Diagnostic real-time PCR assays for the detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks

Cereulide-producing Bacillus cereus can cause an emetic type of food-borne disease that mimics the symptoms provoked by Staphylococcus aureus. Based on the recently discovered genetic background for cereulide formation, a novel 5' nuclease (TaqMan) real-time PCR assay was developed to provide a rapid and sensitive method for the specific detection of emetic B. cereus in food. The TaqMan assay includes an internal amplification control and primers and a probe designed to target a highly specific part of the cereulide synthetase genes. Additionally, a specific SYBR green I assay was developed and extended to create a duplex SYBR green I assay for the one-step identification and discrimination of the two emesis-causing food pathogens B. cereus and S. aureus. The inclusivity and exclusivity of the assay were assessed using a panel of 100 strains, including 23 emetic B. cereus and 14 S. aureus strains. Different methods for DNA isolation from artificially contaminated foods were evaluated, and established real-time assays were used to analyze two recent emetic food poisonings in southern Germany. One of the food-borne outbreaks included 17 children visiting a day care center who vomited after consuming a reheated rice dish, collapsed, and were hospitalized; the other case concerned a single food-poisoning incident occurring after consumption of cauliflower. Within 2 h, the etiological agent of these food poisonings was identified as emetic B. cereus by using the real-time PCR assay.     

Surveillance of siRNA integrity by FRET imaging

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.     

Characterization of social behavior in the spiny mouse,Acomys cahirinus

While there are many species that are commonly used for the study of mammalian social behavior, there remains a need for lab-suitable organisms that are appropriate for examining sociality specifically in non-reproductive contexts (i.e., social behavior not in the context of mating or parenting). The spiny mouse, Acomys cahirinus, is a cooperatively breeding rodent that lives in large groups and is a species that holds great potential for studying a wide range of social behaviors in reproductive and non-reproductive contexts. Here, we characterize the basic social behaviors in male and female A. cahirinus to obtain a foundation for future study. We tested adult A. cahirinus in social approach, social preference, social interaction, social recognition, and group size preference paradigms. Regardless of sex, novelty, or familiarity, we found that both males and females rapidly approach conspecifics demonstrating high social boldness. Additionally, both sexes are significantly more prosocial than aggressive when freely interacting with conspecifics. However, we observed effects of sex on social preferences, such that males exhibit a preference to affiliate with same-sex conspecifics, whereas females exhibit a preference for affiliating with opposite-sex conspecifics. We discuss how this preference may relate to the cooperative breeding system of spiny mice. Lastly, both sexes show a robust preference for affiliating with large over small groups, indicating they may be an ideal species for the study of mammalian gregariousness. These data lay a basic foundation for future studies that seek to assess complex group dynamics and the mechanisms underlying reproductive and non-reproductive social behaviors in a highly social mammal.     

The in vitro antitumour profile of some 1,2-diaminocyclohexane organotin complexes

Platinum compounds containing the ligand 1,2-diaminocyclohexane (DACH) such as tetraplatin [PtCl4(DACH)] have been found to be active in cisplatin-resistant tumour models. In an attempt to develop novel metal-based drugs with a different therapeutic profile to cisplatin, we have synthesised a series of tin compounds containing the DACH ligand, including the Sn analogue of tetraplatin [SnCl4(DACH)], and the di- and monoorganotin complexes [Ph2Sn(OAc)2(DACH)], [Bu2Sn(OAc)2(DACH)], [PhSnCl3(DACH)], [BuSn(OAc)3(DACH)], [BuSnCl3(DACH)], and [PhSn(OCOCF3)3(DACH)]. Mossbauer and IR spectroscopy indicates that the Sn(DACH) complexes are hexacoordinated with a molecular structure similar to that of tetraplatin. These compounds were tested for potential antitumour activity against a panel of human tumour cell lines, (SW620, SW1116 colon carcinoma, ZR-75-1 breast carcinoma, HT1376 bladder carcinoma, SKOV-3, PA-1 ovarian carcinoma). [Ph2Sn(penicillinate)], [Ph2Sn(OCOCH2NCOCH2NH2)], [Ph2Sn(OAc)2] were included for comparison. The results show that whereas [SnCl4(DACH)] and the monoorganotin complexes had limited or no activity, the diorganotin DACH complexes were cytotoxic with an associated increase in potency on going from diphenyl to dibutyltin, with mean IC50 values of 7.26+/-4.09 micromol ml(-1) for [Ph2Sn(OAc)2(DACH)] and 2.58+/-0.83 micromol ml(-1) for [Bu2Sn(OAc)2(DACH)] across the cell line panel. Comparison with [Ph2Sn(OAc)2] (IC50 0.69-0.43 micromol ml(-1)) indicated that addition of the DACH ligand resulted in a decrease in cytotoxicity but increased differential toxicity across the cell line panel. These results indicate that the diorganotin DACH complexes merit further investigation as potential metal-based antitumour drugs.     

Physiological Significance of Network Organization in Fungi

The evolution of multicellularity has occurred in diverse lineages and in multiple ways among eukaryotic species. For plants and fungi, multicellular forms are derived from ancestors that failed to separate following cell division, thus retaining cytoplasmic continuity between the daughter cells. In networked organisms, such as filamentous fungi, cytoplasmic continuity facilitates the long-distance transport of resources without the elaboration of a separate vascular system. Nutrient translocation in fungi is essential for nutrient cycling in ecosystems, mycorrhizal symbioses, virulence, and substrate utilization. It has been proposed that an interconnected mycelial network influences resource translocation, but the theory has not been empirically tested. Here we show, by using mutants that disrupt network formation in Neurospora crassa (Δso mutant, no fusion; ΔPrm-1 mutant, ∼50% fusion), that the translocation of labeled nutrients is adversely affected in homogeneous environments and is even more severely impacted in heterogeneous environments. We also show that the ability to share resources and genetic exchange between colonies (via hyphal fusion) is very limited in mature colonies, in contrast to in young colonies and germlings that readily share nutrients and genetic resources. The differences in genetic/resource sharing between young and mature colonies were associated with variations in colony architecture (hyphal differentiation/diameters, branching patterns, and angles). Thus, the ability to share resources and genetic material between colonies is developmentally regulated and is a function of the age of a colony. This study highlights the necessity of hyphal fusion for efficient nutrient translocation within an N. crassa colony but also shows that established N. crassa colonies do not share resources in a significant manner.     

Stoichiometry of the CD95 death-inducing signaling complex: experimental and modeling evidence for a death effector domain chain model

The CD95 (Fas/APO-1) death-inducing signaling complex (DISC) is essential for the initiation of CD95-mediated apoptotic and nonapoptotic responses. The CD95 DISC comprises CD95, FADD, procaspase-8, procaspase-10, and c-FLIP proteins. Procaspase-8 and procaspase-10 are activated at?the DISC, leading to the formation of active caspases and apoptosis initiation. In this study we analyzed the?stoichiometry of the CD95 DISC. Using quantitative western blots, mass spectrometry, and mathematical modeling, we reveal that the amount of DED proteins procaspase-8/procaspase-10 and c-FLIP at the DISC exceeds that of FADD by several-fold. Furthermore, our findings imply that procaspase-8, procaspase-10, and c-FLIP could form DED chains at the DISC, enabling the formation of dimers and efficient activation of caspase-8. Taken together, our findings provide an enhanced understanding of caspase-8 activation and initiation of apoptosis at the DISC.     

Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe^fat/fat mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe^fat/fat mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe^fat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe^fat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe^fat/fat mice; these peptide processing intermediates with C‐terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.     

Intracerebral accumulation of glutaric and 3-hydroxyglutaric acids secondary to limited flux across the blood-brain barrier constitute a biochemical risk factor for neurodegeneration in glutaryl-CoA dehydrogenase deficiency

Glutaric acid (GA) and 3-hydroxyglutaric acids (3-OH-GA) are key metabolites in glutaryl co-enzyme A dehydrogenase (GCDH) deficiency and are both considered to be potential neurotoxins. As cerebral concentrations of GA and 3-OH-GA have not yet been studied systematically, we investigated the tissue-specific distribution of these organic acids and glutarylcarnitine in brain, liver, skeletal and heart muscle of Gcdh-deficient mice as well as in hepatic Gcdh-/- mice and in C57Bl/6 mice following intraperitoneal loading. Furthermore, we determined the flux of GA and 3-OH-GA across the blood-brain barrier (BBB) using porcine brain microvessel endothelial cells. Concentrations of GA, 3-OH-GA and glutarylcarnitine were significantly elevated in all tissues of Gcdh-/- mice. Strikingly, cerebral concentrations of GA and 3-OH-GA were unexpectedly high, reaching similar concentrations as those found in liver. In contrast, cerebral concentrations of these organic acids remained low in hepatic Gcdh-/- mice and after intraperitoneal injection of GA and 3-OH-GA. These results suggest limited flux of GA and 3-OH-GA across the BBB, which was supported in cultured porcine brain capillary endothelial cells. In conclusion, we propose that an intracerebral de novo synthesis and subsequent trapping of GA and 3-OH-GA should be considered as a biochemical risk factor for neurodegeneration in GCDH deficiency.     

Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.