Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “Polymerase Chain Reaction”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Studying the effect of graphene-ZnO nanocomposites on polymerase chain reaction

An emerging area of research is improving the efficiency of the polymerase chain reaction (PCR) by using nanoparticles. With graphene nano-flakes showing promising results, in this paper we report the effect of Graphene-ZnO nanocomposites on Polymerase Chain reaction (PCR) efficiency. G-ZnO nanocomposites were efficiently synthesized via in situ chemical method. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) image confirms the formation of nanocomposites. ZnO nanoparticles of size range ~20-30 nm are uniformly attached on the graphene sheets. No amplification during PCR indicates inhibitory activity of G-ZnO nanocomposites which points the fingers at ZnO moiety of the G-ZnO composite for no amplification during our PCR reaction. Further work should concentrate on finding out the main inhibitory mechanism involved in inhibition of PCR using G-ZnO composites.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2018

Introducing nano-scale quantitative polymerase chain reaction

Highlights: • qPCR remain the gold-standard-method for measuring and validating gene expression levels and alterations. • qPCR reaction volume can be significantly reduced. • 1 μL versus 10 μL total reaction volumes gave comparable results. • The reduction in reaction volume can greatly reduce sample use and reagent cost. We show that the quantitative PCR reaction volume can be reduced significantly compared to the standard procedures, without compromising data quality. By analyzing dilution series (10{sup 0}–10{sup −7}) we found that measurement in 1 μl reaction volume indeed gave valid results comparable to 10 μL, when using a routine pipetting robot. This may enable a significant cost reduction through cutback of both biological material as well as chemical reagents.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required for PCR.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid

Embodiments of the present invention are directed toward devices, systems, and methods for purifying nucleic acids to conduct polymerase chain reaction (PCR) assays. In one example, a method includes generating complexes of silica beads and nucleic acids in a lysis buffer, transporting the complexes through an immiscible fluid to remove interfering compounds from the complexes, further transporting the complexes into a density medium containing components required for PCR where the nucleic acids disassociate from the silica beads, and thermocycling the contents of the density medium to achieve PCR. Signal may be detected from labeling agents in the components required for PCR.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Polymerase chain reaction for the in vitro detection of the pESI plasmid associated with the globally circulating Salmonella Infantis outbreak strain

Abstract A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.

McMillan, Elizabeth A. (ORCID:000000027503514X)↗
NASA NTRS1993

Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

Wu, Liu-Lai↗
DOE OSTI2019

Rapid viability polymerase chain reaction method for detection of Francisella tularensis

Francisella tularensis, which causes potentially fatal tularemia, has been considered an attractive agent of bioterrorism and biological warfare due to its low infectious dose, reported environmental persistence, and ability to be transmitted to humans via multiple exposure routes. Additionally, due to slow growth on even selective culture media, detection of viable F. tularensis from environmental and drinking water samples, usually takes >3 days. Therefore, a rapid viability polymerase chain reaction (RV-PCR) method was developed to detect and identify viable F. tularensis cells in environmental samples. The method uses a change in PCR response during high throughput (48-well) sample incubation in Brain Heart Infusion/Vitox/Fildes/Histidine growth medium to detect viable F. tularensis presence, which is at least two times faster than the current plate culture-based method. Using the method, 101 to 102 live F. tularensis cells were detected in simulated complex sample matrices containing chemical and biological interferences.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

Development of a rapid viability polymerase chain reaction method for detection of Yersinia pestis

Due to the occurrence of natural plague outbreaks and its historical usage as a biological weapon, Yersinia pestis is considered one of the high-priority biological threat agents. It can remain viable in certain environments including water for >100 days. Because of its slow-growth characteristic, it usually takes three or more days to detect and confirm the identity of viable Y. pestis cells by PCR, serological, or biochemical assays when using the traditional microbiological plate-culture-based analysis, and that too, assuming faster growing microbes present in a water sample do not mask the Y. pestis colonies and interfere with analysis. Therefore, a rapid-viability Polymerase Chain Reaction (RV-PCR) method was developed for detection of Y. pestis . The RV-PCR method combines 24 h-incubation broth culture in a 48-well plate, and pre- and post-incubation differential PCR analyses, thereby allowing for rapid and high-throughput sample analysis compared with the current plate culture method. One chromosomal and two plasmid gene target-based real-time PCR assays were down-selected, showing ca. 10 genome equivalent detection; the chromosomal assay was then used for RV-PCR method development. A 10 1 -cell level (10–99 cells) sensitivity of detection was demonstrated even with complex sample backgrounds including known PCR inhibitors (ferrous sulfate and humic acid), as well as metal oxides and microbes present in Arizona Test Dust (ATD). The method sensitivity was maintained in the presence of dead Y. pestis cells up to 10 4 cells per sample. While affording high-throughput and rapid sample analysis, the 48-well plate format used in this method for sample enrichment significantly reduced labor requirements and generation of BioSafety Level-3 (BSL-3) laboratory waste as compared to the usual microbiological plate-culture-based methods. In conclusion, this method may serve as a model for other vegetative bacterial pathogens.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2019

A Universally Primed-Polymerase Chain Reaction (UP-PCR) Marker to Discriminate Clonostachys rosea ACM941 from Related Strains

Clonostachys rosea strain ACM941 is an effective biocontrol agent against several crop diseases including Fusarium head blight. In anticipation of its increased relevance going forward, the development of a reliable DNA-based molecular marker to track it is essential. Universally primed-PCR (UP-PCR) has been used successfully to differentiate other C. rosea strains. Herein, the development of a UP-PCR marker for ACM941 is described. A combination of two primers (AS15 and L45) produced a ~450 bp fragment that was unique to ACM941 compared to other commercial biocontrol agents. Primers subsequently designed based on the obtained fragment also produced a similarly unique band from ACM941 alone. BLAST analysis of the amplified sequence did not yield any homologous sequence in available online databases or within the closely related C. rosea IK726 and CBS125111 strains’ genomes. The specificity of this marker for ACM941 was validated against ten additional C. rosea strains isolated from Canada, with ACM941 producing the brightest band. Taken together, these results imply that the UP-PCR primers AS15 and L45 and the amplified fragment can be used to detect and monitor the ACM941 strain after its release into the environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Gene-matched enrichment and polymerase chain reaction for rapid detection of microorganisms

A method for amplifying and detecting microorganisms, such as species of Listeria, is described. The method utilizes gene-matched enrichment media and PCR-based detection. The enrichment media is spent media produced using a modified microorganism containing a plurality of mutations in a selected gene such that the modified microorganism does not contain the PCR signature. Thus, PCR detects only the amplified microorganism of interest, not the modified microorganism. Exemplary methods and kits for amplification and detection of Listeria species are described.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Extracellular IL-37 promotes osteogenic and odontogenic differentiation of human dental pulp stem cells via autophagy

The osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) contribute to restoration and regeneration of dental tissue. Previous study indicated that interleukin-37 (IL-37) was an anti-inflammatory factor that affected other pro-inflammatory signals. The aim of this study was to explore the effects of IL-37 on the differentiation of DPSCs. DPSCs were cultured in growth medium with different concentrations of IL-37. We selected the optimal concentration for the following experiments by alkaline phosphatase (ALP) activity analysis, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot. Cell counting kit assay (CCK-8) and 5-Ethynyl-2-Deoxyuridine (EdU) assay were conducted to assess the effects of IL-37 on the proliferation of DPSCs. ALP activity assay and staining, alizarin red S (ARS) staining, qRT‐PCR, Western blot as well as immunofluorescence staining were conducted to assess differentiation ability of DPSCs. Western blot, immunofluorescence staining and transmission electron microscopy (TEM) were utilized to examine cell autophagy. Results showed that IL-37 enhanced the osteogenic and odontogenic differentiation ability of DPSCs with no significant influence on the proliferation of DPSCs. Autophagy in DPSCs was activated by IL-37. Activation of autophagy enhanced osteogenesis and odontogenesis of DPSCs, whereas inhibition of autophagy suppressed DPSCs osteogenic and odontogenic differentiation. In conclusion, IL-37 increased osteogenic and odontogenic differentiation via autophagy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Overexpression of DBC1, correlated with poor prognosis, is a potential therapeutic target for hepatocellular carcinoma

Highlights: • DBC1 was upregulated in HCC tissues and cell lines. • Overexpression of DBC1 was correlated with clinicopathological parameters in HCC. • Overexpression of DBC1 predicts poor disease-free survival (DFS) and overall survival (OS) in HCC. • Knockdown of DBC1 inhibits HCC cell lines proliferation. Deleted in Breast Cancer 1 (DBC1) is a regulatory protein involved in cell metabolism and cancer progression. Nevertheless, the expression and prognostic values of DBC1 in hepatocellular carcinoma (HCC) are still not well understood. The following study investigated the clinical significance and biological function of DBC1 in HCC. Briefly, overexpression of DBC1 at transcriptional and translational levels in human HCC tissues compared to adjacent normal tissues was observed using quantitative real-time polymerase chain reaction (qRT-PCR), western blot (WB) and immunohistochemistry (IHC) approach. Furthermore, upregulated DBC1 was significantly correlated with tumor size (p = 0.005), N stage (p = 0.016), M stage (p = 0.011), tumor differentiation (p in vitro. To conclude, these findings demonstrated that DBC1 was essential in tumorigenesis and proliferation. Moreover, it was identified as a potential therapeutic target for HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Intradiencephalon injection of histamine inhibited the recovery of locomotor function of spinal cord injured zebrafish

Human spinal cord injury (SCI) usually causes irreversible disability beneath the injured site due to poor neural regeneration. On the contrary, zebrafish show significant regenerative ability after SCI, thus is usually worked as an animal model for studying neuroregeneration. Most of the previous SCI studies focused on the local site of SCI, the supraspinal-derived signals were rarely mentioned. Here we showed that intradiencephalon injection of histamine (HA) inhibited the locomotor recovery in adult zebrafish post-SCI. Immunofluorescence results showed that intradiencephalon HA administration increased the activated microglia 3 days post injury (dpi), promoted the proliferation of radial glial cells at 7 dpi and affected the morphology of radial glial cells at 11 dpi. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) results showed that intradiencephalon HA administration also reduced the expression of neurotrophic factors including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor1 (IGF-1) at the lesion site, however, had no effect on the expression of pro-inflammatory factors such as TNF-alpha and IL-1 beta. Hence, our data suggested that exogenous intradiencephalon HA retarded locomotor recovery in spinal cord injured zebrafish via modulating the repair microenvironment. - Highlights: • A novel model to study the effect of supraspinal signals on repair of SCI zebrafish. • Exogenous HA administration suppresses the locomotor function recovery after SCI. • Exogenous HA administration modulates the repair microenvironment after SCI.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Systematic Screening of COVID-19 Disease Based on Chest CT and RT-PCR for Cancer Patients Undergoing Radiation Therapy in a Coronavirus French Hotspot

Patients with cancer are presumed to be more vulnerable to COVID-19. We evaluated a screening strategy combining chest computed tomography (CT) and reverse-transcription polymerase chain reaction (RT-PCR) for patients treated with radiation therapy at our cancer center located in a COVID-19 French hotspot during the first wave of the pandemic.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2017

Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma

Irisin is a newly identified myokine that may be cancer-associated, and its impact on liver cancer is unclear. To understand the roles of irisin in liver cancer, we investigated its effect in HepG2 and SMCC7721 hepatocellular carcinoma cells, and the underlying mechanisms. We determined irisin levels in liver tissues and serum samples obtained from patients by using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Irisin levels in cancerous livers were significantly upregulated compared with those in control livers, but serum irisin levels remained unchanged. Additionally, we evaluated the effects of different concentrations of human recombinant modified and active (glycosylated) irisin (IM) or human recombinant nonmodified irisin (INM) on cell migration, proliferation, viability, and invasiveness. CCK8, transwell, and scratching assays demonstrated that irisin significantly increased cell proliferation, invasion, and migration through activation of the PI3K/AKT pathway. Irisin-induced cell proliferation, migration, and invasion were blocked by a PI3K inhibitor (LY294002). Irisin also decreased the cytotoxicity of doxorubicin in HepG2 cells. These data indicate that increased irisin levels may have protective roles in liver cancer cells through partial activation of the PI3K/AKT pathway, which may facilitate liver cancer progression and decrease the sensitivity to chemotherapy. - Highlights: • Irisin levels are upgraded in patients with hepatocellular carcinoma. • Irisin induces cell proliferation, migration, invasiveness, and inhibit appotosis of cell induced by Dox. • Irisin induces the activation of PI3K/AKT pathway. • Irisin has protective roles in liver cancer cells, inducing disease progression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Application of deoxygenation-aeration cycling to control the predatory bacterium Vampirovibrio chlorellavorus in Chlorella sorokiniana cultures

A previously untested approach was evaluated to enable management of the predatory bacterium, Vampirovibrio chlorellavorus, a pathogen of Chlorella sorokiniana, in suspension cultures grown in a laboratory test reactor. Because V. chlorellavorus is an obligate aerobic bacterium, whereas C. sorokiniana grows under aerobic and anaerobic conditions, deoxygenation of the culture was expected to be detrimental to the pathogen, but not to the algal host. The effect of deoxygenation on the uninfected (healthy) C. sorokiniana suspension cells, compared to the C. sorokiniana-V. chlorellavorus co-culture, was studied in relation to biomass, dissolved oxygen, ratio of C. sorokiniana to V. chlorellavorus DNA, and visual and light microscopic observations. Preliminary experiments were conducted to test the effects of different deoxygenation-aeration cycling regimes on performance of V. chlorellavorus-free C. sorokiniana cultures. To an aerobic culture, pure nitrogen gas was introduced to create anoxic conditions, followed by the injection of ambient air to re-establish an aerobic environment. Under this repeated cycling regime, C. sorokiniana was shown to tolerate the anoxic conditions for extended timespans that ranged from 2 to 8 h over a 5-day test period. The analogous aerobic-anoxic cycling with the C. sorokiniana-V. chlorellavorus co-cultures resulted in ‘near-normal’ growth cycle and harvestable biomass, whereas the continuously-aerated (aerobic) co-cultures that were grown without the deoxygenation step in the cycle collapsed in 3 days. Visual and light microscopic observations revealed intact C. sorokiniana cells were present in the deoxygenated cultures, compared to the aerobically-grown, brown-colored algal cultures consisting of collapsed cells. Quantitative polymerase chain reaction analysis showed continuous increases in the ratio of V. chlorellavorus (16S rDNA) to C. sorokiniana (18S rDNA) DNA in the aerated co-cultures, with greater increases during dark periods, while the pathogen-to-host DNA ratio in the deoxygenated co-cultures was relatively low and algal cells did not collapse, as would be expected following pathogen attack.

Algae, Anoxic, Bomass, Infection, Nitrogen, Quanti↗
DOE OSTI2017

MiR-33a suppresses proliferation of NSCLC cells via targeting METTL3 mRNA

Methyltransferase like 3 (METTL3) was incipiently known as a methyltransferase which was responsible for N{sup 6}-methyladenosine (m{sup 6}A) methylation. METTL3 can promote the expression of several crucial oncoproteins and its high expression enhanced proliferation, survival, and invasion of human lung cancer cells. However, how METTL3 was regulated is seldom understood in non-small-cell lung carcinoma (NSCLC). In the present study, miR-33a was suspicious to target to the 3'-untranslated region (3'UTR) of METTL3 mRNA via in silico prediction. Besides, the expressions of METTL3 were higher in NSCLC tissues than those in adjacent tissues, and METTL3 expressions were positively related to the expressions of miR-33a in NSCLC tissues which confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). MiR-33a can directly target to the 3'UTR of METTL3 mRNA which examined by luciferase reporter gene assay. Moreover, we found that miR-33a can reduce the expression of METTL3 at both mRNA and protein levels using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Functionally, miR-33a can reduce the proliferation of A549 and NCI-H460 cells. Conversely, inhibition of miR-33a by anti-miR-33a can rescue that using 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and 5-ethynyl-2-deoxyuridine (EdU) assay. Similarly, miR-33a can reduce cellular anchorage-independent growth of A549 cells. Additionally, the negative influences of miR-33a on the downstream genes of METTL3 were examined by Western blot analysis. Thus, we concluded that miR-33a can attenuate NSCLC cells proliferation via targeting to the 3'UTR of METTL3 mRNA. Our findings provide new insights into the mechanism of METTL3 regulation by micro RNA, and supports METTL3 as a therapeutic target in NSCLC. - Highlights: • METTL3 expression is negatively correlated with miR-33a. • MiR-33a can directly target to the 3'UTR of METTL3 mRNA. • MiR-33a inhibits NSCLC cells proliferation through decreasing METTL3. • MiR-33a induces the down-regulation of EGFR through targeting METTL3.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2000

Growth and differentiation of human lens epithelial cells in vitro on matrix

PURPOSE: To characterize the growth and maturation of nonimmortalized human lens epithelial (HLE) cells grown in vitro. METHODS: HLE cells, established from 18-week prenatal lenses, were maintained on bovine corneal endothelial (BCE) extracellular matrix (ECM) in medium supplemented with basic fibroblast growth factor (FGF-2). The identity, growth, and differentiation of the cultures were characterized by karyotyping, cell morphology, and growth kinetics studies, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence, and Western blot analysis. RESULTS: HLE cells had a male, human diploid (2N = 46) karyotype. The population-doubling time of exponentially growing cells was 24 hours. After 15 days in culture, cell morphology changed, and lentoid formation was evident. Reverse transcription-polymerase chain reaction (RT-PCR) indicated expression of alphaA- and betaB2-crystallin, fibroblast growth factor receptor 1 (FGFR1), and major intrinsic protein (MIP26) in exponential growth. Western analyses of protein extracts show positive expression of three immunologically distinct classes of crystallin proteins (alphaA-, alphaB-, and betaB2-crystallin) with time in culture. By Western blot analysis, expression of p57(KIP2), a known marker of terminally differentiated fiber cells, was detectable in exponential cultures, and levels increased after confluence. MIP26 and gamma-crystallin protein expression was detected in confluent cultures, by using immunofluorescence, but not in exponentially growing cells. CONCLUSIONS: HLE cells can be maintained for up to 4 months on ECM derived from BCE cells in medium containing FGF-2. With time in culture, the cells demonstrate morphologic characteristics of, and express protein markers for, lens fiber cell differentiation. This in vitro model will be useful for investigations of radiation-induced cataractogenesis and other studies of lens toxicity.

Non-NASA Center↗