Rapid and Sensitive Detection of Shiga Toxin-Producing (STEC) from Food Matrices Using the CANARY Biosensor Assay.

Shiga toxin-producing (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef).

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from .

Shiga-toxin-producing (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology.

Evaluation of the Salmonella CANARY® Zephyr Assay for the Detection of Salmonella enterica on Environmental Surfaces: AOAC Performance Tested MethodSM 042201.

Background: The Salmonella CANARY® Zephyr assay is designed to provide rapid and reliable detection of Salmonella enterica from various types of environmental surfaces, including stainless steel, silicone rubber, high-density polyethylene (HDPE), and glazed ceramic. The assay is using cell- and immuno-based CANARY technology and tested on Smiths Detection's Zephyr platform.

Objective: The objective of this validation study was to evaluate the Salmonella CANARY Zephyr assay for its inclusivity/exclusivity, matrix study for 4 claimed environmental surfaces, consistency/stability, and robustness.

A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices.

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation's food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) Zephyr detects BoNT/A holotoxin at limits of detection (LOD) of 10.

The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis.

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment.

Nuclear localization signal deletion mutants of lamin A and progerin reveal insights into lamin A processing and emerin targeting.

Lamin A is a major component of the lamina, which creates a dynamic network underneath the nuclear envelope. Mutations in the lamin A gene (LMNA) cause severe genetic disorders, one of which is Hutchinson-Gilford progeria syndrome (HGPS), a disease triggered by a dominant mutant named progerin. Unlike the wild-type lamin A, whose farnesylated C-terminus is excised during post-translational processing, progerin retains its farnesyl tail and accumulates on the nuclear membrane, resulting in abnormal nuclear morphology during interphase.

Leishmania-mediated inhibition of iron export promotes parasite replication in macrophages.

Leishmania parasites infect macrophages, cells that play an important role in organismal iron homeostasis. By expressing ferroportin, a membrane protein specialized in iron export, macrophages release iron stored intracellularly into the circulation. Iron is essential for the intracellular replication of Leishmania, but how the parasites compete with the iron export function of their host cell is unknown.

Live imaging assay for assessing the roles of Ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds.

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca(2+) is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca(2+)-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis.

Pathways of iron acquisition and utilization in Leishmania.

Iron is essential for many metabolic pathways, but is toxic in excess. Recent identification of the ferric iron reductase LFR1, the ferrous iron transporter LIT1, and the heme transporter LHR1 greatly advanced our understanding of how Leishmania parasites acquire iron and regulate its uptake. LFR1 and LIT1 have close orthologs in plants, and are required for Leishmania virulence.

Heme uptake mediated by LHR1 is essential for Leishmania amazonensis virulence.

The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire this essential factor from the environment. Previous studies showed that L. amazonensis incorporates heme through the transmembrane protein LHR1 (Leishmania Heme Response 1).

Extracellular amastigotes of Trypanosoma cruzi are potent inducers of phagocytosis in mammalian cells.

The protozoan parasite Trypanosoma cruzi, the aetiological agent of Chagas' disease, has two infective life cycle stages, trypomastigotes and amastigotes. While trypomastigotes actively enter mammalian cells, highly infective extracellular amastigotes (type I T. cruzi) rely on actin-mediated uptake, which is generally inefficient in non-professional phagocytes.

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms.

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections.

Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion.

Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca(2+) transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear.

Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes.

Syt VII is a Ca(2+) sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex.

Functional characterization of the N-terminal domain of subunit H (Vma13p) of the yeast vacuolar ATPase.

The yeast Saccharomyces cerevisiae vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex responsible for acidifying intracellular organelles and is highly regulated. One of the regulatory subunits, subunit H, is encoded by the VMA13 gene in yeast and is composed of two domains, the N-terminal domain (amino acids (aa) 1-352) and the C-terminal domain (aa 353-478). The N-terminal domain is required for the activation of the complex, whereas the C-terminal domain is required for coupling ATP hydrolysis to proton translocation (Liu, M.

PKR1 encodes an assembly factor for the yeast V-type ATPase.

Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells.

Vta1p and Vps46p regulate the membrane association and ATPase activity of Vps4p at the yeast multivesicular body.

Previous two-hybrid analysis of the 17 soluble class E Vps yeast proteins revealed that Vps46p/Did2p interacts with Vta1p and the AAA (ATPase associated with a variety of cellular activities) ATPase Vps4p. Here we report that the binding of Vps46p to Vps4p and Vta1p is direct and not mediated by additional proteins, and the binding of Vps46p to Vps4p is ATP independent. Vps46p regulates the membrane association of Vps4p and is required for the interaction of Vta1p with Vps32p/Snf7p of the ESCRT-III complex.

Topological characterization of the c, c', and c" subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae.

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V(1) and V(0). Hydrolysis of ATP in the V(1) subcomplex is tightly coupled to proton translocation accomplished by the V(0) subcomplex, which is composed of five unique subunits (a, d, c, c', and c").

Structure and assembly of the yeast V-ATPase.

The yeast V-ATPase belongs to a family of V-type ATPases present in all eucaryotic organisms. In Saccharomyces cerevisiae the V-ATPase is localized to the membrane of the vacuole as well as the Golgi complex and endosomes. The V-ATPase brings about the acidification of these organelles by the transport of protons coupled to the hydrolysis of ATP.