Ready-to-use thermally stable mastermix pills for molecular biology applications

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.     

Improvements in single-use bioreactor film material composition leads to robust and reliable Chinese hamster ovary cell performance

Single-use technologies, in particular disposable bioreactor bags, have become integral within the biopharmaceutical community. However, safety concerns arose upon the identification of toxic leachable compounds derived from the plastic materials. Although the leachable bis(2,4-di-tert-butylphenyl)-phosphate (bDtBPP) has been previously shown to inhibit CHO cell growth, it is critical to determine if other compounds like this are still present in subsequent generations of films for industrial application. This study compares the performance of CHO cells, CHO-K1, and CHO-DP12, cultured in media conditioned in an older single-use bioreactor (SUB) film (F-1) and a newer generation film (F-2) from the same vendor. CHO cells cultured in media conditioned for 7 days in the F-1 film demonstrated significantly reduced growth and antibody productivity profiles when compared to controls and media conditioned for the same time period in the newer F-2 film. Proteomic profiling of CHO cells cultured in the F-1 conditioned media identified differentially expressed proteins involved in oxidative stress response as well as compromised ATP synthesis. These potentially metabolically compromised cells exhibited reduced oxidative phosphorylation activity as well as lower glycolytic metabolism, characteristic of slower growing cells. Nonvolatile and metal leachables analysis of film extracts by LC–MS revealed a reduction in the abundance of the analyzed leachates from F-2 films when compared to F-1 films including bDtBPP, potentially explaining improved CHO cell growth in F-2 conditioned media. Furthermore, in vitro endocrine disruptor testing of the known leachable revealed this molecule to possess the potential to act as an androgen antagonist. This study demonstrates an improvement in the materials composition used in modern generations of SUBs for safe application in the bioprocess.     

Genomic insights into the adaptation and evolution of the nautilus,an ancient but evolving “living fossil”

The nautilus, commonly known as a “living fossil,” is endangered and may be at risk of extinction. The lack of genomic information hinders a thorough understanding of its biology and evolution, which can shed light on the conservation of this endangered species. Here, we report the first high-quality chromosome-level genome assembly of Nautilus pompilius. The assembled genome size comprised 785.15 Mb. Comparative genomic analyses indicated that transposable elements (TEs) and large-scale genome reorganizations may have driven lineage-specific evolution in the cephalopods. Remarkably, evolving conserved genes and recent TE insertion activities were identified in N. pompilius, and we speculate that these findings reflect the strong adaptability and long-term survival of the nautilus. We also identified gene families that are potentially responsible for specific adaptation and evolution events. Our study provides unprecedented insights into the specialized biology and evolution of N. pompilius, and the results serve as an important resource for future conservation genomics of the nautilus and closely related species.     

Astressin-amide and astressin-acid are structurally different in dimethylsulfoxide

The C-terminally amidated CRF antagonist astressin binds to CRF-R1 or CRF-R2 receptors with low nanomolar affinity while the corresponding astressin-acid has >100 times less affinity. To understand the role of the amide group in binding, the conformations of astressin-amide and astressin-acid were studied in DMSO using NMR techniques. The 3D NMR structures show that the backbones of both analogs prefer an alpha-helical conformation, with a small kink around Gln(26). However, astressin-amide has a well-defined helical structure from Leu(27) to Ile(41) and a conformation very similar to the bioactive conformation reported by our group (Grace et al., Proc Natl Acad Sci USA 2007, 104, 4858-4863). In contrast, astressin-acid has an irregular helical conformation from Arg(35) onward, including a rearrangement of the side chains in that region. This structural difference highlights the crucial role of the C-terminal amidation for stabilization of astressin's bioactive conformation.     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids

The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.     

Validation of 1,3-butadiene exposure estimates for workers at a synthetic rubber plant

PURPOSE: This investigation assessed the validity of estimates of exposure to 1,3-butadiene (BD) developed for a plant included in a study of mortality among synthetic rubber industry workers. The estimates were developed without using historical measurement data and have not been validated previously. METHODS: Personal BD measurements came from an exposure-monitoring program initiated in 1977. For each job, we computed the year-specific difference between the BD estimate and the mean of BD measurements. We also computed rank correlation coefficients and calculated the mean, across all measurements, of the difference between the estimate and the measurement. RESULTS: The mean BD concentration was 5.2 ppm for 4978 measurements and 4.7 ppm for the corresponding estimates. The mean difference between estimates and measurements was -0.50 ppm (standard deviation, 26.5 ppm) overall and ranged from -227.9 to +27.0 ppm among all 306 job/year combinations. Estimates were correlated with measurements for all 306 combinations (rank correlation coefficient, r=0.45, p<0.0001), for 82 combinations pertaining to jobs that were well-defined by a specific set of tasks and typically found in styrene-BD rubber (SBR) plants (r=0.81, p<0.0001), for 70 combinations pertaining to jobs that were well-defined but not typical (r=0.29, p=0.01) and for 92 combinations pertaining to poorly-defined jobs typically found in SBR plants (r=0.56, <0.0001). Estimates were not correlated with measurements for poorly defined jobs not typically found in SBR plants (r=0.01, p=0.93). For well-defined typical SBR jobs with measurement means that were over 7.0 ppm, estimates were consistently lower than measurements. CONCLUSIONS: Possible reasons for differences between estimates and measurements included faulty assumptions used in developing BD estimates, unstable or nonrepresentive measurements and errors in linking measurement data to the job-exposure matrix. Exposure misclassification may have been more severe for subjects from the validation study plant than for subjects from other plants in the mortality study. BD estimates for typical SBR jobs, which comprise most operations at all but one of the plants in the mortality study, appeared to be useful for ranking workers by cumulative exposure. Uncertainty analyses would enhance the utility of the BD exposure estimates for quantitative risk assessment.     

Cancer risk assessment for 1,3-butadiene: dose-response modeling from an epidemiological perspective

The dose-response assessment of the association between 1,3-butadiene (BD) and leukemia mortality among workers in the North American synthetic rubber industry is explored. Analyses are based on the most recent University of Alabama at Birmingham epidemiological study and exposure estimation. The U.S. EPA Science Advisory Board recommendations of using the most recent data and giving consideration to peak exposures to BD have been followed. If cumulative BD ppm-years is to be used as the predictor of the leukemia rate ratio, then the performance of that predictor is statistically significantly improved if the slope in the predictor is estimated with age and the cumulative number of BD peaks (where a BD peak is any exposure, regardless of duration, to a BD concentration above 100 ppm) added as categorical covariates. After age and the cumulative number of BD peaks are incorporated as categorical covariates in the Poisson regression model, the estimated concentration (EC(001)) corresponding to an excess risk of 0.001 as a result of continuous environmental exposure is 11.2 ppm; however, the estimated slope for BD cumulative ppm-years in the linear rate ratio for leukemia used to derive this EC(001) is not statistically significantly different from zero. Sensitivity analyses using alternative models indicate either essentially no risk or estimated EC(001) values of 9 and 77 ppm. Analyses suggesting the absence of a statistically significant low-dose risk versus cumulative BD ppm-years are presented. Sensitivity analyses of other malignant neoplasms of lymphatic and hematopoietic tissue (specifically, lymphoid and myeloid neoplasms) resulted in conclusions about the dose-response modeling methodology that were supportive of the methodology used for leukemia.     

Two functionally distinct Axin-like proteins regulate canonical Wnt signaling in C. elegans

Axin is a central component of the canonical Wnt signaling pathway that interacts with the adenomatous polyposis coli protein APC and the kinase GSK3beta to downregulate the effector beta-catenin. In the nematode Caenorhabditis elegans, canonical Wnt signaling is negatively regulated by the highly divergent Axin ortholog PRY-1. Mutation of pry-1 leads to constitutive activation of BAR-1/beta-catenin-dependent Wnt signaling and results in a range of developmental defects. The pry-1 null phenotype is however not fully penetrant, indicating that additional factors may partially compensate for PRY-1 function. Here, we report the cloning and functional analysis of a second Axin-like protein, which we named AXL-1. We show that despite considerable sequence divergence with PRY-1 and other Axin family members, AXL-1 is a functional Axin ortholog. AXL-1 functions redundantly with PRY-1 in negatively regulating BAR-1/beta-catenin signaling in the developing vulva and the Q neuroblast lineage. In addition, AXL-1 functions independently of PRY-1 in negatively regulating canonical Wnt signaling during excretory cell development. In contrast to vertebrate Axin and the related protein Conductin, AXL-1 and PRY-1 are not functionally equivalent. We conclude that Axin function in C. elegans is divided over two different Axin orthologs that have specific functions in negatively regulating canonical Wnt signaling.