 21, 18 and 13. By using NIPT, the number of invasive procedures may be reduced significantly compared to maternal age and first-trimester screening. PMID:27689149

  15. Single Nucleotide Polymorphism-Based Analysis of Cell-Free Fetal DNA in 3000 Cases from Germany and Austria

    PubMed Central

    Eiben, B.; Krapp, M.; Borth, H.; Kutur, N.; Kreiselmaier, P.; Glaubitz, R.; Deutinger, J.; Merz, E.

    2015-01-01

    Background & Patient: Data from 3 008 patients, who underwent single-nucleotide-polymorphism (SNP)-based noninvasive prenatal testing (NIPT) are presented. Method: The PanoramaTM test (Natera, San Carlos, CA) was used to analyze cell-free fetal DNA from maternal blood for trisomies 21, 18, and 13, triploidy and sex-chromosome aneuploidies. Result: In 2 942 (97.8%) cases, a result was obtained. The average fetal fraction was 10.2%. A high-risk result for fetal aneuploidy was made for 65 (2.2%) cases. In 59 (90.8%) of these cases, invasive testing confirmed the aneuploidy. There were 6 false-positive cases. In the false-positive group, the fetal fraction was significantly lower. The overall positive predictive value was 90.8%. No false-negative cases were reported but many patients in this study have not delivered yet. Therefore, exact data cannot be given for potential false-negative cases. Conclusion: SNP-based NIPT is a reliable screening method for evaluating the risk of aneuploidies of chromosomes 21, 18 and 13. By using NIPT, the number of invasive procedures may be reduced significantly compared to maternal age and first-trimester screening.

  16. An Economic Analysis of Cell-Free DNA Non-Invasive Prenatal Testing in the US General Pregnancy Population

    PubMed Central

    Benn, Peter; Curnow, Kirsten J.; Chapman, Steven; Michalopoulos, Steven N.; Hornberger, John; Rabinowitz, Matthew

    2015-01-01

    Objective Analyze the economic value of replacing conventional fetal aneuploidy screening approaches with non-invasive prenatal testing (NIPT) in the general pregnancy population. Methods Using decision-analysis modeling, we compared conventional screening to NIPT with cell-free DNA (cfDNA) analysis in the annual US pregnancy population. Sensitivity and specificity for fetal aneuploidies, trisomy 21, trisomy 18, trisomy 13, and monosomy X, were estimated using published data and modeling of both first- and second trimester screening. Costs were assigned for each prenatal test component and for an affected birth. The overall cost to the healthcare system considered screening costs, the number of aneuploid cases detected, invasive procedures performed, procedure-related euploid losses, and affected pregnancies averted. Sensitivity analyses evaluated the effect of variation in parameters. Costs were reported in 2014 US Dollars. Results Replacing conventional screening with NIPT would reduce healthcare costs if it can be provided for $744 or less in the general pregnancy population. The most influential variables were timing of screening entry, screening costs, and pregnancy termination rates. Of the 13,176 affected pregnancies undergoing screening, NIPT detected 96.5% (12,717/13,176) of cases, compared with 85.9% (11,314/13,176) by conventional approaches. NIPT reduced invasive procedures by 60.0%, with NIPT and conventional methods resulting in 24,596 and 61,430 invasive procedures, respectively. The number of procedure-related euploid fetal losses was reduced by 73.5% (194/264) in the general screening population. Conclusion Based on our analysis, universal application of NIPT would increase fetal aneuploidy detection rates and can be economically justified. Offering this testing to all pregnant women is associated with substantial prenatal healthcare benefits. PMID:26158465

  17. National decline in invasive prenatal diagnostic procedures in association with uptake of combined first trimester and cell-free DNA aneuploidy screening.

    PubMed

    Robson, Stephen J; Hui, Lisa

    2015-10-01

    In late 2012, a new screening test for fetal aneuploidy based on circulating cell-free DNA (cfDNA) became available to Australian women. The introduction of this technology in the United States has led to a reduction in invasive diagnostic procedures. Analysis of the number of amniocentesis and chorionic villus sampling (CVS) procedures performed in Australia from 1994 to 2014 shows that the introduction of cfDNA testing has been associated with the most rapid decline in invasive procedures in the last 20 years. This change has important implications for training in, and maintenance of, the procedural skills of amniocentesis and CVS. PMID:26259499

  18. National decline in invasive prenatal diagnostic procedures in association with uptake of combined first trimester and cell-free DNA aneuploidy screening.

    PubMed

    Robson, Stephen J; Hui, Lisa

    2015-10-01

    In late 2012, a new screening test for fetal aneuploidy based on circulating cell-free DNA (cfDNA) became available to Australian women. The introduction of this technology in the United States has led to a reduction in invasive diagnostic procedures. Analysis of the number of amniocentesis and chorionic villus sampling (CVS) procedures performed in Australia from 1994 to 2014 shows that the introduction of cfDNA testing has been associated with the most rapid decline in invasive procedures in the last 20 years. This change has important implications for training in, and maintenance of, the procedural skills of amniocentesis and CVS.

  19. Testing for oncogenic molecular aberrations in cell-free DNA-based liquid biopsies in the clinic: are we there yet?

    PubMed

    Polivka, Jiri; Pesta, Martin; Janku, Filip

    2015-01-01

    The optimal choice of cancer therapy depends upon analysis of the tumor genome for druggable molecular alterations. The spatial and temporal intratumor heterogeneity of cancers creates substantial challenges, as molecular profile depends on time and site of tumor tissue collection. To capture the entire molecular profile, multiple biopsies from primary and metastatic sites at different time points would be required, which is not feasible for ethical or economic reasons. Molecular analysis of circulating cell-free DNA offers a novel, minimally invasive method that can be performed at multiple time-points and plausibly better represents the prevailing molecular profile of the cancer. Molecular analysis of this cell-free DNA offers multiple clinically useful applications, such as identification of molecular targets for cancer therapy, monitoring of tumor molecular profile in real time, detection of emerging molecular aberrations associated with resistance to particular therapy, determination of cancer prognosis and diagnosis of cancer recurrence or progression.

  20. Value of Quantitative and Qualitative Analyses of Circulating Cell-Free DNA as Diagnostic Tools for Hepatocellular Carcinoma

    PubMed Central

    Liao, Wenjun; Mao, Yilei; Ge, Penglei; Yang, Huayu; Xu, Haifeng; Lu, Xin; Sang, Xinting; Zhong, Shouxian

    2015-01-01

    Abstract Qualitative and quantitative analyses of circulating cell-free DNA (cfDNA) are potential methods for the detection of hepatocellular carcinoma (HCC). Many studies have evaluated these approaches, but the results have been variable. This meta-analysis is the first to synthesize these published results and evaluate the use of circulating cfDNA values for HCC diagnosis. All articles that met our inclusion criteria were assessed using QUADAS guidelines after the literature research. We also investigated 3 subgroups in this meta-analysis: qualitative analysis of abnormal concentrations of circulating cfDNA; qualitative analysis of single-gene methylation alterations; and multiple analyses combined with alpha-fetoprotein (AFP). Statistical analyses were performed using the software Stata 12.0. We synthesized these published results and calculated accuracy measures (pooled sensitivity and specificity, positive/negative likelihood ratios [PLRs/NLRs], diagnostic odds ratios [DORs], and corresponding 95% confidence intervals [95% CIs]). Data were pooled using bivariate generalized linear mixed model. Furthermore, summary receiver operating characteristic curves and area under the curve (AUC) were used to summarize overall test performance. Heterogeneity and publication bias were also examined. A total of 2424 subjects included 1280 HCC patients in 22 studies were recruited in this meta-analysis. Pooled sensitivity and specificity, PLR, NLR, DOR, AUC, and CIs of quantitative analysis were 0.741 (95% CI: 0.610–0.840), 0.851 (95% CI: 0.718–0.927), 4.970 (95% CI: 2.694–9.169), 0.304 (95% CI: 0.205–0.451), 16.347 (95% CI: 8.250–32.388), and 0.86 (95% CI: 0.83–0.89), respectively. For qualitative analysis, the values were 0.538 (95% CI: 0.401–0.669), 0.944 (95% CI: 0.889–0.972), 9.545 (95% CI: 5.298–17.196), 0.490 (95% CI: 0.372–0.646), 19.491 (95% CI: 10.458–36.329), and 0.87 (95% CI: 0.84–0.90), respectively. After combining with AFP assay, the

  1. Comparison of HIV type 1 sequences from plasma, cell-free breast milk, and cell-associated breast milk viral populations in treated and untreated women in Mozambique.

    PubMed

    Andreotti, Mauro; Galluzzo, Clementina M; Guidotti, Giovanni; Germano, Paola; Altan, Annamaria Doro; Pirillo, Maria Franca; Marazzi, Maria Cristina; Vella, Stefano; Palombi, Leonardo; Giuliano, Marina

    2009-07-01

    We analyzed the sequences of the HIV viral populations obtained from plasma, cell-free breast milk, and breast milk cells of HAART-treated (23) and untreated (30) HIV-infected women to obtain information about the origin of the breast milk virus. Sequence analyses of viruses were performed using the TruGene HIV-1 assay. Direct sequences of the reverse transcriptase (RT) and protease (PR) genes were analyzed using the Phylip 3.68 suite of sequence analysis program and pairwise evolutionary distances were calculated with the Kimura two parameter model for estimation of distances. We found that the genetic distances between the plasma and the cell-free breast milk viruses and between the cell-free and cell-associated breast milk viruses for RT were higher in HAART-receiving women than in untreated women, suggesting viral evolution under selective drug pressure in breast milk. Our data support the hypothesis of the presence of an actively replicating viral population in the breast milk compartment, distinct from that present in plasma. PMID:19552594

  2. Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system.

    PubMed

    Sun, Zachary Z; Yeung, Enoch; Hayes, Clarmyra A; Noireaux, Vincent; Murray, Richard M

    2014-06-20

    Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models.

  3. Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors.

    PubMed

    Yan, Weixin; Zhang, Aiguo; Powell, Michael J

    2016-07-21

    Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived "driver" and "drug-resistant" alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called "liquid biopsy" allows for the dynamic monitoring of the patients' tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

  4. Noninvasive Prenatal Screening for Genetic Diseases Using Massively Parallel Sequencing of Maternal Plasma DNA.

    PubMed

    Chitty, Lyn S; Lo, Y M Dennis

    2015-01-01

    The identification of cell-free fetal DNA (cffDNA) in maternal plasma in 1997 heralded the most significant change in obstetric care for decades, with the advent of safer screening and diagnosis based on analysis of maternal blood. Here, we describe how the technological advances offered by next-generation sequencing have allowed for the development of a highly sensitive screening test for aneuploidies as well as definitive prenatal molecular diagnosis for some monogenic disorders. PMID:26187875

  5. Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.

    PubMed

    Hsu, M Y; Eagle, S G; Inouye, M; Inouye, S

    1992-07-15

    msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent

  6. Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma.

    PubMed

    De Mattos-Arruda, Leticia; Mayor, Regina; Ng, Charlotte K Y; Weigelt, Britta; Martínez-Ricarte, Francisco; Torrejon, Davis; Oliveira, Mafalda; Arias, Alexandra; Raventos, Carolina; Tang, Jiabin; Guerini-Rocco, Elena; Martínez-Sáez, Elena; Lois, Sergio; Marín, Oscar; de la Cruz, Xavier; Piscuoglio, Salvatore; Towers, Russel; Vivancos, Ana; Peg, Vicente; Ramon y Cajal, Santiago; Carles, Joan; Rodon, Jordi; González-Cao, María; Tabernero, Josep; Felip, Enriqueta; Sahuquillo, Joan; Berger, Michael F; Cortes, Javier; Reis-Filho, Jorge S; Seoane, Joan

    2015-11-10

    Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis.

  7. Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures.

    PubMed

    Hamprecht, K; Mikeler, E; Jahn, G

    1997-12-01

    The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.

  8. Noninvasive prenatal testing using cell-free fetal DNA in maternal plasma.

    PubMed

    Dharajiya, Nilesh; Zwiefelhofer, Tricia; Guan, Xiaojun; Angkachatchai, Vach; Saldivar, Juan-Sebastian

    2015-01-20

    Noninvasive prenatal testing (NIPT) represents an outstanding example of how novel scientific discoveries can be quickly and successfully developed into hugely impactful clinical diagnostic tests. Since the introduction of NIPT to detect trisomy 21 in late 2011, the technology has rapidly advanced to analyze other autosomal and sex chromosome aneuploidies, and now includes the detection of subchromosomal deletion and duplication events. Here we provide a brief overview of how noninvasive prenatal testing using next-generation sequencing is performed.

  9. Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

    PubMed Central

    Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

    2015-01-01

    To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

  10. Prognostic and diagnostic value of eosinopenia, C-reactive protein, procalcitonin, and circulating cell-free DNA in critically ill patients admitted with suspicion of sepsis

    PubMed Central

    2014-01-01

    Introduction The aims of this study were to assess the reliability of circulating cell-free DNA (cf-DNA) concentrations, compared with C-reactive protein (CRP), procalcitonin (PCT) and eosinophil count, in the diagnosis of infections in patients with systemic inflammatory response syndrome (SIRS) and their prognostic values in a cohort of critically ill patients. Methods We conducted a prospective cohort study in a medical-surgical intensive care unit of a university hospital. Eosinophil count and concentrations of cf-DNA, CRP, and PCT were measured in patients who fulfilled SIRS criteria at admission to the intensive care unit (ICU) and a second determination 24 hours later. DNA levels were determined by a PCR method using primers for the human beta-haemoglobin gene. Results One hundred and sixty consecutive patients were included: 43 SIRS without sepsis and 117 with sepsis. Levels of CRP and PCT, but not cf-DNA or eosinophil count, were significantly higher in patients with sepsis than in SIRS-no sepsis group on days 1 and 2. PCT on day 1 achieves the best area under the curve (AUC) for sepsis diagnosis (0.87; 95% confidence interval = 0.81-0.94). Levels of cf-DNA do not predict outcome and the accuracy of these biomarkers for mortality prediction was lower than that shown by APACHE II score. PCT decreases significantly from day 1 to day 2 in survivors in the entire cohort and in patients with sepsis without significant changes in the other biomarkers. Conclusions Our data do not support the clinical utility of cf-DNA measurement in critical care patients with SIRS. PCT is of value especially for infection identification in patients with SIRS at admission to the ICU. PMID:24903083

  11. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    PubMed

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

  12. Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

    PubMed

    Tamminga, Saskia; van Maarle, Merel; Henneman, Lidewij; Oudejans, Cees B M; Cornel, Martina C; Sistermans, Erik A

    2016-01-01

    Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA. PMID:27117661

  13. Accuracy of non-invasive prenatal testing using cell-free DNA for detection of Down, Edwards and Patau syndromes: a systematic review and meta-analysis

    PubMed Central

    Taylor-Phillips, Sian; Freeman, Karoline; Geppert, Julia; Agbebiyi, Adeola; Uthman, Olalekan A; Madan, Jason; Clarke, Angus; Quenby, Siobhan; Clarke, Aileen

    2016-01-01

    Objective To measure test accuracy of non-invasive prenatal testing (NIPT) for Down, Edwards and Patau syndromes using cell-free fetal DNA and identify factors affecting accuracy. Design Systematic review and meta-analysis of published studies. Data sources PubMed, Ovid Medline, Ovid Embase and the Cochrane Library published from 1997 to 9 February 2015, followed by weekly autoalerts until 1 April 2015. Eligibility criteria for selecting studies English language journal articles describing case–control studies with ≥15 trisomy cases or cohort studies with ≥50 pregnant women who had been given NIPT and a reference standard. Results 41, 37 and 30 studies of 2012 publications retrieved were included in the review for Down, Edwards and Patau syndromes. Quality appraisal identified high risk of bias in included studies, funnel plots showed evidence of publication bias. Pooled sensitivity was 99.3% (95% CI 98.9% to 99.6%) for Down, 97.4% (95.8% to 98.4%) for Edwards, and 97.4% (86.1% to 99.6%) for Patau syndrome. The pooled specificity was 99.9% (99.9% to 100%) for all three trisomies. In 100 000 pregnancies in the general obstetric population we would expect 417, 89 and 40 cases of Downs, Edwards and Patau syndromes to be detected by NIPT, with 94, 154 and 42 false positive results. Sensitivity was lower in twin than singleton pregnancies, reduced by 9% for Down, 28% for Edwards and 22% for Patau syndrome. Pooled sensitivity was also lower in the first trimester of pregnancy, in studies in the general obstetric population, and in cohort studies with consecutive enrolment. Conclusions NIPT using cell-free fetal DNA has very high sensitivity and specificity for Down syndrome, with slightly lower sensitivity for Edwards and Patau syndrome. However, it is not 100% accurate and should not be used as a final diagnosis for positive cases. Trial registration number CRD42014014947. PMID:26781507

  14. Coexposure to benzo[a]pyrene and UVA induces DNA damage: first proof of double-strand breaks in a cell-free system.

    PubMed

    Toyooka, Tatsushi; Ibuki, Yuko; Takabayashi, Fumiyo; Goto, Rensuke

    2006-01-01

    DNA damage induced by solar ultraviolet (UV) radiation plays an important role in the induction of skin cancer. Although UVA constitutes the majority of solar UV radiation, it is less damaging to DNA than UVB. The DNA damage produced by UVA radiation, however, can be augmented in the presence of a photosensitizer. We previously used benzo[a]pyrene (BaP), an environmental carcinogenic polycyclic aromatic hydrocarbon, as an exogenous photosensitizer, and demonstrated that combined exposure to BaP and UVA resulted in DNA double-strand breaks (DSBs) in cultured Chinese hamster ovary (CHO-K1) cells. In this study, we investigated whether coexposure to BaP and UVA induces DSBs in a cell-free system and whether reactive oxygen species (ROS) were involved in the generation of the DSBs. DSBs were induced by the coexposure both in the cell-free system (in vitro) and in CHO-K1 cells (in vivo), but not by treatment with BaP or UVA alone. DSB induction in vitro required higher doses of UVA and BaP than were required in vivo, suggesting that the mechanism of DSB induction differed. A similar difference in efficiency also was observed in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by coexposure to BaP and UVA in vitro and in vivo. A singlet oxygen ((1)O2) scavenger (NaN3) effectively inhibited the production of DSBs and 8-oxodG, suggesting that (1)O2 is a principal ROS generated by BaP and UVA both in vitro and in vivo. Furthermore, repair-deficient xrs-5 cells were more sensitive to coexposure with BaP and UVA than were CHO-K1 cells, but the two cell lines were equally sensitive to the combined treatment in the presence of NaN3. This result suggested that the cell death produced by coexposure to BaP and UVA was at least partly due to the DSBs generated by (1)O2. Our findings indicate that coexposure to BaP and UVA effectively induced DNA damage, especially DSBs, which results in phototoxicity and possibly photocarcinogenesis.

  15. Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

    PubMed Central

    1993-01-01

    Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation. PMID:8253833

  16. Quantitative and Qualitative Analysis of Circulating Cell-Free DNA Can Be Used as an Adjuvant Tool for Prostate Cancer Screening: A Meta-Analysis

    PubMed Central

    Luo, Changliang; Hu, Wei; Ding, Xu; Yuan, Chunhui

    2016-01-01

    As part of “liquid biopsy,” lots of literature indicated the potential diagnostic value of circulating cell-free DNA (cfDNA) in the management of prostate cancer (PCa). However, the literature on the accuracy of cfDNA detection in PCa has been inconsistent. Hence, we performed this meta-analysis to assess the diagnostic value of cfDNA in PCa. A total of 19 articles were included in this analysis according to the inclusion and exclusion criteria. We then investigated two main subgroups in this meta-analysis, including qualitative analysis of abnormal level of cfDNA and qualitative analysis of single-gene methylation alterations. Overall, the results of quantitative analysis showed sensitivity of 0.73 (95% CI, 0.62–0.82) and specificity of 0.80 (95% CI, 0.70–0.87), with an area under the curve (AUC) of 0.83 (95% CI, 0.80–0.86). For qualitative assessment, the values were 0.34 (95% CI, 0.22–0.48), 0.99 (95% CI, 0.97–1.00), and 0.91 (95% CI, 0.88–0.93), respectively. Our results suggest the pooled specificity of each subgroup is much higher than the specificity of prostate-specific antigen (PSA). However, they are not recommended for PCa screening alone, because their sensitivities are not higher than the conventional serum biomarkers PSA. We conclude that analysis of cfDNA can be used as an adjuvant tool for PCa screening. PMID:27766004

  17. Circulating cell-free mitochondrial DNA as the probable inducer of early endothelial dysfunction in the prediabetic patient.

    PubMed

    Alvarado-Vásquez, Noé

    2015-09-01

    Recent evidence has shown that 346million people in the world have diabetes mellitus (DM); this number will increase to 439million by 2030. In addition, current data indicate an increase in DM cases in the population between 40 and 59years of age. Diabetes is associated with the development of micro- and macro-vascular complications, derived from chronic hyperglycemia on the endothelium. Some reports demonstrate that people in a prediabetic state have a major risk of developing early endothelial dysfunction (ED). Today, it is accepted that individuals considered as prediabetic patients are in a pro-inflammatory state associated with endothelial and mitochondrial dysfunction. It is important to mention that impaired mitochondrial functionality has been linked to endothelial apoptosis and release of mitochondrial DNA (mtDNA) in patients with sepsis, cardiac disease, or atherosclerosis. This free mtDNA could promote ED, as well as other side effects on the vascular system through the activation of the toll-like receptor 9 (TLR9). TLR9 is expressed in different cell types (e.g., T or B lymphocytes, mastocytes, and epithelial and endothelial cells). It is localized intracellularly and recognizes non-methylated dinucleotides of viral, bacterial, and mitochondrial DNA. Recently, it has been reported that TLR9 is associated with the pathogenesis of lupus erythematosus, rheumatoid arthritis, and autoimmune diabetes. In this work, it is hypothesized that the increase in the levels of circulating mtDNA is the trigger of early ED in the prediabetic patient, and later on in the older patient with diabetes, through activation of the TLR9 present in the endothelium. PMID:26026597

  18. Calcium fluxes across the plasma membrane of Commelina communis L. assayed in a cell-free system

    SciTech Connect

    Siebers, B.; Graef, P.; Weiler, E.W. )

    1990-07-01

    The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous two-phase partitioning was loaded with {sup 45}Ca{sup 2+} through the action of the plasma membrane Ca{sup 2+}-ATPase. Results suggest the presence of a Ca{sup 2+} channel in the plasma membrane of C. communis. The channel is obtained in a Ca{sup 2+}-inactivated state after preparation and Ca{sup 2+}-loading of the vesicles. The inactivation is removed by TFP (trifluoperazine) or W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), presumably due to the Ca{sup 2+}-mobilizing effect of these compounds. The activated Ca{sup 2+} channel is La{sup 3+} sensitive and, in the cell, would allow for passage of Ca{sup 2+} into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed.

  19. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA – Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis

    PubMed Central

    Hatt, Lotte; Aagaard, Mads M.; Graakjaer, Jesper; Bach, Cathrine; Sommer, Steffen; Agerholm, Inge E.; Kølvraa, Steen; Bojesen, Anders

    2015-01-01

    Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE) in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes. PMID:26230497

  20. Microarray-Based Analysis of Methylation Status of CpGs in Placental DNA and Maternal Blood DNA--Potential New Epigenetic Biomarkers for Cell Free Fetal DNA-Based Diagnosis.

    PubMed

    Hatt, Lotte; Aagaard, Mads M; Graakjaer, Jesper; Bach, Cathrine; Sommer, Steffen; Agerholm, Inge E; Kølvraa, Steen; Bojesen, Anders

    2015-01-01

    Epigenetic markers for cell free fetal DNA in the maternal blood circulation are highly interesting in the field of non-invasive prenatal testing since such markers will offer a possibility to quantify the amount of fetal DNA derived from different chromosomes in a maternal blood sample. The aim of the present study was to define new fetal specific epigenetic markers present in placental DNA that can be utilized in non-invasive prenatal diagnosis. We have conducted a high-resolution methylation specific beadchip microarray study assessing more than 450.000 CpG sites. We have analyzed the DNA methylation profiles of 10 maternal blood samples and compared them to 12 1st trimesters chorionic samples from normal placentas, identifying a number of CpG sites that are differentially methylated in maternal blood cells compared to chorionic tissue. To strengthen the utility of these differentially methylated CpG sites to be used with methyl-sensitive restriction enzymes (MSRE) in PCR-based NIPD, we furthermore refined the list of selected sites, containing a restriction sites for one of 16 different methylation-sensitive restriction enzymes. We present a list of markers on chromosomes 13, 18 and 21 with a potential for aneuploidy testing as well as a list of markers for regions harboring sub-microscopic deletion- or duplication syndromes.

  1. Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA.

    PubMed

    Yin, Ai-hua; Peng, Chun-fang; Zhao, Xin; Caughey, Bennett A; Yang, Jie-xia; Liu, Jian; Huang, Wei-wei; Liu, Chang; Luo, Dong-hong; Liu, Hai-liang; Chen, Yang-yi; Wu, Jing; Hou, Rui; Zhang, Mindy; Ai, Michael; Zheng, Lianghong; Xue, Rachel Q; Mai, Ming-qin; Guo, Fang-fang; Qi, Yi-ming; Wang, Dong-mei; Krawczyk, Michal; Zhang, Daniel; Wang, Yu-nan; Huang, Quan-fei; Karin, Michael; Zhang, Kang

    2015-11-24

    Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer. PMID:26554006

  2. Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA.

    PubMed

    Yin, Ai-hua; Peng, Chun-fang; Zhao, Xin; Caughey, Bennett A; Yang, Jie-xia; Liu, Jian; Huang, Wei-wei; Liu, Chang; Luo, Dong-hong; Liu, Hai-liang; Chen, Yang-yi; Wu, Jing; Hou, Rui; Zhang, Mindy; Ai, Michael; Zheng, Lianghong; Xue, Rachel Q; Mai, Ming-qin; Guo, Fang-fang; Qi, Yi-ming; Wang, Dong-mei; Krawczyk, Michal; Zhang, Daniel; Wang, Yu-nan; Huang, Quan-fei; Karin, Michael; Zhang, Kang

    2015-11-24

    Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.

  3. Noninvasive detection of fetal subchromosomal abnormalities by semiconductor sequencing of maternal plasma DNA

    PubMed Central

    Yin, Ai-hua; Peng, Chun-fang; Zhao, Xin; Caughey, Bennett A.; Yang, Jie-xia; Liu, Jian; Huang, Wei-wei; Liu, Chang; Luo, Dong-hong; Liu, Hai-liang; Chen, Yang-yi; Wu, Jing; Hou, Rui; Zhang, Mindy; Ai, Michael; Zheng, Lianghong; Xue, Rachel Q.; Mai, Ming-qin; Guo, Fang-fang; Qi, Yi-ming; Wang, Dong-mei; Krawczyk, Michal; Zhang, Daniel; Wang, Yu-nan; Huang, Quan-fei; Karin, Michael; Zhang, Kang

    2015-01-01

    Noninvasive prenatal testing (NIPT) using sequencing of fetal cell-free DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer. PMID:26554006

  4. Fetal RHD Genotyping Using Real-Time Polymerase Chain Reaction Analysis of Cell-Free Fetal DNA in Pregnancy of RhD Negative Women in South of Iran

    PubMed Central

    Moezzi, Leili; Keshavarz, Zeinab; Ranjbaran, Reza; Aboualizadeh, Farzaneh; Behzad-Behbahani, Abbas; Abdullahi, Masooma; Ramezani, Amin; Samsami, Alamtaj; Sharifzadeh, Sedigheh

    2016-01-01

    Background Maternal-fetal RhD antigen incompatibility causes approximately 50% of clinically significant alloimmunization cases. The routine use of prophylactic anti-D immunoglobulin has dramatically reduced hemolytic disease of the fetus and newborn. Recently, fetal RHD genotyping in RhD negative pregnant women has been suggested for appropriate use of anti-D immunoglobulin antenatal prophylaxis and decrease unnecessary prenatal interventions. Materials and Methods In this prospective cohort study, in order to develop a reliable and non-invasive method for fetal RHD genotyping, cell free fetal DNA (cffD- NA) was extracted from maternal plasma. Real-time quantitative polymerase chain reaction (qPCR) for detection of RHD exons 7, 5, 10 and intron 4 was performed and the results were compared to the serological results of cord blood cells as the gold standard method. SRY gene and hypermethylated Ras-association domain family member 1 (RASSF1A) gene were used to confirm the presence of fetal DNA in male and female fetuses, respectively. Results Out of 48 fetuses between 8 and 32 weeks (wks) of gestational age (GA), we correctly diagnosed 45 cases (93.75%) of RHD positive fetuses and 2 cases (4.16%) of the RHD negative one. Exon 7 was amplified in one sample, while three other RHD gene sequences were not detected; the sample was classified as inconclusive, and the RhD serology result after birth showed that the fetus was RhD-negative. Conclusion Our results showed high accuracy of the qPCR method using cffDNA for fetal RHD genotyping and implicate on the efficiency of this technique to predict the competence of anti-D immunoglobulin administration. PMID:27123202

  5. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    PubMed

    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. PMID:26073472

  6. Routine testing of fetal Rhesus D status in Rhesus D negative women using cell-free fetal DNA: an investigation into the preferences and information needs of women

    PubMed Central

    Oxenford, Kerry; Silcock, Caroline; Hill, Melissa; Chitty, Lyn

    2013-01-01

    Objective The goal of this study is to investigate women's preferences and information needs for routine implementation of fetal Rhesus D (RhD) typing using cell-free fetal DNA. Methods A questionnaire was developed following focus groups and interviews with both health professionals and RhD negative (RhD−) women offered fetal RhD genotyping within a research study and distributed to RhD− women attending routine antenatal appointments in four National Health Service hospitals. Current knowledge of blood types, anti-D administration, fetal RhD genotyping and future practices were explored. Results A total of 19 respondents participated in interviews and focus groups, and 270 respondents completed the questionnaires. Questionnaire respondents overwhelmingly felt that the test should be offered to all RhD− women (92.1%), and 75.9% said that they would accept this test. Most were happy to have the test even if it involved extra blood tests (89.3%) or appointments (79%). The knowledge of blood groups was poor. Although 90.7% knew that the baby could have a different blood group from themselves, only 34% knew that blood groups are inherited from both parents. More than 40% were not aware that anti-D would not be required if their baby was RhD−. Conclusions Women would welcome the introduction of routine fetal RhD genotyping. Information leaflets and training of midwives will be essential for implementation to ensure good understanding regarding testing. © 2013 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd. PMID:23625761

  7. Clinical applications of maternal plasma fetal DNA analysis: translating the fruits of 15 years of research.

    PubMed

    Chiu, Rossa Wai Kwun; Lo, Yuk Ming Dennis

    2013-01-01

    The collection of fetal genetic materials is required for the prenatal diagnosis of fetal genetic diseases. The conventional methods for sampling fetal genetic materials, such as amniocentesis and chorionic villus sampling, are invasive in nature and are associated with a risk of fetal miscarriage. For decades, scientists had been pursuing studies with goals to develop non-invasive methods for prenatal diagnosis. In 1997, the existence of fetal derived cell-free DNA molecules in plasma of pregnant women was first demonstrated. This finding provided a new source of fetal genetic material that could be obtained safely through the collection of a maternal blood sample and provided a new avenue for the development of non-invasive prenatal diagnostic tests. Now 15 years later, the diagnostic potential of circulating fetal DNA analysis has been realized. Fruitful research efforts have resulted in the clinical implementation of a number of non-invasive prenatal tests based on maternal plasma DNA analysis and included tests for fetal sex assessment, fetal rhesus D blood group genotyping and fetal chromosomal aneuploidy detection. Most recently, research groups have succeeded in decoding the entire fetal genome from maternal plasma DNA analysis which paved the way for the achievement of non-invasive prenatal diagnosis of many single gene diseases. A paradigm shift in the practice of prenatal diagnosis has begun.

  8. Circulating cell-free DNA has a high degree of specificity to detect exon 19 deletions and the single-point substitution mutation L858R in non-small cell lung cancer

    PubMed Central

    Wang, Meifang; Lei, Huaiding; Luo, Guoshi; Liu, Xianjun; Xiong, Chang; Liu, Dan; Liu, Jie; Tang, Yijun

    2016-01-01

    Detection of an epidermal growth factor receptor (EGFR) mutation in circulating cell-free DNA (cfDNA) is a noninvasive method to collect genetic information to guide treatment of lung cancer with tyrosine-kinase inhibitors (TKIs). However, the association between cfDNA and detection of EGFR mutations in tumor tissue remains unclear. Here, a meta-analysis was performed to determine whether cfDNA could serve as a substitute for tissue specimens for the detection of EGFR mutations. The pooled sensitivity, specificity, and areas under the curve of cfDNA were 0.60, 0.94, and 0.9208 for the detection of EGFR mutations, 0.64, 0.99, and 0.9583 for detection of the exon 19 deletion, and 0.57, 0.99, and 0.9605 for the detection of the L858R mutation, respectively. Our results showed that cfDNA has a high degree of specificity to detect exon 19 deletions and L858R mutation. Due to its high specificity and noninvasive characteristics, cfDNA analysis presents a promising method to screen for mutations in NSCLC and predict patient response to EGFR-TKI treatment, dynamically assess treatment outcome, and facilitate early detection of resistance mutations. PMID:27081078

  9. Increased levels of cell-free human placental lactogen mRNA at 28-32 gestational weeks in plasma of pregnant women with placenta previa and invasive placenta.

    PubMed

    Kawashima, Akihiro; Sekizawa, Akihiko; Ventura, Walter; Koide, Keiko; Hori, Kyouko; Okai, Takashi; Masashi, Yoshida; Furuya, Kenichi; Mizumoto, Yoshifumi

    2014-02-01

    We compared the levels of cell-free human placental lactogen (hPL) messenger RNA (mRNA) in maternal plasma at 28 to 32 weeks of gestation between women with diagnosis of placenta previa or invasive placenta and women with an uneventful pregnancy. Sensitivity and specificity of hPL mRNA for the prediction of invasive placenta were further explored. Plasma hPL mRNA were quantified by real-time reverse-transcriptase polymerase chain reaction in women with placenta previa (n = 13), invasive placenta (n = 5), and normal pregnancies (n = 92). Median (range) hPL mRNA was significantly higher in women with placenta previa, 782 (10-2301) copies/mL of plasma, and in those with invasive placenta, 615 (522-2102) copies/mL of plasma, when compared to normal pregnancies, 90 (4-4407) copies/mL of plasma, P < .01 and P < .05, respectively. We found a sensitivity of 100% and a specificity of 61.5% for the prediction of invasive placenta among women with placenta previa. In conclusion, expression of hPL mRNA is increased in plasma of women with placenta previa and invasive placenta at 28 to 32 weeks of gestation.

  10. Raman spectroscopic study of plasma-treated salmon DNA

    SciTech Connect

    Lee, Geon Joon; Kim, Yong Hee; Choi, Eun Ha; Kwon, Young-Wan

    2013-01-14

    In this research, we studied the effect of plasma treatment on the optical/structural properties of the deoxyribonucleic acid (DNA) extracted from salmon sperm. DNA-cetyltrimethylammonium (CTMA) films were obtained by complexation of DNA with CTMA. Circular dichroism (CD) and Raman spectra indicated that DNA retained its double helical structure in the solid film. The Raman spectra exhibited several vibration modes corresponding to the nuclear bases and the deoxyribose-phosphate backbones of the DNA, as well as the alkylchains of CTMA. Dielectric-barrier-discharge (DBD) plasma treatment induced structural modification and damage to the DNA, as observed by changes in the ultraviolet-visible absorption, CD, and Raman spectra. The optical emission spectra of the DBD plasma confirmed that DNA modification was induced by plasma ions such as reactive oxygen species and reactive nitrogen species.

  11. The extended version of restriction analysis approach for the examination of the ability of low-molecular-weight compounds to modify DNA in a cell-free system.

    PubMed

    Kołodziejski, Dominik; Brillowska-Dąbrowska, Anna; Bartoszek, Agnieszka

    2015-01-01

    One of the primary requirements in toxicology is the assessment of ability of chemicals to induce DNA covalent modification. There are several well-established methods used for this purpose such as (32)P-Postlabeling or HPLC-MS. However, all of these approaches have difficult to overcome limitations, which prevents their use in genotoxin screening. Here, we describe the simple protocol exploiting specificity of restriction enzymes for the detection of DNA modification. It uses a specifically designed DNA amplicon, which contains two restriction sites recognized by Tru1I or MspI/HapII endonucleases. Modification of a restriction site abolishes its recognition and thus cleavage by the corresponding enzyme. The inhibition of cleavage indicates the occurrence of DNA modification of the restriction site(s), simultaneously pointing at the kind of base pairs (AT or GC) involved in DNA adduct formation. Previously, the application of this method was demonstrated for two antitumor compounds. Current study shows the extended version, that includes different ways of activation of tested compounds. Moreover, we propose an array of applications being of interest in toxicological research such as monitoring the kinetics of DNA adduct formation, detection of oxidative DNA damage, as well as assessment of the ability of antioxidative phytochemicals to prevent the latter DNA lesions.

  12. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  13. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma.

    PubMed

    Lun, Fiona M F; Tsui, Nancy B Y; Chan, K C Allen; Leung, Tak Y; Lau, Tze K; Charoenkwan, Pimlak; Chow, Katherine C K; Lo, Wyatt Y W; Wanapirak, Chanane; Sanguansermsri, Torpong; Cantor, Charles R; Chiu, Rossa W K; Lo, Y M Dennis

    2008-12-16

    Prenatal diagnosis of monogenic diseases, such as cystic fibrosis and beta-thalassemia, is currently offered as part of public health programs. However, current methods based on chorionic villus sampling and amniocentesis for obtaining fetal genetic material pose a risk to the fetus. Since the discovery of cell-free fetal DNA in maternal plasma, the noninvasive prenatal assessment of paternally inherited traits or mutations has been achieved. Due to the presence of background maternal DNA, which interferes with the analysis of fetal DNA in maternal plasma, noninvasive prenatal diagnosis of maternally inherited mutations has not been possible. Here we describe a digital relative mutation dosage (RMD) approach that determines if the dosages of the mutant and wild-type alleles of a disease-causing gene are balanced or unbalanced in maternal plasma. When applied to the testing of women heterozygous for the CD41/42 (-CTTT) and hemoglobin E mutations on HBB, digital RMD allows the fetal genotype to be deduced. The diagnostic performance of digital RMD is dependent on interplay between the fractional fetal DNA concentration and number of DNA molecules in maternal plasma. To achieve fetal genotype diagnosis at lower volumes of maternal plasma, fetal DNA enrichment is desired. We thus developed a digital nucleic acid size selection (NASS) strategy that effectively enriches the fetal DNA without additional plasma sampling or experimental time. We show that digital NASS can work in concert with digital RMD to increase the proportion of cases with classifiable fetal genotypes and to bring noninvasive prenatal diagnosis of monogenic diseases closer to reality.

  14. Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing.

    PubMed

    Lo, Y M Dennis

    2013-12-01

    The discovery of cell-free fetal DNA in maternal plasma in 1997 has stimulated a rapid development of non-invasive prenatal testing. The recent advent of massively parallel sequencing has allowed the analysis of circulating cell-free fetal DNA to be performed with unprecedented sensitivity and precision. Fetal trisomies 21, 18 and 13 are now robustly detectable in maternal plasma and such analyses have been available clinically since 2011. Fetal genome-wide molecular karyotyping and whole-genome sequencing have now been demonstrated in a number of proof-of-concept studies. Genome-wide and targeted sequencing of maternal plasma has been shown to allow the non-invasive prenatal testing of β-thalassaemia and can potentially be generalized to other monogenic diseases. It is thus expected that plasma DNA-based non-invasive prenatal testing will play an increasingly important role in future obstetric care. It is thus timely and important that the ethical, social and legal issues of non-invasive prenatal testing be discussed actively by all parties involved in prenatal care.

  15. Immune Reactivation by Cell-Free Fetal DNA in Healthy Pregnancies Re-Purposed to Target Tumors: Novel Checkpoint Inhibition in Cancer Therapeutics

    PubMed Central

    Enninga, Elizabeth Ann L.; Nevala, Wendy K.; Holtan, Shernan G.; Markovic, Svetomir N.

    2015-01-01

    The role of the immune system in cancer progression has become increasingly evident over the past decade. Chronic inflammation in the promotion of tumorigenesis is well established, and cancer-associated tolerance/immune evasion has long been appreciated. Recent developments of immunotherapies targeting cancer-associated inflammation and immune tolerance, such as cancer vaccines, cell therapies, neutralizing antibodies, and immune checkpoint inhibitors, have shown promising clinical results. However, despite significant therapeutic advances, most patients diagnosed with metastatic cancer still succumb to their malignancy. Treatments are often toxic, and the financial burden of novel therapies is significant. Thus, new methods for utilizing similar biological systems to compare complex biological processes can give us new hypotheses for combating cancer. One such approach is comparing trophoblastic growth and regulation to tumor invasion and immune escape. Novel concepts regarding immune activation in pregnancy, especially reactivation of the immune system at labor through toll like receptor engagement by fetal derived DNA, may be applicable to cancer immunotherapy. This review summarizes mechanisms of inflammation in cancer, current immunotherapies used in the clinic, and suggestions for looking beyond oncology for novel methods to reverse cancer-associated tolerance and immunologic exhaustion utilizing mechanisms encountered in normal human pregnancy. PMID:26379664

  16. Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment.

    PubMed

    Chia, Puey Ling; Do, Hongdo; Morey, Adrienne; Mitchell, Paul; Dobrovic, Alexander; John, Thomas

    2016-08-01

    AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms. Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA. In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse. ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression. PMID:27393503

  17. Epstein-Barr Virus (EBV) DNA in plasma is not encapsidated in patients with EBV-related malignancies.

    PubMed

    Ryan, Julie L; Fan, Hongxin; Swinnen, Lode J; Schichman, Steven A; Raab-Traub, Nancy; Covington, Mary; Elmore, Sandra; Gulley, Margaret L

    2004-06-01

    Epstein-Barr Virus (EBV), a ubiquitous gamma herpes virus, infects more than 95% of the human population before adulthood. Life-long persistence, usually without adverse health consequences, relies on a balance between viral latency, viral replication, and host immune response. Patients with EBV-related disease often have high levels of EBV DNA in their plasma. This study addresses whether this circulating, cell-free EBV DNA is encapsidated in virions or exists as naked genomes. First, an assay was developed, combining DNase I and quantitative real-time PCR, to discriminate encapsidated from naked EBV DNA. EBV DNA was almost always naked in the plasma of AIDS-related lymphoma patients (n = 11) and immunosuppressed/posttransplantation patients (n = 8). In contrast, infectious mononucleosis patients (n = 30) often had a mixture of encapsidated and naked EBV DNA. These findings may be important in understanding how viral load relates to disease status and in predicting response to nucleoside analogs and other antiviral therapies. PMID:15167006

  18. Whole Genome Amplification of Plasma-Circulating DNA Enables Expanded Screening for Allelic Imbalance in Plasma

    PubMed Central

    Li, Jin; Harris, Lyndsay; Mamon, Harvey; Kulke, Matthew H.; Liu, Wei-Hua; Zhu, Penny; Mike Makrigiorgos, G.

    2006-01-01

    Apoptotic and necrotic tumor cells release DNA into plasma, providing an accessible tumor biomarker. Tumor-released plasma-circulating DNA can be screened for tumor-specific genetic changes, including mutation, methylation, or allelic imbalance. However, technical problems relating to the quantity and quality of DNA collected from plasma hinder downstream genetic screening and reduce biomarker detection sensitivity. Here, we present a new methodology, blunt-end ligation-mediated whole genome amplification (BL-WGA), that efficiently amplifies small apoptotic fragments (<200 bp) as well as intermediate and large necrotic fragments (>5 kb) and enables reliable high-throughput analysis of plasma-circulating DNA. In a single-tube reaction, purified double-stranded DNA was blunted with T4 DNA polymerase, self-ligated or cross-ligated with T4 DNA ligase and amplified via random primer-initiated multiple displacement amplification. Using plasma DNA from breast cancer patients and normal controls, we demonstrate that BL-WGA amplified the plasma-circulating genome by ∼1000-fold. Of 25 informative polymorphic sites screened via polymerase chain reaction-denaturating high-performance liquid chromatography, 24 (95%) were correctly determined by BL-WGA to be allelic retention or imbalance compared to 44% by multiple displacement amplification. By enabling target magnification and application of high-throughput genome analysis, BL-WGA improves sensitivity for detection of circulating tumor-specific biomarkers from bodily fluids or for recovery of nucleic acids from suboptimally stored specimens. PMID:16436631

  19. Assessing cellular DNA damage from a helium plasma needle.

    PubMed

    Morales-Ramírez, P; Cruz-Vallejo, V; Peña-Eguiluz, R; López-Callejas, R; Rodríguez-Méndez, B G; Valencia-Alvarado, R; Mercado-Cabrera, A; Muñoz-Castro, A E

    2013-06-01

    The aim of the present study is to determine the deoxyribonucleic acid (DNA) damage by cells exposed to atmospheric pressure non-thermal plasma (APNTP). Mouse leukocytes embedded in agarose were exposed to the plasma at two different distances from a helium plasma needle outlet and during three different exposure periods. Damage was assessed by the single cell gel electrophoresis assay. The results indicate that, at 0.1 cm from the plasma needle, the exposure caused complete DNA fragmentation determined by the presence of so called "clouds". Samples exposed at 0.5 cm from the slide sample surface presented damage proportional to the exposure periods in terms of tail intensity, tail moment and "clouds" frequency. Studies performed with alkaline single cell gel electrophoresis assay to determine DNA breaks and alkali-labile sites, indicates that DNA damage produced by exposure to APNTP was caused mainly by oxidative radicals, rather than by UV light which causes cyclobutane pyrimidine dimers. These results allow us to conclude that plasma needle induced DNA breaks in mice leukocytes proportionally to exposure time.

  20. Correlation between helium atmospheric pressure plasma jet (APPJ) variables and plasma induced DNA damage

    NASA Astrophysics Data System (ADS)

    Adhikari, Ek R.; Ptasinska, Sylwia

    2016-09-01

    A helium atmospheric pressure plasma jet (APPJ) source with a dielectric capillary and two tubular electrodes was used to induce damage in aqueous plasmid DNA. The fraction of different types of DNA damage (i.e., intact or undamaged, double strand breaks (DSBs), and single strand breaks (SSBs)) that occurred as the result of plasma irradiation was quantified through analysis of agarose gel electrophoresis images. The total DNA damage increased with an increase in both flow rate and duration of irradiation, but decreased with an increase in distance between the APPJ and sample. The average power of the plasma was calculated and the length of APPJ was measured for various flow rates and voltages applied. The possible effects of plasma power and reactive species on DNA damage are discussed.

  1. Correlation between helium atmospheric pressure plasma jet (APPJ) variables and plasma induced DNA damage

    NASA Astrophysics Data System (ADS)

    Adhikari, Ek R.; Ptasinska, Sylwia

    2016-09-01

    A helium atmospheric pressure plasma jet (APPJ) source with a dielectric capillary and two tubular electrodes was used to induce damage in aqueous plasmid DNA. The fraction of different types of DNA damage (i.e., intact or undamaged, double strand breaks (DSBs), and single strand breaks (SSBs)) that occurred as the result of plasma irradiation was quantified through analysis of agarose gel electrophoresis images. The total DNA damage increased with an increase in both flow rate and duration of irradiation, but decreased with an increase in distance between the APPJ and sample. The average power of the plasma was calculated and the length of APPJ was measured for various flow rates and voltages applied. The possible effects of plasma power and reactive species on DNA damage are discussed. Contribution to the Topical Issue "Low-Energy Interactions related to Atmospheric and Extreme Conditions", edited by S. Ptasinska, M. Smialek-Telega, A. Milosavljevic, B. Sivaraman.

  2. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  3. Plasma-activated air mediates plasmid DNA delivery in vivo.

    PubMed

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

  4. Comparative study of human and rabbit cell infection with cell-free HTLV-I.

    PubMed

    Yamade, I; Isono, T; Ishiguro, T; Yoshida, Y

    1993-01-01

    Infection of human and rabbit cells with cell-free HTLV-I was studied by PCR analysis. Both human and rabbit PBL were infected similarly by cell-free virus of both human and rabbit cell origin. Cells were infected with the cell-free virus without prior treatment and regardless of the concentration of the culture supernatant containing the virus. Human and rabbit cell lines were also infected similarly by the cell-free virus, the proviral DNA persisting for more than two months. The culture supernatants of HTLV-I-producing cells could thus be a potential cause of laboratory infections. PMID:8423456

  5. Oxygen plasma treated interactive polycarbonate DNA microarraying platform.

    PubMed

    Tamarit-López, Jesús; Morais, Sergi; Puchades, Rosa; Maquieira, Angel

    2011-12-21

    A novel DNA microarrying platform based on oxygen plasma activation of polycarbonate surface of compact disks (DVD) is presented. Carboxylic acid groups are generated in few seconds on polycarbonate in an efficient, fast, and clean way. Following this surface activation strategy, amino-modified oligonucleotide probes were covalently attached, reaching an immobilization density of 2 pmol cm(-2). Atomic force microscopy imaging revealed the nondestructive character of this treatment when applied for short times, allowing for disk scanning in standard DVD drives. DNA assays performed on oxygen plasma treated disks resulted very efficient with maximum hybridization yield of 93% and reaching a low limit of detection (200 pM) for perfect match synthetic oligonucleotide targets when reading the disk with a standard drive as detector. The approach was also evaluated by scoring single nucleotide polymorphisms with a discrimination ratio of 12.8. As proof of concept, the oxygen plasma treated interactive polycarbonate DNA microarraying platform was applied to the detection of PCR products of Salmonella spp., reaching a detection limit of 2 nM that corresponds to a DNA concentration of only 1 c.f.u./mL. The results confirm the suitability of the microarray platform for analysis of biological samples with high sensitivity. PMID:22044406

  6. Cold atmospheric pressure plasma jet interactions with plasmid DNA

    SciTech Connect

    O'Connell, D.; Cox, L. J.; Hyland, W. B.; McMahon, S. J.; Reuter, S.; Graham, W. G.; Gans, T.; Currell, F. J.

    2011-01-24

    The effect of a cold (<40 deg. C) radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. Gel electrophoresis was used to analyze the DNA forms post-treatment. The experimental data are fitted to a rate equation model that allows for quantitative determination of the rates of single and double strand break formation. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks.

  7. Translation in cell-free systems

    SciTech Connect

    Jagus, R.

    1987-01-01

    The simplest, unambiguous identification of a particular mRNA is the identification of its protein product. This can be established by translation of the mRNA of interest in a cell-free protein-synthesizing system. Messenger RNA protein product identification is important in the isolation of a particular mRNA species for cDNA cloning and in the identification of positive cDNA clones. The two high-activity translation systems in common use are those prepared from rabbit reticulocytes and from wheat germ. Both systems are easy to prepare, and both are available commercially. Each has advantages and disadvantages over the other and a choice between the two will depend on the type of mRNAs to be translated, the prejudices of experience, and availability. The main disadvantage of the reticulocyte system is that it requires removal of endogenous mRNA. However, this is a relatively simple procedure. The wheat germ system does not require removal of endogenous mRNA and may translate weakly initiating mRNAs more efficiently. However, ionic optima for translation in the wheat germ system are more sensitive to the nature and concentration of mRNA and may need to be determined for each template. The biggest problem with the use of the wheat germ system is its tendency to produce incomplete translation products due to premature termination.

  8. Fragment Length of Circulating Tumor DNA

    PubMed Central

    Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

    2016-01-01

    Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

  9. Measuring DNA through a Nanopore Fabricated Using Plasma Processing Technology

    NASA Astrophysics Data System (ADS)

    Rossnagel, S. M.

    2009-10-01

    We have been developing a device based on a 2-3 nm diameter pore between two electrolyte volumes for the transit of DNA by means of a potential gradient. The nanopore is configured with 3 electrodes, each about 3 nm thick with 2-3nm dielectric spacers. The nanopore electrodes can be used to trap DNA in-transit, and ideally measure the impedance and hence the identity of each nucleotide as it passes through the nanopore, allowing real time sequencing of the DNA. The goal is to operate at megahertz, allowing sequencing of the entire genome within a few hours a fairly modest cost. This project has lead to numerous new developments in nanoscale fabrication, particularly for nanofluidics. The nanopore devices are fabricated using a number of critical plasma processing steps, both deposition and etch, in our 200mm pilot facility.

  10. Laser Plasma Jet Driven Microparticles for DNA/Drug Delivery

    PubMed Central

    Menezes, Viren; Mathew, Yohan; Takayama, Kazuyoshi; Kanno, Akira; Hosseini, Hamid

    2012-01-01

    This paper describes a microparticle delivery device that generates a plasma jet through laser ablation of a thin metal foil and uses the jet to accomplish particle delivery into soft living targets for transferring biological agents. Pure gold microparticles of 1 µm size were coated with a plasmid DNA, pIG121Hm, and were deposited as a thin layer on one surface of an aluminum foil. The laser (Nd:YAG, 1064 nm wavelength) ablation of the foil generated a plasma jet that carried the DNA coated particles into the living onion cells. The particles could effectively penetrate the target cells and disseminate the DNA, effecting the transfection of the cells. Generation of the plasma jet on laser ablation of the foil and its role as a carrier of microparticles was visualized using a high-speed video camera, Shimadzu HPV-1, at a frame rate of 500 kfps (2 µs interframe interval) in a shadowgraph optical set-up. The particle speed could be measured from the visualized images, which was about 770 m/s initially, increased to a magnitude of 1320 m/s, and after a quasi-steady state over a distance of 10 mm with an average magnitude of 1100 m/s, started declining, which typically is the trend of a high-speed, pulsed, compressible jet. Aluminum launch pad (for the particles) was used in the present study to make the procedure cost-effective, whereas the guided, biocompatible launch pads made of gold, silver or titanium can be used in the device during the actual clinical operations. The particle delivery device has a potential to have a miniature form and can be an effective, hand-held drug/DNA delivery device for biological applications. PMID:23226394

  11. Laser plasma jet driven microparticles for DNA/drug delivery.

    PubMed

    Menezes, Viren; Mathew, Yohan; Takayama, Kazuyoshi; Kanno, Akira; Hosseini, Hamid

    2012-01-01

    This paper describes a microparticle delivery device that generates a plasma jet through laser ablation of a thin metal foil and uses the jet to accomplish particle delivery into soft living targets for transferring biological agents. Pure gold microparticles of 1 µm size were coated with a plasmid DNA, pIG121Hm, and were deposited as a thin layer on one surface of an aluminum foil. The laser (Nd:YAG, 1064 nm wavelength) ablation of the foil generated a plasma jet that carried the DNA coated particles into the living onion cells. The particles could effectively penetrate the target cells and disseminate the DNA, effecting the transfection of the cells. Generation of the plasma jet on laser ablation of the foil and its role as a carrier of microparticles was visualized using a high-speed video camera, Shimadzu HPV-1, at a frame rate of 500 kfps (2 µs interframe interval) in a shadowgraph optical set-up. The particle speed could be measured from the visualized images, which was about 770 m/s initially, increased to a magnitude of 1320 m/s, and after a quasi-steady state over a distance of 10 mm with an average magnitude of 1100 m/s, started declining, which typically is the trend of a high-speed, pulsed, compressible jet. Aluminum launch pad (for the particles) was used in the present study to make the procedure cost-effective, whereas the guided, biocompatible launch pads made of gold, silver or titanium can be used in the device during the actual clinical operations. The particle delivery device has a potential to have a miniature form and can be an effective, hand-held drug/DNA delivery device for biological applications. PMID:23226394

  12. Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer

    PubMed Central

    Karakas, Bedri; Qubbaj, Wafa; Al-Hassan, Saad; Coskun, Serdar

    2015-01-01

    The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders. PMID:25970589

  13. Biomarkers of Adiponectin: Plasma Protein Variation and Genomic DNA Polymorphisms

    PubMed Central

    Gu, Harvest F.

    2009-01-01

    Adiponectin is secreted by white adipose tissue and exists as the most abundant adipokine in the human plasma. Recent research has indicated that plasma adiponectin levels are inversely correlated with body mass index (BMI) and insulin resistance. Reduction of plasma adiponectin levels is commonly observed in the patients with type 2 diabetes (T2D) and/or in those who are obese in comparison with healthy control individuals. The adiponectin (AdipoQ) gene has a moderate linkage disequilibrium (LD), but two small LD blocks are observed, respectively, in the promoter region and the boundary of exon 2-intron 2. Genetic association studies have demonstrated that single nucleotide polymorphisms (SNPs) +45G15G(T/G) in exon 2 and +276G/T in intron 2 of the AdipoQ gene confer the risk susceptibility to the development of T2D, obesity and diabetic nephropathy (DN). The SNPs in the promoter region, including −11426A/G, −11377C/G and −11391G/A, are found to be associated with T2D and DN. Recent research has indicated that the promoter polymorphisms interfere with the AdipoQ promoter activity. The haplotypes constructed by the promoter polymorphisms and SNP +276G/T in intron 2 are associated with circulating adiponectin levels. This review summarises genetic and pathophysiological relevancies of adiponectin and discusses about the biomarkers of adiponectin plasma protein variation and genomic DNA polymorphisms. PMID:20029651

  14. A Cross-Sectional Comparison of Druggable Mutations in Primary Tumors, Metastatic Tissue, Circulating Tumor Cells, and Cell-Free Circulating DNA in Patients with Metastatic Breast Cancer: The MIRROR Study Protocol

    PubMed Central

    Picornell, Antoni C; Alvarez, Enrique L; Martin, Miguel

    2016-01-01

    Background Characterization of the driver mutations in an individual metastatic breast cancer (MBC) patient is critical to selecting effective targeted therapies. Currently, it is believed that the limited efficacy of many targeted drugs may be due to the expansion of drug resistant clones with different genotypes that were already present in the primary tumor. Identifying the genomic alterations of these clones, and introducing combined or sequential targeted drug regimens, could lead to a significant increase in the efficacy of currently available targeted therapies. Objective The primary objective of this study is to assess the concordance/discordance of mutations between the primary tumor and metastatic tissue in MBC patients. Secondary objectives include comparing the genomic profiles of circulating tumor cells (CTCs) and circulating free DNA (cfDNA) from peripheral blood with those of the primary tumor and metastatic tissue for each patient, evaluating these mutations in the signaling pathways that are relevant to the disease, and testing the feasibility of introducing liquid biopsy as a translational laboratory tool in clinical practice. Methods The multicenter, transversal, observational MIRROR study is currently ongoing in three participating hospitals. All consecutive patients with MBC confirmed by radiologic findings will be screened for eligibility, either at first relapse or if tumor regrowth occurs while on treatment for metastatic disease. Results Patient recruitment is currently ongoing. To date, 41 patients have a complete set of tissue samples available (plasma, CTCs, and formalin-fixed, paraffin-embedded primary tumor and metastatic tumor). However, none of these samples have undergone nucleic acids extraction or targeted deep sequencing. Conclusions The results of this study may have a significant influence on the practical management of patients with MBC, and may provide clues to clinicians that lead towards a better stratification of patients

  15. A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma

    PubMed Central

    Zhang, L; Zhang, M; Yang, S; Cao, Y; Bingrong Zhang, S; Yin, L; Tian, Y; Ma, Y; Zhang, A; Okunieff, P; Zhang, L

    2010-01-01

    A simple and accurate method for measuring the biological effects of radiation is of increasing importance, especially in mass casualty scenarios. We have therefore developed a new biodosimetric technique targeting circulating B1 DNA in mouse plasma by branched DNA signal amplification for rapid quantification of plasma DNA. This technology targets repetitive elements of the B1 retrotransposon in the mouse genome, followed by signal amplification using Panomics Quantigene 2.0 reagents. Evaluation was conducted concerning precision, accuracy and linearity. Plasma samples were collected from mice 0–24 h after 0–10 Gy total body irradiation (TBI). The average inter- and intra-assay coefficients of variance were 8.7% and 12.3%, respectively. The average recovery rate of spiked DNA into plasma was 89.5%. This assay revealed that when BALB/c and NIH Swiss mice were exposed to 6 Gy TBI, plasma B1 DNA levels increased significantly at 3 h post-TBI, peaked at 9 h and gradually returned toward baseline levels in 24 h. A dose-dependent change in plasma DNA was observed at 9 h post-TBI; the dose–response relation was monotonic, exhibiting linearity for BALB/c mice from 3 to 6 Gy (r = 0.993) and NIH Swiss mice from 3 to 7 Gy (r = 0.98). This branched DNA-based assay is reliable, accurate and sensitive in detecting plasma B1 DNA quantitatively. A radiation dose-correlated increase in plasma B1 DNA was demonstrated in BALB/c and NIH Swiss mice in the dose range from 3 to 6 Gy, suggesting that plasma B1 DNA has potential as a biomarker for radiation biological effect. PMID:20675464

  16. Increased Levels of Plasma Epstein Barr Virus DNA Identify a Poor-Risk Subset of Patients With Advanced Stage Cutaneous T-Cell Lymphoma

    PubMed Central

    Haverkos, Bradley M.; Gru, Alejandro A.; Geyer, Susan M.; Bingman, Anissa K.; Hemminger, Jessica A.; Mishra, Anjali; Wong, Henry K.; Pancholi, Preeti; Freud, Aharon G.; Caligiuri, Michael A.; Baiocchi, Robert A.; Porcu, Pierluigi

    2016-01-01

    Discovering prognostic factors that simultaneously describe tumor characteristics and improve risk stratification is a priority in cutaneous T-cell lymphoma (CTCL). More than a third of advanced stage CTCL patients in this cohort had detectable cell free plasma Epstein–Barr virus (EBV)-DNA (pEBVd) using quantitative real-time polymerase chain reaction. An increased level of pEBVd was highly concordant with EBV (ie, Epstein–Barr virus RNAs) in tumor tissue and was associated with inferior survival. Introduction Outcomes in advanced stage (AS) cutaneous T-cell lymphomas (CTCL) are poor but with great variability. Epstein–Barr virus (EBV) is associated with a subset of non-Hodgkin lymphomas. Frequency of plasma EBV-DNA (pEBVd) detection, concordance with EBV RNA (EBER) in tumor tissue, codetection of plasma cytomegalovirus DNA (pCMVd), and prognostic effect in AS CTCL are unknown. Patients and Methods Patients (n = 46; 2006–2013) with AS CTCL (≥IIB) were retrospectively studied. pEBVd and pCMVd were longitudinally measured using quantitative real-time polymerase chain reaction. EBER in situ hybridization (ISH) was performed on tumor samples. Survival from time of diagnosis (ToD) and time of progression to AS was assessed. Results Plasma EBV-DNA and pCMVd were detected in 37% (17 of 46) and 17% (8 of 46) of AS CTCL patients, respectively. pCMVd detection was significantly more frequent in pEBVd-positive (pEBVd+) than pEBVd− patients (35% vs. 7%; P = .038). Tumor tissue for EBER-ISH was available in 14 of 17 pEBVd+ and 22 of 29 pEBVd− patients; 12 of 14 (85.7%) pEBVd+ patients were EBER+ versus 0 of 22 pEBVd− patients. Frequency of large cell transformation (LCT) tended to be greater in pEBVd+ patients, but was not significant (10 of 14 pEBVd+ vs. 10 of 23 pEBVd−; P = .17). No notable differences in rates of increased levels of serum lactate dehydrogenase (LDH) were observed (17 of 17 pEBVd+ vs. 27 of 29 pEBVd−). pEBVd detection was associated with

  17. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    SciTech Connect

    Kim, G. J.; Lee, J. K.; Kim, W.; Kim, K. T.

    2010-01-11

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  18. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    NASA Astrophysics Data System (ADS)

    Kim, G. J.; Kim, W.; Kim, K. T.; Lee, J. K.

    2010-01-01

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  19. Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

    SciTech Connect

    Cao Hongbin; Banh, Alice; Kwok, Shirley; Shi Xiaoli; Wu, Simon; Krakow, Trevor; Khong, Brian; Bavan, Brindha; Bala, Rajeev; Pinsky, Benjamin A.; Colevas, Dimitrios; Pourmand, Nader; Koong, Albert C.; Kong, Christina S.; Le, Quynh-Thu

    2012-03-01

    Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16{sup INK4a} staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using {sup 18}F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  20. Plasma levels of mitochondrial and nuclear DNA in patients with massive pulmonary embolism in the emergency department: a prospective cohort study

    PubMed Central

    2013-01-01

    Introduction Cell-free plasma mitochondrial DNA (mt-DNA) and nuclear DNA (n-DNA) are biomarkers with prognostic utility in conditions associated with a high rate of cell death. This exploratory study aimed to determine the plasma levels of both nucleic acids in patients with massive and submassive pulmonary embolism (PE) and to compare them with other biomarkers, such as heart-type fatty acid-binding protein (H-FABP) and troponin I (Tn-I) Methods This was a prospective observational study of 37 consecutive patients with massive PE, 37 patients with submassive PE, and 37 healthy subjects. Quantifications of plasma mt-DNA and n-DNA with real-time quantitative polymerase chain reaction (PCR), and plasma H-FABP and Tn-I by commercial assays, were done on blood samples drawn within 4 hours after presentation at the emergency department. Results Plasma mt-DNA and n-DNA concentrations were much higher in patients with massive PE (median, 2,970 GE/ml; interquartile range (IQR), 1,050 to 5,485; and 3,325 GE/ml, IQR: 1,080 to 5,790, respectively) than in patients with submassive PE (870 GE/ml and 1,245 GE/ml, respectively; P < 0.01) or controls (185 GE/ml and 520 GE/ml, respectively). Eighteen patients with massive PE died of a PE-related cause by day 15 of observation. Plasma mt-DNA and n-DNA values were 2.3-fold and 1.9-fold higher in the subgroup of nonsurviving patients than in survivors. H-FABP and Tn-I values were also higher in patients with massive PE who died (7.3 ng/ml and 0.023 ng/ml, respectively) than in those who survived (6.4 ng/ml, and 0.016 ng/ml, respectively). By receiver operating curve (ROC) analysis, the best cutoff values for predicting 15-day mortality were 3,380 GE/ml for mt-DNA, 6.8 ng/ml for H-FABP, 3,625 GE/ml for n-DNA, and 0.020 ng/ml for Tn-I, based on the calculated areas under the curve (AUCs) of 0.89 (95% confidence interval (CI), 0.78 to 0.99), 0.76 (95% CI, 0.69 to 093), 0.73 (95% CI, 0.58 to 0.91), and 0.59 (95% CI, 0.41 to 0

  1. Effect of cold atmospheric pressure He-plasma jet on DNA change and mutation

    NASA Astrophysics Data System (ADS)

    Yaopromsiri, C.; Yu, L. D.; Sarapirom, S.; Thopan, P.; Boonyawan, D.

    2015-12-01

    Cold atmospheric pressure plasma jet (CAPPJ) effect on DNA change was studied for assessment of its safety. The experiment utilized a home-developed CAPPJ using 100% helium to directly treat naked DNA plasmid pGFP (plasmid green fluorescent protein). A traversal electric field was applied to separate the plasma components and both dry and wet sample conditions were adopted to investigate various factor roles in changing DNA. Plasma species were measured by using optical emission spectroscopy. DNA topological form change was analyzed by gel electrophoresis. The plasma jet treated DNA was transferred into bacterial Escherichia coli cells for observing mutation. The results show that the He-CAPPJ could break DNA strands due to actions from charge, radicals and neutrals and potentially cause genetic modification of living cells.

  2. Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

    SciTech Connect

    Yan Xu; He Guangyuan; Shi Mengjun; Gao Xuan; Li Yin; Ma Fengyun; Yu Men; Wang Changdong; Wang Yuesheng; Yang Guangxiao; Zou Fei; Lu Xinpei; Xiong Qing; Xiong Zilan

    2009-08-24

    The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

  3. Identification of DNA-binding proteins on human umbilical vein endothelial cell plasma membrane.

    PubMed Central

    Chan, T M; Frampton, G; Cameron, J S

    1993-01-01

    The binding of anti-DNA antibodies to the endothelial cell is mediated through DNA, which forms a bridge between the immunoglobulin and the plasma membrane. We have shown that 32P-labelled DNA bound to the plasma membrane of human umbilical vein endothelial cells (HUVEC) by a saturable process, which could be competitively inhibited by non-radiolabelled DNA. In addition, DNA-binding was enhanced in HUVEC that had been treated with IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha). DNA-binding proteins of mol. wt 46,000, 92,000, and 84,000 were identified by the binding of 32P-labelled DNA to plasma membrane proteins separated on SDS-PAGE. DNA-binding proteins of mol. wt 46,000 and 84,000 were also present in the cytosol and nucleus. Murine anti-DNA MoAb410 bound to a single band, at mol. wt 46,000, of plasma membrane protein, in the presence of DNA. Our results showed that DNA-binding proteins are present in different cellular fractions of endothelial cells. DNA-binding proteins on the cell membrane could participate in the in situ formation of immune deposits; and their presence in the cell nucleus suggests a potential role in the modulation of cell function. Images Fig. 3 Fig. 4 PMID:8419070

  4. A novel method for the detection of plasma jet boundaries by exploring DNA damage

    NASA Astrophysics Data System (ADS)

    Bahnev, Blagovest; Bowden, Mark D.; Stypczyńska, Agnieszka; Ptasińska, Sylwia; Mason, Nigel J.; Braithwaite, Nicholas St. J.

    2014-06-01

    In this study we have investigated the plasma-air boundary of an atmospheric pressure discharge by exposing samples of dry plasmid DNA to a plasma jet. While visible emission from excited plasma species suggested that the plasma jet had dimensions approximately 5.5 cm long and 0.4 cm wide, damage to DNA samples was detected at distances of up to 20 cm from the tip of the jet with observable effects far outside the luminous plasma volume. Reactive oxygen species were identified as the most likely cause of DNA damage at these long distances. These results provide a novel method for determining the extent of any kind of plasma jet generated in the open atmosphere.

  5. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    SciTech Connect

    Han, Xu; Ptasinska, Sylwia; Klas, Matej; Liu, Yueying; Sharon Stack, M.

    2013-06-10

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  6. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    NASA Astrophysics Data System (ADS)

    Han, Xu; Klas, Matej; Liu, Yueying; Sharon Stack, M.; Ptasinska, Sylwia

    2013-06-01

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  7. Low-energy plasma immersion ion implantation to induce DNA transfer into bacterial E. coli

    NASA Astrophysics Data System (ADS)

    Sangwijit, K.; Yu, L. D.; Sarapirom, S.; Pitakrattananukool, S.; Anuntalabhochai, S.

    2015-12-01

    Plasma immersion ion implantation (PIII) at low energy was for the first time applied as a novel biotechnology to induce DNA transfer into bacterial cells. Argon or nitrogen PIII at low bias voltages of 2.5, 5 and 10 kV and fluences ranging from 1 × 1012 to 1 × 1017 ions/cm2 treated cells of Escherichia coli (E. coli). Subsequently, DNA transfer was operated by mixing the PIII-treated cells with DNA. Successes in PIII-induced DNA transfer were demonstrated by marker gene expressions. The induction of DNA transfer was ion-energy, fluence and DNA-size dependent. The DNA transferred in the cells was confirmed functioning. Mechanisms of the PIII-induced DNA transfer were investigated and discussed in terms of the E. coli cell envelope anatomy. Compared with conventional ion-beam-induced DNA transfer, PIII-induced DNA transfer was simpler with lower cost but higher efficiency.

  8. Plasma Concentration of Parasite DNA as a Measure of Disease Severity in Falciparum Malaria

    PubMed Central

    Imwong, Mallika; Woodrow, Charles J.; Hendriksen, Ilse C. E.; Veenemans, Jacobien; Verhoef, Hans; Faiz, M. Abul; Mohanty, Sanjib; Mishra, Saroj; Mtove, George; Gesase, Samwel; Seni, Amir; Chhaganlal, Kajal D.; Day, Nicholas P. J.; Dondorp, Arjen M.; White, Nicholas J.

    2015-01-01

    In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples. PMID:25344520

  9. Flexible Programming of Cell-Free Protein Synthesis Using Magnetic Bead-Immobilized Plasmids

    PubMed Central

    Lee, Ka-Young; Lee, Kyung-Ho; Park, Ji-Woong; Kim, Dong-Myung

    2012-01-01

    The use of magnetic bead-immobilized DNA as movable template for cell-free protein synthesis has been investigated. Magnetic microbeads containing chemically conjugated plasmids were used to direct cell-free protein synthesis, so that protein generation could be readily programmed, reset and reprogrammed. Protein synthesis by using this approach could be ON/OFF-controlled through repeated addition and removal of the microbead-conjugated DNA and employed in sequential expression of different genes in a same reaction mixture. Since the incubation periods of individual template plasmids are freely controllable, relative expression levels of multiple proteins can be tuned to desired levels. We expect that the presented results will find wide application to the flexible design and execution of synthetic pathways in cell-free chassis. PMID:22470570

  10. Noninvasive prenatal testing by maternal plasma DNA analysis: current practice and future applications.

    PubMed

    Chiu, Rossa W K

    2014-01-01

    Prenatal screening of fetal chromosomal aneuploidies and some common genetic diseases is an integral part of antenatal care. Definitive prenatal diagnosis is conventionally achieved by the sampling of fetal genetic material by amniocentesis or chorionic villus sampling. Due to the invasiveness of those procedures, they are associated with a 1 in 200 chance of fetal miscarriage. Hence, researchers have been exploring noninvasive ways to sample fetal genetic material. The presence of cell-free DNA released by the fetus into the circulation of its mother was demonstrated in 1997. Circulating fetal DNA is therefore obtainable through the collection of a blood sample from the pregnant woman without posing any physical harm to the fetus. By analyzing this source of fetal genetic material, researchers have succeeded in developing DNA-based noninvasive tests for the assessment of Down syndrome and single gene diseases. Since the end of 2011, tests for the noninvasive assessment of chromosomal aneuploidies have become commercially available in parts of the world. Recommendations from professional groups have since been made regarding how these tests could be incorporated into the framework of existing prenatal screening programs. More recently, cell-free circulating fetal DNA analysis have been shown to be applicable to the deciphering of the fetal molecular karyotype, genome and methylome. It is envisioned that an increasing number of the noninvasive prenatal tests will become clinically available. The ethical, social and legal implications of the introduction of some of these tests would need to be discussed in the context of different cultures, societal values and the legal framework. PMID:25083893

  11. Cytolytic activity of Naegleria fowleri cell-free extract.

    PubMed

    Fulford, D E; Marciano-Cabral, F

    1986-11-01

    The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.

  12. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy.

  13. Plasma cell differentiation is coupled to division-dependent DNA hypomethylation and gene regulation.

    PubMed

    Barwick, Benjamin G; Scharer, Christopher D; Bally, Alexander P R; Boss, Jeremy M

    2016-10-01

    The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.

  14. Synergistic effects of atmospheric pressure plasma-emitted components on DNA oligomers: a Raman spectroscopic study.

    PubMed

    Edengeiser, Eugen; Lackmann, Jan-Wilm; Bründermann, Erik; Schneider, Simon; Benedikt, Jan; Bandow, Julia E; Havenith, Martina

    2015-11-01

    Cold atmospheric-pressure plasmas have become of increasing importance in sterilization processes especially with the growing prevalence of multi-resistant bacteria. Albeit the potential for technological application is obvious, much less is known about the molecular mechanisms underlying bacterial inactivation. X-jet technology separates plasma-generated reactive particles and photons, thus allowing the investigation of their individual and joint effects on DNA. Raman spectroscopy shows that particles and photons cause different modifications in DNA single and double strands. The treatment with the combination of particles and photons does not only result in cumulative, but in synergistic effects. Profilometry confirms that etching is a minor contributor to the observed DNA damage in vitro. Schematics of DNA oligomer treatment with cold atmospheric-pressure plasma.

  15. [The cell-free protein synthesis-based protein microarray technology].

    PubMed

    Lu, Linli; Lin, Bicheng

    2010-12-01

    The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay. PMID:21375003

  16. Noninvasive prenatal diagnosis by analysis of fetal DNA in maternal plasma.

    PubMed

    Chiu, Rossa W K; Lo, Y M Dennis

    2006-01-01

    Prenatal diagnosis has become a standard part of obstetrics care. Genetic diagnoses are established prenatally through the sampling of fetal genetic material by invasive methods such as amniocentesis or chorionic villus sampling, which are associated with a risk of fetal loss. Hence, the recent discovery of the presence of fetal DNA in maternal plasma has led to exciting possibilities of noninvasive prenatal diagnosis. Numerous applications have since been reported for the analysis of circulating fetal DNA. The accuracy of fetal DNA assessment from maternal plasma is dependent on the appropriate preanalytical handling of maternal blood samples, an efficient fetal DNA extraction protocol, and a sensitive and specific detection method. The protocol that has been applied regularly in the laboratory of the authors for the reliable detection and quantification of circulating fetal DNA is presented in this chapter.

  17. Products of DNA, protein and lipid oxidative damage in relation to vitamin C plasma concentration.

    PubMed

    Krajcovicová-Kudlácková, M; Dusinská, M; Valachovicová, M; Blazícek, P; Pauková, V

    2006-01-01

    Oxidative stress plays an important role in the pathogenesis of numerous chronic age-related free radical-induced diseases. Improved antioxidant status minimizes oxidative damage to DNA, proteins, lipids and other biomolecules. Diet-derived antioxidants such as vitamin C, vitamin E, carotenoids and related plant pigments are important in antioxidative defense and maintaining health. The results of long-term epidemiological and clinical studies suggest that protective vitamin C plasma concentration for minimum risk of free radical disease is higher than 50 micromol/l. Products of oxidative damage to DNA (DNA strand breaks with oxidized purines and pyrimidines), proteins (carbonyls) and lipids (conjugated dienes of fatty acids, malondialdehyde) were estimated in a group of apparently healthy adult non-smoking population in dependence on different vitamin C plasma concentrations. Under conditions of protective plasma vitamin C concentrations (>50 micromol/l) significantly lower values of DNA, protein and lipid oxidative damage were found in comparison with the vitamin C-deficient group (<50 micromol/l). The inhibitory effect of higher fruit and vegetable consumption (leading to higher vitamin C intake and higher vitamin C plasma concentrations) on oxidation of DNA, proteins and lipids is also expressed by an inverse significant correlation between plasma vitamin C and products of oxidative damage. The results suggest an important role of higher and frequent consumption of protective food (fruit, vegetables, vegetable oils, nuts, seeds and cereal grains) in prevention of free radical disease.

  18. Characterization of Atmospheric Pressure Plasma Jet (APPJ) and Its Effect on Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Adhikari, Ek; Ptasinska, Sylwia

    2015-09-01

    A helium atmospheric pressure plasma jet (APPJ) source was constructed and then characterized by monitoring a deflected current on a high voltage electrode and a potential difference between two electrodes. The deflected current was also monitored for the APPJ source with varied electrical and fed gas composition e.g. admixtures of He and water vapor. The deflected power per cycle for gas admixtures was decreased with the increase in fraction of water vapor. In addition, this APPJ source was used to induce damage to aqueous plasmid DNA. The fraction of supercoiled, single-strand breaks and double-strand breaks in DNA were quantified by using agarose gel electrophoresis. The number of DNA strand breaks increased as a function of plasma irradiation time and decrease as a distance between APPJ and DNA sample increased. The APPJ with the gas admixture, in which the fraction of water vapor was varied, was also used to induce damage to aqueous DNA samples. The damage level decreased with the increase in a fraction of water vapor under specific experimental conditions. The change in numbers of DNA strand breaks irradiated by a pure He plasma and a plasma with a gas admixture is predicted by different physical and chemical process in the APPJ. This material is based upon work supported by the U.S. Department of Energy Office of Science, Office of Basic Energy Sciences under Award Number DE-FC02-04ER15533.

  19. Relationship of seminal plasma level and extender type to sperm motility and DNA integrity.

    PubMed

    Love, C C; Brinsko, S P; Rigby, S L; Thompson, J A; Blanchard, T L; Varner, D D

    2005-04-01

    The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sperm/mL in either CST or KMT (10% CST; 10% KMT); centrifuged to remove virtually all seminal plasma and resuspended in either CST or KMT (0% CST-Cent; 0% KMT-Cent); centrifuged semen to remove virtually all seminal plasma and resuspended with previously filtered seminal plasma from the same stallion in either CST or KMT to a final concentration of 20% seminal plasma (20% CST-Cent; 20% KMT-Cent). Sperm motion characteristics were determined by CASA and DNA integrity (%COMP, percent of cells outside the main population) evaluated by the Sperm Chromatin Structure Assay prior to cooling, and after 24 and 48 h cooled-storage at 5 degrees C. After 48 h of storage at 5 degrees C, extenders with 0% seminal plasma (0% CST-Cent, 0% KMT-Cent) maintained highest quality DNA (P < 0.05), but 0% KMT-Cent maintained higher velocity measures (P < 0.05) than 0% CST-Cent. Total sperm motility was highest (P < 0.05) in 0% CST-Cent, 0% KMT-Cent, 10% CST, 20% CST-Cent, and 20% CST compared to the other treatment groups. Progressive sperm motility was highest (P < 0.05) after 48 h of storage in the treatment with 10% seminal plasma in Kenney extender (10% CST), despite a reduction in DNA integrity. Regardless of extender type, addition of 20% seminal plasma following centrifugation resulted in almost a two-fold increase in %COMP(alpha t), even though one of the treatments (20% CST-Cent) maintained

  20. Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

    PubMed

    Jiang, Haojun; Xie, Yifan; Li, Xuchao; Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

    2016-01-01

    Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

  1. Exercise-induced oxidatively damaged DNA in humans: evaluation in plasma or urine?

    PubMed

    Karpouzi, Christina; Nikolaidis, Stefanos; Kabasakalis, Athanasios; Tsalis, George; Mougios, Vassilis

    2016-01-01

    Physical exercise can induce oxidative damage in humans. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a widely known biomarker of DNA oxidation, which can be determined in blood and urine. The aim of the present study was to compare these two biological fluids in terms of which is more suitable for the estimation of the oxidative damage of DNA by measuring the concentration of 8-OHdG one hour after maximal exercise by enzyme immunoassay. The concentration of 8-OHdG increased with exercise only in plasma (p < 0.001), and values differed between exercise tests in both plasma and urine (p < 0.05). In conclusion, plasma appears to be more sensitive to exercise-induced 8-OHdG changes than urine and, hence, a more appropriate medium for assessing oxidative damage of DNA, although the poor repeatability of the measurement needs to be addressed in future studies. PMID:26849281

  2. DNA, histones and neutrophil extracellular traps exert anti-fibrinolytic effects in a plasma environment.

    PubMed

    Varjú, Imre; Longstaff, Colin; Szabó, László; Farkas, Ádám Zoltán; Varga-Szabó, Veronika Judit; Tanka-Salamon, Anna; Machovich, Raymund; Kolev, Krasimir

    2015-06-01

    In response to various inflammatory stimuli, neutrophils secrete neutrophil extracellular traps (NETs), web-like meshworks of DNA, histones and granular components forming supplementary scaffolds in venous and arterial thrombi. Isolated DNA and histones are known to promote thrombus formation and render fibrin clots more resistant to mechanical forces and tissue-type plasminogen activator (tPA)-induced enzymatic digestion. The present study extends our earlier observations to a physiologically more relevant environment including plasma clots and NET-forming neutrophils. A range of techniques was employed including imaging (scanning electron microscopy (SEM), confocal laser microscopy, and photoscanning of macroscopic lysis fronts), clot permeability measurements, turbidimetric lysis and enzyme inactivation assays. Addition of DNA and histones increased the median fibre diameter of plasma clots formed with 16 nM thrombin from 108 to 121 and 119 nm, respectively, and decreased their permeability constant from 6.4 to 3.1 and 3.7×10(-9) cm(2). Histones effectively protected thrombin from antithrombin-induced inactivation, while DNA inhibited plasminogen activation on the surface of plasma clots and their plasmin-induced resolution by 20 and 40 %, respectively. DNA and histones, as well as NETs secreted by phorbol-myristate-acetate-activated neutrophils, slowed down the tPA-driven lysis of plasma clots and the latter effect could be reversed by the addition of DNase (streptodornase). SEM images taken after complete digestion of fibrin in NET-containing plasma clots evidenced retained NET scaffold that was absent in DNase-treated clots. Our results show that DNA and histones alter the fibrin architecture in plasma clots, while NETs contribute to a decreased lytic susceptibility that can be overcome by DNase.

  3. Plasma induced DNA damage: Comparison with the effects of ionizing radiation

    SciTech Connect

    Lazović, S.; Maletić, D.; Puač, N.; Malović, G.; Petrović, Z. Lj.; Leskovac, A.; Filipović, J.; Joksić, G.

    2014-09-22

    We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of the treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.

  4. Plasma induced DNA damage: Comparison with the effects of ionizing radiation

    NASA Astrophysics Data System (ADS)

    Lazović, S.; Maletić, D.; Leskovac, A.; Filipović, J.; Puač, N.; Malović, G.; Joksić, G.; Petrović, Z. Lj.

    2014-09-01

    We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of the treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.

  5. The comparison of DNA damage induced by micro DBD plasma and low energy electron for curing human diseases

    NASA Astrophysics Data System (ADS)

    Park, Yeunsoo

    2015-09-01

    It is well known that low energy electrons (LEE, especially below 10 eV) can generate DNA damage via indirect action named dissociative electron attachment (DEA). We can now explain some parts of the exact mechanism on DNA damage by LEE collision with direct ionization effect when cancer patients get the radiotherapy. It is kind of remarkable information in the field of radiation therapy. However, it is practically very difficult to directly apply this finding to human disease cure due to difficulty of LEE therapy actualization and request of further clinical studies. Recently, there is a novel challenge in plasma application, that is, how we can apply plasma technology to diagnosis and treatment of many serious diseases like cancer. Cold atmospheric pressure plasma (CAPP) is a very good source to apply to plasma medicine and bio-applications because of low temperature, low cost, and easy handling. Some scientists have already reported good results related to clinical plasma application. The purposes of this study are to further find out exact mechanisms of DNA damage by LEE at the molecular level, to verify new DNA damage like structural alteration on DNA subunits and to compare DNA damage by LEE and plasma source. We will keep expanding our study to DNA damage by plasma source to develop plasma-based new medical and biological applications. We will show some recent results, DNA damage by LEE and non-thermal plasma.

  6. Atmospheric Nonthermal Plasma-Treated PBS Inactivates Escherichia coli by Oxidative DNA Damage.

    PubMed

    Yost, Adam D; Joshi, Suresh G

    2015-01-01

    We recently reported that phosphate-buffered saline (PBS) treated with nonthermal dielectric-barrier discharge plasma (plasma) acquires strong antimicrobial properties, but the mechanisms underlying bacterial inactivation were not known. The goal of this study is to understand the cellular responses of Escherichia coli and to investigate the properties of plasma-activated PBS. The plasma-activated PBS induces severe oxidative stress in E. coli cells and reactive-oxygen species scavengers, α-tocopherol and catalase, protect E. coli from cell death. Here we show that the response of E. coli to plasma-activated PBS is regulated by OxyR and SoxyRS regulons, and mediated predominantly through the expression of katG that deactivates plasma-generated oxidants. During compensation of E. coli in the absence of both katG and katE, sodA and sodB are significantly overexpressed in samples exposed to plasma-treated PBS. Microarray analysis found that up-regulation of genes involved in DNA repair, and E. coli expressing recA::lux fusion was extremely sensitive to the SOS response upon exposure to plasma-treated PBS. The cellular changes include rapid loss of E. coli membrane potential and membrane integrity, lipid peroxidation, accumulation of 8-hydroxy-deoxyguinosine (8OHdG), and severe oxidative DNA damage; reveal ultimate DNA disintegration, and cell death. Together, these data suggest that plasma-treated PBS contains hydrogen peroxide and superoxide like reactive species or/and their products which lead to oxidative changes to cell components, and are eventually responsible for cell death. PMID:26461113

  7. Atmospheric Nonthermal Plasma-Treated PBS Inactivates Escherichia coli by Oxidative DNA Damage

    PubMed Central

    Yost, Adam D.; Joshi, Suresh G.

    2015-01-01

    We recently reported that phosphate-buffered saline (PBS) treated with nonthermal dielectric-barrier discharge plasma (plasma) acquires strong antimicrobial properties, but the mechanisms underlying bacterial inactivation were not known. The goal of this study is to understand the cellular responses of Escherichia coli and to investigate the properties of plasma-activated PBS. The plasma-activated PBS induces severe oxidative stress in E. coli cells and reactive-oxygen species scavengers, α-tocopherol and catalase, protect E. coli from cell death. Here we show that the response of E. coli to plasma-activated PBS is regulated by OxyR and SoxyRS regulons, and mediated predominantly through the expression of katG that deactivates plasma-generated oxidants. During compensation of E. coli in the absence of both katG and katE, sodA and sodB are significantly overexpressed in samples exposed to plasma-treated PBS. Microarray analysis found that up-regulation of genes involved in DNA repair, and E. coli expressing recA::lux fusion was extremely sensitive to the SOS response upon exposure to plasma-treated PBS. The cellular changes include rapid loss of E. coli membrane potential and membrane integrity, lipid peroxidation, accumulation of 8-hydroxy-deoxyguinosine (8OHdG), and severe oxidative DNA damage; reveal ultimate DNA disintegration, and cell death. Together, these data suggest that plasma-treated PBS contains hydrogen peroxide and superoxide like reactive species or/and their products which lead to oxidative changes to cell components, and are eventually responsible for cell death. PMID:26461113

  8. Oxidized Extracellular DNA as a Stress Signal in Human Cells

    PubMed Central

    Ermakov, Aleksei V.; Konkova, Marina S.; Kostyuk, Svetlana V.; Izevskaya, Vera L.; Veiko, Natalya N.

    2013-01-01

    The term “cell-free DNA” (cfDNA) was recently coined for DNA fragments from plasma/serum, while DNA present in in vitro cell culture media is known as extracellular DNA (ecDNA). Under oxidative stress conditions, the levels of oxidative modification of cellular DNA and the rate of cell death increase. Dying cells release their damaged DNA, thus, contributing oxidized DNA fragments to the pool of cfDNA/ecDNA. Oxidized cell-free DNA could serve as a stress signal that promotes irradiation-induced bystander effect. Evidence points to TLR9 as a possible candidate for oxidized DNA sensor. An exposure to oxidized ecDNA stimulates a synthesis of reactive oxygen species (ROS) that evokes an adaptive response that includes transposition of the homologous loci within the nucleus, polymerization and the formation of the stress fibers of the actin, as well as activation of the ribosomal gene expression, and nuclear translocation of NF-E2 related factor-2 (NRF2) that, in turn, mediates induction of phase II detoxifying and antioxidant enzymes. In conclusion, the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells and, possibly, in the human body; in particular, it might contribute to systemic abscopal effects of localized irradiation treatments. PMID:23533696

  9. DNA combing on low-pressure oxygen plasma modified polysilsesquioxane substrates for single-molecule studies

    PubMed Central

    Sriram, K. K.; Chang, Chun-Ling; Rajesh Kumar, U.; Chou, Chia-Fu

    2014-01-01

    Molecular combing and flow-induced stretching are the most commonly used methods to immobilize and stretch DNA molecules. While both approaches require functionalization steps for the substrate surface and the molecules, conventionally the former does not take advantage of, as the latter, the versatility of microfluidics regarding robustness, buffer exchange capability, and molecule manipulation using external forces for single molecule studies. Here, we demonstrate a simple one-step combing process involving only low-pressure oxygen (O2) plasma modified polysilsesquioxane (PSQ) polymer layer to facilitate both room temperature microfluidic device bonding and immobilization of stretched single DNA molecules without molecular functionalization step. Atomic force microscopy and Kelvin probe force microscopy experiments revealed a significant increase in surface roughness and surface potential on low-pressure O2 plasma treated PSQ, in contrast to that with high-pressure O2 plasma treatment, which are proposed to be responsible for enabling effective DNA immobilization. We further demonstrate the use of our platform to observe DNA-RNA polymerase complexes and cancer drug cisplatin induced DNA condensation using wide-field fluorescence imaging. PMID:25332730

  10. A New Blood Collection Device Minimizes Cellular DNA Release During Sample Storage and Shipping When Compared to a Standard Device

    PubMed Central

    Norton, Sheila E; Luna, Kristin K; Lechner, Joel M; Qin, Jianbing; Fernando, M Rohan

    2013-01-01

    Background Cell-free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K3EDTA tubes, when subjected to conditions that may occur during sample storage and shipping. Methods Blood samples were drawn from healthy donors into K3EDTA and Cell-Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real-time polymerase chain reaction (qPCR). Results Shaking and shipping blood in K3EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K3EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable. Conclusions BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations. PMID:23852790

  11. Probing cell-free gene expression noise in femtoliter volumes.

    PubMed

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta; Collier, C Patrick; Simpson, Michael L

    2013-09-20

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell-relevant 20 fL volumes (between the volumes of Escherichia coli and Saccharomyces cerevisiae ), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely among different containers, likely due to non-Poisson distribution of expression machinery at the observed scale. Previously, this phenomenon has been observed only in liposomes. In addition, we analyze gene expression noise. This analysis is facilitated by our use of cell-free systems, which allow the mapping of the measured noise properties to intrinsic noise models. In contrast, previous live cell noise analysis efforts have been complicated by multiple noise sources. Noise analysis reveals signatures of translational bursting, while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  12. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    NASA Astrophysics Data System (ADS)

    Han, Xu; Klas, Matej; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

    2013-09-01

    The nitrogen atmospheric pressure plasma jet (APPJ) has been shown to effectively induce DNA double strand breaks in SCC-25 oral cancer cells. The APPJ source constructed in our laboratory consists of two external electrodes wrapping around a quartz tube and nitrogen as a feed gas and operates based on dielectric barrier gas discharge. Generally, it is more challenging to ignite plasma in N2 atmosphere than in noble gases. However, this design provides additional advantages such as lower costs compared to the noble gases for future clinical operation. Different parameters of the APPJ configuration were tested in order to determine radiation dosage. To explore the effects of delayed damage and cell self-repairing, various incubation times of cells after plasma treatment were also performed. Reactive species generated in plasma jet and in liquid environment are essential to be identified and quantified, with the aim of unfolding the mystery of detailed mechanisms for plasma-induced cell apoptosis. Moreover, from the comparison of plasma treatment effect on normal oral cells OKF6T, an insight to the selectivity for cancer treatment by APPJ can be explored. All of these studies are critical to better understand the damage responses of normal and abnormal cellular systems to plasma radiation, which are useful for the development of advanced plasma therapy for cancer treatment at a later stage.

  13. Multi-purpose utility of circulating plasma DNA testing in patients with advanced cancers.

    PubMed

    Perkins, Geraldine; Yap, Timothy A; Pope, Lorna; Cassidy, Amy M; Dukes, Juliet P; Riisnaes, Ruth; Massard, Christophe; Cassier, Philippe A; Miranda, Susana; Clark, Jeremy; Denholm, Katie A; Thway, Khin; Gonzalez De Castro, David; Attard, Gerhardt; Molife, L Rhoda; Kaye, Stan B; Banerji, Udai; de Bono, Johann S

    2012-01-01

    Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5-1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical 'hot-spot' mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid

  14. Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.

    PubMed

    Belcheva, Antoaneta; Ivanova-Kicheva, Maria; Tzvetkova, Petia; Marinov, Mihail

    2004-10-01

    Cigarette smoking is a serious health problem of our society. It is known that cigarette smoke is a cell mutagen and carcinogen, and that it may affect adversely male fertility. The possible detrimental effects on sperm cells are of great interest but the data available to support this statement are somewhat elusive. To approach this problem we examined conventional semen parameters, plasma membrane translocation of phosphatidylserine (PS) (annexin V/6-CFDA cell staining) and sperm DNA integrity (comet assay) in a group of healthy man smoking cigarettes on a regular basis. The results of the study were compared with the results of the same tests in healthy non-smoking donors. Significant difference in standard sperm parameters between the two groups was not found. Intensive expression of PS on the sperm plasma membrane surface (assayed by annexin V positive staining) was detected in the smokers group. There is a significant increase of population of apoptotic spermatozoa in ejaculates of smokers. Albeit DNA damages (high frequencies of double- and single- stranded DNA breaks) in spermatozoa of smokers are increased compared with non-smokers, but this difference is not statistically significant. Sperm DNA integrity of healthy smokers remains in the normal range, but a clear negative trend is observed, especially in respect of disturbance of plasma membrane phospholipid asymmetry.

  15. GEC Student Award for Excellence Finalist: Interaction of Non-Thermal Dielectric Barrier Discharge Plasma with DNA inside Cells

    NASA Astrophysics Data System (ADS)

    Kalghatgi, Sameer; Kelly, Crystal; Fridman, Gregory; Clifford-Azizkhan, Jane; Fridman, Alexander; Friedman, Gary

    2008-10-01

    Direct non-thermal plasma is now being widely considered for various medical applications, viz; cancer treatment, coagulation, wound healing. However, the understanding of the interaction between non-thermal plasma and cells is lacking. Here we study the possibility that effects of the plasma treatment can penetrate though cellular membranes without destroying them. One of the most important of such effects to investigate would be DNA double strand breaks (DSB's) since these are some of the important events in a cell's life cycle. We measured DNA DSB's in mammalian cells using immunofluorescence and western blots. Hydrogen peroxide treatment was used as a positive control since it is known to induce massive DNA double strand breaks. The results indicate that short (5 seconds) direct plasma treatment at low power (0.2 W/cm^2) does produce DNA DSB's in mammalian cells. This means that somehow plasma penetrates inside the cells. Several questions arise about what is the mechanism of penetration and do the cells repair the DNA DSB's. We show that the cells do repair the DNA DSB's produced by short exposure of low power plasma. Although the detailed mechanisms are being investigated we confirmed that reactive oxygen species mediate interaction between plasma and DNA.

  16. Low Temperature Plasma Causes Double-Strand Break DNA Damage in Primary Epithelial Cells Cultured from a Human Prostate Tumour

    PubMed Central

    Hirst, Adam M.; Frame, Fiona M.; Maitland, Norman J.; O’Connell, Deborah

    2016-01-01

    Research in the new field of plasma medicine continues to demonstrate the efficacy of low temperature plasmas for numerous biomedical applications. Responses such as reduction in cell viability and cell death for cancer therapy, cell proliferation for wound healing, and bacterial inactivation have been observed as a result of plasma treatment. In this study we applied low temperature plasma to prostate cancer primary cells and tissue to inflict irreparable DNA damage. PMID:26819484

  17. Probing cell-free gene expression noise in femtoliter volumes

    SciTech Connect

    Karig, David K; Jung, Seung-Yong; Srijanto, Bernadeta R; Collier, Pat; Simpson, Michael L

    2013-01-01

    Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell relevant 20 fL volumes (between the volumes of E. coli and S. cerevisiae), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely at this volume, and we analyze gene expression noise. Noise analysis reveals signatures of translational bursting while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.

  18. Extracellular Self-DNA (esDNA), but Not Heterologous Plant or Insect DNA (etDNA), Induces Plasma Membrane Depolarization and Calcium Signaling in Lima Bean (Phaseolus lunatus) and Maize (Zea mays)

    PubMed Central

    Barbero, Francesca; Guglielmotto, Michela; Capuzzo, Andrea; Maffei, Massimo E.

    2016-01-01

    Extracellular self-DNA (esDNA) is produced during cell and tissue damage or degradation and has been shown to induce significant responses in several organisms, including plants. While the inhibitory effects of esDNA have been shown in conspecific individuals, little is known on the early events involved upon plant esDNA perception. We used electrophysiology and confocal laser scanning microscopy calcium localization to evaluate the plasma membrane potential (Vm) variations and the intracellular calcium fluxes, respectively, in Lima bean (Phaseolus lunatus) and maize (Zea mays) plants exposed to esDNA and extracellular heterologous DNA (etDNA) and to etDNA from Spodoptera littoralis larvae and oral secretions. In both species, esDNA induced a significant Vm depolarization and an increased flux of calcium, whereas etDNA was unable to exert any of these early signaling events. These findings confirm the specificity of esDNA to induce plant cell responses and to trigger early signaling events that eventually lead to plant response to damage. PMID:27690017

  19. Clinical relevance of circulating cell-free microRNAs in ovarian cancer.

    PubMed

    Nakamura, Koji; Sawada, Kenjiro; Yoshimura, Akihiko; Kinose, Yasuto; Nakatsuka, Erika; Kimura, Tadashi

    2016-01-01

    Ovarian cancer is the leading cause of death among gynecologic malignancies. Since ovarian cancer develops asymptomatically, it is often diagnosed at an advanced and incurable stage. Despite many years of research, there is still a lack of reliable diagnostic markers and methods for early detection and screening. Recently, it was discovered that cell-free microRNAs (miRNAs) circulate in the body fluids of healthy and diseased patients, suggesting that they may serve as a novel diagnostic marker. This review summarizes the current knowledge regarding the potential clinical relevance of circulating cell-free miRNA for ovarian cancer diagnosis, prognosis, and therapeutics. Despite the high levels of ribonucleases in many types of body fluids, most of the circulating miRNAs are packaged in microvesicles, exosomes, or apoptotic bodies, are binding to RNA-binding protein such as argonaute 2 or lipoprotein complexes, and are thus highly stable. Cell-free miRNA signatures are known to be parallel to those from the originating tumor cells, indicating that circulating miRNA profiles accurately reflect the tumor profiles. Since it is well established that the dysregulation of miRNAs is involved in the tumorigenesis of ovarian cancer, cell-free miRNAs circulating in body fluids such as serum, plasma, whole blood, and urine may reflect not only the existence of ovarian cancer but also tumor histology, stage, and prognoses of the patients. Several groups have successfully demonstrated that serum or plasma miRNAs are able to discriminate patients with ovarian cancer patients from healthy controls, suggesting that the addition of these miRNAs to current testing regimens may improve diagnosis accuracies for ovarian cancer. Furthermore, recent studies have revealed that changes in levels of cell-free circulating miRNAs are associated with the condition of cancer patients. Discrepancies between the results across studies due to the lack of an established endogenous miRNA control to

  20. Losartan reduces oxidative damage to renal DNA and conserves plasma antioxidant capacity in diabetic rats

    PubMed Central

    Bigagli, Elisabetta; Tarantini, Francesca; Di Serio, Claudia; Raimondi, Laura

    2015-01-01

    Increased reactive oxygen species (ROS) levels produced by hyperglycemia and angiotensin-II (AT-II) are considered among the pathogenic factors in the malignant transformation of diabetic renal cells. We aimed to investigate the potential role of AT-II in the increased cancer risk seen in diabetes; measuring oxidative damage to renal DNA and protective antioxidant defenses, including adiponectin (Adp) and plasma antioxidant capacity by the Ferric Reducing Ability of Plasma (FRAP) method. In the kidney of streptozotocin (STZ)-induced (55 mg/kg) diabetic rats either treated or not treated for 3 weeks with losartan, an AT-II type 1 receptor antagonist (20 mg/kg/day); we measured 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels, as an index of oxidative DNA damage, circulating Adp and FRAP. Diabetic rats showed significantly higher 8-oxodGuo levels in renal DNA (8.48 ± 0.98 × 10−6 dG, mean ± SEM n = 11) than normoglycemic ones (1.18 ± 0.04 × 10−6 dG, mean ± SEM, n=7) and lower plasma Adp and FRAP levels in comparison to normoglycemics. The treatment of diabetic rats with losartan significantly (P < 0.01) reduced 8-oxodGuo levels (5.4 ± 0.58 × 10−6 dG, mean ± SEM n=9) in renal DNA and conserved FRAP values. Moreover, an inverse correlation was found between 8-oxodGuo in kidney DNA and circulating Adp levels in normoglycemic and diabetic rats. Losartan treatment preserves FRAP levels, reduces DNA oxidative injury and thus the carcinogenesis risk. Furthermore, our results indicate that Adp plasma levels are a further marker of oxidative injury to the kidney and confirm that it is an important part of the plasma antioxidant defense. PMID:25710927

  1. Losartan reduces oxidative damage to renal DNA and conserves plasma antioxidant capacity in diabetic rats.

    PubMed

    Lodovici, Maura; Bigagli, Elisabetta; Tarantini, Francesca; Di Serio, Claudia; Raimondi, Laura

    2015-11-01

    Increased reactive oxygen species (ROS) levels produced by hyperglycemia and angiotensin-II (AT-II) are considered among the pathogenic factors in the malignant transformation of diabetic renal cells. We aimed to investigate the potential role of AT-II in the increased cancer risk seen in diabetes; measuring oxidative damage to renal DNA and protective antioxidant defenses, including adiponectin (Adp) and plasma antioxidant capacity by the Ferric Reducing Ability of Plasma (FRAP) method. In the kidney of streptozotocin (STZ)-induced (55 mg/kg) diabetic rats either treated or not treated for 3 weeks with losartan, an AT-II type 1 receptor antagonist (20 mg/kg/day); we measured 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels, as an index of oxidative DNA damage, circulating Adp and FRAP. Diabetic rats showed significantly higher 8-oxodGuo levels in renal DNA (8.48 ± 0.98 × 10(-6) dG, mean ± SEM n = 11) than normoglycemic ones (1.18 ± 0.04 × 10(-6) dG, mean ± SEM, n=7) and lower plasma Adp and FRAP levels in comparison to normoglycemics. The treatment of diabetic rats with losartan significantly (P < 0.01) reduced 8-oxodGuo levels (5.4 ± 0.58 × 10(-6) dG, mean ± SEM n=9) in renal DNA and conserved FRAP values. Moreover, an inverse correlation was found between 8-oxodGuo in kidney DNA and circulating Adp levels in normoglycemic and diabetic rats. Losartan treatment preserves FRAP levels, reduces DNA oxidative injury and thus the carcinogenesis risk. Furthermore, our results indicate that Adp plasma levels are a further marker of oxidative injury to the kidney and confirm that it is an important part of the plasma antioxidant defense.

  2. A cell free system for functional centromere and kinetochore assembly Authors

    PubMed Central

    Guse, Annika; Fuller, Colin J.; Straight, Aaron F.

    2013-01-01

    Summary This protocol describes a cell free system to study vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell free system lends itself to protein immunodepletion, complementation and drug inhibition as tools to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification, and protein post-translational modification. This system provides a distinct advantage over cell-based investigations where perturbing centromere and kinetochore function often results in lethality. Reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins after incubation in egg extract that locally stabilize microtubules and, upon microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment occupies 3 days. PMID:23018190

  3. Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study

    PubMed Central

    Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

    2016-01-01

    Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future. PMID:27631491

  4. Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

    PubMed

    Jiang, Haojun; Xie, Yifan; Li, Xuchao; Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

    2016-01-01

    Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future. PMID:27631491

  5. The emerging age of cell-free synthetic biology.

    PubMed

    Smith, Mark Thomas; Wilding, Kristen M; Hunt, Jeremy M; Bennett, Anthony M; Bundy, Bradley C

    2014-08-25

    The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.

  6. [Transcripts of satellite DNA in blood plasma: probable markers of tumor growth].

    PubMed

    Kondratova, V N; Botezatu, I V; Shelepov, V P; Likhtenshtein, A V

    2014-01-01

    A recent study of human normal and tumor tissues revealed a high transcriptional activity of pericentromeric satellite DNA repeats (they produce half of all transcripts in tumor cells that is many times higher than in normal ones). It was found also that the two subtypes of satellite DNA (HSATII and GSATII) are transcribed reciprocally, i.e. there is a sharp prevalence of HSATII transcription in tumors, while GSATII transcription prevails in the corresponding normal tissues. As different RNAs are present in blood plasma, and some of them serve as effectivetumor markers, we attempted for the first time to evaluate satellite HSATII and GSATII RNAs in the blood plasma of healthy donors and cancer patients. The RT-PCR protocol designed for this purpose allowed us to detect transcripts of both HSATII and GSATII repeats. As it has been shown, HSATII transcripts are more abundant than GSATII ones in plasma of healthy donors and vice versa in plasma of cancer patients; these ratios being diametrically opposed to those that exist within the cells. Some suggestions concerning origins of circulating satellite RNAs and their probable role as tumor markers are discussed.

  7. Creating a completely "cell-free" system for protein synthesis.

    PubMed

    Smith, Mark Thomas; Bennett, Anthony M; Hunt, Jeremy M; Bundy, Bradley C

    2015-01-01

    Cell-free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell-free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell-free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell-free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell-free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems' protein synthesis capabilities. Lyophilization provides the additional benefit of long-term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely "cell-free" systems.

  8. Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation.

    PubMed

    Korzeneva, Inna B; Kostuyk, Svetlana V; Ershova, Liza S; Osipov, Andrian N; Zhuravleva, Veronika F; Pankratova, Galina V; Porokhovnik, Lev N; Veiko, Natalia N

    2015-09-01

    The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly - at survival of the cells, including the cells with the damaged DNA. PMID:26113293

  9. DNA damage in oral cancer and normal cells induced by nitrogen atmospheric pressure plasma jets

    NASA Astrophysics Data System (ADS)

    Han, Xu; Kapaldo, James; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

    2015-09-01

    Nitrogen atmospheric pressure plasma jets (APPJs) have been shown to effectively induce DNA double strand breaks in SCC25 oral cancer cells. The APPJ source constructed in our laboratory operates based on dielectric barrier discharge. It consists of two copper electrodes alternatively wrapping around a fused silica tube with nitrogen as a feed gas. It is generally more challenging to ignite plasma in N2 atmosphere than in noble gases. However, N2 provides additional advantages such as lower costs compared to noble gases, thus this design can be beneficial for the future long-term clinical use. To compare the effects of plasma on cancer cells (SCC25) and normal cells (OKF), the cells from both types were treated at the same experimental condition for various treatment times. The effective area with different damage levels after the treatment was visualized as 3D maps. The delayed damage effects were also explored by varying the incubation times after the treatment. All of these studies are critical for a better understanding of the damage responses of cellular systems exposed to the plasma radiation, thus are useful for the development of the advanced plasma cancer therapy. The research described herein was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Basic Energy Sciences, Office of Science, United States Department of Energy through Grant No. DE-FC02-04ER15533.

  10. In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission.

    PubMed

    Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

    2015-11-11

    Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell-cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105-106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT-BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.

  11. In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

    PubMed Central

    Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

    2015-01-01

    Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105–106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies. PMID:26569290

  12. Circulating DNA in plasma and serum: biology, preanalytical issues and diagnostic applications.

    PubMed

    Lui, Yanni Y N; Dennis, Y M L

    2002-10-01

    The recent studies in circulating nucleic acids have brought about a new dimension to medical diagnostics. In oncology, various tumor-associated molecular alterations have been detected in the plasma/serum of cancer patients. These findings have important implications for the diagnosis, prognostication and monitoring of many types of malignancies. In pregnancy, the discovery of fetal DNA in maternal circulation has opened up a new source of fetal genetic material for noninvasive analysis for numerous fetal conditions and detection of certain pregnancy-associated disorders. The measurement of circulating DNA has also found potential application in the post-treatment monitoring of transplant patients and the assessment and prognostication of trauma patients. Although much attention has focused on circulating DNA, the knowledge of its biology is still at an early stage. For example, the origin and mechanisms of release of circulating DNA remain to be elucidated. The eventual clinical application of circulating DNA technology would also require the thorough elucidation of preanalytical factors that may affect its measurement in clinical laboratories.

  13. Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

    PubMed Central

    Hrebien, Sarah; O’Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Turner, Nicholas

    2016-01-01

    Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research. PMID:27760227

  14. Fluorescent In Situ Folding Control for Rapid Optimization of Cell-Free Membrane Protein Synthesis

    PubMed Central

    Müller-Lucks, Annika; Bock, Sinja; Wu, Binghua; Beitz, Eric

    2012-01-01

    Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality. PMID:22848743

  15. A cell-free expression and purification process for rapid production of protein biologics.

    PubMed

    Sullivan, Challise J; Pendleton, Erik D; Sasmor, Henri H; Hicks, William L; Farnum, John B; Muto, Machiko; Amendt, Eric M; Schoborg, Jennifer A; Martin, Rey W; Clark, Lauren G; Anderson, Mark J; Choudhury, Alaksh; Fior, Raffaella; Lo, Yu-Hwa; Griffey, Richard H; Chappell, Stephen A; Jewett, Michael C; Mauro, Vincent P; Dresios, John

    2016-02-01

    Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

  16. A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery.

    PubMed

    Karim, Ashty S; Jewett, Michael C

    2016-07-01

    Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms. PMID:26996382

  17. DNA strand breaks induced by soft X-ray pulses from a compact laser plasma source

    NASA Astrophysics Data System (ADS)

    Adjei, Daniel; Wiechec, Anna; Wachulak, Przemyslaw; Ayele, Mesfin Getachew; Lekki, Janusz; Kwiatek, Wojciech M.; Bartnik, Andrzej; Davídková, Marie; Vyšín, Luděk; Juha, Libor; Pina, Ladislav; Fiedorowicz, Henryk

    2016-03-01

    Application of a compact laser plasma source of soft X-rays in radiobiology studies is demonstrated. The source is based on a laser produced plasma as a result of irradiation of a double-stream gas puff target with nanosecond laser pulses from a commercially available Nd:YAG laser. The source allows irradiation of samples with soft X-ray pulses in the "water window" spectral range (wavelength: 2.3-4.4 nm; photon energy: 280-560 eV) in vacuum or a helium atmosphere at very high-dose rates and doses exceeding the kGy level. Single-strand breaks (SSB) and double-strand breaks (DBS) induced in DNA plasmids pBR322 and pUC19 have been measured. The different conformations of the plasmid DNA were separated by agarose gel electrophoresis. An exponential decrease in the supercoiled form with an increase in linear and relaxed forms of the plasmids has been observed as a function of increasing photon fluence. Significant difference between SSB and DSB in case of wet and dry samples was observed that is connected with the production of free radicals in the wet sample by soft X-ray photons and subsequent affecting the plasmid DNA. Therefore, the new source was validated to be useful for radiobiology experiments.

  18. [Genetic diagnosis of thalassemia mutations with free fetal DNA in pregnant plasma].

    PubMed

    Lin, Xiao-Rong; You, Liu-Xia; Chen, Yong

    2013-10-01

    This research was aimed to develop a simple, rapid, accurate and non-invasive method by means of flow-through hybridization technology, which can be used for molecular screening and early prenatal diagnosis for detecting common β-thalassemias mutational genotypes. By using PCR technology combined with flow-through hybridization of low-density gene chip technology, the 6 sets of PCR primer single tube multiplex PCR system and 29 types of DNA probes were designed, then the mutational thalassemias in foetus DNA was rapidly detected in total of 60 anaemia pregnant women plasma. The results showed that 4 cases with deletional α-thalassemias, 3 cases with β-thalassemias, 1 case with mixed type of α & β-thalassemias were detected in foetus DNA of 60 pregnant women plasmas. It is concluded that the method presented in this study is easy to handle, rapid, reliable and cost-effective for detecting 3 common deletional α-thalassemias and 17 common mutational β-thalassemia.

  19. Mechanisms of a novel anticancer therapeutic strategy involving atmospheric pressure plasma-mediated apoptosis and DNA strand break formation.

    PubMed

    Chung, Woo-Hyun

    2016-01-01

    Atmospheric pressure plasma has been developed for a variety of biomedical applications due to its chemically reactive components. Recently, the plasma has emerged as a promising novel cancer therapy based on its ability to selectively ablate cancer cells while leaving normal cells essentially unaffected. The therapeutic effect of plasma is attributed to intracellular generation of reactive oxygen/nitrogen species (ROS/RNS) leading to mitochondria-mediated apoptosis and to activation of the DNA damage checkpoint signaling pathway via severe DNA strand break formation. However, the biochemical mechanisms responsible for appropriate activation of these physiological events and which pathway is more crucial for plasma-mediated cytotoxicity have not been clarified. Understanding the molecular link between ROS/RNS-mediated apoptosis and DNA damage-involved chromosome instability is critical for the development of more efficacious therapeutic strategies for selective killing of diverse cancer cells.

  20. Cell-Free Synthetic Biology: Thinking Outside the Cell

    PubMed Central

    Hodgman, C. Eric; Jewett, Michael C.

    2011-01-01

    Cell-free synthetic biology is emerging as a powerful technology aimed to understand, harness, and expand the capabilities of natural biological systems without using intact cells. Cell-free systems bypass cell walls and remove genetic regulation to enable direct access to the inner workings of the cell. The unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the rapid development of engineering foundations for cell-free systems in recent years. These efforts have led to programmed circuits, spatially organized pathways, co-activated catalytic ensembles, rational optimization of synthetic multi-enzyme pathways, and linear scalability from the micro-liter to the 100-liter scale. It is now clear that cell-free systems offer a versatile test-bed for understanding why nature’s designs work the way they do and also for enabling biosynthetic routes to novel chemicals, sustainable fuels, and new classes of tunable materials. While challenges remain, the emergence of cell-free systems is poised to open the way to novel products that until now have been impractical—if not impossible—to produce by other means. PMID:21946161

  1. Use of molecular beacons for the rapid analysis of DNA damage induced by exposure to an atmospheric pressure plasma jet

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Miyachika, Saki; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2015-12-01

    A rapid method for evaluating the damage caused to DNA molecules upon exposure to plasma is demonstrated. Here, we propose the use of a molecular beacon for rapid detection of DNA strand breaks induced by atmospheric pressure plasma jet (APPJ) irradiation. Scission of the molecular beacon by APPJ irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the DNA strand breaks. The results show that the increase in fluorescence intensity is proportional to the exposure time and the rate of fluorescence increase is proportional to the discharge power. This simple and rapid method allows the estimation of DNA damage induced by exposure to a non-thermal plasma.

  2. Use of molecular beacons for the rapid analysis of DNA damage induced by exposure to an atmospheric pressure plasma jet

    SciTech Connect

    Kurita, Hirofumi E-mail: mizuno@ens.tut.ac.jp; Miyachika, Saki; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira E-mail: mizuno@ens.tut.ac.jp

    2015-12-28

    A rapid method for evaluating the damage caused to DNA molecules upon exposure to plasma is demonstrated. Here, we propose the use of a molecular beacon for rapid detection of DNA strand breaks induced by atmospheric pressure plasma jet (APPJ) irradiation. Scission of the molecular beacon by APPJ irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the DNA strand breaks. The results show that the increase in fluorescence intensity is proportional to the exposure time and the rate of fluorescence increase is proportional to the discharge power. This simple and rapid method allows the estimation of DNA damage induced by exposure to a non-thermal plasma.

  3. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  4. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H. Craig

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  5. Method of producing a cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1998-05-26

    Bacteria which produce large amounts of cellulose-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  6. Cellulase-containing cell-free fermentate produced from microorganism ATCC 55702

    DOEpatents

    Dees, H.C.

    1997-12-16

    Bacteria which produce large amounts of cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  7. Production of eukaryotic cell-free lysate from Leishmania tarentolae.

    PubMed

    Johnston, Wayne A; Alexandrov, Kirill

    2014-01-01

    In this chapter, we describe the production and application of a eukaryotic cell-free expression system based on Leishmania tarentolae. This single-celled flagellate allows straightforward and inexpensive cultivation in flasks or bioreactors. Unlike many other Leishmania species, it is nonpathogenic to humans and does not require special laboratory precautions. An additional reason it is a convenient source organism for cell-free lysate production is that all endogenous protein expression can be suppressed by a single antisense oligonucleotide targeting splice leader sequence on the 5'-end of all protein coding RNAs. We describe simple procedures for cell disruption and lysate processing starting from bioreactor culture. We also describe introduction of genetic information via vectors containing species-independent translation initiation sites (SITS). We consider that such an inexpensive eukaryotic cell-free production system has many advantages when expressing multi-subunit proteins or difficult to express proteins. PMID:24395406

  8. Determination of phosphorus impurity that directly affects quantification of microbial genomic DNA using inductively coupled plasma optical emission spectrometry.

    PubMed

    Yang, Hyo-Jin; Yang, Inchul; Choi, Jun-Hyuk; Kang, Dukjin; Han, Myung-Sub; Kim, Sook-Kyung

    2014-04-01

    We prepared genomic DNA from human placenta, Escherichia coli, and Bacillus subtilis using various DNA extraction methods and quantified the genomic DNA using ultraviolet (UV) spectrophotometry, capillary electrophoresis (CE), and inductively coupled plasma optical emission spectrometry (ICP-OES). Application of ICP-OES unexpectedly led to a serious overestimation of phosphorus in B. subtilis genomic DNA prepared using cetyltrimethyl ammonium bromide (CTAB). Further investigations using reversed-phase high-performance liquid chromatography (RP-HPLC), ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and (31)P nuclear magnetic resonance (NMR) identified the phosphorus impurity as lipoteichoic acid (LTA). PMID:24486318

  9. Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients.

    PubMed

    Riediger, Anja Lisa; Dietz, Steffen; Schirmer, Uwe; Meister, Michael; Heinzmann-Groth, Ingrid; Schneider, Marc; Muley, Thomas; Thomas, Michael; Sültmann, Holger

    2016-01-01

    Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods. PMID:27640882

  10. Mutation analysis of circulating plasma DNA to determine response to EGFR tyrosine kinase inhibitor therapy of lung adenocarcinoma patients

    PubMed Central

    Riediger, Anja Lisa; Dietz, Steffen; Schirmer, Uwe; Meister, Michael; Heinzmann-Groth, Ingrid; Schneider, Marc; Muley, Thomas; Thomas, Michael; Sültmann, Holger

    2016-01-01

    Long-lasting success in lung cancer therapy using tyrosine kinase inhibitors (TKIs) is rare since the tumors develop resistance due to the occurrence of molecularly altered subclones. The aim of this study was to monitor tumors over time based on the quantity of mutant plasma DNA and to identify early indications for therapy response and tumor progression. Serial plasma samples from lung adenocarcinoma patients treated with TKIs were used to quantify EGFR and KRAS mutations in circulating DNA by digital PCR. Mutant DNA levels were compared with the courses of responses to treatment with TKIs, conventional chemotherapy, radiotherapy, or combinations thereof. Variations in plasma DNA mutation levels over time were found in 15 patients. We categorize three major courses: First, signs of therapy response are associated with a fast clearing of plasma DNA mutations within a few days. Second, periods of stable disease are accompanied by either absence of mutations or fluctuation at low levels. Finally, dramatic increase of mutational load is followed by rapid tumor progression and poor patient survival. In summary, the serial assessment of EGFR mutations in the plasma of NSCLC patients allows conclusions about controlled disease and tumor progression earlier than currently available methods. PMID:27640882

  11. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    PubMed Central

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  12. Lack of KIs virus DNA in plasma and cerebrospinal fluid in Italy.

    PubMed

    Macera, Lisa; Focosi, Daniele; Manzin, Aldo; Ceccherini Nelli, Luca; Pistello, Mauro; Maggi, Fabrizio

    2015-10-01

    Dear Sirs, Satoh et al. recently screened 516 Japanese blood donors with PCR using primers constructed from the consensus domain of the helicase of positive-stranded RNA viruses. They reported a novel enveloped virus with a circular double-stranded DNA genome (tentatively named KIs virus, KIs-V) (Satoh et al., 2011) occurring in 36 out of the 100 hepatitis E (HEV) antibody-positive donors with elevated alanine aminotransferase (ALT) levels (>60 IU/L). More recently, Biagini et al. failed to find KIs-V in plasma from 576 French blood donors with unknown HEV serostatus and unknown ALT values (Biagini et al., 2012). Based on an HEV seroprevalence of 3-52% in France, the authors suggested an uncommon frequency of KIs-V infection in healthy persons in France. To date, no information has been available on the prevalence of KIs-V DNA in Italy. In the present paper, we analyzed KIs-V in 242 plasma samples of blood donors, transplant recipients, and patients with chronic viral infections, and in 52 cerebrospinal fluid (CSF) samples of patients with different neurological disorders. Informed consent was obtained from all patients and the study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its amendments. Viral DNA extraction was carried out on 200 μl of plasma or 200 μl of CSF by using QIAamp DNA blood kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Extracted nucleic acids were amplified for KIs-V DNA with the nested PCR protocol developed by Satoh et al. (2011) and used for screening Japanese blood donors. The first and second PCR rounds were designed on 458 and 304 nt-length fragments, respectively. To validate the amplification process, positive controls obtained from plasma dilutions of a synthetic template corresponding to the target sequence were run in each PCR. PCR sensitivity was less than 5 copies of target sequence. Fourteen liver and 16 kidney and/or pancreas transplant

  13. Lack of KIs virus DNA in plasma and cerebrospinal fluid in Italy.

    PubMed

    Macera, Lisa; Focosi, Daniele; Manzin, Aldo; Ceccherini Nelli, Luca; Pistello, Mauro; Maggi, Fabrizio

    2015-10-01

    Dear Sirs, Satoh et al. recently screened 516 Japanese blood donors with PCR using primers constructed from the consensus domain of the helicase of positive-stranded RNA viruses. They reported a novel enveloped virus with a circular double-stranded DNA genome (tentatively named KIs virus, KIs-V) (Satoh et al., 2011) occurring in 36 out of the 100 hepatitis E (HEV) antibody-positive donors with elevated alanine aminotransferase (ALT) levels (>60 IU/L). More recently, Biagini et al. failed to find KIs-V in plasma from 576 French blood donors with unknown HEV serostatus and unknown ALT values (Biagini et al., 2012). Based on an HEV seroprevalence of 3-52% in France, the authors suggested an uncommon frequency of KIs-V infection in healthy persons in France. To date, no information has been available on the prevalence of KIs-V DNA in Italy. In the present paper, we analyzed KIs-V in 242 plasma samples of blood donors, transplant recipients, and patients with chronic viral infections, and in 52 cerebrospinal fluid (CSF) samples of patients with different neurological disorders. Informed consent was obtained from all patients and the study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its amendments. Viral DNA extraction was carried out on 200 μl of plasma or 200 μl of CSF by using QIAamp DNA blood kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Extracted nucleic acids were amplified for KIs-V DNA with the nested PCR protocol developed by Satoh et al. (2011) and used for screening Japanese blood donors. The first and second PCR rounds were designed on 458 and 304 nt-length fragments, respectively. To validate the amplification process, positive controls obtained from plasma dilutions of a synthetic template corresponding to the target sequence were run in each PCR. PCR sensitivity was less than 5 copies of target sequence. Fourteen liver and 16 kidney and/or pancreas transplant

  14. Sealable Femtoliter Chamber Arrays for Cell-free Biology

    PubMed Central

    Norred, Sarah Elizabeth; Caveney, Patrick M.; Retterer, Scott T.; Boreyko, Jonathan B.; Fowlkes, Jason D.; Collier, Charles Patrick; Simpson, Michael L.

    2015-01-01

    Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment. PMID:25867144

  15. Sealable femtoliter chamber arrays for cell-free biology

    DOE PAGES

    Retterer, Scott T.; Fowlkes, Jason Davidson; Collier, Charles Patrick; Simpson, Michael L.; Norred, Sarah Elizabeth; Caveney, Patrick M.; Boreyko, Jonathan B.

    2015-03-11

    Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g. global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) devicemore » contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Lastly, we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques to characterize CFPS gene circuits and their interactions with the cell-free environment.« less

  16. Sealable femtoliter chamber arrays for cell-free biology

    SciTech Connect

    Retterer, Scott T.; Fowlkes, Jason Davidson; Collier, Charles Patrick; Simpson, Michael L.; Norred, Sarah Elizabeth; Caveney, Patrick M.; Boreyko, Jonathan B.

    2015-03-11

    Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g. global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Lastly, we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques to characterize CFPS gene circuits and their interactions with the cell-free environment.

  17. Does rat fetal DNA induce preeclampsia in pregnant rats?

    PubMed

    Konečná, B; Borbélyová, V; Celec, P; Vlková, B

    2015-02-01

    Cell-free fetal DNA in maternal circulation is higher during preeclampsia. It is unclear whether it is the cause or the consequence of the disease. The aim of this study was to prove whether injected rat fetal DNA induces preeclampsia-like symptoms in pregnant Wistar rats. They received daily i.p. injections of water or rat fetal DNA (400 μg) from gestation day 14 to 18. Blood pressure, proteinuria, placental and fetal weight were measured at gestation day 19. Plasma DNase activity, proteinuria and creatinine clearance were assessed. There was no significant difference in any of the measured parameters. The results of this study do not confirm the hypothesis that fetal DNA might induce preeclampsia. This is in contrast to others using human fetal DNA in mice. Further studies should be focused on the effects of fetal DNA from the same species protected from DNase activity.

  18. Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.

    PubMed

    Arabi, M

    2004-10-01

    Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r = -0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA. PMID:15458549

  19. Experiment and mathematical modeling of gene expression dynamics in a cell-free system.

    PubMed

    Stögbauer, Tobias; Windhager, Lukas; Zimmer, Ralf; Rädler, Joachim O

    2012-05-01

    Cell-free in vitro expression is increasingly important for high-throughput expression screening, high yield protein production and synthetic biology applications. Yet its potential for quantitative investigation of gene expression and regulatory circuits is limited by the availability of data on composition, kinetic rate constants and standardized computational tools for modeling. Here we report on calibration measurements and mathematical modeling of a reconstituted in vitro expression system. We measured a series of GFP expression and mRNA transcription time courses under various initial conditions and established the translation step as the bottle neck of in vitro protein synthesis. Cell-free translation was observed to expire after 3 h independent of initial template DNA concentration. We developed a minimalistic rate equation model and optimized its parameters by performing a concurrent fit to measured time courses. The model predicts the dependence of protein yield not only on template DNA concentration, but also on experimental timing and hence is a valuable tool to optimize yield strategies. PMID:22481223

  20. Electrochemical Characterization of O2 Plasma Functionalized Multi-Walled Carbon Nanotube Electrode for Legionella pneumophila DNA Sensor

    NASA Astrophysics Data System (ADS)

    Park, Eun Jin; Lee, Jun-Yong; Hyup Kim, Jun; Kug Kim, Sun; Lee, Cheol Jin; Min, Nam Ki

    2010-08-01

    An electrochemical DNA sensor for Legionella pneumophila detection was constructed using O2 plasma functionalized multi-walled carbon nanotube (MWCNT) film as a working electrode (WE). The cyclic voltammetry (CV) results revealed that the electrocatalytic activity of plasma functionalized MWCNT (pf-MWCNT) significantly changed depending on O2 plasma treatment time due to some oxygen containing functional groups on the pf-MWCNT surface. Scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra were also presented the changes of their surface morphologies and oxygen composition before and after plasma treatment. From a comparison study, it was found that the pf-MWCNT WEs had higher electrocatalytic activity and more capability of probe DNA immobilization: therefore, electrochemical signal changes by probe DNA immobilization and hybridization on pf-MWCNT WEs were larger than on Au WEs. The pf-MWCNT based DNA sensor was able to detect a concentration range of 10 pM-100 nM of target DNA to detect L. pneumophila.

  1. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    DOEpatents

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  2. Effects of Erythrocyte Deformability and Aggregation on the Cell Free Layer and Apparent Viscosity of Microscopic Blood Flows

    PubMed Central

    Zhang, Junfeng; Johnson, Paul C.; Popel, Aleksander S.

    2010-01-01

    Concentrated erythrocyte (i.e., red blood cell) suspensions flowing in microchannels have been simulated with an immersed-boundary lattice Boltzmann algorithm, to examine the cell layer development process and the effects of cell deformability and aggregation on hemodynamic and hemorheological behaviors. The cells are modeled as two-dimensional deformable biconcave capsules and experimentally measured cell properties have been utilized. The aggregation among cells is modeled by a Morse potential. The flow development process demonstrates how red blood cells migrate away from the boundary toward the channel center, while the suspending plasma fluid is displaced to the cell free layer regions left by the migrating cells. Several important characteristics of microscopic blood flows observed experimentally have been well reproduced in our model, including the cell free layer, blunt velocity profile, changes in apparent viscosity, and the Fahraeus effect. We found that the cell free layer thickness increases with both cell deformability and aggregation strength. Due to the opposing effects of the cell free layer lubrication and the high viscosity of cell-concentrated core, the influence of aggregation is complex but. The lubrication effect appears to dominate, causing the relative apparent viscosity to decrease with aggregation. It appears therefore that the immersed-boundary lattice Boltzmann numerical model may be useful in providing valuable information on microscopic blood flows in various microcirculation situations. PMID:19323969

  3. Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo).

    PubMed

    Słowińska, Mariola; Olczak, Mariusz; Wojtczak, Mariola; Glogowski, Jan; Jankowski, Jan; Watorek, Wiesław; Amarowicz, Ryszard; Ciereszko, Andrzej

    2008-06-01

    The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

  4. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    PubMed

    Catherine, Christy; Lee, Seung-Won; Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  5. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

    PubMed Central

    Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

    2015-01-01

    Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

  6. Longitudinal Bank for Serum, Plasma and DNA for Detection of Biomarkers

    SciTech Connect

    Ward, David C

    2009-01-31

    With the support of this DOE appropriation, NVCI has established a biorepository for serum, plasma, DNA and urine specimens. Over 2,500 patients have been consented which is over 90% of all new patients seen at NVCI. The specimens have been coded, centrifuged, aliquoted and frozen at -80°C in a rapid manner so that they are all processed in less than 1 hour from the acquisition. There are over 28,000 aliquoted, coded tubes in our inventory. Specimens from 200 control volunteer subjects without any history of cancer also have been banked. The patient specimens are encoded and the demographics and therapeutic treatments are linked to the Oncore software. This computer program catalogues the specimens and provides a rapid conduit between the biorepository and the NVCI electronic medical record.

  7. An optimized sensitive method for quantitation of DNA/RNA viruses in heparinized and cryopreserved plasma

    PubMed Central

    Ding, Ming; Bullotta, Arlene; Caruso, Lori; Gupta, Phalguni; Rinaldo, Charles R.; Chen, Yue

    2011-01-01

    Sodium heparin, an anticoagulant used widely for blood collection, has been known to inhibit DNA polymerase activity in polymerase chain reaction (PCR) assays. However, all cryopreserved plasma samples collected in the 1980s and early 1990s at the Multicenter AIDS Cohort Study were from heparin-treated blood, which poses a problem in quantifying the target nucleic acids contained in those samples by PCR assay. In this study, a nucleic acid extraction procedure was optimized to remove the heparin from extracted nucleic acids. Using this optimized method, similar human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV) loads of these viruses that were added to normal donor blood from ethylenediaminetetraacetic acid (EDTA), acid citrate dextrose (ACD) or sodium heparin tubes were detected by reverse transcriptase (RT) real-time PCR and real-time PCR. Comparable HIV-1 and CMV loads were also detected in the blood of persons with active HIV-1 and CMV infections collected in EDTA-, ACD- or sodium heparin-treated tubes by RT real-time and real-time PCR. The findings showed that the optimized nucleic acid extraction procedure efficiently removes the heparin inhibition effect on the performance of real-time PCR. This method could be used to extract nucleic acids from archived, heparinized plasma for PCR based quantitation of target molecules. PMID:21645549

  8. Application of fetal DNA in maternal plasma for noninvasive prenatal diagnosis.

    PubMed

    Chiu, Rossa W K; Lo, Y M Dennis

    2002-01-01

    Prenatal diagnosis of fetal genetic conditions is a standard part of modern obstetric care. Many of the current methods rely on invasive methods and are associated with an inherent risk of fetal loss. Consequently, there has been a long-term goal for development of noninvasive prenatal diagnostic methods. In 1997, the presence of fetal DNA in maternal plasma was first discovered through the detection of Y-chromosome-specific sequences in the plasma of women conceived with male fetuses. This discovery has opened up new possibilities in the development of noninvasive prenatal diagnostic methods through a source of fetal genetic material that could be conveniently accessible simply through the collection of a maternal peripheral blood sample. To date, there have been numerous reported applications, including fetal RhD genotyping, prenatal diagnosis of sex-linked disorders, paternally inherited genetic diseases and some pregnancy-associated conditions, including preeclampsia. More recently, there have been significant new developments with expanding number of potential applications.

  9. Association between sperm DNA integrity and seminal plasma antioxidant levels in health workers occupationally exposed to ionizing radiation

    SciTech Connect

    Kumar, Dayanidhi; Salian, Sujith Raj; Kalthur, Guruprasad; Uppangala, Shubhashree; Kumari, Sandhya; Challapalli, Srinivas; Chandraguthi, Shrinidhi Gururajarao; Jain, Navya; Krishnamurthy, Hanumanthappa; Kumar, Pratap; Adiga, Satish Kumar

    2014-07-15

    There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers. - Highlights: • Seminal plasma antioxidants were measured in men occupationally exposed to radiation. • Sperm chromatin integrity was significantly affected in the exposed group. • Glutathione and total antioxidant capacity was significantly higher in exposed group. • Sperm DNA damage in exposed subjects

  10. Hepatitis B virus DNA stability in plasma samples under short-term storage at 42°C.

    PubMed

    Almeida, R W de; Espírito-Santo, M P; Sousa, P S F; Almeida, A J de; Lampe, E; Lewis-Ximenez, L L

    2015-06-01

    We evaluated the stability of hepatitis B virus (HBV) DNA in plasma samples stored at 42°C for external quality assessment (EQA) panels of viral load. To assess the stability of plasma samples containing different concentrations of HBV DNA, serial dilutions of HBV-infected samples with a viral load of 6.40 log(10) IU/mL were made to yield viral loads of 5, 4, and 3 log(10) IU/mL. These were incubated at 42°C for up to 7 days and then frozen at -70°C. Viral load testing for HBV DNA was performed for all samples using COBAS® AmpliPrep/COBAS® TaqMan® HBV Test (v.2.0, Roche, Switzerland). Results were compared with fresh frozen plasma samples as a benchmark to establish acceptable measurements on the days following sample collection. Although the results of this study demonstrated a decrease in HBV DNA viral load ranging from 0.005 to 0.30 log(10) IU/mL after storage at 42°C for up to 7 days, these values did not exceed 0.5 log(10), which is the estimated intra-assay variation for molecular tests. Thus, the insignificant decrease in viral load suggests that shipment of HBV in plasma samples at temperatures of up to 42°C is permissible if they are frozen within 7 days.

  11. Hepatitis B virus DNA stability in plasma samples under short-term storage at 42°C

    PubMed Central

    de Almeida, R.W.; Espírito-Santo, M.P.; Sousa, P.S.F.; de Almeida, A.J.; Lampe, E.; Lewis-Ximenez, L.L.

    2015-01-01

    We evaluated the stability of hepatitis B virus (HBV) DNA in plasma samples stored at 42°C for external quality assessment (EQA) panels of viral load. To assess the stability of plasma samples containing different concentrations of HBV DNA, serial dilutions of HBV-infected samples with a viral load of 6.40 log(10) IU/mL were made to yield viral loads of 5, 4, and 3 log(10) IU/mL. These were incubated at 42°C for up to 7 days and then frozen at -70°C. Viral load testing for HBV DNA was performed for all samples using COBAS¯ AmpliPrep/COBAS¯ TaqMan¯ HBV Test (v.2.0, Roche, Switzerland). Results were compared with fresh frozen plasma samples as a benchmark to establish acceptable measurements on the days following sample collection. Although the results of this study demonstrated a decrease in HBV DNA viral load ranging from 0.005 to 0.30 log(10) IU/mL after storage at 42°C for up to 7 days, these values did not exceed 0.5 log(10), which is the estimated intra-assay variation for molecular tests. Thus, the insignificant decrease in viral load suggests that shipment of HBV in plasma samples at temperatures of up to 42°C is permissible if they are frozen within 7 days. PMID:25790101

  12. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

  13. Comparison of the prognostic impact of serum anti-EBV antibody and plasma EBV DNA assays in nasopharyngeal carcinoma

    SciTech Connect

    Twu, C.-W.; Wang, W.-Y.; Liang, W.-M.; Jan, J.-S.; Jiang, R.-S.; Chao, Jeffrey; Jin, Y.-T.; Lin, J.-C. . E-mail: jclin@vghtc.gov.tw

    2007-01-01

    Purpose: Nasopharyngeal carcinoma (NPC) has been proven as an Epstein-Barr virus (EBV)-associated cancer. Serum anti-EBV antibodies and plasma EBV DNA have been investigated as surrogate markers for NPC. A comparison of the prognostic impacts of both assays has never been reported. Methods and Materials: Paired serum and plasma samples from 114 previously untreated NPC patients were collected and subjected to an immunofluorescence assay for immunoglobulin (Ig)A and IgG antibodies against the viral capsid antigen (VCA) and a real-time quantitative polymerase chain reaction assay for EBV DNA measurement. The effects of both assays on patient prognosis were thoroughly investigated. Results: Relapsed patients had significantly higher pretreatment EBV DNA concentration than patients without relapse (p 0.0006). No associations of VCA-IgA (p = 0.9669) or VCA-IgG (p = 0.6125) were observed between patients with and without relapse. The 4-year overall survival (60.3% vs. 93.1%, p < 0.0001) and relapse-free survival rates (54.4% vs. 77.9%, p = 0.0009) were significantly lower in patients with higher pretreatment EBV DNA load than in those with lower EBV DNA load. Patients with persistently detectable EBV DNA after treatment had significantly worse 4-year overall (30.8% vs. 84.6%, p < 0.0001) and relapse-free survival rates (15.4% vs. 74.0%, p < 0.0001) than those with undetectable EBV DNA. The VCA-IgA and VCA-IgG titer could not predict survivals (all p > 0.1). Cox multivariate analyses also showed the same results. Conclusion: Plasma EBV DNA is superior to serum EBV VCA antibodies in prognostic predictions for NPC.

  14. A practical and novel method to extract genomic DNA from blood collection kits for plasma protein preservation.

    PubMed

    Waters, Jon; Dhere, Vishal; Benjamin, Adam; Sekar, Arvind; Kumar, Archana; Prahalad, Sampath; Okou, David T; Kugathasan, Subra

    2013-05-18

    Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources. The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes(1), to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes(2-4). Challenges from these methods were mainly

  15. A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

    PubMed Central

    Waters, Jon; Dhere, Vishal; Benjamin, Adam; Sekar, Arvind; Kumar, Archana; Prahalad, Sampath; Okou, David T.; Kugathasan, Subra

    2013-01-01

    Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources. The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly

  16. Expression of c-myc and induction of DNA synthesis by platelet-poor plasma in human diploid fibroblasts

    SciTech Connect

    Ferrari, S.; Calabretta, B.; Battini, R.; Cosenza, S.C.; Owen, T.A.; Soprano, K.J.; Baserga, R. )

    1988-01-01

    When WI-38 human diploid fibroblasts become confluent, they stop synthesizing DNA and dividing. Addition of serum causes the quiescent cell to reenter the cell cycle. Prolonged quiescence after confluence decreases and delays the response to serum. For a few days after reaching confluence. WI-38 cells also respond to platelet-poor plasma. During this period, although not cycling, WI-38 cells still express c-myc and other growth-regulated genes, as measured by steady-state RNA levels. If the quiescence is prolonged further, c-myc expression (and that of two other growth-regulated genes) is no longer detectable, and its disappearance coincides with a loss of response to platelet-poor plasma. These results suggest that, also under physiological conditions, the expression of c-myc and other growth-regulated genes can cooperate with platelet-poor plasma in inducing cellular DNA synthesis in human diploid fibroblasts.

  17. B1 Sequence-Based Real-Time Quantitative PCR: A Sensitive Method for Direct Measurement of Mouse Plasma DNA Levels After Gamma Irradiation

    SciTech Connect

    Zhang Hengshan; Zhang, Steven B.; Sun Weimin; Yang Shanmin; Zhang Mei; Wang Wei; Liu Chaomei; Zhang Kunzhong; Swarts, Steven; Fenton, Bruce M.; Keng, Peter; Maguire, David; Okunieff, Paul Zhang Lurong

    2009-08-01

    Purpose: Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA. Methods and Materials: Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation ({sup 137}Cs {gamma}-ray source at {approx}1.86 Gy/min; homogeneity: {+-} 6.5%). Results: The correlation coefficient between DNA levels and the threshold cycle value (C{sub T}) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples. Conclusions: A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.

  18. Plant RNA processing: soybean pre-mRNA in a pea cell-free extract

    SciTech Connect

    Schuler, M.A.; Hanley, B.A.

    1987-05-01

    Using a pea cell-free extract they have demonstrated the splicing of an SP6 fusion transcript containing an intron derived from the soybean seed storage protein ..beta..-subunit gene. Intron 115 from the conglycinin gene was cloned into a SP6 vector and transcribed using standard recombinant DNA techniques. Incubation of radioactively labeled fusion transcripts in the cell-free system produced a number of products which were identified by primer extension and S1 nuclease analysis. All the products are linear RNA molecules. Lariat intermediates, similar to those found in the yeast and HeLa cell RNA processing systems, have not been detected. The linear RNA products detected in their plant in vitro processing system have various portions of the intron removed which suggests that alternative splice sites are used in processing of this plant intron due to activation of cryptic splice sites or creation of splice sites in the fusion construction. The kinetics of the reactions and parameters of the extract are similar to those determined for the HeLa cell system. Sucrose gradient analysis has demonstrated that the plant RNA products sedimented in a 30S particle, similar in size to that found for the spliceosome of the HeLa cell system.

  19. Canine Systemic Lupus Erythematosus. TRANSMISSION OF SEROLOGIC ABNORMALITIES BY CELL-FREE FILTRATES

    PubMed Central

    Lewis, Robert M.; Andre-Schwartz, Janine; Harbis, Gerald S.; Hirsch, Martin S.; Black, Paul H.; Schwartz, Robert S.

    1973-01-01

    The presence of viruses was sought in a colony of dogs bred from parents with systemic lupus crythematosus (SLE). Cell-free filtrates prepared from the spleens of these animals were injected into newborn dogs, mice, and rats. The canine recipients developed antinuclear antibody (ANA) and positive lupus erythematosus (LE) cell tests: ANA and, in some cases, antinative DNA antibodies were produced by the murine recipients: no abnormalities were detected in the rats. Serial passage of spleen cells or cell-free filtrates of spleen tissue in syngeneic mice reduced the time required for appearance of ANA from 9 to 4 mo. Some murine recipients of the canine filtrate developed malignant lymphomas. Murine leukemia viruses were identified in these tumors by electron microscopic, virologic, and serologic technics. These neoplasms, but not other tumors known to contain murine leukemia viruses, were associated with the production of ANA. Puppies inoculated with the canine filtrate-induced mouse lymphoma developed ANA and positive LE cell tests within 4 mo. The results were interpreted to indicate the presence in canine SLE of a virus capable of: (a) inducing the serologic abnormalities of SLE in normal dogs and mice: (b) activating latent murine leukemia viruses: and (c) spreading by both horizonal and vertical routes. Images PMID:4124208

  20. High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format.

    PubMed

    Quast, Robert B; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041

  1. High-yield cell-free synthesis of human EGFR by IRES-mediated protein translation in a continuous exchange cell-free reaction format

    PubMed Central

    Quast, Robert B.; Sonnabend, Andrei; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems derived from eukaryotic sources often provide comparatively low amounts of several μg per ml of de novo synthesized membrane protein. In order to overcome this, we herein demonstrate the high-yield cell-free synthesis of the human EGFR in a microsome-containing system derived from cultured Sf21 cells. Yields were increased more than 100-fold to more than 285 μg/ml by combination of IRES-mediated protein translation with a continuous exchange cell-free reaction format that allowed for prolonged reaction lifetimes exceeding 24 hours. In addition, an orthogonal cell-free translation system is presented that enabled the site-directed incorporation of p-Azido-L-phenylalanine by amber suppression. Functionality of cell-free synthesized receptor molecules is demonstrated by investigation of autophosphorylation activity in the absence of ligand and interaction with the cell-free synthesized adapter molecule Grb2. PMID:27456041

  2. Cell-free construction of disarmed Abutilon mosaic virus-based gene silencing vectors.

    PubMed

    Krenz, Björn; Wege, Christina; Jeske, Holger

    2010-10-01

    The bipartite Abutilon mosaic virus (AbMV) was engineered as a versatile silencing vector in which the coat protein gene of DNA A was deleted and replaced by sequences of interest. Plants transgenic for the dimeric AbMV DNA B component were used as test hosts to minimize the risk of unintended release of the recombinant DNA. The vector construct was stable genetically upon systemic infection and, in common with the parental virus, the vector remained phloem-limited. For virus-induced gene silencing (VIGS), a phytoene desaturase gene fragment was isolated from Nicotiana benthamiana (NbPDS) and inserted into the vector. After agroinfection, phytoene desaturase silencing was triggered efficiently in all leaf tissues without interference by viral symptoms. In order to facilitate further the use of the system, a technique for cell-free construction of recombinants was established using rolling circle amplification and biolistic inoculation of DNA B-transgenic plants. This novel procedure provides a convenient and safe way for delivering VIGS constructs for functional genomics. PMID:20638413

  3. [Methylation of FHIT gene promoter region in DNA from plasma of patients with myelodysplastic syndromes and demethylating effect of decitabine].

    PubMed

    Deng, Yin-Fen; Zhang, Lei; Zhang, Xiu-Qun; Hu, Ming-Qiu; Dai, Dan; Zhang, Xue-Zhong; Xu, Yan-Li

    2012-10-01

    This study was aimed to detect the methylation status of FHIT gene promoter region in the DNA from plasma of patients with myelodysplastic syndrome (MDS), and to investigate the demethylating effect of decitabine. Methylation-specific PCR method was used to detect the methylation status of FHIT gene promoter region in the DNA from plasma of 4 patients with MDS before and after treatment with decitabine plus semis CAG therapy (among them, 1 case of newly diagnosed MDS, 3 cases progressed into acute leukemia). The results indicated that 3 cases were found to have an increased methylation in the promoter region. After treatment with decitabine plus semis CAG, increased methylation was reversed in 2 cases. In 4 cases, 2 cases displayed clinical response. It is concluded that FHIT gene hypermethylation is associated with MDS pathogenesis. Decitabine has demethylating effect on the FHIT gene hypermethylation of plasma from MDS patients. Detecting the methylation status of FHIT gene in DNA from plasma may play a role in MDS auxiliary diagnosis or prognosis.

  4. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells.

    PubMed

    Caron, Gersende; Hussein, Mourad; Kulis, Marta; Delaloy, Céline; Chatonnet, Fabrice; Pignarre, Amandine; Avner, Stéphane; Lemarié, Maud; Mahé, Elise A; Verdaguer-Dot, Núria; Queirós, Ana C; Tarte, Karin; Martín-Subero, José I; Salbert, Gilles; Fest, Thierry

    2015-11-01

    Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-?1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

  5. HIV DNA loads, plasma residual viraemia and risk of virological rebound in heavily treated, virologically suppressed HIV-infected patients.

    PubMed

    Gianotti, N; Canducci, F; Galli, L; Cossarini, F; Salpietro, S; Poli, A; Nozza, S; Spagnuolo, V; Clementi, M; Sampaolo, M; Ceresola, E R; Racca, S; Lazzarin, A; Castagna, A

    2015-01-01

    In this single-centre, retrospective study, we analyzed data of 194 patients receiving antiretroviral therapy with <50 human immunodeficiency virus (HIV) RNA copies/mL in plasma and 318 HIV RNA/DNA paired samples. By kinetic polymerase chain reaction (kPCR) molecular system analysis, 104 (54%) subjects had undetectable HIV RNA and 90 (46%) had residual viraemia. Median (interquartile range) HIV DNA load was 780 (380-1930) copies/10(6) peripheral blood lymphocytes (PBL), and HIV DNA loads were independently associated with residual viraemia (p 0.002). Virological rebound occurred in 29/194 (15%) patients over a median (interquartile range) follow-up of 17.5 (13.5-31.5) months. Residual viraemia (p 0.002), but not HIV DNA load, was independently associated with virological rebound.

  6. Detection of DNA of Lymphotropic Herpesviruses in Plasma of Human Immunodeficiency Virus-Infected Patients: Frequency and Clinical Significance

    PubMed Central

    Broccolo, Francesco; Bossolasco, Simona; Careddu, Anna M.; Tambussi, Giuseppe; Lazzarin, Adriano; Cinque, Paola

    2002-01-01

    The frequency and clinical significance of detection of DNA of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), HHV-7, and HHV-8 in plasma were investigated by PCR. The plasma was obtained from 120 selected human immunodeficiency virus (HIV)-infected patients, of whom 75 had AIDS-related manifestations, 32 had primary HIV infection (PHI), and 13 had asymptomatic infections. Nested PCR analysis revealed that none of the lymphotropic herpesviruses tested were found in patients with PHI, in asymptomatic HIV-positive individuals, or in HIV-negative controls. By contrast, DNA of one or more of the viruses was found in 42 (56%) of 75 patients with AIDS-related manifestations, including CMV disease (CMV-D) or AIDS-related tumors. The presence of CMV DNA in plasma was significantly associated with CMV-D (P < 0.001). By contrast, EBV detection was not significantly associated with AIDS-related lymphomas (P = 0.31). Interestingly, the presence of HHV-8 DNA in plasma was significantly associated with Kaposi's sarcoma (KS) disease (P < 0.001) and with the clinical status of KS patients (P < 0.001). CMV (primarily), EBV, and HHV-8 were the viruses most commonly reactivated in the context of severe immunosuppression (P < 0.05). In contrast, HHV-6 and HHV-7 infections were infrequent at any stage of disease. In conclusion, plasma PCR was confirmed to be useful in the diagnosis of CMV-D but not in that of tumors or other conditions possibly associated with EBV, HHV-6, and HHV-7. Our findings support the hypothesis of a direct involvement of HHV-8 replication in KS pathogenesis, thus emphasizing the usefulness of sensitive and specific diagnostic tests to monitor HHV-8 infection. PMID:12414753

  7. cfDNA analysis from blood in melanoma.

    PubMed

    Molina-Vila, Miguel A; de-Las-Casas, Clara Mayo; Bertran-Alamillo, Jordi; Jordana-Ariza, Nuria; González-Cao, María; Rosell, Rafael

    2015-11-01

    Testing of tumor tissue remains the recommended method for detecting the presence of somatic mutations in human malignancies. V600E is the most frequent somatic point mutation in metastatic melanoma, providing a unique molecular marker for this malignancy. In addition, tumors carrying this mutation are primary candidates for BRAF-targeted therapy. Although metastatic melanoma patients usually have sufficient tumor tissue available for genetic analyses, the detection of V600E in blood can have prognostic and predictive value. In addition, patients are rarely re-biopsied and genetic testing in blood can be useful for monitoring response to therapy. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA), RNA associated to platelets and circulating tumor cells (CTCs) are some of the materials that can be derived from the blood of cancer patients. cfDNA can be easily purified from serum and plasma and contains DNA fragments of tumor origin. For this reason, it is the most widely used material for the detection of somatic mutations in blood. Several methodologies have been used to determine V600E status in the cfDNA of metastatic melanoma and some studies have demonstrated that the identification and follow-up of V600E in cfDNA can have prognostic and predictive value.

  8. Cell-Free Protein Expression under Macromolecular Crowding Conditions

    PubMed Central

    Ge, Xumeng; Luo, Dan; Xu, Jianfeng

    2011-01-01

    Background Cell-free protein expression (CFPE) comprised of in vitro transcription and translation is currently manipulated in relatively dilute solutions, in which the macromolecular crowding effects present in living cells are largely ignored. This may not only affect the efficiency of protein synthesis in vitro, but also limit our understanding of the functions and interactions of biomolecules involved in this fundamental biological process. Methodology/Principal Findings Using cell-free synthesis of Renilla luciferase in wheat germ extract as a model system, we investigated the CFPE under macromolecular crowding environments emulated with three different crowding agents: PEG-8000, Ficoll-70 and Ficoll-400, which vary in chemical properties and molecular size. We found that transcription was substantially enhanced in the macromolecular crowding solutions; up to 4-fold increase in the mRNA production was detected in the presence of 20% (w/v) of Ficoll-70. In contrast, translation was generally inhibited by the addition of each of the three crowding agents. This might be due to PEG-induced protein precipitation and non-specific binding of translation factors to Ficoll molecules. We further explored a two-stage CFPE in which transcription and translation was carried out under high then low macromolecular crowding conditions, respectively. It produced 2.2-fold higher protein yield than the coupled CFPE control. The macromolecular crowding effects on CFPE were subsequently confirmed by cell-free synthesis of an approximately two-fold larger protein, Firefly luciferase, under macromolecular crowding environments. Conclusions/Significance Three macromolecular crowding agents used in this research had opposite effects on transcription and translation. The results of this study should aid researchers in their choice of macromolecular crowding agents and shows that two-stage CFPE is more efficient than coupled CFPE. PMID:22174874

  9. Cold atmospheric pressure plasma jets: Interaction with plasmid DNA and tailored electron heating using dual-frequency excitation

    SciTech Connect

    Niemi, K.; O'Neill, C.; Cox, L. J.; Waskoenig, J.; Hyland, W. B.; McMahon, S. J.; Reuter, S.; Currell, F. J.; Graham, W. G.; O'Connell, D.; Gans, T.

    2012-05-25

    Recent progress in plasma science and technology has enabled the development of a new generation of stable cold non-equilibrium plasmas operating at ambient atmospheric pressure. This opens horizons for new plasma technologies, in particular in the emerging field of plasma medicine. These non-equilibrium plasmas are very efficient sources for energy transport through reactive neutral particles (radicals and metastables), charged particles (ions and electrons), UV radiation, and electro-magnetic fields. The effect of a cold radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks. Plasma manipulation techniques for controlled energy delivery are highly desirable. Numerical simulations are employed for detailed investigations of the electron dynamics, which determines the generation of reactive species. New concepts based on nonlinear power dissipation promise superior strategies to control energy transport for tailored technological exploitations.

  10. Comparison of two molecular assays for detection of cytomegalovirus DNA in whole blood and plasma samples from transplant recipients.

    PubMed

    Costa, Cristina; Sidoti, Francesca; Mantovani, Samantha; Gregori, Gabriella; Proietti, Alex; Ghisetti, Valeria; Cavallo, Rossana

    2016-09-01

    In immunosuppressed patients, pre-emptive therapy and a strict follow-up of CMV infection are the standard of care for the prevention of CMV disease. Several real-time PCR assays for CMV DNA quantification on whole blood (WB) and plasma (PL) are commercially available. This study compared and correlated CMV viral loads obtained by the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform on plasma specimens with those obtained on corresponding whole blood specimens by the real-time PCR assay (ELITe MGB-CMV) in 185 sequential samples from 41 immunosuppressed patients. Correlation between the two assays was good. Kinetics of CMV DNA within the same patient was similar, but PL viral load was constantly 1 log lower than WB. In patients under antiviral therapy, low level of CMV DNA persisted in WB, while it was absent in PL. The good correlation between CMV DNA detected on both PL and WB supports the reliability of the two matrices for viral monitoring and the therapeutic management of CMV infection. Nevertheless, due to significant quantification differences between PL and WB CMV DNA, the same biological specimen should be used for a sequential and reliable follow-up of patients at high risk of CMV infection. PMID:27602416

  11. Cell-Free Metabolic Engineering: Biomanufacturing beyond the cell

    PubMed Central

    Dudley, Quentin M.; Karim, Ashty S.; Jewett, Michael C.

    2014-01-01

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L−1 hr−1, reaction scales of >100L, and new directions in protein purification, spatial organization and enzyme stability. In the coming years, CFME will offer exciting opportunities to (i) debug and optimize biosynthetic pathways, (ii) carry out design-build-test iterations without re-engineering organisms, and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility. PMID:25319678

  12. Cell-free metabolic engineering: Biomanufacturing beyond the cell

    SciTech Connect

    Dudley, QM; Karim, AS; Jewett, MC

    2014-10-15

    Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low-cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state-of-the-art include low volumetric productivities, build-up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell-free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell-free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L-1 h(-1), reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design-build-test iterations without re-engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility.

  13. Circulating Tumor DNA Detection in Early-Stage Non-Small Cell Lung Cancer Patients by Targeted Sequencing

    PubMed Central

    Chen, Ke-Zhong; Lou, Feng; Yang, Fan; Zhang, Jing-Bo; Ye, Hua; Chen, Wei; Guan, Tian; Zhao, Ming-Yu; Su, Xue-Xia; Shi, Rong; Jones, Lindsey; Huang, Xue F.; Chen, Si-Yi; Wang, Jun

    2016-01-01

    Circulating tumor DNA (ctDNA) isolated from peripheral blood has recently been shown to be an alternative source to detect gene mutations in primary tumors; however, most previous studies have focused on advanced stage cancers, and few have evaluated ctDNA detection in early-stage lung cancer. In the present study, blood and tumor samples were collected prospectively from 58 early-stage non-small lung cancer (NSCLC) patients (stages IA, IB, and IIA) and a targeted sequencing approach was used to detect somatic driver mutations in matched tumor DNA (tDNA) and plasma ctDNA. We identified frequent driver mutations in plasma ctDNA and tDNA in EGFR, KRAS, PIK3CA, and TP53, and less frequent mutations in other genes, with an overall study concordance of 50.4% and sensitivity and specificity of 53.8% and 47.3%, respectively. Cell-free (cfDNA) concentrations were found to be significantly associated with some clinical features, including tumor stage and subtype. Importantly, the presence of cfDNA had a higher positive predictive value than that of currently used protein tumor biomarkers. This study demonstrates the feasibility of identifying plasma ctDNA mutations in the earliest stage lung cancer patients via targeted sequencing, demonstrating a potential utility of targeted sequencing of ctDNA in the clinical management of NSCLC. PMID:27555497

  14. Cell-free and cell-associated viremia in pigs after oronasal infection with Aujeszky's disease virus.

    PubMed

    Nawynck, H J; Pensaert, M B

    1995-03-01

    Nine pigs were examined for the presence of viremia during the first week after oronasal inoculation of 10(8.0) TCID50 Aujeszky's disease virus (ADV). Blood was taken at 1, 2, 3, 5 and 7 days post inoculation (PI) and the presence of cell-free ADV in plasma and of ADV-infected mononuclear cells was examined by titration and by cocultivation with permissive cells, respectively. The mononuclear cells of 6 of the 9 pigs, collected at 3 and 5 days PI were further separated into subpopulations of enriched monocytes and enriched lymphocytes. Both subpopulations were cocultivated. Nasal secretions were collected from 4 of the 9 pigs for the determination of virus titers and interferon concentrations. Both infected mononuclear cells and cell-free ADV were demonstrated in 5 pigs, infected mononuclear cells only were found in 2 pigs, and neither cell-associated or cell-free ADV were detected in 2 pigs. Two of the 7 viremic animals were positive on one single day, 3 on 2 days, 1 on 3 days and 1 on 4 days. The number of infected cells was approximately 5 times higher in monocytes than in lymphocytes. The highest virus titers were present in those nasal fluids with the lowest alpha-interferon concentration. A correlation between the titer of locally produced ADV in the nose and the presence of a viremia was not found. In conclusion, we can state that a viremia regularly occurs under both cell-free and cell-associated form after an oronasal inoculation of ADV and that monocytes are the most susceptible mononuclear cells.

  15. Enterococcus faecium LKE12 Cell-Free Extract Accelerates Host Plant Growth via Gibberellin and Indole-3-Acetic Acid Secretion.

    PubMed

    Lee, Ko-Eun; Radhakrishnan, Ramalingam; Kang, Sang-Mo; You, Young-Hyun; Joo, Gil-Jae; Lee, In-Jung; Ko, Jae-Hwan; Kim, Jin-Ho

    2015-09-01

    The use of microbial extracts containing plant hormones is a promising technique to improve crop growth. Little is known about the effect of bacterial cell-free extracts on plant growth promotion. This study, based on phytohormonal analyses, aimed at exploring the potential mechanisms by which Enterococcus faecium LKE12 enhances plant growth in oriental melon. A bacterial strain, LKE12, was isolated from soil, and further identified as E. faecium by 16S rDNA sequencing and phylogenetic analysis. The plant growth-promoting ability of an LKE12 bacterial culture was tested in a gibberellin (GA)-deficient rice dwarf mutant (waito-C) and a normal GA biosynthesis rice cultivar (Hwayongbyeo). E. faecium LKE12 significantly improved the length and biomass of rice shoots in both normal and dwarf cultivars through the secretion of an array of gibberellins (GA1, GA3, GA7, GA8, GA9, GA12, GA19, GA20, GA24, and GA53), as well as indole-3-acetic acid (IAA). To the best of our knowledge, this is the first study indicating that E. faecium can produce GAs. Increases in shoot and root lengths, plant fresh weight, and chlorophyll content promoted by E. faecium LKE12 and its cell-free extract inoculated in oriental melon plants revealed a favorable interaction of E. faecium LKE12 with plants. Higher plant growth rates and nutrient contents of magnesium, calcium, sodium, iron, manganese, silicon, zinc, and nitrogen were found in cell-free extract-treated plants than in control plants. The results of the current study suggest that E. faecium LKE12 promotes plant growth by producing GAs and IAA; interestingly, the exogenous application of its cell-free culture extract can be a potential strategy to accelerate plant growth.

  16. Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

    PubMed

    Anderson, Mark J; Stark, Jessica C; Hodgman, C Eric; Jewett, Michael C

    2015-07-01

    Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 μg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (μg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.

  17. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  18. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H. Craig

    1998-01-01

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques.

  19. Detergent composition comprising a cellulase containing cell-free fermentate produced from microorganism ATCC 55702 or mutant thereof

    DOEpatents

    Dees, H.C.

    1998-07-14

    Bacteria which produce large amounts of a cellulase-containing cell-free fermentate have been identified. The original bacterium (ATCC 55703) was genetically altered using nitrosoguanidine (MNNG) treatment to produce the enhanced cellulase producing bacterium (ATCC 55702), which was identified through replicate plating. ATCC 55702 has improved characteristics and qualities for the degradation of cellulosic waste materials for fuel production, food processing, textile processing, and other industrial applications. ATCC 55702 is an improved bacterial host for genetic manipulations using recombinant DNA techniques, and is less likely to destroy genetic manipulations using standard mutagenesis techniques. 5 figs.

  20. Spatial Dependence of DNA Damage in Bacteria due to Low-Temperature Plasma Application as Assessed at the Single Cell Level

    PubMed Central

    Privat-Maldonado, Angela; O’Connell, Deborah; Welch, Emma; Vann, Roddy; van der Woude, Marjan W.

    2016-01-01

    Low temperature plasmas (LTPs) generate a cocktail of reactive nitrogen and oxygen species (RNOS) with bactericidal activity. The RNOS however are spatially unevenly distributed in the plasma. Here we test the hypothesis that this distribution will affect the mechanisms underpinning plasma bactericidal activity focussing on the level of DNA damage in situ. For the first time, a quantitative, single cell approach was applied to assess the level of DNA damage in bacteria as a function of the radial distance from the centre of the plasma jet. Salmonella enterica on a solid, dry surface was treated with two types of LTP: an atmospheric-pressure dielectric barrier discharge plasma jet (charged and neutral species) and a radio-frequency atmospheric-pressure plasma jet (neutral species). In both cases, there was an inverse correlation between the degree of DNA damage and the radial distance from the centre of the plasma, with the highest DNA damage occurring directly under the plasma. This trend was also observed with Staphylococcus aureus. LTP-generated UV radiation was eliminated as a contributing factor. Thus valuable mechanistic information can be obtained from assays on biological material, which can inform the development of LTP as a complementary or alternative therapy for (topical) bacterial infections. PMID:27759098

  1. Plasma Mitochondrial DNA Levels as a Biomarker of Lipodystrophy Among HIV-infected Patients Treated with Highly Active Antiretroviral Therapy (HAART).

    PubMed

    Dai, Z; Cai, W; Hu, F; Lan, Y; Li, L; Chung, C; Caughey, B; Zhang, K; Tang, X

    2015-01-01

    Lipodystrophy is a common complication in HIV-infected patients taking highly active antiretroviral therapy. Its early diagnosis is crucial for timely modification of antiretroviral therapy. We hypothesize that mitochondrial DNA in plasma may be a potential marker of LD in HIV-infected individuals. In this study, we compared plasma mitochondrial DNA levels in HIV-infected individuals and non-HIV-infected individuals to investigate its potential diagnostic value. Total plasma DNA was extracted from 67 HIV-infected patients at baseline and 12, 24 and 30 months after initiating antiretroviral therapy. Real-time quantitative PCR was used to determine the mitochondrial DNA levels in plasma. Lipodystrophy was defined by the physician-assessed presence of lipoatrophy or lipohypertrophy in one or more body regions. The mitochondrial DNA levels in plasma were significantly higher at baseline in HIV-infected individuals than in non-HIV-infected individuals (p<0.05). At month 30, 33 out of 67 patients (49.2%) showed at least one sign of lipodystrophy. The mean plasma mitochondrial DNA levels in lipodystrophy patients were significantly higher compared to those without lipodystrophy at month 24 (p<0.001). The receiver operating curve analysis demonstrated that using plasma mitochondrial DNA level (with cut-off value <5.09 log10 copies/ml) as a molecular marker allowed identification of patients with lipodystrophy with a sensitivity of 64.2% and a specificity of 73.0%. Our data suggest that mitochondrial DNA levels may help to guide therapy selection with regards to HIV lipodystrophy risk.

  2. Plasma Epstein–Barr virus and Hepatitis B virus in non-Hodgkin lymphomas: Two lymphotropic, potentially oncogenic, latently occurring DNA viruses

    PubMed Central

    Sinha, Mahua; Rao, Clementina Rama; Premalata, C. S.; Shafiulla, Mohammed; Lakshmaiah, K. C.; Jacob, Linu Abraham; Babu, Govind K.; Viveka, B. K.; Appaji, L.; Subramanyam, Jayshree R.

    2016-01-01

    Context: There is a need to study potential infective etiologies in lymphomas. Lymphocyte-transforming viruses can directly infect lymphocytes, disrupt normal cell functions, and promote cell division. Epstein–Barr virus (EBV) is known to be associated with several lymphomas, especially Hodgkin lymphomas (HLs). And recently, the lymphocyte-transforming role of hepatitis B virus (HBV) has been emphasized. Aims: The aim of this study was to elucidate the association of two potentially oncogenic, widely prevalent latent DNA viruses, EBV and HBV, in non-HL (NHL). Settings and Design: In this prospective study, we estimated plasma EBV and HBV DNA in NHL patients. Materials and Methods: Peripheral blood was obtained from newly diagnosed, treatment na ïve, histologically confirmed NHL patients. Plasma EBV DNA was quantified by real-time polymerase chain reaction (PCR) targeting Epstein–Barr Nucleic acid 1 while the plasma HBV DNA was detected using nested PCR targeting HBX gene. In a small subset of patients, follow-up plasma samples post-anticancer chemotherapy were available and retested for viral DNA. Results: Of the 110 NHL patients, ~79% were B-cell NHL and ~21% were T-cell NHL. Plasma EBV-DNA was detected in 10% NHLs with a higher EBV association in Burkitt lymphoma (33.3%) than other subtypes. Pretherapy HBV DNA was detected in 21% NHLs; most of them being diffuse large B-cell lymphoma (DLBCL). Moreover, 42% of DLBCL patients had HBV DNA in plasma. Since all patients were HBV surface antigen seronegative at diagnosis, baseline plasma HBV-DNAemia before chemotherapy was indicative of occult hepatitis B infection. Conclusions: Our findings indicate a significant association of HBV with newly diagnosed DLBCL.

  3. The crosstalk of telomere dysfunction and inflammation through cell-free TERRA containing exosomes.

    PubMed

    Wang, Zhuo; Lieberman, Paul M

    2016-08-01

    Telomeric repeats-containing RNA (TERRA) are telomere-derived non-coding RNAs that contribute to telomere function in protecting chromosome ends. We recently identified a cell-free form of TERRA (cfTERRA) enriched in extracellular exosomes. These cfTERRA-containing exosomes stimulate inflammatory cytokines when incubated with immune responsive cells. Here, we report that cfTERRA levels were increased in exosomes during telomere dysfunction induced by the expression of the dominant negative TRF2. The exosomes from these damaged cells also enriched with DNA damage marker γH2AX and fragmented telomere repeat DNA. Purified cfTERRA stimulated inflammatory cytokines, but the intact membrane-associated nucleoprotein complexes produced a more robust cytokine activation. Therefore, we propose cfTERRA-containing exosomes transport a telomere-associated molecular pattern (TAMP) and telomere-specific alarmin from dysfunctional telomeres to the extracellular environment to elicit an inflammatory response. Since cfTERRA can be readily detected in human serum it may provide a useful biomarker for the detection of telomere dysfunction in the early stage of cancers and aging-associated inflammatory disease. PMID:27351774

  4. The crosstalk of telomere dysfunction and inflammation through cell-free TERRA containing exosomes.

    PubMed

    Wang, Zhuo; Lieberman, Paul M

    2016-08-01

    Telomeric repeats-containing RNA (TERRA) are telomere-derived non-coding RNAs that contribute to telomere function in protecting chromosome ends. We recently identified a cell-free form of TERRA (cfTERRA) enriched in extracellular exosomes. These cfTERRA-containing exosomes stimulate inflammatory cytokines when incubated with immune responsive cells. Here, we report that cfTERRA levels were increased in exosomes during telomere dysfunction induced by the expression of the dominant negative TRF2. The exosomes from these damaged cells also enriched with DNA damage marker γH2AX and fragmented telomere repeat DNA. Purified cfTERRA stimulated inflammatory cytokines, but the intact membrane-associated nucleoprotein complexes produced a more robust cytokine activation. Therefore, we propose cfTERRA-containing exosomes transport a telomere-associated molecular pattern (TAMP) and telomere-specific alarmin from dysfunctional telomeres to the extracellular environment to elicit an inflammatory response. Since cfTERRA can be readily detected in human serum it may provide a useful biomarker for the detection of telomere dysfunction in the early stage of cancers and aging-associated inflammatory disease.

  5. DNA methylation biomarkers: cancer and beyond.

    PubMed

    Mikeska, Thomas; Craig, Jeffrey M

    2014-01-01

    Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient's response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease.

  6. DNA Methylation Biomarkers: Cancer and Beyond

    PubMed Central

    Mikeska, Thomas; Craig, Jeffrey M.

    2014-01-01

    Biomarkers are naturally-occurring characteristics by which a particular pathological process or disease can be identified or monitored. They can reflect past environmental exposures, predict disease onset or course, or determine a patient’s response to therapy. Epigenetic changes are such characteristics, with most epigenetic biomarkers discovered to date based on the epigenetic mark of DNA methylation. Many tissue types are suitable for the discovery of DNA methylation biomarkers including cell-based samples such as blood and tumor material and cell-free DNA samples such as plasma. DNA methylation biomarkers with diagnostic, prognostic and predictive power are already in clinical trials or in a clinical setting for cancer. Outside cancer, strong evidence that complex disease originates in early life is opening up exciting new avenues for the detection of DNA methylation biomarkers for adverse early life environment and for estimation of future disease risk. However, there are a number of limitations to overcome before such biomarkers reach the clinic. Nevertheless, DNA methylation biomarkers have great potential to contribute to personalized medicine throughout life. We review the current state of play for DNA methylation biomarkers, discuss the barriers that must be crossed on the way to implementation in a clinical setting, and predict their future use for human disease. PMID:25229548

  7. Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications.

    PubMed

    Chong, Shaorong

    2014-10-01

    During the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology.

  8. Fetal RhD genotyping from maternal plasma.

    PubMed

    Lo, Y M

    1999-10-01

    The prenatal diagnosis of fetal rhesus D (RhD) status is useful for the management of RhD-negative women with partners heterozygous for the RHD gene. Conventional methods for prenatal fetal RhD status determination involve invasive procedures such as fetal blood sampling and amniocentesis. The recent demonstration of the existence of cell-free fetal DNA in maternal plasma and serum opens up the possibility of determining fetal RhD status by analysis of maternal plasma or serum DNA. This possibility has recently been realized by three independent groups of investigators. This development represents an important step towards the routine application of noninvasive fetal blood group diagnosis in sensitized pregnancies and may become a model for developing safer noninvasive prenatal tests for other single-gene disorders.

  9. The influence of hypoxic physical activity on cfDNA as a new marker of vascular inflammation

    PubMed Central

    Zembron-Lacny, Agnieszka; Baldy-Chudzik, Katarzyna; Orysiak, Joanna; Sitkowski, Dariusz; Banach, Maciej

    2015-01-01

    The phenomenon of circulating cell-free DNA (cfDNA) is important for many biomedical disciplines including the field of exercise biochemistry and physiology. It is likely that cfDNA is released into the plasma by apoptosis of endothelial cells and circulating endothelial progenitor cells (EPCs), and/or by NETosis of immune cells induced by strenuous exercise. Increases of cfDNA are described to be a potential hallmark for the overtraining syndrome, and might be related to aseptic vascular inflammation in athletes. Yet, the relevance of systemic inflammation and cfDNA with endothelial dysfunction in athletes still remains unclear. In this review article, we provide a current overview of exercise-induced cfDNA release to the circulation with special emphasis on its relationship with apoptosis and NETosis and the effect of hypoxic physical activity on vascular inflammation in athletes. PMID:26788076

  10. The influence of hypoxic physical activity on cfDNA as a new marker of vascular inflammation.

    PubMed

    Pokrywka, Andrzej; Zembron-Lacny, Agnieszka; Baldy-Chudzik, Katarzyna; Orysiak, Joanna; Sitkowski, Dariusz; Banach, Maciej

    2015-12-10

    The phenomenon of circulating cell-free DNA (cfDNA) is important for many biomedical disciplines including the field of exercise biochemistry and physiology. It is likely that cfDNA is released into the plasma by apoptosis of endothelial cells and circulating endothelial progenitor cells (EPCs), and/or by NETosis of immune cells induced by strenuous exercise. Increases of cfDNA are described to be a potential hallmark for the overtraining syndrome, and might be related to aseptic vascular inflammation in athletes. Yet, the relevance of systemic inflammation and cfDNA with endothelial dysfunction in athletes still remains unclear. In this review article, we provide a current overview of exercise-induced cfDNA release to the circulation with special emphasis on its relationship with apoptosis and NETosis and the effect of hypoxic physical activity on vascular inflammation in athletes. PMID:26788076

  11. Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B

    PubMed Central

    Tajik, Zahra; Keyvani, Hossein; Bokharaei-Salim, Farah; Zolfaghari, Mohammad Reza; Fakhim, Shahin; Keshvari, Maryam; Alavian, Seyed Moayed

    2015-01-01

    Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a marker of HBV replication in the liver of patients infected with HBV. Objectives: This study aimed to investigate the association between the presence of cccDNA in the plasma samples of Iranian treatment-naive patients with chronic hepatitis B infection and HBV viral load and HBsAg levels. Patients and Methods: From April 2012 to May 2015, 106 treatment-naive patients with chronic hepatitis B infection were enrolled in this cross-sectional study. The HBsAg titer was measured by the Roche HBsAg II assay on the Cobas e411 system, and HBV DNA quantitation was performed using the COBAS TaqMan 48 kit. Real-time polymerase chain reaction was performed for the detection of HBV cccDNA. Results: The mean (SD) age of the patients was 41.1 ± 12.4 years (range, 20 - 62 years). From a total of 106 study participants, 67 (63.2%) were males. The HBV cccDNA was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccDNA in plasma sample of males (23.9%) and females (7.7%) (P = 0.039). Also, a significant correlation was found between the presence of cccDNA in plasma sample of the patients and HBV viral load level (P < 0.0001) and HBsAg titer (P = 0.0043). Conclusions: This study showed that cccDNA can be detected in the plasma specimen of 17.9% of Iranian treatment-naive patients with chronic hepatitis B infection. Therefore, designing prospective studies focusing on the detection of cccDNA in these patients would provide more information. PMID:26504471

  12. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  13. Heterogeneity of guinea-pig caseins synthesized and sequestered by cell-free protein-synthesizing systems.

    PubMed Central

    Pascall, J C; Boulton, A P; Parker, D; Hall, L; Craig, R K

    1981-01-01

    1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:7316995

  14. Plasma Levels of Bacterial DNA Correlate with Immune Activation and the Magnitude of Immune Restoration in Persons with Antiretroviral-Treated HIV Infection

    PubMed Central

    Jiang, Wei; Lederman, Michael M.; Hunt, Peter; Sieg, Scott F.; Haley, Kathryn; Rodriguez, Benigno; Landay, Alan; Martin, Jeffrey; Sinclair, Elizabeth; Asher, Ava I.; Deeks, Steven G.; Douek, Daniel C.; Brenchley, Jason M.

    2009-01-01

    The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)–infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4+ T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection. PMID:19265479

  15. Cell-free protein synthesis and assembly on a biochip

    NASA Astrophysics Data System (ADS)

    Heyman, Yael; Buxboim, Amnon; Wolf, Sharon G.; Daube, Shirley S.; Bar-Ziv, Roy H.

    2012-06-01

    Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.

  16. Cell-free Circulating miRNA Biomarkers in Cancer

    PubMed Central

    Mo, Meng-Hsuan; Chen, Liang; Fu, Yebo; Wang, Wendy; Fu, Sidney W.

    2012-01-01

    Considerable attention and an enormous amount of resources have been dedicated to cancer biomarker discovery and validation. However, there are still a limited number of useful biomarkers available for clinical use. An ideal biomarker should be easily assayed with minimally invasive medical procedures but possess high sensitivity and specificity. Commonly used circulating biomarkers are proteins in serum, most of which require labor-intensive analysis hindered by low sensitivity in early tumor detection. Since the deregulation of microRNA (miRNA) is associated with cancer development and progression, profiling of circulating miRNAs has been used in a number of studies to identify novel minimally invasive miRNA biomarkers. In this review, we discuss the origin of the circulating cell-free miRNAs and their carriers in blood. We summarize the clinical use and function of potentially promising miRNA biomarkers in a variety of different cancers, along with their downstream target genes in tumor initiation and development. Additionally, we analyze some technical challenges in applying miRNA biomarkers to clinical practice. PMID:23074383

  17. Probiotic Properties of Lyophilized Cell Free Extract of Lactobacillus casei

    PubMed Central

    Saadatzadeh, Afrooz; Fazeli, Mohamma Reza; Jamalifar, Hossein; Dinarvand, Rassoul

    2013-01-01

    Background In recent years there have been considerable interests in the use of probiotic live cells for nutritional and therapeutic purposes. This strategy can be concomitant with some limitations such as survival of live cell during the GI-transit and their effective delivery to target tissues upon ingestion. Several attempts have been made to overcome these limitations such as their microencapsulation, spray-drying and lyophilization. Objectives In this study extract of cultured probiotics without cells was evaluated for its antimicrobial effects, antioxidant activity, and its stability. Materials and Methods In this work the potential of lyophilized-cell-free-probiotic-extract (LPE) as a suitable alternative strategy for the preparation of probiotic-products was investigated. The main aim of this study was to find out the antibacterial and antioxidant activity of LPE and also its stability. LPE was obtained by centrifugation and subsequent lyophilization of the collected supernatant from culture media of Lactobacillus casei. An enzymatic reagent-kit was used for detection of its content of lactic acid. Antibacterial test was performed using agar cup-plat-method, the DPPH scavenging -assay was used to determine its antioxidant activity and during a storage course, LPE was under a long-term stability study. Results Results showed that, LPE had more antipathogenic effects, antioxidant activity, and stability during storage-time when compared to fresh probiotic-extract. Conclusions Employing the LPE as a new approach, gives novel concept of probiotic-products in food and medical marketing. PMID:24624202

  18. Diurnal Variations of Human Circulating Cell-Free Micro-RNA.

    PubMed

    Heegaard, Niels H H; Carlsen, Anting Liu; Lilje, Berit; Ng, Kim Lee; Rønne, Mette E; Jørgensen, Henrik L; Sennels, Henriette; Fahrenkrug, Jan

    2016-01-01

    A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks). The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA) that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79) of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system) exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks. PMID:27494182

  19. Diurnal Variations of Human Circulating Cell-Free Micro-RNA

    PubMed Central

    Heegaard, Niels H. H.; Carlsen, Anting Liu; Lilje, Berit; Ng, Kim Lee; Rønne, Mette E.; Jørgensen, Henrik L.; Sennels, Henriette; Fahrenkrug, Jan

    2016-01-01

    A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks). The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA) that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79) of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system) exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks. PMID:27494182

  20. Haplotype-based approach for noninvasive prenatal diagnosis of congenital adrenal hyperplasia by maternal plasma DNA sequencing.

    PubMed

    Ma, Dingyuan; Ge, Huijuan; Li, Xuchao; Jiang, Tao; Chen, Fang; Zhang, Yanyan; Hu, Ping; Chen, Shengpei; Zhang, Jingjing; Ji, Xiuqing; Xu, Xun; Jiang, Hui; Chen, Minfeng; Wang, Wei; Xu, Zhengfeng

    2014-07-10

    Prenatal diagnosis of congenital adrenal hyperplasia (CAH) is of clinical significance because in utero treatment is available to prevent virilization of an affected female fetus. However, traditional prenatal diagnosis of CAH relies on genetic testing of fetal genomic DNA obtained using amniocentesis or chorionic villus sampling, which is associated with an increased risk of miscarriage. The aim of this study was to demonstrate the feasibility of a new haplotype-based approach for the noninvasive prenatal testing of CAH due to 21-hydroxylase deficiency. Parental haplotypes were constructed using target-region sequencing data of the parents and the proband. With the assistance of the parental haplotypes, we recovered fetal haplotypes using a hidden Markov model (HMM) through maternal plasma DNA sequencing. In the genomic region around the CYP21A2 gene, the fetus inherited the paternal haplotype '0' alleles linked to the mutant CYP21A2 gene, but the maternal haplotype '1' alleles linked to the wild-type gene. The fetus was predicted to be an unaffected carrier of CAH, which was confirmed by genetic analysis of fetal genomic DNA from amniotic fluid cells. This method was further validated by comparing the inferred SNP genotypes with the direct sequencing data of fetal genomic DNA. The result showed an accuracy of 96.41% for the inferred maternal alleles and an accuracy of 97.81% for the inferred paternal alleles. The haplotype-based approach is feasible for noninvasive prenatal testing of CAH.

  1. Cell-free transfer of sterols by plant fractions

    SciTech Connect

    Morre, D.J.; Wilkinson, F.E.; Morre, D.M. ); Moreau, P. ); Sandelius, A.S. ); Penel, C.; Greppin, H. )

    1990-05-01

    Microsomes from etiolated hypocotyls of soybean or leaves of light-grown spinach radiolabeled in vivo with ({sup 3}H)acetate or in vitro with ({sup 3}H)squalene or ({sup 3}H)cholesterol as donor transferred radioactivity to unlabeled acceptor membranes immobilized on nitrocellulose. Most efficient transfer was with plasma membrane or tonoplast as the acceptor. The latter were highly purified by aqueous two-phase partition (plasma membrane) and preparative free-flow electrophoresis (tonoplast and plasma membrane). Plasma membrane- and tonoplast-free microsomes and purified mitochondria were less efficient acceptors. Sterol transfer was verified by thin-layer chromatography of extracted lipids. Transfer was time- and temperature-dependent, required ATP but was not promoted by cytosol. The nature of the donor (endoplasmic reticulum, Golgi apparatus or both) and of the transfer mechanism is under investigation.

  2. Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer.

    PubMed

    Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Morizane, Chigusa; Nara, Satoshi; Hama, Natsuko; Suzuki, Masami; Furukawa, Eisaku; Kato, Mamoru; Hayashi, Hideyuki; Kohno, Takashi; Ueno, Hideki; Shimada, Kazuaki; Okusaka, Takuji; Nakagama, Hitoshi; Shibata, Tatsuhiro; Yachida, Shinichi

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients. PMID:26669280

  3. Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer

    PubMed Central

    Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Morizane, Chigusa; Nara, Satoshi; Hama, Natsuko; Suzuki, Masami; Furukawa, Eisaku; Kato, Mamoru; Hayashi, Hideyuki; Kohno, Takashi; Ueno, Hideki; Shimada, Kazuaki; Okusaka, Takuji; Nakagama, Hitoshi; Shibata, Tatsuhiro; Yachida, Shinichi

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients. PMID:26669280

  4. Oral and Intraperitoneal Administration of Quercetin Decreased Lymphocyte DNA Damage and Plasma Lipid Peroxidation Induced by TSA In Vivo

    PubMed Central

    Chan, Shu-Ting; Shiau, Rong-Jen; Liao, Jiunn-Wang; Yeh, Shu-Lan

    2014-01-01

    Our previous study showed that quercetin enhances the anticancer effect of trichostatin A (TSA) in xenograft mice given quercetin intraperitoneally (10 mg/kg, 3 times/week). Herein, we investigate whether quercetin administered orally exerts such an effect and prevents the cytotoxic side effects of TSA. We found that quercetin given orally (20 and 100 mg/kg, 3 times/week) failed to enhance the antitumor effect of TSA although it increased the total quercetin concentration more than quercetin administered intraperitoneally in the plasma. The compound quercetin-3-glucuronide (Q3G) increased the most. However, quercetin administered intraperitoneally increased the total quercetin level in tumor tissues more than oral quercetin. Oral and intraperitoneal administration of quercetin similarly decreased lymphocyte DNA damage and plasma lipid peroxidation level induced by TSA. Furthermore, we found that the enhancing effect of Q3G on the antitumor effect of TSA and the incorporation of Q3G was less than that of quercetin in A549 cells. However, we found that A549 cells possessed the ability to convert Q3G to quercetin. In conclusion, different from quercetin administered intraperitoneally, quercetin administered orally failed to enhance the antitumor effect of TSA because of its metabolic conversion. However, it prevented TSA-induced DNA damage and lipid peroxidation. PMID:24868531

  5. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    DOE PAGES

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

  6. Toward Microfluidic Reactors for Cell-Free Protein Synthesis at the Point-of-Care.

    PubMed

    Timm, Andrea C; Shankles, Peter G; Foster, Carmen M; Doktycz, Mitchel J; Retterer, Scott T

    2016-02-10

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro and nanofluidic architectures, CFPS can be optimized for point-of-care use. Here, the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care, is described. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel "reactor" and "feeder" channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, and can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. It has been shown that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor. PMID:26690885

  7. Impact of stochastic fluctuations in the cell free layer on nitric oxide bioavailability

    PubMed Central

    Park, Sang-Woo; Intaglietta, Marcos; Tartakovsky, Daniel M.

    2015-01-01

    A plasma stratum (cell free layer or CFL) generated by flowing blood interposed between the red blood cell (RBC) core and the endothelium affects generation, consumption, and transport of nitric oxide (NO) in the microcirculation. CFL width is a principal factor modulating NO diffusion and vessel wall shears stress development, thus significantly affecting NO bioavailability. Since the CFL is bounded by the surface formed by the chaotically moving RBCs and the stationary but spatially non-uniform endothelial surface, its width fluctuates randomly in time and space. We analyze how these stochastic fluctuations affect NO transport in the CFL and NO bioavailability. We show that effects due to random boundaries do not average to zero and lead to an increase of NO bioavailability. Since endothelial production of NO is significantly enhanced by temporal variability of wall shear stress, we posit that stochastic shear stress stimulation of the endothelium yields the baseline continual production of NO by the endothelium. The proposed stochastic formulation captures the natural continuous and microscopic variability, whose amplitude is measurable and is of the scale of cellular dimensions. It provides a realistic model of NO generation and regulation. PMID:26578944

  8. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    SciTech Connect

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.

  9. Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative.

    PubMed

    Loeffler, Juergen; Mengoli, Carlo; Springer, Jan; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Klingspor, Lena; Lagrou, Katrien; Melchers, Willem J G; Morton, C Oliver; Barnes, Rosemary A; Donnelly, J Peter; White, P Lewis

    2015-09-01

    The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing.

  10. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

    PubMed

    Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

    2016-06-20

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  11. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing

    PubMed Central

    Ståhlberg, Anders; Krzyzanowski, Paul M.; Jackson, Jennifer B.; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E.

    2016-01-01

    Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. PMID:27060140

  12. Detection of cell-free, liver-specific mRNAs in peripheral blood from rats with hepatotoxicity: a potential toxicological biomarker for safety evaluation.

    PubMed

    Miyamoto, Makoto; Yanai, Mariko; Ookubo, Shingo; Awasaki, Naoko; Takami, Kenji; Imai, Ryoetsu

    2008-12-01

    To verify the concept that cell-free organ/tissue-specific mRNAs leaking from drug-damaged organs/tissues into peripheral blood could be toxicological biomarkers for identification of the target organs of drug toxicity, we attempted to detect liver-specific mRNAs in peripheral blood from rats with chemical-induced hepatotoxicity. We selected alpha(1)-microglobulin/bikunin precursor (Ambp) and albumin mRNAs as tentative liver-specific biomarkers and successfully detected them by reverse transcription (RT)-PCR in peripheral blood 24 h after D-galactosamine HCl (D-gal) or acetaminophen administration. Moreover, albumin mRNA was detected 2 h after D-gal administration, although plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were still unchanged. On the other hand, in peripheral blood from rat with bupivacaine HCl-induced skeletal muscle damage, neither Ambp nor albumin mRNA was detectable while plasma creatine kinase, ALT, and AST levels prominently increased 2 or 12 h after dosing. Furthermore, Ambp mRNA was also detectable in filtered plasma from rats with liver damage, indicating that cell-free Ambp mRNA can be present in peripheral blood. In conclusion, cell-free, liver-specific Ambp, and albumin mRNAs were detectable in peripheral blood from rats with chemical-induced liver damage. It is believed that the detection of cell-free organ/tissue-specific mRNA in peripheral blood is a promising approach in the survey of toxicological biomarkers. PMID:18779383

  13. Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids

    PubMed Central

    Imamura, Taisuke; Komatsu, Shuhei; Ichikawa, Daisuke; Kawaguchi, Tsutomu; Miyamae, Mahito; Okajima, Wataru; Ohashi, Takuma; Arita, Tomohiro; Konishi, Hirotaka; Shiozaki, Atsushi; Morimura, Ryo; Ikoma, Hisashi; Okamoto, Kazuma; Otsuji, Eigo

    2016-01-01

    Despite recent advances in surgical techniques and perioperative management, the prognosis of pancreatic cancer (PCa) remains extremely poor. To provide optimal treatment for each patient with Pca, superior biomarkers are urgently needed in all phases of management from early detection to staging, treatment monitoring, and prognosis. In the blood of patients with cancer, circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs), such as DNA, mRNA, and noncoding RNA have been recognized. In the recent years, their presence in the blood has encouraged researchers to investigate their potential use as novel blood biomarkers, and numerous studies have demonstrated their potential clinical utility as a biomarker for certain types of cancer. This concept, called “liquid biopsy” has been focused on as a less invasive, alternative approach to cancer tissue biopsy for obtaining genetic and epigenetic aberrations that contribute to oncogenesis and cancer progression. In this article, we review the available literature on CTCs and cfNAs in patients with cancer, particularly focusing on PCa, and discuss future perspectives in this field. PMID:27433079

  14. Cell-Free Transcription of Mammalian Chromatin: Transcription of Globin Messenger RNA Sequences from Bone-Marrow Chromatin with Mammalian RNA Polymerase

    PubMed Central

    Steggles, A. W.; Wilson, G. N.; Kantor, J. A.; Picciano, D. J.; Falvey, A. K.; Anderson, W. F.

    1974-01-01

    A mammalian cell-free transcriptional system was developed in which mammalian RNA polymerase synthesizes globin messenger RNA sequences from bone-marrow chromatin. The messenger RNA sequences are detected by measurement of the ability of the transcribed RNA to hybridize with globin complementary DNA. The globin complementary DNA is synthesized by the enzyme from avian myeloblastosis virus, RNA-directed DNA polymerase, with purified globin messenger RNA as template. The specificity of the globin complementary DNA in annealing reactions was verified by preparing DNA complementary to liver messenger RNA and showing that the globin and liver complementary DNAs are specific for their own messenger RNAs. Both DNA-dependent RNA polymerase II from sheep liver and RNA polymerase from Escherichia coli can transcribe globin messenger RNA sequences from rabbit bone-marrow chromatin; however, the mammalian enzyme appears to be more specific in that globin gene sequences represent a higher proportion of the RNA synthesized. Neither polymerase can transcribe globin messenger RNA sequences from rabbit-liver chromatin. This cell-free assay system should be useful in searching for mammalian transcriptional regulatory factors. PMID:4364529

  15. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  16. Acrolein and chloroacetaldehyde: an examination of the cell and cell-free biomarkers of toxicity.

    PubMed

    MacAllister, Stephanie L; Martin-Brisac, Nicolas; Lau, Vincent; Yang, Kai; O'Brien, Peter J

    2013-02-25

    Cyclophosphamide and ifosfamide are two commonly used DNA-alkylating agents in cancer chemotherapy that undergo biotransformation to several toxic and non-toxic metabolites, including acrolein and chloroacetaldehyde (CAA). Acrolein and CAA toxicities occur by several different mechanisms, including ROS formation and protein damage (oxidation), however, these pathways of toxicity and protecting agents used to prevent them have yet to be compared and ranked in a single study. This research focused on the molecular targets of acrolein and CAA toxicities and strategies to decrease toxicities. Hepatocyte viability (cytotoxicity) was assessed using Trypan blue uptake; formation of reactive oxygen species (ROS) and endogenous H2O2 were also assessed in the hepatocyte model. In cell-free models (bovine serum albumin and hepatic microsomes), protein carbonylation was the measurement of toxicity. The present study demonstrated that acrolein was a more potent toxin than CAA for freshly isolated rat hepatocytes, bovine serum albumin and rat hepatic microsomes. Acrolein protein carbonylation was dependent on its concentration; as acrolein concentration increased, protein carbonylation increased in a linear trend, whereas, CAA deviated from the trend and did not cause protein carbonylation at lower concentrations (<400 μM). Aldehyde dehydrogenase (ALDH) is a major pathway for detoxifying pathway for CAA in hepatocytes, as a 3-fold increase in cytotoxicity occurred when cells were incubated with cyanamide, an ALDH inhibitor. Inhibiting ALDH or depleting GSH in hepatocytes increased cytotoxicity by about 3-fold in acrolein-treated hepatocytes. The overall effectiveness of protecting agents to prevent or suppress acrolein or CAA toxicities in cell and cell-free models were ranked in order of most effective to least effective: reducing agents (sodium borohydride, sodium bisulfite)>thiol-containing compounds (N-acetylcysteine, cysteine, glutathione, 2-mercaptoethane sulfonate [MESNA

  17. Developmental strategies and regulation of cell-free enzyme system for ethanol production: a molecular prospective.

    PubMed

    Khattak, Waleed Ahmad; Ullah, Muhammad Wajid; Ul-Islam, Mazhar; Khan, Shaukat; Kim, Minah; Kim, Yeji; Park, Joong Kon

    2014-12-01

    Most biomanufacturing systems developed for the production of biocommodities are based on whole-cell systems. However, with the advent of innovative technologies, the focus has shifted from whole-cell towards cell-free enzyme system. Since more than a century, researchers are using the cell-free extract containing the required enzymes and their respective cofactors in order to study the fundamental aspects of biological systems, particularly fermentation. Although yeast cell-free enzyme system is known since long ago, it is rarely been studied and characterized in detail. In this review, we hope to describe the major pitfalls encountered by whole-cell system and introduce possible solutions to them using cell-free enzyme systems. We have discussed the glycolytic and fermentative pathways and their regulation at both transcription and translational levels. Moreover, several strategies employed for development of cell-free enzyme system have been described with their potential merits and shortcomings associated with these developmental approaches. We also described in detail the various developmental approaches of synthetic cell-free enzyme system such as compartmentalization, metabolic channeling, protein fusion, and co-immobilization strategies. Additionally, we portrayed the novel cell-free enzyme technologies based on encapsulation and immobilization techniques and their development and commercialization. Through this review, we have presented the basics of cell-free enzyme system, the strategies involved in development and operation, and the advantages over conventional processes. Finally, we have addressed some potential directions for the future development and industrialization of cell-free enzyme system.

  18. Optimization of kinetic parameters for the degradation of plasmid DNA in rat plasma

    NASA Astrophysics Data System (ADS)

    Chaudhry, Q. A.

    2014-12-01

    Biotechnology is a rapidly growing area of research work in the field of pharmaceutical sciences. The study of pharmacokinetics of plasmid DNA (pDNA) is an important area of research work. It has been observed that the process of gene delivery faces many troubles on the transport of pDNA towards their target sites. The topoforms of pDNA has been termed as super coiled (S-C), open circular (O-C) and linear (L), the kinetic model of which will be presented in this paper. The kinetic model gives rise to system of ordinary differential equations (ODEs), the exact solution of which has been found. The kinetic parameters, which are responsible for the degradation of super coiled, and the formation of open circular and linear topoforms have a great significance not only in vitro but for modeling of further processes as well, therefore need to be addressed in great detail. For this purpose, global optimization techniques have been adopted, thus finding the optimal results for the said model. The results of the model, while using the optimal parameters, were compared against the measured data, which gives a nice agreement.

  19. Creating highly dense and uniform protein and DNA microarrays through photolithography and plasma modification of glass substrates.

    PubMed

    Malainou, A; Petrou, P S; Kakabakos, S E; Gogolides, E; Tserepi, A

    2012-04-15

    We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(®)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection.

  20. Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

    PubMed

    Nozawa, Akira; Tozawa, Yuzuru

    2014-01-01

    Functional proteomics of plant membrane proteins is an important approach to understand the comprehensive architecture of each metabolic pathway in plants. One bottleneck in the characterization of membrane proteins is the difficulty in producing sufficient quantities of functional protein for analysis. Here, we describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system. Owing to the open nature of cell-free synthesis reaction, protein synthesis can be modified with components necessary to produce functional protein. In this way we have developed modifications to a wheat germ cell-free system for the production of functional membrane proteins. Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins. Furthermore, supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins. These modified cell-free synthesis methods facilitate the preparation and subsequent functional analyses of a wide variety of membrane proteins. PMID:24136528

  1. Non-Invasive Prenatal Diagnosis of Lethal Skeletal Dysplasia by Targeted Capture Sequencing of Maternal Plasma

    PubMed Central

    Wang, Yaoshen; Chen, Chao; Gao, Changxin; Yu, Song; Liu, Yan; Song, Wei; Asan; Zhu, Hongmei; Yang, Ling; Deng, Hongmei; Su, Yue; Yi, Xin

    2016-01-01

    Background Since the discovery of cell-free foetal DNA in the plasma of pregnant women, many non-invasive prenatal testing assays have been developed. In the area of skeletal dysplasia diagnosis, some PCR-based non-invasive prenatal testing assays have been developed to facilitate the ultrasound diagnosis of skeletal dysplasias that are caused by de novo mutations. However, skeletal dysplasias are a group of heterogeneous genetic diseases, the PCR-based method is hard to detect multiple gene or loci simultaneously, and the diagnosis rate is highly dependent on the accuracy of the ultrasound diagnosis. In this study, we investigated the feasibility of using targeted capture sequencing to detect foetal de novo pathogenic mutations responsible for skeletal dysplasia. Methodology/Principal Findings Three families whose foetuses were affected by skeletal dysplasia and two control families whose foetuses were affected by other single gene diseases were included in this study. Sixteen genes related to some common lethal skeletal dysplasias were selected for analysis, and probes were designed to capture the coding regions of these genes. Targeted capture sequencing was performed on the maternal plasma DNA, the maternal genomic DNA, and the paternal genomic DNA. The de novo pathogenic variants in the plasma DNA data were identified using a bioinformatical process developed for low frequency mutation detection and a strict variant interpretation strategy. The causal variants could be specifically identified in the plasma, and the results were identical to those obtained by sequencing amniotic fluid samples. Furthermore, a mean of 97% foetal specific alleles, which are alleles that are not shared by maternal genomic DNA and amniotic fluid DNA, were identified successfully in plasma samples. Conclusions/Significance Our study shows that capture sequencing of maternal plasma DNA can be used to non-invasive detection of de novo pathogenic variants. This method has the potential

  2. In vivo reduction of cell-free methemoglobin to oxyhemoglobin results in vasoconstriction in canines

    PubMed Central

    Wang, Dong; Piknova, Barbora; Solomon, Steven B.; Cortes-Puch, Irene; Kern, Steven J.; Sun, Junfeng; Kanias, Tamir; Gladwin, Mark T.; Helms, Christine; Kim-Shapiro, Daniel B.; Schechter, Alan. N; Natanson, Charles

    2013-01-01

    BACKGROUND Cell-free hemoglobin (Hb) in the vasculature leads to vasoconstriction and injury. Proposed mechanisms have been based on nitric oxide (NO) scavenging by oxyhemoglobin (oxyHb) or processes mediated by oxidative reactions of methemoglobin (metHb). To clarify this, we tested the vascular effect and fate of oxyHb or metHb infusions. STUDY DESIGN AND METHODS Twenty beagles were challenged with 1 h similar infusions of (200uM) metHb (n=5), oxyHb (n=5), albumin (n=5), or saline (n=5). Measurements were taken over 3 h. RESULTS Infusions of the two pure Hb species resulted in increases in mean arterial blood pressure (MAP), systemic vascular resistance index, and NO consumption capacity of plasma (all p<0.05) with the effects of oxyHb being greater than that from metHb (MAP; increase 0 to 3h; 27±6 % vs.7±2 %, respectively) (all p<0.05). The significant vasoconstrictive response of metHb (vs. albumin and saline controls) was related to in vivo auto-reduction of metHb to oxyHb, and the vasoactive Hb species that significantly correlated with MAP was always oxyHb, either from direct infusion or after in vivo reduction from metHb. Clearance of total Hb from plasma was faster after metHb than oxyHb infusion (p<0.0001). CONCLUSION These findings indicate that greater NO consumption capacity makes oxyHb more vasoactive than metHb. Additionally, metHb is reduced to oxyHb post-infusion and cleared faster or is less stable than oxyHb. Although we found no direct evidence that metHb itself is involved in acute vascular effects, in aggregate, these studies suggest that metHb is not inert and its mechanism of vasoconstriction is due to its delayed conversion to oxyHb by plasma-reducing agents. PMID:23488474

  3. A Novel Targeted Approach for Noninvasive Detection of Paternally Inherited Mutations in Maternal Plasma.

    PubMed

    van den Oever, Jessica M E; van Minderhout, Ivonne J H M; Harteveld, Cornelis L; den Hollander, Nicolette S; Bakker, Egbert; van der Stoep, Nienke; Boon, Elles M J

    2015-09-01

    The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. We have developed a sensitive, mutation-specific, and fast alternative for next-generation sequencing-mediated noninvasive prenatal diagnosis using a PCR-based method. For this proof-of-principle study, noninvasive fetal paternally inherited mutation detection was performed using cell-free DNA from maternal plasma. Preferential amplification of the paternally inherited allele was accomplished through a personalized approach using a blocking probe against maternal sequences in a high-resolution melting curve analysis-based assay. Enhanced detection of the fetal paternally inherited mutation was obtained for both an autosomal dominant and a recessive monogenic disorder by blocking the amplification of maternal sequences in maternal plasma. PMID:26162331

  4. Preparation of a Saccharomyces cerevisiae cell-free extract for in vitro translation.

    PubMed

    Wu, Cheng; Sachs, Matthew S

    2014-01-01

    Eukaryotic cell-free in vitro translation systems have been in use since the 1970s. These systems can faithfully synthesize polypeptides when programmed with mRNA, enabling the production of polypeptides for analysis as well as permitting analyses of the cis- and trans-acting factors that regulate translation. Here we describe the preparation and use of cell-free translation systems from the yeast Saccharomyces cerevisiae.

  5. Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns

    PubMed Central

    Joubert, Bonnie R.; den Dekker, Herman T.; Felix, Janine F.; Bohlin, Jon; Ligthart, Symen; Beckett, Emma; Tiemeier, Henning; van Meurs, Joyce B.; Uitterlinden, Andre G.; Hofman, Albert; Håberg, Siri E.; Reese, Sarah E.; Peters, Marjolein J.; Kulle Andreassen, Bettina; Steegers, Eric A. P.; Nilsen, Roy M.; Vollset, Stein E.; Midttun, Øivind; Ueland, Per M.; Franco, Oscar H.; Dehghan, Abbas; de Jongste, Johan C.; Wu, Michael C.; Wang, Tianyuan; Peddada, Shyamal D.; Jaddoe, Vincent W. V.; Nystad, Wenche; Duijts, Liesbeth; London, Stephanie J.

    2016-01-01

    Folate is vital for fetal development. Periconceptional folic acid supplementation and food fortification are recommended to prevent neural tube defects. Mechanisms whereby periconceptional folate influences normal development and disease are poorly understood: epigenetics may be involved. We examine the association between maternal plasma folate during pregnancy and epigenome-wide DNA methylation using Illumina's HumanMethyl450 Beadchip in 1,988 newborns from two European cohorts. Here we report the combined covariate-adjusted results using meta-analysis and employ pathway and gene expression analyses. Four-hundred forty-three CpGs (320 genes) are significantly associated with maternal plasma folate levels during pregnancy (false discovery rate 5%); 48 are significant after Bonferroni correction. Most genes are not known for folate biology, including APC2, GRM8, SLC16A12, OPCML, PRPH, LHX1, KLK4 and PRSS21. Some relate to birth defects other than neural tube defects, neurological functions or varied aspects of embryonic development. These findings may inform how maternal folate impacts the developing epigenome and health outcomes in offspring. PMID:26861414

  6. Impact of Cu(2+) ions on the structure of colistin and cell-free system nucleic acid degradation.

    PubMed

    Stokowa-Sołtys, Kamila; Kasprowicz, Aleksandra; Wrzesiński, Jan; Ciesiołka, Jerzy; Gaggelli, Nicola; Gaggelli, Elena; Valensin, Gianni; Jeżowska-Bojczuk, Małgorzata

    2015-10-01

    Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV-vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health.

  7. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

    PubMed Central

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-01-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. PMID:26478227

  8. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems.

    PubMed

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-11-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field.

  9. The Human Endogenous Protection System against Cell-Free Hemoglobin and Heme Is Overwhelmed in Preeclampsia and Provides Potential Biomarkers and Clinical Indicators

    PubMed Central

    Johansson, Maria E.; Edström-Hägerwall, Anneli; Larsson, Irene; Jälmby, Maya; Hansson, Stefan R.; Åkerström, Bo

    2015-01-01

    Preeclampsia (PE) complicates 3–8% of all pregnancies and manifests clinically as hypertension and proteinuria in the second half of gestation. The pathogenesis of PE is not fully understood but recent studies have described the involvement of cell-free fetal hemoglobin (HbF). Hypothesizing that PE is associated with prolonged hemolysis we have studied the response of the cell-free Hb- and heme defense network. Thus, we have investigated the levels of cell-free HbF (both free, denoted HbF, and in complex with Hp, denoted Hp-HbF) as well as the major human endogenous Hb- and heme-scavenging systems: haptoglobin (Hp), hemopexin (Hpx), α1-microglobulin (A1M) and CD163 in plasma of PE women (n = 98) and women with normal pregnancies (n = 47) at term. A significant increase of the mean plasma HbF concentration was observed in women with PE. Plasma levels of Hp and Hpx were statistically significantly reduced, whereas the level of the extravascular heme- and radical scavenger A1M was significantly increased in plasma of women with PE. The Hpx levels significantly correlated with maternal blood pressure. Furthermore, HbF and the related scavenger proteins displayed a potential to be used as clinical biomarkers for more precise diagnosis of PE and are candidates as predictors of identifying pregnancies with increased risk of obstetrical complications. The results support that PE pathophysiology is associated with increased HbF-concentrations and an activation of the physiological Hb-heme defense systems. PMID:26368565

  10. Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer

    PubMed Central

    Tie, J.; Kinde, I.; Wang, Y.; Wong, H. L.; Roebert, J.; Christie, M.; Tacey, M.; Wong, R.; Singh, M.; Karapetis, C. S.; Desai, J.; Tran, B.; Strausberg, R. L.; Diaz, L. A.; Papadopoulos, N.; Kinzler, K. W.; Vogelstein, B.; Gibbs, P.

    2015-01-01

    Background Early indicators of treatment response in metastatic colorectal cancer (mCRC) could conceivably be used to optimize treatment. We explored early changes in circulating tumor DNA (ctDNA) levels as a marker of therapeutic efficacy. Patients and methods This prospective study involved 53 mCRC patients receiving standard first-line chemotherapy. Both ctDNA and CEA were assessed in plasma collected before treatment, 3 days after treatment and before cycle 2. Computed tomography (CT) scans were carried out at baseline and 8–10 weeks and were centrally assessed using RECIST v1.1 criteria. Tumors were sequenced using a panel of 15 genes frequently mutated in mCRC to identify candidate mutations for ctDNA analysis. For each patient, one tumor mutation was selected to assess the presence and the level of ctDNA in plasma samples using a digital genomic assay termed Safe-SeqS. Results Candidate mutations for ctDNA analysis were identified in 52 (98.1%) of the tumors. These patient-specific candidate tissue mutations were detectable in the cell-free DNA from the plasma of 48 of these 52 patients (concordance 92.3%). Significant reductions in ctDNA (median 5.7-fold; P < 0.001) levels were observed before cycle 2, which correlated with CT responses at 8–10 weeks (odds ratio = 5.25 with a 10-fold ctDNA reduction; P = 0.016). Major reductions (≥10-fold) versus lesser reductions in ctDNA precycle 2 were associated with a trend for increased progression-free survival (median 14.7 versus 8.1 months; HR = 1.87; P = 0.266). Conclusions ctDNA is detectable in a high proportion of treatment naïve mCRC patients. Early changes in ctDNA during first-line chemotherapy predict the later radiologic response. PMID:25851626

  11. Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine.

    PubMed

    Holmes, Emily Eva; Jung, Maria; Meller, Sebastian; Leisse, Annette; Sailer, Verena; Zech, Julie; Mengdehl, Martina; Garbe, Leif-Alexander; Uhl, Barbara; Kristiansen, Glen; Dietrich, Dimo

    2014-01-01

    DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of

  12. Comparison of nested PCR for detection of DNA in plasma with pp65 leukocytic antigenemia procedure for diagnosis of human cytomegalovirus infection.

    PubMed Central

    Freymuth, F; Gennetay, E; Petitjean, J; Eugene, G; Hurault de Ligny, B; Ryckelynck, J P; Legoff, C; Hazera, P; Bazin, C

    1994-01-01

    A nested PCR was used for the detection of human cytomegalovirus (HCMV) DNA in plasma. The presence of HCMV DNA and its correlation to pp65 leukocytic antigenemia were investigated with 299 blood samples from 45 organ transplant recipients and 63 AIDS patients. Of the 53 samples positive by nested PCR, 52 (98%) were also positive for leukocytic antigenemia and 23 had high levels of antigenemia (> 50 positive cells per 2 x 10(5) leukocytes). Of the 246 samples negative in PCR, only 3 (1.2%) had highly positive antigenemia. For 15 patients having a high antigenemia level in the course of their disease, consecutive blood samples were studied and also assessed for viremia in culture. The extent to which HCMV DNA, detected by PCR, was present in plasma correlated with increased levels of HCMV leukocytic antigenemia for six of the eight AIDS patients and for all the organ transplant recipients. Positivity for HCMV DNA in PCR and for viremia in cell culture was usually restricted to the highest antigenemia levels. From a total of 69 blood samples, PCR and culture gave positive results, respectively, for 17 of 32 samples (53%) and 14 of 32 samples (43%) from transplant recipients and for 15 of 37 samples (40%) and 9 of 37 samples (24%) from AIDS patients. Our findings have shown a strong correlation between high levels of leukocytic antigenemia and HCMV DNA in plasma. The detection of HCMV DNA in plasma by this nested PCR can prove HCMV dissemination in blood, but it lacks the rapidity and simplicity of the leukocytic pp65 antigenemia procedure. PMID:8077418

  13. Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

    PubMed

    Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

    2015-06-01

    A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.

  14. Human herpesvirus infections of the central nervous system: laboratory diagnosis based on DNA detection by nested PCR in plasma and cerebrospinal fluid samples.

    PubMed

    Rimério, Carla Aparecida Tavares; De Oliveira, Renato Souza; de Almeida Bonatelli, Murilo Queiroz; Nucci, Anamarli; Costa, Sandra Cecília Botelho; Bonon, Sandra Helena Alves

    2015-04-01

    Infections of the central nervous systems (CNS) present a diagnostic problem for which an accurate laboratory diagnosis is essential. Invasive practices, such as cerebral biopsy, have been replaced by obtaining a polymerase chain reaction (PCR) diagnosis using cerebral spinal fluid (CSF) as a reference method. Tests on DNA extracted from plasma are noninvasive, thus avoiding all of the collateral effects and patient risks associated with CSF collection. This study aimed to determine whether plasma can replace CSF in nested PCR analysis for the detection of CNS human herpesvirus (HHV) diseases by analysing the proportion of patients whose CSF nested PCR results were positive for CNS HHV who also had the same organism identified by plasma nested PCR. In this study, CSF DNA was used as the "gold standard," and nested PCR was performed on both types of samples. Fifty-two patients with symptoms of nervous system infection were submitted to CSF and blood collection. For the eight HHV, one positive DNA result-in plasma and/or CSF nested PCR-was considered an active HHV infection, whereas the occurrence of two or more HHVs in the same sample was considered a coinfection. HHV infections were positively detected in 27/52 (51.9%) of the CSF and in 32/52 (61.5%) of the plasma, difference not significant, thus nested PCR can be performed on plasma instead of CSF. In conclusion, this findings suggest that plasma as a useful material for the diagnosis of cases where there is any difficulty to perform a CSF puncture.

  15. Longitudinal Bank for Serum, Plasma, and DNA for Detection of Biomarkers

    SciTech Connect

    Vogelzang, Nicolas; Fink, Louis

    2007-11-12

    The discovery of genetic or biochemical markers to discriminate malignant cancers from normal or benign disease states, markers to stage cancer or monitor disease progression and markers that provide an early indication of an individual’s response to chemotherapy have become a major research objectives of the oncology community over the past few years. The goal of the project is to create a patient speciment bank of serum, plasma, urine and tissues from approximately 1500 individuals. The collection of samples from individuals on a longitudinal basis provided proteomic and biochemical data to be correlated with clinical endpoints. This greatly enhanced our ability to identify biiomarkers for staging different cancers and to detect patient responsiveness to therapy at an early state in the treatment process.

  16. Quantitation of human immunodeficiency virus type 1 DNA forms with the second template switch in peripheral blood cells predicts disease progression independently of plasma RNA load.

    PubMed

    Kostrikis, Leondios G; Touloumi, Giota; Karanicolas, Rose; Pantazis, Nikos; Anastassopoulou, Cleo; Karafoulidou, Anastasia; Goedert, James J; Hatzakis, Angelos

    2002-10-01

    There are several forms of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood T cells and lymph nodes in untreated HIV-1-infected individuals and in patients whose plasma HIV-1 RNA levels are suppressed by long-term combination antiretroviral therapy. However, it remains to be established whether the concentration of HIV-1 DNA in cells predicts the clinical outcome of HIV-1 infection. In this report, we measured the concentration of HIV-1 DNA forms which has undergone the second template switch (STS DNA) and 2-long-terminal-repeat DNA circles in peripheral blood mononuclear cell (PBMC) samples. To do this, we used molecular-beacon-based real-time PCR assays and studied 130 patients with hemophilia in the Multicenter Hemophilia Cohort Study. We assessed the influence of baseline HIV-1 STS DNA levels on the progression of HIV-1 disease in the absence of combination antiretroviral therapy by Kaplan-Meier and Cox regression analysis. Among the patients who progressed to AIDS, the median levels (interquartile ranges) of STS HIV-1 DNA in PBMC were significantly higher than those of patients who remained AIDS free during the 16 years of follow-up (1,017 [235 to 6,059] and 286 [31 to 732] copies per 10(6) PBMC, respectively; P < 0.0001). Rates of progression to death and development of AIDS varied significantly (log rank P < 0.001) by quartile distribution of HIV-1 STS DNA levels. After adjustment for age at seroconversion, baseline CD4(+) T-cell counts, plasma viral load, and T-cell-receptor excision circles, the relative hazards (RH) of death and AIDS were significantly increased with higher HIV-1 STS DNA levels (adjusted RH, 1.84 [95% confidence interval (CI), 1.30 to 2.59] and 2.62 [95% CI, 1.75 to 3.93] per 10-fold increase per 10(6) PBMC, respectively). HIV-1 STS DNA levels in each individual remained steady in longitudinal PBMC samples during 16 years of follow-up. Our findings show that the concentration of HIV-1 STS DNA in PBMC complements the HIV

  17. Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14.

    PubMed

    Serrano, Edith; Pérez-Pé, Rosaura; Calleja, Lucía; Guillén, Natalia; Casao, Adriana; Hurtado-Guerrero, Ramón; Muiño-Blanco, Teresa; Cebrián-Pérez, José A

    2013-05-01

    In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76-85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality. PMID:23462333

  18. Expression Optimization and Inducible Negative Feedback in Cell-Free Systems

    SciTech Connect

    Karig, David K; Iyer, Sukanya; Simpson, Michael L; Doktycz, Mitchel John

    2012-01-01

    Synthetic biology offers great promise to a variety of applications through the forward engineering of biological function. Most efforts in this field have focused on employing living cells. Cell-free approaches, on the other hand, offer simpler and more flexible contexts, but few synthetic systems based on cell-free protein expression have been constructed. Here, we evaluate cell-free regulatory systems based on T7 promoter driven expression, and we demonstrate negative feedback, an essential motif in many natural and engineered systems. First, we characterize variants of TetR and LacI repressible T7 promoters in a cell-free context and examine sequence elements that determine expression efficiency. Then, we explore different approaches for composing regulatory systems, leading to the implementation of inducible negative feedback in E. coli extracts and in the minimal PURE system, which consists of purified proteins necessary for transcription and translation. Our quantitative cell-free component characterizations and demonstration of negative feedback embody important steps on the path to harnessing biological function in a bottom up fashion.

  19. Rapid cell-free forward engineering of novel genetic ring oscillators

    PubMed Central

    Niederholtmeyer, Henrike; Sun, Zachary Z; Hori, Yutaka; Yeung, Enoch; Verpoorte, Amanda; Murray, Richard M; Maerkl, Sebastian J

    2015-01-01

    While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior. DOI: http://dx.doi.org/10.7554/eLife.09771.001 PMID:26430766

  20. Cell-free Expression and In Meso Crystallisation of an Integral Membrane Kinase for Structure Determination

    PubMed Central

    Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-01-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a 3-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipidic mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28 Å resolution. The quality of cellular and cell-free expressed kinase samples have been evaluated systematically by comparing i) spectroscopic properties, ii) purity and oligomer formation, iii) lipid content and iv) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698

  1. Identification of tissue-specific cell death using methylation patterns of circulating DNA.

    PubMed

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J; Wasserfall, Clive H; Schatz, Desmond A; Greenbaum, Carla J; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K; Golan, Talia; Ben Sasson, Shmuel A; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A M James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-03-29

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

  2. Identification of tissue-specific cell death using methylation patterns of circulating DNA

    PubMed Central

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-01-01

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

  3. Identification of tissue-specific cell death using methylation patterns of circulating DNA.

    PubMed

    Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J; Wasserfall, Clive H; Schatz, Desmond A; Greenbaum, Carla J; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K; Golan, Talia; Ben Sasson, Shmuel A; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A M James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

    2016-03-29

    Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

  4. Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease

    PubMed Central

    Olsson, Eleonor; Winter, Christof; George, Anthony; Chen, Yilun; Howlin, Jillian; Tang, Man-Hung Eric; Dahlgren, Malin; Schulz, Ralph; Grabau, Dorthe; van Westen, Danielle; Fernö, Mårten; Ingvar, Christian; Rose, Carsten; Bendahl, Pär-Ola; Rydén, Lisa; Borg, Åke; Gruvberger-Saal, Sofia K; Jernström, Helena; Saal, Lao H

    2015-01-01

    Metastatic breast cancer is usually diagnosed after becoming symptomatic, at which point it is rarely curable. Cell-free circulating tumor DNA (ctDNA) contains tumor-specific chromosomal rearrangements that may be interrogated in blood plasma. We evaluated serial monitoring of ctDNA for earlier detection of metastasis in a retrospective study of 20 patients diagnosed with primary breast cancer and long follow-up. Using an approach combining low-coverage whole-genome sequencing of primary tumors and quantification of tumor-specific rearrangements in plasma by droplet digital PCR, we identify for the first time that ctDNA monitoring is highly accurate for postsurgical discrimination between patients with (93%) and without (100%) eventual clinically detected recurrence. ctDNA-based detection preceded clinical detection of metastasis in 86% of patients with an average lead time of 11 months (range 0–37 months), whereas patients with long-term disease-free survival had undetectable ctDNA postoperatively. ctDNA quantity was predictive of poor survival. These findings establish the rationale for larger validation studies in early breast cancer to evaluate ctDNA as a monitoring tool for early metastasis detection, therapy modification, and to aid in avoidance of overtreatment. PMID:25987569

  5. In vitro translation in a hybrid cell free lysate with exogenous cellular ribosomes.

    PubMed

    Panthu, Baptiste; Décimo, Didier; Balvay, Laurent; Ohlmann, Théophile

    2015-05-01

    Cell free protein synthesis systems (CFPS) have been widely used to express proteins and to explore the pathways of gene expression. In the present manuscript, we describe the design of a novel adaptable hybrid in vitro translation system which is assembled with ribosomes isolated from many different origins. We first show that this hybrid system exhibits all important features such as efficiency, sensitivity, reproducibility and the ability to translate specialized mRNAs in less than 1 h. In addition, the unique design of this cell free assay makes it highly adaptable to utilize ribosomes isolated from many different organs, tissues or cell types.

  6. Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

    PubMed Central

    Shi, J; Dixon, R A; Gonzales, R A; Kjellbom, P; Bhattacharyya, M K

    1995-01-01

    An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein. Images Fig. 1 Fig. 2 PMID:7753826

  7. Targeted Next-Generation Sequencing of Plasma DNA from Cancer Patients: Factors Influencing Consistency with Tumour DNA and Prospective Investigation of Its Utility for Diagnosis

    PubMed Central

    Kaisaki, Pamela J.; Cutts, Anthony; Popitsch, Niko; Camps, Carme; Pentony, Melissa M.; Wilson, Gareth; Page, Suzanne; Kaur, Kulvinder; Vavoulis, Dimitris; Henderson, Shirley; Gupta, Avinash; Middleton, Mark R.; Karydis, Ioannis; Talbot, Denis C.; Schuh, Anna; Taylor, Jenny C.

    2016-01-01

    Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient. PMID:27626278

  8. Targeted Next-Generation Sequencing of Plasma DNA from Cancer Patients: Factors Influencing Consistency with Tumour DNA and Prospective Investigation of Its Utility for Diagnosis.

    PubMed

    Kaisaki, Pamela J; Cutts, Anthony; Popitsch, Niko; Camps, Carme; Pentony, Melissa M; Wilson, Gareth; Page, Suzanne; Kaur, Kulvinder; Vavoulis, Dimitris; Henderson, Shirley; Gupta, Avinash; Middleton, Mark R; Karydis, Ioannis; Talbot, Denis C; Schuh, Anna; Taylor, Jenny C

    2016-01-01

    Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient. PMID:27626278

  9. A Low-Cost Efficient Multiplex PCR for Prenatal Sex Determination in Bovine Fetus Using Free Fetal DNA in Maternal Plasma

    PubMed Central

    Davoudi, Arash; Seighalani, Ramin; Aleyasin, Seyed Ahmad; Tarang, Alireza; Salehi, Abdolreza Salehi; Tahmoressi, Farideh

    2012-01-01

    Background In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction (PCR) analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. Materials and Methods In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 µl maternal plasma. Two primer pairs for bovine amelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. Results The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. Conclusion The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses. PMID:25505511

  10. Identification of Circulating Tumor DNA for the Early Detection of Small-cell Lung Cancer.

    PubMed

    Fernandez-Cuesta, Lynnette; Perdomo, Sandra; Avogbe, Patrice H; Leblay, Noemie; Delhomme, Tiffany M; Gaborieau, Valerie; Abedi-Ardekani, Behnoush; Chanudet, Estelle; Olivier, Magali; Zaridze, David; Mukeria, Anush; Vilensky, Marta; Holcatova, Ivana; Polesel, Jerry; Simonato, Lorenzo; Canova, Cristina; Lagiou, Pagona; Brambilla, Christian; Brambilla, Elisabeth; Byrnes, Graham; Scelo, Ghislaine; Le Calvez-Kelm, Florence; Foll, Matthieu; McKay, James D; Brennan, Paul

    2016-08-01

    Circulating tumor DNA (ctDNA) is emerging as a key potential biomarker for post-diagnosis surveillance but it may also play a crucial role in the detection of pre-clinical cancer. Small-cell lung cancer (SCLC) is an excellent candidate for early detection given there are no successful therapeutic options for late-stage disease, and it displays almost universal inactivation of TP53. We assessed the presence of TP53 mutations in the cell-free DNA (cfDNA) extracted from the plasma of 51 SCLC cases and 123 non-cancer controls. We identified mutations using a pipeline specifically designed to accurately detect variants at very low fractions. We detected TP53 mutations in the cfDNA of 49% SCLC patients and 11.4% of non-cancer controls. When stratifying the 51 initial SCLC cases by stage, TP53 mutations were detected in the cfDNA of 35.7% early-stage and 54.1% late-stage SCLC patients. The results in the controls were further replicated in 10.8% of an independent series of 102 non-cancer controls. The detection of TP53 mutations in 11% of the 225 non-cancer controls suggests that somatic mutations in cfDNA among individuals without any cancer diagnosis is a common occurrence, and poses serious challenges for the development of ctDNA screening tests. PMID:27377626

  11. Characterizing and prototyping genetic networks with cell-free transcription-translation reactions.

    PubMed

    Takahashi, Melissa K; Hayes, Clarmyra A; Chappell, James; Sun, Zachary Z; Murray, Richard M; Noireaux, Vincent; Lucks, Julius B

    2015-09-15

    A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative 'design-build-test' cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription-translation (TX-TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX-TL system that utilizes the native Escherichia coli TX-TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX-TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX-TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems.

  12. Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis.

    PubMed

    Meng, Qinglong; Zhang, Yanfei; Ju, Xiaozhi; Ma, Chunling; Ma, Hongwu; Chen, Jiuzhou; Zheng, Ping; Sun, Jibin; Zhu, Jun; Ma, Yanhe; Zhao, Xueming; Chen, Tao

    2016-05-20

    5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production.

  13. New bioproduction systems: from molecular circuits to novel reactor concepts in cell-free biotechnology.

    PubMed

    Rupp, Steffen

    2013-01-01

    : The last decades witnessed a strong growth in several areas of biotechnology, especially in fields related to health, as well as in industrial biotechnology. Advances in molecular engineering now enable biotechnologists to design more efficient pathways in order to convert a larger spectrum of renewable resources into industrially used biofuels and chemicals as well as into new pharmaceuticals and therapeutic proteins. In addition material sciences advanced significantly making it more and more possible to integrate biology and engineering. One of the key questions currently is how to develop new ways of engineering biological systems to cope with the complexity and limitations given by the cell. The options to integrate biology with classical engineering advanced cell free technologies in the recent years significantly. Cell free protein production using cellular extracts is now a well-established universal technology for production of proteins derived from many organisms even at the milligram scale. Among other applications it has the potential to supply the demand for a multitude of enzymes and enzyme variants facilitating in vitro metabolic engineering. This review will briefly address the recent achievements and limitations of cell free conversions. Especially, the requirements for reactor systems in cell free biotechnology, a currently underdeveloped field, are reviewed and some perspectives are given on how material sciences and biotechnology might be able to advance these new developments in the future.

  14. Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.

    PubMed

    Sonar, S; Patel, N; Fischer, W; Rothschild, K J

    1993-12-21

    Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.

  15. Multi-input regulation and logic with T7 promoters in cells and cell free systems

    SciTech Connect

    Iyer, Sukanya; Karig, David K; Norred, Sarah E; Simpson, Michael L; Doktycz, Mitchel John

    2014-01-01

    Engineered gene circuits offer an opportunity to harness biological systems for biotechnological and biomedical applications. However, reliance on host E. coli promoters for the construction of circuit elements, such as logic gates, makes implementation of predictable, independently functioning circuits difficult. In contrast, T7 promoters offer a simple orthogonal expression system for use in a variety of cellular backgrounds and even in cell free systems. Here we develop a T7 promoter system that can be regulated by two different transcriptional repressors for the construction of a logic gate that functions in cells and in cell free systems. We first present LacI repressible T7lacO promoters that are regulated from a distal lac operator site for repression. We next explore the positioning of a tet operator site within the T7lacO framework to create T7 promoters that respond to tet and lac repressors and realize an IMPLIES gate. Finally, we demonstrate that these dual input sensitive promoters function in a commercially available E. coli cell-free protein expression system. Together, our results contribute to the first demonstration of multi-input regulation of T7 promoters and expand the utility of T7 promoters in cell based as well as cell-free gene circuits.

  16. Cell-free synthesis system suitable for disulfide-containing proteins

    SciTech Connect

    Matsuda, Takayoshi; Watanabe, Satoru; Kigawa, Takanori

    2013-02-08

    Highlights: ► Cell-free synthesis system suitable for disulfide-containing proteins is proposed. ► Disulfide bond formation was facilitated by the use of glutathione buffer. ► DsbC catalyzed the efficient shuffling of incorrectly formed disulfide bonds. ► Milligram quantities of functional {sup 15}N-labeled BPTI and lysozyme C were obtained. ► Synthesized proteins were both catalytically functional and properly folded. -- Abstract: Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional {sup 15}N-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses.

  17. Antibacterial Nitroacridine, Nitroakridin 3582: Binding to Nucleic Acids In Vitro and Effects on Selected Cell-Free Model Systems of Macromolecular Biosynthesis

    PubMed Central

    Wolfe, Alan D.; Cook, Thomas M.; Hahn, Fred E.

    1971-01-01

    Nitroakridin 3582 (NA) formed complexes with native deoxyribonucleic acid (DNA) and with transfer ribonucleic acid (tRNA) species from Escherichia coli. Spectrophotometric titrations of NA with these nucleic acids produced numerical results from which nonlinear adsorption isotherms were derived. These curves indicated the existence of more than one class of binding sites on the polymers to which NA was bound by more than one process. The stoichiometry of strong binding of NA to double helical DNA was in agreement with a conventional value (1 ligand molecule per 4.2 component nucleotides) for complete intercalation binding. NA inhibited the DNA-dependent DNA polymerase I and RNA polymerase reactions, the first strongly and the second appreciably. These inhibitions corresponded to the extents to which NA inhibits DNA and RNA biosyntheses in vivo. Evidently, NA interferes with the template function of DNA. The drug also inhibited the polymerization of phenylalanine in a cell-free E. coli ribosome-polyuridylic acid [poly (U)] system. The effect paralleled an inhibition of the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. Ethidium bromide acted similarly. The antimalarial drug, chloroquine, stimulated polyphenylalanine synthesis, apparently as a result of stimulating the poly (U)-directed binding of phenylalanyl tRNA to ribosomes. PMID:4945180

  18. In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

    PubMed

    Sekiguchi, J M; Kmiec, E B

    1992-02-25

    Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.

  19. Plasma genetic and genomic abnormalities predict treatment response and clinical outcome in advanced prostate cancer

    PubMed Central

    Du, Meijun; Dittmar, Rachel L.; Lee, Adam; Nandy, Debashis; Yuan, Tiezheng; Guo, Yongchen; Wang, Yuan; Tschannen, Michael R.; Worthey, Elizabeth; Jacob, Howard; See, William; Kilari, Deepak; Wang, Xuexia; Hovey, Raymond L.; Huang, Chiang-Ching; Wang, Liang

    2015-01-01

    Liquid biopsies, examinations of tumor components in body fluids, have shown promise for predicting clinical outcomes. To evaluate tumor-associated genomic and genetic variations in plasma cell-free DNA (cfDNA) and their associations with treatment response and overall survival, we applied whole genome and targeted sequencing to examine the plasma cfDNAs derived from 20 patients with advanced prostate cancer. Sequencing-based genomic abnormality analysis revealed locus-specific gains or losses that were common in prostate cancer, such as 8q gains, AR amplifications, PTEN losses and TMPRSS2-ERG fusions. To estimate tumor burden in cfDNA, we developed a Plasma Genomic Abnormality (PGA) score by summing the most significant copy number variations. Cox regression analysis showed that PGA scores were significantly associated with overall survival (p < 0.04). After androgen deprivation therapy or chemotherapy, targeted sequencing showed significant mutational profile changes in genes involved in androgen biosynthesis, AR activation, DNA repair, and chemotherapy resistance. These changes may reflect the dynamic evolution of heterozygous tumor populations in response to these treatments. These results strongly support the feasibility of using non-invasive liquid biopsies as potential tools to study biological mechanisms underlying therapy-specific resistance and to predict disease progression in advanced prostate cancer. PMID:25915538

  20. The clinical significance of EBV DNA in the plasma and peripheral blood mononuclear cells of patients with or without EBV diseases.

    PubMed

    Kanakry, Jennifer A; Hegde, Aparna M; Durand, Christine M; Massie, Allan B; Greer, Amy E; Ambinder, Richard F; Valsamakis, Alexandra

    2016-04-21

    Epstein-Barr virus (EBV) is a ubiquitous virus that establishes a latent infection within the host and in some cases can lead to the development of EBV-associated lymphomas, lymphoproliferative disorders, hemophagocytic lymphohistiocytosis, solid tumors, and other diseases. We studied the clinical significance of detecting EBV DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of 2146 patients who had blood specimens sent to the Johns Hopkins Hospital clinical laboratory for viral quantitative real-time polymerase chain reaction assay over a 5-year period. Within this largely immunocompromised and hospitalized cohort, 535 patients (25%) had EBV detected in plasma or PBMCs. When EBV was detected in the absence of an EBV(+)disease (n = 402), it was present only in PBMCs in 69% of cases. Immunocompromised patients were less likely to have EBV in plasma than in PBMCs in the absence of EBV(+)disease. In patients with active, systemic EBV(+)diseases (n = 105), EBV was detected in plasma in 99% of cases but detected in PBMCs in only 54%. Across a range of copy number cutoffs, EBV in plasma had higher specificity and sensitivity for EBV(+)disease as compared with EBV in PBMCs. EBV copy number in plasma distinguished untreated, EBV(+)lymphoma from EBV(+)lymphoma in remission and EBV(-)lymphoma, and also distinguished untreated, EBV(+)posttransplantation lymphoproliferative disorder (PTLD) from EBV(+)PTLD in remission and EBV(-)PTLD. EBV copy number quantification is a useful diagnostic marker across the spectrum of EBV(+)diseases, even among immunocompromised patients, with plasma specimens more indicative of EBV(+)disease than PBMCs.

  1. A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging

    PubMed Central

    Cerqueira, Carla; Pang, Yuk-Ying S.; Day, Patricia M.; Thompson, Cynthia D.; Buck, Christopher B.; Lowy, Douglas R.

    2015-01-01

    ABSTRACT We have established a cell-free in vitro system to study human papillomavirus type 16 (HPV16) assembly, a poorly understood process. L1/L2 capsomers, obtained from the disassembly of virus-like particles (VLPs), were incubated with nuclear extracts to provide access to the range of cellular proteins that would be available during assembly within the host cell. Incorporation of a reporter plasmid “pseudogenome” was dependent on the presence of both nuclear extract and ATP. Unexpectedly, L1/L2 VLPs that were not disassembled prior to incubation with a reassembly mixture containing nuclear extract also encapsidated a reporter plasmid. As with HPV pseudoviruses (PsV) generated intracellularly, infection by cell-free particles assembled in vitro required the presence of L2 and was susceptible to the same biochemical inhibitors, implying the cell-free assembled particles use the infectious pathway previously described for HPV16 produced in cell culture. Using biochemical and electron microscopy analyses, we observed that, in the presence of nuclear extract, intact VLPs partially disassemble, providing a mechanistic explanation to how the exogenous plasmid was packaged by these particles. Further, we provide evidence that capsids containing an <8-kb pseudogenome are resistant to the disassembly/reassembly reaction. Our results suggest a novel size discrimination mechanism for papillomavirus genome packaging in which particles undergo iterative rounds of disassembly/reassembly, seemingly sampling DNA until a suitably sized DNA is encountered, resulting in the formation of a stable virion structure. IMPORTANCE Little is known about papillomavirus assembly biology due to the difficulties in propagating virus in vitro. The cell-free assembly method established in this paper reveals a new mechanism for viral genome packaging and will provide a tractable system for further dissecting papillomavirus assembly. The knowledge gained will increase our understanding of

  2. Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance

    PubMed Central

    Nishimoto, Sachiko; Fukuda, Daiju; Higashikuni, Yasutomi; Tanaka, Kimie; Hirata, Yoichiro; Murata, Chie; Kim-Kaneyama, Joo-ri; Sato, Fukiko; Bando, Masahiro; Yagi, Shusuke; Soeki, Takeshi; Hayashi, Tetsuya; Imoto, Issei; Sakaue, Hiroshi; Shimabukuro, Michio; Sata, Masataka

    2016-01-01

    Obesity stimulates chronic inflammation in adipose tissue, which is associated with insulin resistance, although the underlying mechanism remains largely unknown. Here we showed that obesity-related adipocyte degeneration causes release of cell-free DNA (cfDNA), which promotes macrophage accumulation in adipose tissue via Toll-like receptor 9 (TLR9), originally known as a sensor of exogenous DNA fragments. Fat-fed obese wild-type mice showed increased release of cfDNA, as determined by the concentrations of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in plasma. cfDNA released from degenerated adipocytes promoted monocyte chemoattractant protein-1 (MCP-1) expression in wild-type macrophages, but not in TLR9-deficient (Tlr9−/−) macrophages. Fat-fed Tlr9−/− mice demonstrated reduced macrophage accumulation and inflammation in adipose tissue and better insulin sensitivity compared with wild-type mice, whereas bone marrow reconstitution with wild-type bone marrow restored the attenuation of insulin resistance observed in fat-fed Tlr9−/− mice. Administration of a TLR9 inhibitory oligonucleotide to fat-fed wild-type mice reduced the accumulation of macrophages in adipose tissue and improved insulin resistance. Furthermore, in humans, plasma ssDNA level was significantly higher in patients with computed tomography–determined visceral obesity and was associated with homeostasis model assessment of insulin resistance (HOMA-IR), which is the index of insulin resistance. Our study may provide a novel mechanism for the development of sterile inflammation in adipose tissue and a potential therapeutic target for insulin resistance. PMID:27051864

  3. Circulating free DNA concentration is an independent prognostic biomarker in lung cancer.

    PubMed

    Tissot, Claire; Toffart, Anne-Claire; Villar, Stéphanie; Souquet, Pierre-Jean; Merle, Patrick; Moro-Sibilot, Denis; Pérol, Maurice; Zavadil, Jiri; Brambilla, Christian; Olivier, Magali; Couraud, Sébastien

    2015-12-01

    Plasma circulating cell-free (cf)DNA is of interest in oncology because it has been shown to contain tumour DNA and may thus be used as liquid biopsy. In nonsmall cell lung cancer (NSCLC), cfDNA quantification has been proposed for the monitoring and follow-up of patients. However, available studies are limited and need to be confirmed by studies with larger sample sizes and including patients who receive more homogenous treatments. Our objective was to assess the predictive and prognostic value of plasma cfDNA concentration in a large series of patients with NSCLC and treated with a standard chemotherapy regimen.We included samples from lung cancer patients recruited into the Pharmacogenoscan study. The cfDNA of 218 patients was extracted and quantified by fluorometry before and after two or three cycles of platinum-based chemotherapy. The association between baseline and post-chemotherapy concentrations and treatment response, assessed by RECIST (response evaluation criteria in solid tumours) or patient survival was analysed.Patients with high cfDNA concentrations (highest tertile) at baseline had a significantly worse disease-free and overall survival than those with lower concentrations (lowest and middle tertiles) (median overall survival 10 months (95% CI 10.7-13.9) versus 14.2 months (95% CI 12.6-15.8), respectively; p=0.001). In multivariate analysis, increased baseline concentration of cfDNA was an independent prognostic factor. However, we did not find any association between cfDNA concentration and response to treatment.cfDNA may be a biomarker for the assessment of prognosis in NSCLC. However, total concentration of cfDNA does not appear to predict chemotherapy response.

  4. 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.

  5. The Mechanism of Proinflammatory HDL Generation in Sickle Cell Disease Is Linked to Cell-Free Hemoglobin via Haptoglobin

    PubMed Central

    Ji, Xiang; Feng, Yimin; Tian, Hui; Meng, Wei; Wang, Weiling; Liu, Na; Zhang, Jun; Wang, Lingshu; Wang, Jian; Gao, Haiqing

    2016-01-01

    In sickle cell disease (SCD), the inflammatory properties of high-density lipoprotein (HDL) can be changed by cell-free hemoglobin (Hb), which is released into the blood during hemolysis. Hb in the plasma of SCD patients or mice can bind with HDL specifically inducing an inflammatory reaction. In our study, we found increased amounts of inflammatory factor proteins in the chronic oxidative state of SCD with higher levels of Hb, haptoglobin (Hp) and hemopexin (Hx) in the apolipoprotein A-I (ApoA-1) particles of HDL and the role of HDL is changed from being anti-inflammatory to proinflammatory. Our results also suggest Hp and Hx, the scavengers of Hb in HDL, are positively associated with inflammatory levels in SCD patients. HDL retained its inflammatory inhibition role in Hp−/− mice, with less Hb accumulation. Hx may further prevent inflammatory reaction because its level will be even higher when lack of Hx. We therefore demonstrated that Hp is indispensable during the process whereby Hb associates with HDL and plays a clear proinflammatory role. Therefore, it is essential to break the binding between Hb and Hp for treatment. The dissociation of Hb/Hp/Hx complexes may also play an important role in the study of other inflammatory angiogenesis-related diseases. PMID:27716784

  6. A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

    PubMed

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2013-08-01

    Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy. PMID:23851421

  7. Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

    PubMed Central

    Lewis, Daniel D.; Villarreal, Fernando D.; Wu, Fan; Tan, Cheemeng

    2014-01-01

    As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems. PMID:25538941

  8. Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies.

    PubMed

    Rues, Ralf-Bernhardt; Henrich, Erik; Boland, Coilin; Caffrey, Martin; Bernhard, Frank

    2016-01-01

    The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments. PMID:27485326

  9. Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

    PubMed Central

    2015-01-01

    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins. PMID:24937763

  10. Synthetic biology outside the cell: linking computational tools to cell-free systems.

    PubMed

    Lewis, Daniel D; Villarreal, Fernando D; Wu, Fan; Tan, Cheemeng

    2014-01-01

    As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

  11. Cytoprotective effect of preparations from various parts of Punica granatum L. fruits in oxidatively injured mammalian cells in comparison with their antioxidant capacity in cell free systems.

    PubMed

    Sestili, Piero; Martinelli, Chiara; Ricci, Donata; Fraternale, Daniele; Bucchini, Anahi; Giamperi, Laura; Curcio, Rosanna; Piccoli, Giovanni; Stocchi, Vilberto

    2007-07-01

    Pomegranate (Punica granatum L.) juice (PJ) is being increasingly proposed as a nutritional supplement to prevent atherosclerosis in humans. This therapeutically valuable potential has been attributed to PJ antioxidant capacity which has been mostly tested by means of cell-free assays: indeed, to the best of our knowledge, no study has focused on the direct antioxidant capacity of PJ in cultured cells. Here, the antioxidant capacity in cell free-systems of preparations from various parts of pomegranate has been compared with their cytoprotective -bona fide antioxidant--activity in cultured human cells (U937 promonocytes and HUVEC endothelial cells) exposed to an array of oxidizing agents. Pomegranate derivatives were PJ, arils only juice (AJ) and aqueous rinds extract (RE). In cell-free assays--1,1-diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence luminol/xanthine/xanthine oxidase and lipoxygenase assays--all the preparations displayed good antioxidant capacity, the relative potency order being RE>PJ>AJ. On the contrary, only RE was capable of preventing the deleterious effects--cytotoxicity, DNA damage and depletion of non-protein sulphydrils (NPSH) pool--caused by treatment of cells with H(2)O(2), tert-butylhydroperoxide (tB-OOH) or oxidized lipoproteins (Ox-LDL) via a mechanism which is likely to involve both direct scavenging of radical species and iron chelation. Surprisingly, AJ and PJ slightly sensitized cells to the cytotoxic effects of the three agents. Then it would appear that AJ, the major and tasty part of PJ, does not contain ellagic acid and punicalagin (i.e. the polyphenols highly represented in RE which are reputed to be responsible for the antioxidant capacity) in amounts sufficient to exert cytoprotection in oxidatively injured, living cells. Based on these results, the development and evaluation of rinds-only based derivatives for antiatherogenic preventive purposes in humans should be encouraged. PMID:17376699

  12. Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

    PubMed Central

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-01-01

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a α-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins. PMID:18840687

  13. Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

    PubMed

    Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Latif Ibrahim, Abdul; Cass, Anthony E G

    2015-01-01

    The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant. PMID:25723061

  14. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

    PubMed

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-10-14

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

  15. Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

    PubMed

    Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Latif Ibrahim, Abdul; Cass, Anthony E G

    2015-01-01

    The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

  16. Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

    PubMed Central

    Reymond, P; Kunz, B; Paul-Pletzer, K; Grimm, R; Eckerskorn, C; Farmer, E E

    1996-01-01

    Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication. PMID:8989883

  17. Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates.

    PubMed

    Shrestha, Prashanta; Smith, Mark Thomas; Bundy, Bradley Charles

    2014-01-25

    Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation.

  18. Cell-free expression of a functional pore-only sodium channel

    PubMed Central

    Kovácsová, Gabriela; Gustavsson, Emil; Wang, Jiajun; Kreir, Mohamed; Peuker, Sebastian; Westenhoff, Sebastian

    2015-01-01

    Voltage-gated sodium channels participate in the propagation of action potentials in excitable cells. Eukaryotic Navs are pseudo homotetrameric polypeptides, comprising four repeats of six transmembrane segments (S1–S6). The first four segments form the voltage-sensing domain and S5 and S6 create the pore domain with the selectivity filter. Prokaryotic Navs resemble these characteristics, but are truly tetrameric. They can typically be efficiently synthesized in bacteria, but production in vitro with cell-free synthesis has not been demonstrated. Here we report the cell-free expression and purification of a prokaryotic tetrameric pore-only sodium channel. We produced milligram quantities of the functional channel protein as characterized by size-exclusion chromatography, infrared spectroscopy and electrophysiological recordings. Cell-free expression enables advanced site-directed labelling, post-translational modifications, and special solubilization schemes. This enables next-generation biophysical experiments to study the principle of sodium ion selectivity and transport in sodium channels. PMID:25770647

  19. Cell-free methods to produce structurally intact mammalian membrane proteins

    PubMed Central

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1–1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  20. In vitro Fab display: a cell-free system for IgG discovery.

    PubMed

    Stafford, Ryan L; Matsumoto, Marissa L; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R; Baliga, Ramesh; Murray, Christopher J; Thanos, Christopher D; Hallam, Trevor J; Sato, Aaron K

    2014-04-01

    Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF.

  1. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening. PMID:24395411

  2. Cloning-independent expression and screening of enzymes using cell-free protein synthesis systems.

    PubMed

    Kwon, Yong-Chan; Song, Jae-Kwang; Kim, Dong-Myung

    2014-01-01

    We present a strategy for expression and screening of microbial enzymes without involving cloning procedures. Libraries of putative ω-transaminases (ω-TA) and mutated Candida antarctica lipase B (CalB) are PCR-amplified from bacterial colonies and directly expressed in an Escherichia coli-based cell-free protein synthesis system. The open nature of cell-free protein synthesis system also allows streamlined analysis of the enzymatic activity of the expressed enzymes, which greatly shortens the time required for enzyme screening. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function, in order to accelerate the discovery of novel enzymes. The proposed strategy can also serve as a viable option for the rapid and precise tuning of enzyme molecules, not only for analytical purposes, but also for industrial applications. This is accomplished via large-scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.

  3. Cell-free methods to produce structurally intact mammalian membrane proteins.

    PubMed

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  4. A sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells.

    PubMed

    Shao, Lijian; Feng, Wei; Lee, Kyung-Jong; Chen, Benjamin P C; Zhou, Daohong

    2012-01-01

    Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 µg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34(+) hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34(+)CD38(-) cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34(+)CD38(+) cells). In contrast, mouse quiescent HSCs (Pyronin-Y(low) LKS(+) cells) and cycling HSCs (Pyronin-Y(hi) LKS(+) cells) repaired the damage more efficiently than HPCs (LKS(-) cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs.

  5. Long-Term Prognostic Effects of Plasma Epstein-Barr Virus DNA by Minor Groove Binder-Probe Real-Time Quantitative PCR on Nasopharyngeal Carcinoma Patients Receiving Concurrent Chemoradiotherapy

    SciTech Connect

    Lin, J.-C. . E-mail: jclin@vghtc.gov.tw; Wang, W.-Y.; Liang, W.-M.; Chou, H.-Y.; Jan, J.-S.; Jiang, R.-S.; Wang, J.-Y.; Twu, C.-W.; Liang, K.-L.; Chao, Jeffrey; Shen, W.-C.

    2007-08-01

    Purpose: To evaluate the long-term prognostic impact of plasma Epstein-Barr virus (EBV) DNA concentration measured by real-time quantitative polymerase chain reaction (RTQ-PCR) in nasopharyngeal carcinoma (NPC) patients receiving concurrent chemoradiotherapy (CCRT). Methods and Materials: Epstein-Barr virus DNA was retrospectively measured from stock plasma of 152 biopsy-proven NPC patients with Stage II-IV (M0) disease with a RTQ-PCR using the minor groove binder-probe. All patients received CCRT with a median follow-up of 78 months. We divided patients into three subgroups: (1) low pretreatment EBV DNA (<1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-L/post-U) (2) high pretreatment EBV DNA ({>=}1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-H/post-U), and (3) low or high pretreatment EBV DNA and detectable posttreatment EBV DNA (pre-L or H/post-D) for prognostic analyses. Results: Epstein-Barr virus DNA (median concentration, 573 copies/mL; interquartile range, 197-3,074) was detected in the pretreatment plasma of 94.1% (143/152) of patients. After treatment, plasma EBV DNA decreased or remained 0 for all patients and was detectable in 31 patients (20.4%) with a median concentration 0 copy/mL (interquartile range, 0-0). The 5-year overall survival rates of the pre-L/post-U, pre-H/post-U, and pre-L or H/post-D subgroups were 87.2%, 71.0%, and 38.7%, respectively (p < 0.0001). The relapse-free survival showed similar results with corresponding rates of 85.6%, 75.9%, and 26.9%, respectively (p < 0.0001). Multivariate Cox analysis confirmed the superior effects of plasma EBV DNA compared to other clinical parameters in prognosis prediction. Conclusion: Plasma EBV DNA is the most valuable prognostic factor for NPC. More chemotherapy should be considered for patients with persistently detectable EBV DNA after CCRT.

  6. Lyophilized Escherichia coli-based cell-free systems for robust, high-density, long-term storage.

    PubMed

    Smith, Mark Thomas; Berkheimer, Scott D; Werner, Christopher J; Bundy, Bradley C

    2014-01-01

    Cell-free protein synthesis (CFPS) is a versatile tool for rapid recombinant protein production and engineering. One drawback of cell-free technology is the necessity to store the major components-cell extracts and energy systems-below freezing in bulky aqueous solutions. Here we describe simple methods for lyophilizing extracts and preparing powdered energy systems for CFPS. These techniques allow for high-density storage of cell-free systems that are more robust against temperature and bacterial degradation. Our methods have the potential to decrease storage expenses, allow for longer shelf-life of cell extracts at room temperature, and enable durable portable protein production technologies.

  7. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    PubMed

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  8. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    PubMed

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  9. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA

    PubMed Central

    Lin, Linhua; Yin, Xuyang; Wang, Jun; Chen, Dayang; Chen, Fang; Jiang, Hui; Ren, Jinghui; Wang, Wei

    2016-01-01

    Objectives The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT) for fetal copy number variants (CNVs) in clinical samples, using a whole-genome sequencing method. Method A total of 919 archived maternal plasma samples with karyotyping/microarray results, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads) whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS) to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated. Results 33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results in the study. Ten false positive results and two false negative results were obtained. The sensitivity and specificity of detection deletions/duplications were 84.21% and 98.42%, respectively. Conclusion Whole-genome sequencing-based NIPT has high performance in detecting genome-wide CNVs, in particular >10Mb CNVs using the current FCAPS algorithm. It is possible to implement the current method in NIPT to prenatally screening for fetal CNVs. PMID:27415003

  10. Detection and quantification of male-specific fetal DNA in the serum of pregnant cynomolgus monkeys (Macaca fascicularis).

    PubMed

    Yasmin, Lubna; Takano, Jun-Ichiro; Nagai, Yasushi; Otsuki, Junko; Sankai, Tadashi

    2015-02-01

    Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175-131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys. PMID:25730760

  11. Haplotype-assisted accurate non-invasive fetal whole genome recovery through maternal plasma sequencing

    PubMed Central

    2013-01-01

    Background The applications of massively parallel sequencing technology to fetal cell-free DNA (cff-DNA) have brought new insight to non-invasive prenatal diagnosis. However, most previous research based on maternal plasma sequencing has been restricted to fetal aneuploidies. To detect specific parentally inherited mutations, invasive approaches to obtain fetal DNA are the current standard in the clinic because of the experimental complexity and resource consumption of previously reported non-invasive approaches. Methods Here, we present a simple and effective non-invasive method for accurate fetal genome recovery-assisted with parental haplotypes. The parental haplotype were firstly inferred using a combination strategy of trio and unrelated individuals. Assisted with the parental haplotype, we then employed a hidden Markov model to non-invasively recover the fetal genome through maternal plasma sequencing. Results Using a sequence depth of approximately 44X against a an approximate 5.69% cff-DNA concentration, we non-invasively inferred fetal genotype and haplotype under different situations of parental heterozygosity. Our data show that 98.57%, 95.37%, and 98.45% of paternal autosome alleles, maternal autosome alleles, and maternal chromosome X in the fetal haplotypes, respectively, were recovered accurately. Additionally, we obtained efficient coverage or strong linkage of 96.65% of reported Mendelian-disorder genes and 98.90% of complex disease-associated markers. Conclusions Our method provides a useful strategy for non-invasive whole fetal genome recovery. PMID:23445748

  12. Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

    PubMed Central

    1993-01-01

    We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis. PMID:8408207

  13. Cell-free protein synthesis in a microchamber revealed the presence of an optimum compartment volume for high-order reactions.

    PubMed

    Okano, Taiji; Matsuura, Tomoaki; Suzuki, Hiroaki; Yomo, Tetsuya

    2014-06-20

    The application of microelectromechanical systems (MEMS) to chemistry and biochemistry allows various reactions to be performed in microscale compartments. Here, we aimed to use the glass microchamber to study the compartment size dependency of the protein synthesis, one of the most important reactions in the cell. By encapsulating the cell-free protein synthesis system with different reaction orders in femtoliter microchambers, chamber size dependency of the reaction initiated with a constant copy number of DNA was investigated. We were able to observe the properties specific to the high order reactions in microcompartments with high precision and found the presence of an optimum compartment volume for a high-order reaction using real biological molecules.

  14. In vitro nonsense suppression in [psi+] and [psi-] cell-free lysates of Saccharomyces cerevisiae.

    PubMed Central

    Tuite, M F; Cox, B S; McLaughlin, C S

    1983-01-01

    An homologous in vitro assay for yeast nonsense suppressors was used to examine the effect of the cytoplasmically inherited genetic determinant [psi] on the efficiency of in vitro nonsense suppression. The efficiency of all three types of yeast tRNA-mediated nonsense suppressor (ochre, amber, and UGA) is much greater in cell-free lysates prepared from a sup+ [psi+] strain than in lysates prepared from an isogeneic sup+ [psi-] strain. Lysates prepared from a [psi-] strain, into which the [psi+] determinant was reintroduced by kar1-mediated cytoduction, support efficient suppression. Evidence is also presented that [psi-] lysates contain an inhibitor of in vitro nonsense suppression. Images PMID:6344070

  15. Continuous cell-free protein synthesis using glycolytic intermediates as energy sources.

    PubMed

    Kim, Ho-Cheol; Kim, Tae-Wan; Park, Chang-Gil; Oh, In-Seok; Park, Kyungmoon; Kim, Dong-Myung

    2008-05-01

    In this work, we demonstrate that glycolytic intermediates can serve as efficient energy sources to regenerate ATP during continuous-exchange cell-free (CECF) protein synthesis reactions. Through the use of an optimal energy source, approximately 10 mg/ml of protein was generated from CECF protein synthesis reaction at greatly reduced reagent costs. Compared with the conventional reactions utilizing phosphoenol pyruvate as an energy source, the described method yields 10-fold higher productivity per unit reagent cost, making the techniques of CECF protein synthesis more realistic alternative for rapid protein production.

  16. Conformational Antibody Binding to a Native, Cell-Free Expressed GPCR in Block Copolymer Membranes

    PubMed Central

    de Hoog, Hans-Peter M.; Lin JieRong, Esther M.; Banerjee, Sourabh; Décaillot, Fabien M.; Nallani, Madhavan

    2014-01-01

    G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4. PMID:25329156

  17. Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes.

    PubMed

    Kim, Ho-Cheol; Kim, Kwang-Soo; Kang, Taek-Jin; Choi, Jong Hyun; Song, Jae Jun; Choi, Yun Hee; Kim, Byung-Gee; Kim, Dong-Myung

    2015-12-01

    Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into γ-aminobutyric acid (GABA), an engineered glutamate decarboxylase (GADβ) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity.

  18. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    PubMed Central

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  19. A new cell-free bandage-type artificial skin for cutaneous wounds.

    PubMed

    Aoki, Shigehisa; Takezawa, Toshiaki; Ikeda, Satoshi; Narisawa, Yutaka; Oshikata-Miyazaki, Ayumi; Miyauchi, Syohei; Hirayama, Hiroshi; Sawaguchi, Tomoya; Chimuro, Tomoyuki; Toda, Shuji

    2015-01-01

    Engineered skin substitutes are widely used in skin wound management. However, no currently available products satisfy all the criteria of usability in emergency situations, easy handling, and minimal scar formation. To overcome these shortcomings, we designed a cell-free bandage-type artificial skin, named "VitriBand" (VB), using adhesive film dressing, silicone-coated polyethylene terephthalate film, and collagen xerogel membrane defined as a dried collagen vitrigel membrane without free water. We analyzed its advantages over in-line products by comparing VB with hydrocolloid dressing and collagen sponge. For evaluation, mice inflicted with full-thickness skin defects were treated with VB, hydrocolloid dressing, and collagen sponge. A plastic film group treated only with adhesive film dressing and silicone-coated polyethylene terephthalate film, and a no treatment group were also compared. VB promoted epithelization while inhibiting the emergence of myofibroblasts and inflammation in the regenerating tissue more effectively than the plastic film, hydrocolloid dressing, and collagen sponge products. We have succeeded in establishing a cell-free bandage-type artificial skin that could serve as a promising first-line medical biomaterial for emergency treatment of skin injuries in various medical situations.

  20. Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis.

    PubMed

    Hodgman, C Eric; Jewett, Michael C

    2013-10-01

    Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL(-1) with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10(-4) to ∼4 × 10(-1)  µg $(-1) , which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries.

  1. Formation of Lignans(-)-Secoisolariciresinol and (-)-Matairesinol with Forsythia intermedia Cell-Free Extracts

    NASA Technical Reports Server (NTRS)

    Umezawa, Toshiaki; Davin, Laurence B.; Lewis, Norman G.

    1991-01-01

    In vivo labeling experiments of Forsythia intermedia plant tissue with [8-(C-14)]- and [9,9-(2)H2,OC(2)H3]coniferyl alcohols revealed that the lignans, (-)-secoisolariciresinol and (-)-matairesinol, were derived from two coniferyl alcohol molecules; no evidence for the formation of the corresponding (+)-enantiomers was found. Administration of (+/-)-[Ar-(H-3)] secoisolariciresinols to excised shoots of F.intermedia resulted in a significant conversion into (-)-matairesinol; again, the (+)-antipode was not detected. Experiments using cell-free extracts of F.intermedia confirmed and extended these findings. In the presence of NAD(P)H and H2O2, the cell-free extracts catalyzed the formation of (-)- secoisolariciresinol, with either [8-(C-14)]- or [9,9-(2)H2,OC(2)H3]coniferyl alcohols as substrates. The (+)- enantiomer was not formed. Finally, when either (-)-[Ar-(H-3)] or (+/-)-[Ar-(H-2)]secoisolariciresinols were used as substrates, in the presence of NAD(P), only (-)- and not (+)-matairesinol formation occurred. The other antipode, (+)-secoisolariciresinol, did not serve as a substrate for the formation of either (+)- or (-)-matairesinol. Thus, in F.intermedia, the formation of the lignan, (-)-secoisolariciresinol, occurs under strict stereochemical control, in a reaction or reactions requiring NAD(P)H and H2O2 as cofactors. This stereoselectivity is retained in the subsequent conversion into (-)-matairesinol, since (+)-secoisolariciresinol is not a substrate. These are the first two enzymes to be discovered in lignan formation.

  2. Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

    PubMed

    Li, Bo; Makino, Shin-Ichi; Beebe, Emily T; Urano, Daisuke; Aceti, David J; Misenheimer, Tina M; Peters, Jonathan; Fox, Brian G; Jones, Alan M

    2016-10-01

    Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

  3. A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis.

    PubMed

    Schellhaus, Anna K; Magalska, Adriana; Schooley, Allana; Antonin, Wolfram

    2015-12-19

    During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

  4. Neutrophilic Cell-Free Exudate Induces Antinociception Mediate by the Protein S100A9

    PubMed Central

    Pagano, Rosana L.; Mariano, Mario; Giorgi, Renata

    2006-01-01

    Calcium-binding protein S100A9 (MRP-14) induces antinociceptive effect in an experimental model of painful sensibility and participates of antinociception observed during neutrophilic peritonitis induced by glycogen or carrageenan in mice. In this study, the direct antinociceptive role of the protein S100A9 in neutrophilic cell-free exudates obtained of mice injected with glycogen was investigated. Mice were intraperitoneally injected with a glycogen solution, and after 4, 8, 24, and 48 hours, either the pattern of cell migration of the peritoneal exudate or the nociceptive response of animals was evaluated. The glycogen-induced neutrophilic peritonitis evoked antinociception 4 and 8 hours after inoculation of the irritant. Peritoneal cell-free exudates, collected in different times after the irritant injection, were transferred to naive animals which were submitted to the nociceptive test. The transference of exudates also induced antinociceptive effect, and neutralization of S100A9 activity by anti-S100A9 monoclonal antibody totally reverted this response. This effect was not observed when experiments were made 24 or 48 hours after glycogen injection. These results clearly indicate that S100A9 is secreted during glycogen-induced neutrophilic peritonitis, and that this protein is responsible by antinociception observed in the initial phase of inflammatory reaction. Thus, these data reinforce the hypothesis that the calcium-binding protein S100A9 participates of the endogenous control of inflammatory pain. PMID:17047291

  5. Effects of a novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional stabilizing reagents.

    PubMed

    Das, Kausik; Fernando, M Rohan; Basiaga, Sara; Wigginton, Stephanie M; Williams, Tom

    2014-01-01

    Stabilization of nucleated blood cells by cell stabilizing reagent (BCT reagent) present in the Cell-Free DNA BCT blood collection device and consequent prevention of cell-free DNA contamination by cellular DNA during sample storage and shipping have previously been reported. This study was conducted to investigate the effect of this novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional cell stabilizing reagents, formaldehyde and glutaraldehyde. A 787 bp long DNA fragment from human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified by PCR and used as model system. DNA samples and blood samples were treated with BCT reagent, 0.1% formaldehyde or 0.1% glutaraldehyde at room temperature. DNA amplification was studied using conventional and real-time quantitative PCR. Results indicate that exposure of DNA to the BCT reagent for up to 14 days had no effect on DNA amplification by PCR as compared to the untreated control DNA. However, there was statistically significant decrease in DNA amplification in the DNA samples treated with formaldehyde and glutaraldehyde. We conclude that the BCT reagent used in Cell-Free DNA BCT blood collection device to prevent cell-free DNA contamination by cellular DNA had no effect on DNA amplification by PCR.

  6. DNA methylation analysis determines the high frequency of genic hypomethylation and low frequency of hypermethylation events in plasma cell tumors.

    PubMed

    Salhia, Bodour; Baker, Angela; Ahmann, Gregory; Auclair, Daniel; Fonseca, Rafael; Carpten, John

    2010-09-01

    Multiple myeloma (MM) is a plasma cell malignancy of the bone marrow, which evolves from a premalignant stage called monoclonal gammopathy of undetermined significance (MGUS). In some patients, an intermediate stage referred to as smoldering multiple myeloma (SMM) is clinically recognized, with the full-bore malignancy termed MM. We conducted a study to assess differential CpG methylation at 1,500 genic loci during MM progression and profiled CD138(+) plasma cells from MGUS, SMM, and MM specimens; human myeloma cell lines; and normal plasma cell (NPC) samples. We showed that the number of differentially methylated loci (DML) increased with tumor grade, and the vast majority were due to hypomethylation. Hierarchical clustering analysis revealed samples that coclustered tightly with NPC. These cases, referred to as "normal-like," contained significantly fewer DML when compared with their non-normal-like counterparts and displayed overall methylation levels resembling NPC. This study represents one of the first methylome interrogation studies in MM and points toward global hypomethylation at genic CpG loci as an important and early mechanism driving myelomagenesis. Determining the set of critical genes and pathways based on the myeloma methylome is expected to lead to an improved understanding of biological mechanisms involved in myelomagenesis. PMID:20736376

  7. Reduction in plasma human immunodeficiency virus ribonucleic acid after dideoxynucleoside therapy as determined by the polymerase chain reaction.

    PubMed

    Holodniy, M; Katzenstein, D A; Israelski, D M; Merigan, T C

    1991-11-01

    Cell-free HIV RNA in plasma was detected and quantitated after antiviral therapy by the polymerase chain reaction. RNA was extracted from plasma, reverse transcribed to cDNA, amplified by polymerase chain reaction, and quantitated by absorbance based on an enzyme-linked affinity assay. 72 HIV antibody-positive subjects had one plasma sample taken. 39 who were not receiving antiretroviral therapy at the time had a mean plasma HIV RNA copy number of 690 +/- 360 (mean +/- SEM) per 200 microliters of plasma, while 33 subjects who had been receiving zidovudine therapy for a minimum of 3 mo had a mean copy number of 134 +/- 219 (P less than 0.05). 27 additional HIV antibody-positive patients had two plasma samples taken before and 1 mo after initiating dideoxynucleoside therapy. Plasma HIV RNA copy number fell from 540 +/- 175 to 77 +/- 35 (P less than 0.05). Finally, nine of these subjects had two baseline samples obtained before initiating therapy and two posttreatment samples 1 and 2 mo after therapy was begun. Mean plasma RNA copy number declined from 794 +/- 274 to less than 40 (below the lower limit of sensitivity) after 1 mo of therapy, with suppression maintained after 2 mo of therapy. These results suggest that gene amplification can be used to detect and quantitate changes in plasma HIV RNA after dideoxynucleoside therapy. Plasma HIV polymerase chain reaction may be a more sensitive marker to monitor antiviral therapy, particularly in asymptomatic patients where measurement of p24 antigen or quantitative plasma cultures are negative.

  8. Genetic variants and cell-free hemoglobin processing in sickle cell nephropathy

    PubMed Central

    Saraf, Santosh L.; Zhang, Xu; Shah, Binal; Kanias, Tamir; Gudehithlu, Krishnamurthy P.; Kittles, Rick; Machado, Roberto F.; Arruda, Jose A.L.; Gladwin, Mark T.; Singh, Ashok K.; Gordeuk, Victor R.

    2015-01-01

    Intravascular hemolysis and hemoglobinuria are associated with sickle cell nephropathy. ApoL1 is involved in cell-free hemoglobin scavenging through association with haptoglobin-related protein. APOL1 G1/G2 variants are the strongest genetic predictors of kidney disease in the general African-American population. A single report associated APOL1 G1/G2 with sickle cell nephropathy. In 221 patients with sickle cell disease at the University of Illinois at Chicago, we replicated the finding of an association of APOL1 G1/G2 with proteinuria, specifically with urine albumin concentration (β=1.1, P=0.003), observed an even stronger association with hemoglobinuria (OR=2.5, P=4.3×10−6), and also replicated the finding of an association with hemoglobinuria in 487 patients from the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy study (OR=2.6, P=0.003). In 25 University of Illinois sickle cell disease patients, concentrations of urine kidney injury molecule-1 correlated with urine cell-free hemoglobin concentrations (r=0.59, P=0.002). Exposing human proximal tubular cells to increasing cell-free hemoglobin led to increasing concentrations of supernatant kidney injury molecule-1 (P=0.01), reduced viability (P=0.01) and induction of HMOX1 and SOD2. HMOX1 rs743811 associated with chronic kidney disease stage (OR=3.0, P=0.0001) in the University of Illinois cohort and end-stage renal disease (OR=10.0, P=0.0003) in the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy cohort. Longer HMOX1 GT-tandem repeats (>25) were associated with lower estimated glomerular filtration rate in the University of Illinois cohort (P=0.01). Our findings point to an association of APOL1 G1/G2 with kidney disease in sickle cell disease, possibly through increased risk of hemoglobinuria, and associations of HMOX1 variants with kidney disease, possibly through reduced protection of the kidney from hemoglobin

  9. Genetic variants and cell-free hemoglobin processing in sickle cell nephropathy.

    PubMed

    Saraf, Santosh L; Zhang, Xu; Shah, Binal; Kanias, Tamir; Gudehithlu, Krishnamurthy P; Kittles, Rick; Machado, Roberto F; Arruda, Jose A L; Gladwin, Mark T; Singh, Ashok K; Gordeuk, Victor R

    2015-10-01

    Intravascular hemolysis and hemoglobinuria are associated with sickle cell nephropathy. ApoL1 is involved in cell-free hemoglobin scavenging through association with haptoglobin-related protein. APOL1 G1/G2 variants are the strongest genetic predictors of kidney disease in the general African-American population. A single report associated APOL1 G1/G2 with sickle cell nephropathy. In 221 patients with sickle cell disease at the University of Illinois at Chicago, we replicated the finding of an association of APOL1 G1/G2 with proteinuria, specifically with urine albumin concentration (β=1.1, P=0.003), observed an even stronger association with hemoglobinuria (OR=2.5, P=4.3×10(-6)), and also replicated the finding of an association with hemoglobinuria in 487 patients from the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy study (OR=2.6, P=0.003). In 25 University of Illinois sickle cell disease patients, concentrations of urine kidney injury molecule-1 correlated with urine cell-free hemoglobin concentrations (r=0.59, P=0.002). Exposing human proximal tubular cells to increasing cell-free hemoglobin led to increasing concentrations of supernatant kidney injury molecule-1 (P=0.01), reduced viability (P=0.01) and induction of HMOX1 and SOD2. HMOX1 rs743811 associated with chronic kidney disease stage (OR=3.0, P=0.0001) in the University of Illinois cohort and end-stage renal disease (OR=10.0, P=0.0003) in the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy cohort. Longer HMOX1 GT-tandem repeats (>25) were associated with lower estimated glomerular filtration rate in the University of Illinois cohort (P=0.01). Our findings point to an association of APOL1 G1/G2 with kidney disease in sickle cell disease, possibly through increased risk of hemoglobinuria, and associations of HMOX1 variants with kidney disease, possibly through reduced protection of the kidney from hemoglobin

  10. DNA Polymer Brush Patterning through Photocontrollable Surface-Initiated DNA Hybridization Chain Reaction.

    PubMed

    Huang, Fujian; Zhou, Xiang; Yao, Dongbao; Xiao, Shiyan; Liang, Haojun

    2015-11-18

    The fabrication of DNA polymer brushes with spatial resolution onto a solid surface is a crucial step for biochip research and related applications, cell-free gene expression study, and even artificial cell fabrication. Here, for the first time, a DNA polymer brush patterning method is reported based on the photoactivation of an ortho-nitrobenzyl linker-embedded DNA hairpin structure and a subsequent surface-initiated DNA hybridization chain reaction (HCR). Inert DNA hairpins are exposed to ultraviolet light irradiation to generate DNA duplexes with two active sticky ends (toeholds) in a programmable manner. These activated DNA duplexes can initiate DNA HCR to generate multifunctional patterned DNA polymer brushes with complex geometrical shapes. Different multifunctional DNA polymer brush patterns can be fabricated on certain areas of the same solid surface using this method. Moreover, the patterned DNA brush surface can be used to capture target molecules in a desired manner.

  11. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    PubMed

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms.

  12. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

    PubMed</