Kenneth D. Cole

Trophic transfer of nanoparticles in a simplified invertebrate food web

The unique chemical and physical properties of engineered nanomaterials that make them attractive for numerous applications also contribute to their unexpected behaviour in the environment and biological systems. The potential environmental risks, including their impact on aquatic organisms, have been a central argument for regulating the growth of the nanotechnology sector. Here we show in a simplified food web that carboxylated and biotinylated quantum dots can be transferred to higher trophic organisms (rotifers) through dietary uptake of ciliated protozoans. Quantum dot accumulation from the surrounding environment (bioconcentration) was limited in the ciliates and no quantum dot enrichment (biomagnification) was observed in the rotifers. Our findings indicate that dietary uptake of nanomaterials should be considered for higher trophic aquatic organisms. However, limited bioconcentration and lack of biomagnification may impede the detection of nanomaterials in invertebrate species.

Organization of Serpin Gene-1 from Manduca sexta EVOLUTION OF A FAMILY OF ALTERNATE EXONS ENCODING THE REACTIVE SITE LOOP

Manduca sexta serpin gene-1 encodes a family of serpins whose amino acid sequences are identical in their amino-terminal 336 residues but variable in their carboxyl-terminal 39-46 residues, which includes the reactive site loop (Jiang, H., Wang, Y., and Kanost, M. R. (1994) J. Biol. Chem. 269, 55-58). Here, we report the genes complete nucleotide sequence and exon-intron structure. A unique characteristic of this gene is its exon 9, which is present in 12 alternate forms between exons 8 and 10. Isolation and characterization of cDNA clones containing exons 9C, 9H, and 9I, which were not found previously, indicate that all 12 alternate forms of exon 9 can be utilized to generate 12 different serpins. The splicing pathway apparently allows inclusion of only one exon 9 per molecule of mature serpin-1 mRNA. Analysis of exon-intron border sequences reveals unique features that may be involved in regulation of RNA splicing. The exon 9 region has apparently evolved through rounds of exon duplication and sequence divergence. The exons near the center of the region may have evolved recently, whereas the outermost exons are the most ancient. Exons 9G and 9H were duplicated as a pair from exons 9E and 9F, an event that may have occurred more than once in the history of this gene.

Human CD4+ lymphocytes for antigen quantification: Characterization using conventional flow cytometry and mass cytometry

To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto‐Trol and PBMC–National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto‐Trol is lower and the value on PBMC–NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4+ cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4+ cells from lyophilized PBMC–NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach. Published 2012 Wiley Periodicals, Inc.

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.

Breast cancer biomarker measurements and standards.

Cancer is a heterogeneous disease characterized by changes in the levels and activities of important cellular proteins, including oncogenes and tumor suppressors. Genetic mutations cause changes in protein activity and protein expression levels that result in the altered metabolism, proliferation, and metastasis seen in cancer cells. The identification of the critical biochemical changes in cancer has led to advances in its detection and treatment. An important example of this is the measurement of human epidermal growth factor receptor 2 (HER2), where increased expression occurs in approximately 20–30% of breast cancer tumors. HER2 is a member of the epidermal growth factor receptor family and is an important biomarker expressed on the cell surface. Measurement of the HER2 levels in tumor cells provides diagnostic, prognostic, and treatment information, because a targeted therapeutic is available. The most common methods to measure HER2 levels are immunohistochemistry and in situ hybridization assays. The accurate and reliable measurements of the specific changes in protein biomarkers for detection and treatment of cancer are important challenges. This review is focused on efforts to improve the quantitation and reliability of cancer biomarkers by using standards and reference materials.

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.

A Multistate Model for the Fluorescence Response of R‐Phycoerythrin

Abstract Although strong fluorescence makes the R-phycoerythrin (R-PE) proteins increasingly useful in biological and clinical assays, they are subject to nonlinear effects including transitions to collective dark states and photodegradation, which complicate quantitative applications. We report measurements of R-PE fluorescence intensity as a function of incident power, duration of illumination and temperature. Emission intensity in the band at 570 nm is proportional to incident power for low power levels. At higher incident power, the emission at 570 nm is smaller than expected from a linear dependence on power. We propose that R-PE undergoes both reversible emission cessation on a millisecond time scale attributed to transitions to a collective dark state, and irreversible photodegradation on a time scale of minutes. Singlet oxygen scavengers such as dithiothreitol and n-propyl gallate have protective effects against the latter effect but not the former. Electrophoretic analysis of irradiated solutions of R-PE indicates that significant noncovalent aggregation is correlated with photodegradation. A multistate model based on fluorescence measurements and geometric analysis is proposed for the fluorophores in R-PE. The phycobilin fluorophores are partitioned into three groups: the phycourobilins (PUB) absorbing at 490 nm, one group of phycoerythobilins (PEB) absorbing at 530 nm (PEB-530) and another group of PEB absorbing at 560 nm (PEB-560). The two processes that result in the loss of fluorescence intensity are most likely associated with the PEB-560 group.

Electrophoretic capture of circular DNA in gels

Results on electrophoretic capture of circular DNA in porous gels are reviewed. Processes which cause arrest of circular forms of DNA during electrophoresis can provide very efficient separation mechanism for the purification of plasmids and bacterial artificial chromosomes if the corresponding linear form is not trapped and therefore removed by the electric field. Two types of such topological traps have been proposed, impalement and lobster traps, and we here review the present experimental support for the existence of these two circle‐specific mechanisms. Experiments designed to characterize the traps are discussed, regarding the concentration of the traps as well as their efficiency and capacity to trap both relaxed and supercoiled circular DNA. Studies of the dynamics of the capture process show that the average capture time is on the order of 10 s at 20 V/cm, by which time the circles have migrated several hundred micrometers and have passed hundreds of traps. We also review results on attempts to improve the capacity and efficiency of the trapping process by modification of the gels either by enzymatic treatment or by cogelation of neutral polymers.

Preparative electrochromatography of proteins in various types of porous media.

The performance of commercial and enzymatically modified size‐exclusion (SE) gels in electrochromatography was compared for preparative protein separations. Dextran and agarose‐based SE gels were subjected to enzymatic digestion under mild conditions. This treatment partially hydrolyzed the gel matrix modifying its pore size distribution. Enzymatic treatment of agarose‐based SE gels was found to increase the resolution of the separation. Successful separation of preparative amounts of the A and B forms of β‐lactoglobulin (difference in electrophoretic mobility of 8.5%) was achieved with a high degree of purity using agarose‐based SE gels. The four major whey proteins, β‐lactoglobulin, α‐lactalbumin, BSA and immunoglobulins, were purified from an acid whey preparation. The degree of retention of a protein in electrochromatography followed their free‐solution electrophoretic mobility (μ) when the protein was able to enter the gel pores and the ratio of diffusion/μ when the protein was excluded.