Comprehensive laboratory evaluation of a highly specific lateral flow assay for the presumptive identification of ricin in suspicious white powders and environmental samples.

Ricin, a heterodimeric toxin that is present in the seeds of the Ricinus communis plant, is the biothreat agent most frequently encountered by law enforcement agencies in the United States. Even in untrained hands, the easily obtainable seeds can yield a highly toxic product that has been used in various types of threats, including "white-powder" letters. Although the vast majority of these threats are hoaxes, an impediment to accurate hazard assessments by first responders is the unreliability of rapid detection assays for ricin, such as lateral flow assays (LFAs). One of the complicating factors associated with LFAs is the incorporation of antibodies of poor specificity that cross-react with near-neighbors or with plant lectins that are capable of nonspecifically cross-linking the capture and detector antibodies. Because of the compelling and critical need to promote the interests of public safety and public health, the Department of Homeland Security conducted a comprehensive laboratory evaluation study of a commercial LFA for the rapid detection of ricin. This study was conducted using comprehensive inclusivity and exclusivity panels of ricin and near-neighbor plant materials, along with panels of lectins and "white-powders," to determine the specificity, sensitivity, limits of detection, dynamic range, and repeatability of the assay for the specific intended use of evaluating suspicious white powders and environmental samples in the field.

Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E.

The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

Draft Genome Sequence of a Clostridium botulinum Isolate from Water Used for Cooling at a Plant Producing Low-Acid Canned Foods.

Clostridium botulinum is a pathogen of concern for low-acid canned foods. Here we report draft genomes of a neurotoxin-producing C. botulinum strain isolated from water samples used for cooling low-acid canned foods at a canning facility. The genome sequence confirmed that this strain belonged to C. botulinum serotype B1, albeit with major differences, including thousands of unique single nucleotide polymorphisms (SNPs) compared to other genomes of the same serotype.

Protein Domain Analysis of C. botulinum Type A Neurotoxin and Its Relationship with Other Botulinum Serotypes.

Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication.

Heterologous expression of polycystin-1 inhibits endoplasmic reticulum calcium leak in stably transfected MDCK cells.

We previously found that polycystin-1 accelerated the decay of ligand-activated cytoplasmic calcium transients through enhanced reuptake of calcium into the endoplasmic reticulum (ER; Hooper KM, Boletta A, Germino GG, Hu Q, Ziegelstein RC, Sutters M. Am J Physiol Renal Physiol 289: F521-F530, 2005). Calcium flux across the ER membrane is determined by the balance of active uptake and passive leak. In the present study, we show that polycystin-1 inhibited calcium leak across the ER membrane, an effect that would explain the capacity of this protein to accelerate clearance of calcium from the cytoplasm following a calcium release response. Calcium leak was detected by measurement of the accumulation of calcium in the cytoplasm following treatment with thapsigargin. Heterologous polycystin-1, stably expressed in Madin-Darby canine kidney cells, attenuated the thapsigargin-induced calcium peak with no effect on basal calcium stores, mitochondrial calcium uptake, or extrusion of calcium across the plasma membrane. The capacity of polycystin-1 to limit the rate of decay of ER luminal calcium following inhibition of the pump was shown indirectly using the calcium ionophore ionomycin, and directly by loading the ER with a low-affinity calcium indicator. We conclude that disruption of ER luminal calcium homeostasis may contribute to the cyst phenotype in autosomal dominant polycystic kidney disease.

The isolated polycystin-1 COOH-terminal can activate or block polycystin-1 signaling.

Much of what is known of the activities of polycystin-1 has been inferred from the effects of the isolated cytoplasmic COOH-terminal domain, but it is not clear whether the truncation acts like polycystin-1, as a dominant negative, or in unrelated pathways. To address this question, we have examined functional interactions between the intact and truncated forms of polycystin-1 in one cell system. In cells expressing only native polycystin-1, introduction of the truncation replicated the activity of the full-length protein. Conversely, when background levels of polycystin-1 were modestly elevated, the truncation acted as a dominant negative. Hence, the truncation acts in the polycystin pathway, but with effects that depend upon the background level of polycystin-1 expression. Our data raise the possibility that the cytoplasmic carboxyl terminus, either through cleavage products or intramolecular interactions, might feed back to modulate the activity of parent or intact polycystin-1.