Positive and negative regulation of vertebrate separase by Cdk1-cyclin B1 may explain why securin is dispensable.

Sister chromatid cohesion is established during replication by entrapment of both dsDNAs within the cohesin ring complex. It is dissolved in anaphase when separase, a giant cysteine endopeptidase, cleaves the Scc1/Rad21 subunit of cohesin, thereby triggering chromosome segregation. Separase is held inactive by association with securin until this anaphase inhibitor is destroyed at the metaphase-to-anaphase transition by ubiquitin-dependent degradation. The relevant ubiquitin ligase, the anaphase-promoting complex/cyclosome, also targets cyclin B1, thereby causing inactivation of Cdk1 and mitotic exit. Although separase is essential, securin knock-out mice are surprisingly viable and fertile. Capitalizing on our previous finding that Cdk1-cyclin B1 can also bind and inhibit separase, we investigated whether this kinase might be suitable to maintain faithful timing and execution of anaphase in the absence of securin. We found that, similar to securin, Cdk1-cyclin B1 regulates separase in both a positive and negative manner. Although securin associates with nascent separase to co-translationally assist proper folding, Cdk1-cyclin B1 acts on native state separase. Upon entry into mitosis, Cdk1-cyclin B1-dependent phosphorylation of Ser-1126 renders separase prone to inactivation by aggregation/precipitation. Stable association of Cdk1-cyclin B1 with phosphorylated separase counteracts this tendency and stabilizes separase in an inhibited yet activatable state. These opposing effects are suited to prevent premature cleavage of cohesin in early mitosis while ensuring timely activation of separase by anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. Coupling sister chromatid separation with subsequent exit from mitosis by this simplified mode might have been the common scheme of mitotic control prior to the evolution of securin.

A lateral flow assay for identification of Escherichia coli by ribosomal RNA hybridisation.

Existing technologies for analysis of microbiological contaminants in food or clinical samples are often expensive and require laboratory settings and trained personnel. Here we present a lateral flow assay employing gold nanoparticle-oligodeoxynucleotide conjugates and four-component sandwich hybridisation for direct detection of specific sequences in bacterial 16S ribosomal RNA. Combined with rapid "one step" lysis the developed procedure allows detection of 5 × 10(4) colony forming units per mL Escherichia coli within less than 25 minutes. Several Escherichia coli strains were detected successfully, whereas non-related as well as closely related bacterial species produced no signal. The developed nucleic acid lateral flow assay is inexpensive, rapid to perform and requires no nucleic acid amplification step.

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

Electrochemical detection of microRNAs via gap hybridization assay.

MicroRNAs have recently been associated with cancer development by acting as tumor suppressors or oncogenes and could therefore be applied as molecular markers for early diagnosis of cancer. In this work, we established a rapid, selective, and sensitive gap hybridization assay for detection of mature microRNAs based on four components DNA/RNA hybridization and electrochemical detection using esterase 2-oligodeoxynucleotide conjugates. Complementary binding of microRNA to a gap built of capture and detector oligodeoxynucleotide, the reporter enzyme is brought to the vicinity of the electrode and produces enzymatically an electrochemical signal. In the absence of microRNA, the gap between capture and detector oligodeoxynucleotide is not filled, and missing base stacking energy destabilizes the hybridization complex. The gap hybridization assay demonstrates selective detection of miR-16 within a mixture of other miRNAs, including the feasibility of single mismatch discrimination. Applying the biosensor assay, a detection limit of 2 pM or 2 amol of miR-16 was obtained. Using isolated total RNA from human breast adenocarcinoma MCF-7 cells, the assay detected specifically miR-21 and miR-16 in parallel, and higher expression of oncogene miR-21 compared to miR-16 was demonstrated. Including RNA isolation, the gap hybridization assay was developed with a total assay time of 60 min and without the need for reverse transcription PCR amplification of the sample. The characteristics of the assay developed in this work could satisfy the need for rapid and easy methods for early cancer marker detection in clinical diagnostics.

Vaccinia virus-mediated inhibition of type I interferon responses is a multifactorial process involving the soluble type I interferon receptor B18 and intracellular components.

Poxviruses such as virulent vaccinia virus (VACV) strain Western Reserve encode a broad range of immune modulators that interfere with host responses to infection. Upon more than 570 in vitro passages in chicken embryo fibroblasts (CEF), chorioallantois VACV Ankara (CVA) accumulated mutations that resulted in highly attenuated modified vaccinia virus Ankara (MVA). MVA infection of mice and of dendritic cells (DC) induced significant type I interferon (IFN) responses, whereas infection with VACV alone or in combination with MVA did not. These results implied that VACV expressed an IFN inhibitor(s) that was functionally deleted in MVA. To further characterize the IFN inhibitor(s), infection experiments were carried out with CVA strains isolated after 152 (CVA152) and 386 CEF passages (CVA386). Interestingly, neither CVA152 nor CVA386 induced IFN-alpha, whereas the latter variant did induce IFN-beta. This pattern suggested a consecutive loss of inhibitors during MVA attenuation. Similar to supernatants of VACV- and CVA152-infected DC cultures, recombinantly expressed soluble IFN decoy receptor B18, which is encoded in the VACV genome, inhibited MVA-induced IFN-alpha but not IFN-beta. In the same direction, a B18R-deficient VACV variant triggered only IFN-alpha, confirming B18 as the soluble IFN-alpha inhibitor. Interestingly, VACV infection inhibited IFN responses induced by a multitude of different stimuli, including oligodeoxynucleotides containing CpG motifs, poly(I:C), and vesicular stomatitis virus. Collectively, the data presented show that VACV-mediated IFN inhibition is a multistep process involving secreted factors such as B18 plus intracellular components that cooperate to efficiently shut off systemic IFN-alpha and IFN-beta responses.

Detection of Escherichia coli in meat with an electrochemical biochip.

Detection of foodborne pathogenic and spoilage bacteria by RNA-DNA hybridization is an alternative to traditional microbiological procedures. To achieve high sensitivity with RNA-DNA-based methods, efficient bacterial lysis and release of nucleic acids from bacteria are needed. Here we report the specific detection of the hygiene indicator microorganism Escherichia coli in meat by use of electrochemical biochips. We improved RNA isolation from bacteria in meat juice from pork and beef. Samples, either naturally or artificially contaminated by E. coli, were enriched by incubation in full or minimal medium. A combined treatment of the samples with lysozyme, proteinase K, and sonication resulted in efficient cell disruption and high total RNA yields. Together with optimization of enrichment time, this ensures high sensitivity of electrochemical measurements on biochips. A short enrichment period and the triple-lysis regimen in combination with electrochemical biochip measurement were tested with 25 meat samples. The lower limit of detection of the biochip was approximately 2,000 CFU of E. coli per ml. The entire analysis procedure (5 h of enrichment, triple lysis, and biochip detection) has a lower limit of detection of 1 CFU of E. coli per ml within a total time needed for analysis of 7 h.

Multiplex Immunoassay Techniques for On-Site Detection of Security Sensitive Toxins.

Biological toxins are a heterogeneous group of high molecular as well as low molecular weight toxins produced by living organisms. Due to their physical and logistical properties, biological toxins are very attractive to terrorists for use in acts of bioterrorism. Therefore, among the group of biological toxins, several are categorized as security relevant, e.g., botulinum neurotoxins, staphylococcal enterotoxins, abrin, ricin or saxitoxin. Additionally, several security sensitive toxins also play a major role in natural food poisoning outbreaks. For a prompt response to a potential bioterrorist attack using biological toxins, first responders need reliable, easy-to-use and highly sensitive methodologies for on-site detection of the causative agent. Therefore, the aim of this review is to present on-site immunoassay platforms for multiplex detection of biological toxins. Furthermore, we introduce several commercially available detection technologies specialized for mobile or on-site identification of security sensitive toxins.

An Electrochemical Fiveplex Biochip Assay Based on Anti-Idiotypic Antibodies for Fast On-Site Detection of Bioterrorism Relevant Low Molecular Weight Toxins.

Modern threats of bioterrorism force the need for multiple detection of biothreat agents to determine the presence or absence of such agents in suspicious samples. Here, we present a rapid electrochemical fiveplex biochip screening assay for detection of the bioterrorism relevant low molecular weight toxins saxitoxin, microcystin-LR, T-2 toxin, roridin A and aflatoxin B1 relying on anti-idiotypic antibodies as epitope-mimicking reagents. The proposed method avoids the use of potentially harmful toxin-protein conjugates usually mandatory for competitive immunoassays. The biochip is processed and analyzed on the automated and portable detection platform pBDi within 13.4 min. The fiveplex biochip assay revealed toxin group specificity to multiple congeners. Limits of detection were 1.2 ng/mL, 1.5 ng/mL, 0.4 ng/mL, 0.5 ng/mL and 0.6 ng/mL for saxitoxin, microcystin-LR, T-2 toxin, roridin A or aflatoxin B1, respectively. The robustness of the fiveplex biochip for real samples was demonstrated by detecting saxitoxin, microcystin-LR, HT-2 toxin, roridin A and aflatoxin B1 in contaminated human blood serum without elaborate sample preparation. Recovery rates were between 52-115% covering a wide concentration range. Thus, the developed robust fiveplex biochip assay can be used on-site to quickly detect one or multiple low molecular weight toxins in a single run.

Electrochemical Biochip Assays Based on Anti-idiotypic Antibodies for Rapid and Automated On-Site Detection of Low Molecular Weight Toxins.

Phycotoxins and mycotoxins, such as paralytic shellfish poisoning toxins, type A trichothecenes, and aflatoxins are among the most toxic low molecular weight toxins associated with human poisoning incidents through the consumption of naturally contaminated food. Therefore, there is an utmost need for rapid and sensitive on-site detection systems. Herein, an electrochemical biochip for fast detection of saxitoxin, T-2 toxin as well as aflatoxin M1 and their corresponding congeners, respectively, using a portable and fully automated detection platform (pBDi, portable BioDetector integrated) was developed. Toxin analysis is facilitated upon the biochip via an indirect competitive immunoassay using toxin-specific antibodies combined with anti-idiotypic antibodies. The developed biochips enable detection in the low ng/mL-range within 17 min. Moreover, the assays cover a wide linear working range of 2-3 orders of magnitude above the limit of detection with an inter-chip coefficient of variation lower than 15%. The broad specificity of the employed antibodies which react with a large number of congeners within the respective toxin group allows efficient screening of contaminated samples for the presence of these low molecular weight toxins. With respect to the analysis of human urine samples, we focused here on the detection of saxitoxin, HT-2 toxin, and aflatoxin M1, all known as biomarkers of acute toxin exposure. Overall, it was proved that the developed biochip assays can be used to rapidly and reliably identify severe intoxications caused by these low molecular weight toxins.

Enhancement of electrochemical signal on gold electrodes by polyvalent esterase-dendrimer clusters.

5'-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterase-catalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3-and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.

Detection of bacterial 16S rRNA using multivalent dendrimer-reporter enzyme conjugates.

Novel enzyme-oligodeoxynucleotide conjugate was synthesized to improve sensitivity of Escherichia coli 16S rRNA detection on gold electrodes. Thermostable esterase 2 from Alicyclobacillus acidocaldarius was multiply conjugated to a polyamidoamine dendrimer functionalized by one universal detector oligodeoxynucleotide. Three components rRNA/DNA hybridization between capture oligodeoxynucleotide covalently immobilized on a gold electrode, 16S rRNA and the multivalent esterase-dendrimer cluster was used for detection of E. coli. The linear dependence of the electrochemical signals to analyte concentration revealed a detection limit of 50 colony forming units E. coli, which represents a tenfold signal enhancement if compared to the detection limit achieved with monovalent esterase-oligodeoxynucleotide conjugate.

Rapid, specific and sensitive electrochemical detection of foodborne bacteria.

Electrochemical biochips are an emerging tool for point-of-care diagnostic systems in medicine, food and environmental monitoring. In the current study, a thermostable reporter enzyme, esterase 2 (EST2) from Alicyclobacillus acidocaldarius, is used for specific and sensitive detection of bacteria by one-step rRNA/DNA hybridization between a bacterium-specific capture oligodeoxynucleotide (ODN), bacterial 16S rRNA and an uniform EST2-ODN reporter conjugate. The detection limit corresponds to approximately 500 colony forming units (cfu) Escherichia coli. Beside high sensitivity, the application of electrochemical biochips allows discrimination of two gram-negative and two gram-positive bacteria demonstrating the specificity and the potential for parallel detection of microorganisms. The feasibility of identification of foodborne bacteria was studied with meat juice contaminated with E. coli. This detection system has the capability to be applied for monitoring of bacterial food contamination.