Jamie L. Almeida

Association and decontamination of Bacillus spores in a simulated drinking water system

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log10 increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log10 reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.

Standards for Cell Line Authentication and Beyond.

Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines.

Mouse cell line authentication

The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, few assays are available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.

Bacillus anthracis spore suspensions: determination of stability and comparison of enumeration techniques.

Aim:  To determine the stability and variability in concentration of spore suspensions of Bacillus anthracis (BA) spore suspensions by comparing different methods of enumeration and to detect changes, if any, under different storage conditions.

Authentication of African green monkey cell lines using human short tandem repeat markers.

BackgroundTools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.ResultsThe following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.ConclusionsA functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.

Requirements for the Development of Bacillus Anthracis Spore Reference Materials Used to Test Detection Systems

Bacillus anthracis spores have been used as biological weapons and the possibility of their further use requires surveillance systems that can accurately and reliably detect their presence in the environment. These systems must collect samples from a variety of matrices, process the samples, and detect the spores. The processing of the sample may include removal of inhibitors, concentration of the target, and extraction of the target in a form suitable for detection. Suitable reference materials will allow the testing of each of these steps to determine the sensitivity and specificity of the detection systems. The development of uniform and well-characterized reference materials will allow the comparison of different devices and technologies as well as assure the continued performance of detection systems. This paper discusses the special requirements of reference materials for Bacillus anthracis spores that could be used for testing detection systems. The detection of Bacillus anthracis spores is based on recognition of specific characteristics (markers) on either the spore surface or in the nucleic acids (DNA). We have reviewed the specific markers and their relevance to characterization of reference materials. We have also included the approach for the characterization of candidate reference materials that we are developing at the NIST laboratories. Additional applications of spore reference materials would include testing sporicidal treatments, techniques for sampling the environment, and remediation of spore-contaminated environments.

Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

It is important to develop rapid and reliable processes to monitor the decontamination of toxins released to the environment. The inactivation of the protein toxin ricin by the disinfectants bleach (sodium hypochlorite) and monochloramine was measured by the effect on mammalian cell cytotoxicity. The effect of the disinfectants on the native fluorescence (due mainly to tryptophan and to a lesser extent tyrosine) of ricin was also measured in parallel. Reactions of the disinfectants resulted in a decrease in the native fluorescence that was measured in real time in a noninvasive manner. We compared the inactivation of two well‐characterized model enzymes to the behavior of ricin. The model enzymes studied were lysozyme, a small basic enzyme stabilized with internal disulfide bonds, and heart‐muscle‐type lactate dehydrogenase (LDH), a large protein composed of four subunits. The biological activities of the model enzymes were measured in parallel with their fluorescence. Gel electrophoresis showed a large number of modifications of the proteins caused by the disinfectants reflected in changes in mobility and the formation of higher‐order aggregates. Size‐exclusion chromatography showed that the disinfectants did not break down the subunit structure of ricin but instead resulted in an increased size and heterogeneity of the protein. Size‐exclusion chromatography of LDH indicated that the subunits were dissociated and that higher‐order aggregates were also formed. Bleach caused a rapid inactivation of biological activity correlated with a rapid decrease in the fluorescence. Monochloramine required much higher concentrations for significant effects and the kinetics of the reactions were slow, with half‐life values of the decrease on the order of minutes. Each protein showed individual differences in responses to the disinfectants, but there was a consistent correlation between the loss of fluorescence and the decrease in biological activity. These results indicate that the monitoring the fluorescence is a useful process with limitations that can be used to monitor the inactivation of toxins using disinfectants.

Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods

The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 is a set of genomic DNA samples prepared from five breast cancer cell lines with certified values for the ratio of the HER2 gene copy number to the copy numbers of reference genes determined by real-time quantitative PCR and digital PCR. Targeted-amplicon, whole-exome, and whole-genome sequencing measurements were used with the reference material to compare the performance of both the laboratory steps and the bioinformatic approaches of the different methods using a range of amplification ratios. Although good reproducibility was observed in each next-generation sequencing method, slightly different HER2 copy numbers associated with platform-specific biases were obtained. This study clearly demonstrates the value of Standard Reference Materials 2373 as reference material and as a calibrator for evaluating assay performance as well as for increasing confidence in reporting HER2 amplification for clinical applications.

Development of NIST standard reference material 2373: Genomic DNA standards for HER2 measurements

NIST standard reference material (SRM) 2373 was developed to improve the measurements of the HER2 gene amplification in DNA samples. SRM 2373 consists of genomic DNA extracted from five breast cancer cell lines with different amounts of amplification of the HER2 gene. The five components are derived from the human cell lines SK-BR-3, MDA-MB-231, MDA-MB-361, MDA-MB-453, and BT-474. The certified values are the ratios of the HER2 gene copy numbers to the copy numbers of selected reference genes DCK, EIF5B, RPS27A, and PMM1. The ratios were measured using quantitative polymerase chain reaction and digital PCR, methods that gave similar ratios. The five components of SRM 2373 have certified HER2 amplification ratios that range from 1.3 to 17.7. The stability and homogeneity of the reference materials were shown by repeated measurements over a period of several years. SRM 2373 is a well characterized genomic DNA reference material that can be used to improve the confidence of the measurements of HER2 gene copy number.

A Mechanistically Relevant Cytotoxicity Assay Based on the Detection of Cellular Green Fluorescent Protein

Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.