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Wetlab-2 - Quantitative PCR Tools for Spaceflight Studies of Gene Expression Aboard the International Space Station
Wetlab-2 is a research platform for conducting real-time quantitative gene expression analysis aboard the International Space Station. The system enables spaceflight genomic studies involving a wide variety of biospecimen types in the unique microgravity environment of space. Currently, gene expression analyses of space flown biospecimens must be conducted post flight after living cultures or frozen or chemically fixed samples are returned to Earth from the space station. Post-flight analysis is limited for several reasons. First, changes in gene expression can be transient, changing over a timescale of minutes. The delay between sampling on Earth can range from days to months, and RNA may degrade during this period of time, even in fixed or frozen samples. Second, living organisms that return to Earth may quickly re-adapt to terrestrial conditions. Third, forces exerted on samples during reentry and return to Earth may affect results. Lastly, follow up experiments designed in response to post-flight results must wait for a new flight opportunity to be tested.
A versatile oscillating‐flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis
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A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits
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Investigation of PMA-PCR technique for rapid detection of antimicrobial drug resistant bacteria.
Abstract not provided.
Multiplexed linking PCR
The invention provides compositions and methods for rapid assembly of one or more assembled polynucleotides from a plurality of component polynucleotides, as well as methods of designing polynucleotide primer and linker sets useful in the assembly methods of the invention.
Phosphocreatine attenuates endoplasmic reticulum stress-mediated hepatocellular apoptosis ameliorates insulin resistance in diabetes model
Highlights: • PCr decrease blood glucose level improve body weight. • PCr protected liver tissues against STZ/MGO caused apoptosis. • PCr attenuates hepatic endoplasmic reticulum stress markers. • PCr ameliorates insulin resistance. Diabetes mellitus (DM) associated liver damage is a major health burden. Hepatocellular-damage in DM characterized with elevated endoplasmic reticulum stress (ER) and may enhanced insulin-resistance. Phosphocreatine (PCr) a rapidly high-energy-reserve molecule of phosphates naturally occurs in liver, brain and skeletal muscle. This study aimed to investigate the protective effect of PCr on the liver-injury-associated with DM and to report the mechanism involved. Wistar rat's diabetes model was induced using streptozotocin (STZ), and the animals were treated with 20 mg/kg, or 50 mg/kg PCr injection. Blood glucose level, and body wt were recorded. Liver tissues homogenate were analyzed for liver damage markers alanine transaminase (ALT), aspartate transaminase (AST). Liver tissues proteins further evaluated for apoptosis, endoplasmic reticulum stress (ER), and insulin resistance biomarkers using western blotting. Our results revealed that PCr reduced blood glucose level, improved body wt, ameliorates liver function enzymes. Furthermore, PCr upregulates anti-apoptotic Bcl2 proteins expression, and down-regulates significantly pro-apoptotic casp3 and Bax proteins expression in vivo and invitro. Moreover, ER stress CHOP, GRP78 and ATF4 biomarkers level were significantly attenuated in PCr treated animals comparing to STZ diabetes associated liver-damage model with significant improving in insulin-resistance Akt and IRS-1. Our results revealed that treating with PCr in diabetes-associated liver injury models decreased blood glucose level and possess protective effect in-vitro and in-vivo, which could be suggested as potential therapeutic strategy for diabetes associated liver injury patients.
The Prognostic Value of a Pathologic Complete Response After Neoadjuvant Therapy for Digestive Cancer: Systematic Review and Meta-Analysis of 21 Studies
Background: Neoadjuvant therapy (NAT) before radical excision has become the preferred initial option for locally advanced digestive cancers such as esophageal cancer (EC), esophagogastric junction adenocarcinoma (EGJAC), gastric adenocarcinoma (GAC), rectal cancer (RC), and pancreatic cancer (PC). Although some patients reportedly achieve a pathologic complete response (pCR) after neoadjuvant therapy, the published data are inconsistent regarding whether pCR yields a survival benefit. The current meta-analysis was performed to assess the potential prognostic value of pCR after preoperative therapy for patients with digestive cancers. Methods: An extensive electronic search in PubMed, Web of Science, and the Cochrane Library was performed for relevant articles, from which data relative to independent correlations of pCR with overall survival (OS) and disease-free survival (DFS) were extracted for analysis. A random-effects model was used to calculate the pooled hazard ratios (HRs) with their corresponding 95% confidence intervals (CIs). Results: The study identified 6780 patients who met the inclusion and exclusion criteria. The results showed that pCR was significantly correlated with better OS (HR, 0.50; 95% CI, 0.43–0.58; P < 0.001) and DFS (HR, 0.49; 95% CI, 0.40–0.60; P < 0.001) for the digestive cancer patients who achieved pCR than for those who did not achieve pCR. Subgroup analysis showed that the correlation of pCR with OS was significant in EC (HR, 0.57; 95% CI, 0.47–0.69; P < 0.001), EGJAC/GAC (HR, 0.38; 95% CI, 0.17–0.86; P = 0.02), RC (HR, 0.48; 95% CI, 0.28–0.81; P = 0.006), and PC (HR, 0.41; 95% CI, 0.17–0.97; P = 0.04). In addition, the survival benefit for pCR patients was of similar magnitude, irrespective of the type of study, type of NAT, or ethnicity. Conclusions: A pCR is correlated with favorable survival outcomes compared with a non-pCR for digestive cancer patients after NAT.
Molecular Technique to Understand Deep Microbial Diversity
Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.
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.
Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice
Phosphocreatine (PCr) depletion during isometric twitch stimulation at 5 Hz was measured by (31)P-NMR spectroscopy in gastrocnemius muscles of pentobarbital-anesthetized MM creatine kinase knockout (MMKO) vs. wild-type C57B (WT) mice. PCr depletion after 2 s of stimulation, estimated from the difference between spectra gated to times 200 ms and 140 s after 2-s bursts of contractions, was 2.2 +/- 0.6% of initial PCr in MMKO muscle vs. 9.7 +/- 1.6% in WT muscles (mean +/- SE, n = 7, P < 0.001). Initial PCr/ATP ratio and intracellular pH were not significantly different between groups, and there was no detectable change in intracellular pH or ATP in either group after 2 s. The initial difference in net PCr depletion was maintained during the first minute of continuous 5-Hz stimulation. However, there was no significant difference in the quasi-steady-state PCr level approached after 80 s (MMKO 36.1 +/- 3.5 vs. WT 35.5 +/- 4.4% of initial PCr; n = 5-6). A kinetic model of ATPase, creatine kinase, and adenylate kinase fluxes during stimulation was consistent with the observed PCr depletion in MMKO muscle after 2 s only if ADP-stimulated oxidative phosphorylation was included in the model. Taken together, the results suggest that cytoplasmic ADP more rapidly increases and oxidative phosphorylation is more rapidly activated at the onset of contractions in MMKO compared with WT muscles.
Near Infrared Spectroscopy Calibration for Wood Chemistry: Which Chemometric Technique Is Best for Prediction and Interpretation?
This paper addresses the precision in factor loadings during partial least squares (PLS) and principal components regression (PCR) of wood chemistry content from near infrared reflectance (NIR) spectra. The precision of the loadings is considered important because these estimates are often utilized to interpret chemometric models or selection of meaningful wavenumbers. Standard laboratory chemistry methods were employed on a mixed genus/species hardwood sample set. PLS and PCR, before and after 1st derivative pretreatment, was utilized for model building and loadings investigation. As demonstrated by others, PLS was found to provide better predictive diagnostics. However, PCR exhibited a more precise estimate of loading peaks which makes PCR better for interpretation. Application of the 1st derivative appeared to assist in improving both PCR and PLS loading precision, but due to the small sample size, the two chemometric methods could not be compared statistically. This work is important because to date most research works have committed to PLS because it yields better predictive performance. But this research suggests there is a tradeoff between better prediction and model interpretation. Future work is needed to compare PLS and PCR for a suite of spectral pretreatment techniques.
Development Status of the WetLab-2 Project: New Tools for On-orbit Real-time Quantitative Gene Expression.
The primary objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform to facilitate gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens grown or cultured on orbit. The WetLab-2 equipment will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. In addition to the logistical benefits of in-situ sample processing and analysis, conducting qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms. The system can also validate terrestrial analyses of samples returned from ISS by providing quantitative on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experimental parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Finally, WetLab-2 can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-5 in Aug. 2014.Progress to date: The WetLab-2 project completed a thorough study of commercially available qRT-PCR systems and performed a downselect based on both scientific and engineering requirements. The selected instrument, the Cepheid SmartCycler, has advantages including modular design (16 independent PCR modules), low power consumption, and rapid ramp times. The SmartCycler has multiplex capabilities, assaying up to four genes of interest in each of the 16 modules. The WetLab-2 team is currently working with Cepheid to modify the unit for housing within an EXPRESS rack locker on the ISS. This will enable the downlink of data to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project is currently designing a module that will lyse the cells and extract RNA of sufficient quality for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. Current testing focuses on two promising commercial products and chemistries that allow for RNA extraction with minimal complexity and crew time.
WetLab-2: Tools for Conducting On-Orbit Quantitative Real-Time Gene Expression Analysis on ISS
The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015.
On-Orbit Quantitative Real-Time Gene Expression Analysis Using the Wetlab-2 System
NASA Ames Research Center's WetLab-2 Project enables on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The resulting RNA is dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The project selected the Cepheid SmartCycler (TradeMark), a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, because of its advantages including rugged modular design, low power consumption, rapid thermal ramp times and four-color multiplex detection. Single tube multiplex assays can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to conduct qRT-PCR and generate results on-orbit is an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also eliminate the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples and provide on-orbit gene expression benchmarking prior to sample return. Finally, the system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver launched on SpaceX-7 in June 2015 and will remain on the ISS National Laboratory.
Preoperative Chemoradiation Therapy in Combination With Panitumumab for Patients With Resectable Esophageal Cancer: The PACT Study
Purpose: Preoperative chemoradiation therapy (CRT) has become the standard treatment strategy for patients with resectable esophageal cancer. This multicenter phase 2 study investigated the efficacy of the addition of the epidermal growth factor receptor (EGFR) inhibitor panitumumab to a preoperative CRT regimen with carboplatin, paclitaxel, and radiation therapy in patients with resectable esophageal cancer. Methods and Materials: Patients with resectable cT1N1M0 or cT2-3N0 to -2M0 tumors received preoperative CRT consisting of panitumumab (6 mg/kg) on days 1, 15, and 29, weekly administrations of carboplatin (area under the curve [AUC] = 2), and paclitaxel (50 mg/m{sup 2}) for 5 weeks and concurrent radiation therapy (41.4 Gy in 23 fractions, 5 days per week), followed by surgery. Primary endpoint was pathologic complete response (pCR) rate. We aimed at a pCR rate of more than 40%. Furthermore, we explored the predictive value of biomarkers (EGFR, HER 2, and P53) for pCR. Results: From January 2010 until December 2011, 90 patients were enrolled. Patients were diagnosed predominantly with adenocarcinoma (AC) (80%), T3 disease (89%), and were node positive (81%). Three patients were not resected due to progressive disease. The primary aim was unmet, with a pCR rate of 22%. Patients with AC and squamous cell carcinoma reached a pCR of 14% and 47%, respectively. R0 resection was achieved in 95% of the patients. Main grade 3 toxicities were rash (12%), fatigue (11%), and nonfebrile neutropenia (11%). None of the biomarkers was predictive for response. Conclusions: The addition of panitumumab to CRT with carboplatin and paclitaxel was safe and well tolerated but could not improve pCR rate to the preset criterion of 40%.
Electronic transport characterization of silicon wafers by spatially resolved steady-state photocarrier radiometric imaging
Spatially resolved steady-state photocarrier radiometric (PCR) imaging technique is developed to characterize the electronic transport properties of silicon wafers. Based on a nonlinear PCR theory, simulations are performed to investigate the effects of electronic transport parameters (the carrier lifetime, the carrier diffusion coefficient, and the front surface recombination velocity) on the steady-state PCR intensity profiles. The electronic transport parameters of an n-type silicon wafer are simultaneously determined by fitting the measured steady-state PCR intensity profiles to the three-dimensional nonlinear PCR model. The determined transport parameters are in good agreement with the results obtained by the conventional modulated PCR technique with multiple pump beam radii.
Accurate determination of electronic transport properties of silicon wafers by nonlinear photocarrier radiometry with multiple pump beam sizes
In this paper, photocarrier radiometry (PCR) technique with multiple pump beam sizes is employed to determine simultaneously the electronic transport parameters (the carrier lifetime, the carrier diffusion coefficient, and the front surface recombination velocity) of silicon wafers. By employing the multiple pump beam sizes, the influence of instrumental frequency response on the multi-parameter estimation is totally eliminated. A nonlinear PCR model is developed to interpret the PCR signal. Theoretical simulations are performed to investigate the uncertainties of the estimated parameter values by investigating the dependence of a mean square variance on the corresponding transport parameters and compared to that obtained by the conventional frequency-scan method, in which only the frequency dependences of the PCR amplitude and phase are recorded at single pump beam size. Simulation results show that the proposed multiple-pump-beam-size method can improve significantly the accuracy of the determination of the electronic transport parameters. Comparative experiments with a p-type silicon wafer with resistivity 0.1–0.2 Ω·cm are performed, and the electronic transport properties are determined simultaneously. The estimated uncertainties of the carrier lifetime, diffusion coefficient, and front surface recombination velocity are approximately ±10.7%, ±8.6%, and ±35.4% by the proposed multiple-pump-beam-size method, which is much improved than ±15.9%, ±29.1%, and >±50% by the conventional frequency-scan method. The transport parameters determined by the proposed multiple-pump-beam-size PCR method are in good agreement with that obtained by a steady-state PCR imaging technique.