Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS.

Enzyme-based error correction is a key step in de novo DNA synthesis, yet the inherent instability of error-correction enzymes such as MutS has hindered the throughput and efficiency of DNA synthesis workflows. Here we introduce a process called Improved MICC (iMICC), in which all error-correction steps of oligos and fragments within a complete gene-synthesis cycle are completed in a simple, efficient, and low-cost manner via a MutS protein engineered for high durability. By establishing a disulfide bond of L157C-G233C, full-activity shelf life of E. coli MutS (eMutS) was prolonged from 7 to 49 days and was further extended to 63 days via cellulose-bound 4 °C storage. In synthesis of 10 Cas9 homologues in-solution and 10 xylose reductase (XR) homologues on-chip, iMICC reduced error frequency to 0.64/Kb and 0.41/Kb, respectively, with 72.1% and 86.4% of assembled fragments being error-free. By elevating base accuracy by 37.6-fold while avoiding repetitive preparation of fresh enzymes, iMICC is more efficient and robust than the wild-type eMutS, and it is 6.6-fold more accurate and 26.7-fold cheaper than CorrectASE. These advantages promise its broad applications in industrial DNA synthesis.

[Synthesizing a human genome?] / HGP-write : après la lecture, l'écriture ? - Chroniques génomiques.

The recently proposed « HGP-write ¼ project aims to synthetize a full human genome and to introduce it into cells. This ambitious endeavour is fraught with financial and technical uncertainties and, if successful, would make « synthetic humans ¼ a definite possibility even though this is not part of its announced goals. Accordingly, it has not been received with enthusiasm.

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4',6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Assessment of protein synthesis in highly aerobic canine species at the onset and during exercise training.

Canis lupus familiaris, the domesticated dog, is capable of extreme endurance performance. The ability to perform sustained aerobic exercise is dependent on a well-developed mitochondrial reticulum. In this study we examined the cumulative muscle protein and DNA synthesis in groups of athletic dogs at the onset of an exercise training program and following a strenuous exercise training program. We hypothesized that both at the onset and during an exercise training program there would be greater mitochondrial protein synthesis rates compared with sedentary control with no difference in mixed or cytoplasmic protein synthesis rates. Protein synthetic rates of three protein fractions and DNA synthesis were determined over 1 wk using (2)H2O in competitive Alaskan Huskies and Labrador Retrievers trained for explosive device detection. Both groups of dogs had very high rates of skeletal muscle protein synthesis in the sedentary state [Alaskan Huskies: Mixed = 2.28 ± 0.12, cytoplasmic (Cyto) = 2.91 ± 0.10, and mitochondrial (Mito) = 2.62 ± 0.07; Labrador Retrievers: Mixed = 3.88 ± 0.37, Cyto = 3.85 ± 0.06, and Mito = 2.92 ± 0.20%/day]. Mitochondrial (Mito) protein synthesis rates did not increase at the onset of an exercise training program. Exercise-trained dogs maintained Mito protein synthesis during exercise training when mixed (Mixed) and cytosolic (Cyto) fractions decreased, and this coincided with a decrease in p-RpS6 but also a decrease in p-ACC signaling. Contrary to our hypothesis, canines did not have large increases in mitochondrial protein synthesis at the onset or during an exercise training program. However, dogs have a high rate of protein synthesis compared with humans that perhaps does not necessitate an extra increase in protein synthesis at the onset of aerobic exercise training.

Next generation 1536-well oligonucleotide synthesizer with on-the-fly dispense.

Here we report the development of our Next Generation Automated Multiplexed Oligonucleotide Synthesizer (NG-1536-AMOS), capable of producing 1536 samples in a single run using a multi-well filtered titer plate. With the potential to synthesize up to 3456 samples per plate, we converted the BioRAPTR Flying Reagent Dispenser into an open-well system where spent reagents are drained to waste under vacuum. During synthesis, reagents are delivered on-the-fly to each micro-titer well at volumes ≤5 µl with plate speeds up to 150 mm/s. Using gas-phase cleavage and deprotection, a full plate of 1536 60 mers may be processed with same-day turnaround with an average yield per well at 3.5 nmol. Final product at only $0.00277/base is eluted into a low-volume collection plate for immediate use in downstream application (e.g. Biomek FX for versatile sample handling). Also, crude oligonucleotide quality is comparable to that of commercial synthesis instrumentation, with an error rate on the NG-1536-AMOS platform of 1.53/717 bases. Furthermore, mass spectral analysis on strands synthesized up to 80 bases showed high purity with an average coupling efficiency of 99.5%.

DNA synthesis security.

It is generally assumed that genetic engineering advances will, inevitably, facilitate the misapplication of biotechnology toward the production of biological weapons. Unexpectedly, however, some of these very advances in the areas of DNA synthesis and sequencing may enable the implementation of automated and nonintrusive safeguards to avert the illicit applications of biotechnology. In the case of DNA synthesis, automated DNA screening tools could be built into DNA synthesizers in order to block the synthesis of hazardous agents. In addition, a comprehensive safety and security regime for dual-use genetic engineering research could include nonintrusive monitoring of DNA sequencing. This is increasingly feasible as laboratories outsource this service to just a few centralized sequencing factories. The adoption of automated, nonintrusive monitoring and surveillance of the DNA synthesis and sequencing pipelines may avert many risks associated with dual-use biotechnology. Here, we describe the historical background and current challenges associated with dual-use biotechnologies and propose strategies to address these challenges.

A comprehensive assessment of mitochondrial protein synthesis and cellular proliferation with age and caloric restriction.

It is proposed that caloric restriction (CR) increases mitochondrial biogenesis. However, it is not clear why CR increases an energetically costly biosynthetic process. We hypothesized that 40% CR would decrease mitochondrial protein synthesis and would be regulated by translational rather than transcriptional mechanisms. We assessed cumulative mitochondrial protein synthesis over 6 weeks and its transcriptional and translational regulation in the liver, heart, and skeletal muscle of young (6 month), middle (12 month), and old (24 month) male B6D2F1 mice that were lifelong CR or ad lib (AL) controls. Mitochondrial protein synthesis was not different between AL and CR (fractional synthesis over 6 weeks (range): liver, 91-100%; heart, 74-85%; skeletal muscle, 53-72%) despite a decreased cellular proliferation in liver and heart with CR. With CR, there was an increase in AMP-activated protein kinase phosphorylation/total (P:T) in heart and liver, and an increase in peroxisome proliferator-activated receptor gamma coactivator 1-α mRNA in all tissues, but not protein. Ribosomal protein S6 was decreased with CR. In conclusion, CR maintained mitochondrial protein synthesis while decreasing cellular proliferation during a time of energetic stress, which is consistent with the concept that CR increases somatic maintenance. Alternative mechanisms to global translation initiation may be responsible for selective translation of mitochondrial proteins.

A model for segregation of chromatin after replication: segregation of identical flexible chains in solution.

We study the segregation of two long chains from parallel but randomly twisted start conformations under good solvent conditions using Monte Carlo simulations to mimic chromatin segregation after replication in eukaryotic cells in the end of prophase. To measure the segregation process, we consider the center-of-mass separation between the two chains and the average square distance between the monomers which were connected before segregation starts. We argue that segregation is dominated by free diffusion of the chains, assuming that untwisting can be achieved by Rouse-like fluctuations on the length scale of a twisted loop. Using scaling analysis, we find that chain dynamics is in very good agreement with the free diffusion hypothesis, and segregation dynamics follows this scaling nearly. Long chains, however, show retardation effects that can be described by a new (to us) dynamical exponent, which is slightly larger than the dynamical exponent for Rouse-like diffusion. Our results indicate that nearly free diffusion of chains during a timescale of a few Rouse-times can lead to segregation of chains. A main obstacle during segregation by free diffusion is random twists between daughter strands. We have calculated the number of twists formed by the daughter strands in the start conformations, which turns out to be rather low and increases only with the square-root of the chain length.

Validation of an in vitro model for assessment of androstenedione hepatotoxicity using the rat liver cell line clone-9.

Androstenedione, a naturally occurring steroid hormone, has been used to enhance athletic performance. Little is known, however, about its hepatotoxicity. Clone-9 cells, a non-transformed epithelial cell line that was originally isolated from normal liver of a 4-week old Sprague-Dawley rat, were used as an in vitro model to assess the hepatotoxic potential of androstenedione. The cultures were treated with androstenedione for 24 h at 37 degrees C in 5% CO(2) at concentrations of 0-100 microg ml(-1). After the treatment period, the cells and the culture supernatants were assayed for markers of cytotoxicity which included: release of liver enzymes, cell viability, cellular double-stranded DNA content, oxidative stress, steatosis, cellular ATP content, caspase-3 activity, the mitochondrial permeability transition and induction of cytochrome P450 activity. Significant concentration-dependent differences from control were observed in some endpoints at medium concentrations of 10 microg ml(-1) and above. These in vitro findings were compared with comparable endpoints obtained from an in vivo study of androstenedione toxicity in female Sprague-Dawley rats. Of the eight endpoints that could be compared between the two studies, only three (lipid accumulation, ATP depletion and P450 activity) appeared to be concordant. This suggests that, under the experimental conditions used, the clone-9 cells were not a good model for androstenedione hepatotoxicity.

Vernonia amygdalina: anticancer activity, authentication, and adulteration detection.

Evidence suggests that most chemotherapeutic agents are less effective as treatment in patients with estrogen receptor-negative (ER-) breast carcinomas compared to those with estrogen receptor-positive (ER+) breast carcinomas. Moreover, African American Women (AAW) is disproportionately diagnosed with ER- breast cancer compared to their white counterparts. Novel therapies effective against ER- breast carcinomas are urgently needed to ameliorate the health disparity. Previous reports show that low concentrations (microgram/ml) of water-soluble leaf extracts of a Nigerian edible plant, V. amygdalina (VA), potently retards the proliferative activities of ER+ human breast cancerous cells (MCF-7) in vitro in a concentration-dependent fashion. However, the anti-proliferative activities of VA in either ductal or ER- carcinoma cells have not been characterized. The exposure of BT-549 to increasing concentrations of VA (10, 100, and 1000 microg/mL) inhibited cell growth by approximately 14 % (P<0.05), 22 % (p<0.05), and 50 % (p<0.005) respectively. The cell count studies were corroborated by DNA synthesis studies. Treatments of BT-549 with 10, 100, and 1000 microg/mL VA inhibited DNA synthesis in a concentration dependent fashion by 22 %, 76 % (P<0.05), and 86 % (p<0.01) respectively. BT-549 cells were insensitive to 10 and 100 nM paclitaxel (TAX) treatments. Isolation of DNA from dried VA leaves yielded approximately 12.2 and 1 kbp genomic DNA that were Eco RI-insensitive but Hind III and Bam HI-sensitive. These pieces of information may be used to enhance the safety of medicinal botanical VA through authentication, and adulteration detection.

Production of clinical-grade plasmid DNA for human Phase I clinical trials and large animal clinical studies.

The use of plasmid DNA as vaccines for the treatment of cancer and infectious diseases is on the rise. In order to facilitate the manufacture of clinical-grade plasmid DNA for Phase I clinical trials, we developed a process whereby >200 mg plasmid could be produced in a single production run under Good Manufacturing Practices. A dedicated cleanroom (Class 10,000 with Class 100 biosafety cabinet) is utilized for production of the bacterial cell bank, fermentation, harvest/lysis of the biomass, and downstream purification. Fermentation requires three 16-18 h runs (approximately 12 L each) in shaker-flasks, yielding approximately 60 g bacterial paste following batch centrifugation. The biomass is alkaline-lysed, pooled, and the resulting flocculent precipitate is separated by a novel vacuum step, followed by depth-filtration. Downstream processing includes anion-exchange chromatography, utilizing Qiagen silica-based resin, and precipitation with isopropanol. Following precipitation, the DNA is harvested by centrifugation, dried, formulated, and sterile-filtered using a Sartorius Sartobran 150 filter prior to Final-Filling. All processing steps utilize sterilized, single-use components. This process results in a product manufactured according to regulatory guidelines. The plasmid DNA is sterile with >or=95% supercoiled DNA, an A260/A280 ratio>or=1.9, undetectable or extremely low residual endotoxin, RNA, genomic DNA, protein, and antibiotic. Residual solvent levels are negligible. The product yields the predicted profile upon restriction-enzyme digestion, is biologically active upon transfection and remains stable for several years at -20 degrees C. We have therefore developed a reproducible and cost effective process to manufacture clinical-grade plasmid DNA. This process can be adapted by other academic centers for human or large animal clinical trials.

Statistical inference for quantitative polymerase chain reaction using a hidden markov model: a Bayesian approach.

Quantitative Polymerase Chain Reaction (Q-PCR) aims at determining the initial quantity of specific nucleic acids from the observation of the number of amplified DNA molecules. The most widely used technology to monitor the number of DNA molecules as they replicate is based on fluorescence chemistry. Considering this measurement technique, the observation of DNA amplification by PCR contains intrinsically two kinds of variability. On the one hand, the number of replicated DNA molecules is random, and on the other hand, the measurement of the fluorescence emitted by the DNA molecules is collected with some random error. Relying on a stochastic model of these two types of variability, we aim at providing estimators of the parameters arising in the proposed model, and, more specifically, of the initial amount of molecules. The theory of branching processes is classically used to model the evolution of the number of DNA molecules at each replication cycle. The model is a binary splitting Galton-Watson branching process. Its unknown parameters are the initial number of DNA molecules and the reaction efficiency of PCR, which is defined as the probability of replication of a DNA molecule. The number of DNA molecules is indirectly observed through noisy fluorescence measurements resulting in a so-called Hidden Markov Model. We aim at inference of the parameters of the underlying branching process, and the parameters of the noise from the fluorescence measurements in a Bayesian framework. Using simulations and experimental data, we investigate the performance of the Bayesian estimators obtained by Markov Chain Monte Carlo methods.

Real-time quantitative PCR for assessment of antiviral drug effects against Epstein-Barr virus replication and EBV late mRNA expression.

This study assesses the ability of quantitative real-time PCR to measure the effects of virus DNA polymerase inhibitors on EBV DNA and late mRNAs syntheses in EBV-producing cell lines. In-house real-time quantitative PCRs were used to measure EBV DNA (thymidine kinase) and mRNAs (BLLF1 gene/gp350/220, BVRF2 gene/protease) in P3HR-1 and B95-8 cells induced for EBV production by PMA and exposed to ganciclovir, cidofovir and foscarnet. The calculated 50% effective concentrations (EC(50)) for viral DNA replication inhibition in P3HR-1 cells after 7 days of drug exposure were 0.28+/-0.06, 0.29+/-0.01 and 13.6+/-0.17 microg/mL for ganciclovir, cidofovir and foscarnet, respectively. The EC(50) for B95-8 cells were 0.44+/-0.02, 0.70+/-0.06 and 46.8+/-0.5 microg/mL, respectively. The quantitation of the late viral mRNAs showed a decrease of 79-89% in the mRNA amount after 4 days of antiviral treatment. Nevertheless, a substantial amount of mRNA still remained detectable after drug exposure. The real-time PCR is an improvement in the attempt to simplify EBV DNA-quantitation for antiviral assays. The quantitation of late mRNA does not appear as more informative than DNA quantitation for the assessment of the DNA polymerase inhibitor activity, but it may be useful to assess the antiviral activity of drugs acting by another mechanism.

A time- and cost-efficient system for high-level protein production in mammalian cells.

Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline.

A comparison of colorimetric and DNA quantification assays for the assessment of meniscal fibrochondrocyte proliferation in microcarrier culture.

We have developed and refined a rapid, reliable method for the evaluation of attachment and proliferation of ovine meniscal chondrocytes in microcarrier culture. Assays measuring both mitochondrial activity, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], and DNA synthesis with a PicoGreen assay were compared. The MTT assay was the most sensitive at lower cell concentrations and enabled accurate assessment of cell proliferation over 14 day culture.

Monte Carlo simulation of base and nucleotide excision repair of clustered DNA damage sites. I. Model properties and predicted trends.

DNA is constantly damaged through endogenous processes and by exogenous agents, such as ionizing radiation. Base excision repair (BER) and nucleotide excision repair (NER) help maintain the stability of the genome by removing many different types of DNA damage. We present a Monte Carlo excision repair (MCER) model that simulates key steps in the short-patch and long-patch BER pathways and the NER pathway. The repair of both single and clustered damages, except double-strand breaks (DSBs), is simulated in the MCER model. Output from the model includes estimates of the probability that a cluster is repaired correctly, the fraction of the clusters converted into DSBs through the action of excision repair enzymes, the fraction of the clusters repaired with mutations, and the expected number of repair cycles needed to completely remove a clustered damage site. The quantitative implications of alternative hypotheses regarding the postulated repair mechanisms are investigated through a series of parameter sensitivity studies. These sensitivity studies are also used to help define the putative repair characteristics of clustered damage sites other than DSBs.

A Markov model approach shows a large variation in the length of S phase in MCF-7 breast cancer cells.

BACKGROUND: The potential doubling time of a tumor has been suggested to be a measurement of tumor aggressiveness; therefore, it is of interest to find reliable methods to estimate this time. Because of variability in length of the various cell cycle phases, stochastic modeling of the cell cycle might be a suitable approach. METHODS: The relative movement curve and the DNA synthesis time were estimated by using local polynomial regression methods. Further, the rate of nucleotide incorporation was estimated by using a Markov pure birth process with one absorbing state to model the progression of the DNA distribution through S phase. RESULTS: An estimate of the DNA synthesis time, with confidence intervals, was obtained from the relative movement curve. The Markov approach provided an estimate of the distribution of the time to complete S phase given the initial distribution. Using the Markov approach we also made an estimate of the mean number of active replicons during S phase. CONCLUSIONS: A Markov pure birth process has shown to be useful to model the progression of cells through S phase and to increase knowledge about the variability in the length of S phase and a large variation is shown.

Thiazide-like diuretics attenuate agonist-induced vasoconstriction by calcium desensitization linked to Rho kinase.

Lowering blood pressure using thiazide-like diuretics, including chlorthalidone and hydrochlorothiazide, has been proven to be effective in clinical studies. However, the mechanisms by which thiazide-like diuretics lower blood pressure are still poorly understood. To evaluate whether thiazide-like diuretics cause calcium desensitization in smooth muscle cells, we measured their effects on agonist-induced increase of blood pressure in Wistar rats in vivo and on agonist-induced vasoconstriction of aortic rings, DNA synthesis, and protein synthesis, RhoA, Rho kinase, and intracellular calcium in vascular smooth muscle cells in vitro. Thiazide-like diuretics significantly attenuated angiotensin II-induced or norepinephrine-induced increase of systolic blood pressure in rats. Thiazide-like diuretics inhibited agonist-induced vasoconstriction of aortic rings in a concentration-dependent manner in the presence and absence of endothelium. The inhibitory effects of thiazide-like diuretics were similar to that of the specific Rho kinase inhibitor Y27632. RT-PCR and immunoblotting showed that RhoA and Rho kinase were significantly reduced in vascular smooth muscle cells after administration of thiazide-like diuretics. In contrast, thiazide-like diuretics did not affect protein tyrosine phosphatase-2 (SHP-2) expression. Agonist-induced changes of intracellular calcium were not affected by thiazide-like diuretics. The study indicates that thiazide-like diuretics inhibit agonist-induced vasoconstriction by calcium desensitization in smooth muscle cells linked to the Rho-Rho kinase pathway.