Membrane-based on-line optical analysis system for rapid detection of bacteria and spores

We report here the adaptation of our electronic microchip technology towards the development of a new method for detecting and enumerating bacterial cells and spores. This new approach is based on the immuno-localization of bacterial spores captured on a membrane filter microchip placed within a flow cell. A combination of microfluidic, optical, and software components enables the integration of staining of the bacterial species with fully automated assays. The quantitation of the analyte signal is achieved through the measurement of a collective response or alternatively through the identification and counting of individual spores and particles. This new instrument displays outstanding analytical characteristics, and presents a limit of detection of approximately 500 spores when tested with Bacillus globigii (Bg), a commonly used simulant for Bacillus anthracis (Ba), with a total analysis time of only 5 min. Additionally, the system performed well when tested with real postal dust samples spiked with Bg in the presence of other common contaminants. This new approach is highly customizable towards a large number of relevant toxic chemicals, environmental factors, and analytes of relevance to clinical chemistry applications.     

Evolution of the relaxin-like peptide family

Background  

The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.     

An insulator-based (electrodeless) dielectrophoretic concentrator for microbes in water

Dielectrophoresis (DEP), the motion of a particle caused by an applied electric field gradient, can concentrate microorganisms non-destructively. In insulator-based dielectrophoresis (iDEP) insulating microstructures produce non-uniform electric fields to drive DEP in microsystems. This article describes the performance of an iDEP device in removing and concentrating bacterial cells, spores and viruses while operated with a DC applied electric field and pressure gradient. Such a device can selectively trap particles when dielectrophoresis overcomes electrokinesis or advection. The dielectrophoretic trapping behavior of labeled microorganisms in a glass-etched iDEP device was observed over a wide range of DC applied electric fields. When fields higher than a particle-specific threshold are applied, particles are reversibly trapped in the device. Experiments with Bacillus subtilis spores and the Tobacco Mosaic Virus (TMV) exhibited higher trapping thresholds than those of bacterial cells. The iDEP device was characterized in terms of concentration factor and removal efficiency. Under the experimental conditions used in this study with an initial dilution of 1 x 105 cells/ml, concentration factors of the order of 3000x and removal efficiencies approaching 100% were observed with Escherichia coli cells. These results are the first characterization of an iDEP device for the concentration and removal of microbes in water.     

Evolution of sexual dimorphism and male dimorphism in the expression of beetle horns: phylogenetic evidence for modularity, evolutionary lability, and constraint

Beetle horns are enlarged outgrowths of the head or thorax that are used as weapons in contests over access to mates. Horn development is typically confined to males (sexual dimorphism) and often only to the largest males (male dimorphism). Both types of dimorphism result from endocrine threshold mechanisms that coordinate cell proliferation near the end of the larval period. Here, we map the presence/absence of each type of dimorphism onto a recent phylogeny for the genus Onthophagus (Coleoptera: Scarabaeidae) to explore how horn development has changed over time. Our results provide empirical support for several recent predictions regarding the evolutionary lability of developmental thresholds, including uncoupled evolution of alternative phenotypes and repeated fixation of phenotypes. We also report striking evidence of a possible developmental constraint. We show that male dimorphism and sexual dimorphism map together on the phylogeny; whenever small males have horns, females also have horns (and vice versa). We raise the possibility that correlated evolution of these two phenomena results from a shared element in their endocrine regulatory mechanisms rather than a history of common selection pressures. These results illustrate the type of insight that can be gained only from the integration of developmental and evolutionary perspectives.     

Calcium phosphate and calcium oxalate crystal handling is dependent upon CLC-5 expression in mouse collecting duct cells

Defects in an intracellular chloride channel CLC-5 cause Dent's disease, an inherited kidney stone disorder. Using a collecting duct model, mIMCD-3 cells, we show expression of dimeric mCLC-5. Transient transfection of antisense CLC-5 reduces CLC-5 protein expression. Binding of both calcium phosphate (hydroxyapatite) and calcium oxalate monohydrate (COM) crystals overlaid onto mIMCD-3 cultures was affected by altered CLC-5 expression. Calcium phosphate crystal agglomerations (>10 microm) were minimal in control (9%) and sense (13%) CLC-5-transfected cells, compared to 66% of antisense CLC-5-transfected cells (P<0.001). Small calcium phosphate crystals (<10 microm) were found associated with 45% of sense CLC-5-treated cells, of which the majority (11/14 cells) appeared to be internalised within the cell. Calcium oxalate agglomerations (>10 microm) were also largely absent for controls or sense mCLC-5 transfectants (11% and 9% of cells, respectively) with COM crystal agglomerates predominating in antisense CLC-5 transfectants (66%, P<0.0001). We conclude that collecting duct cells with reduced CLC-5 expression lead to a tendency to form calcium crystal agglomeration, which may help explain the nephrocalcinosis and nephrolithiasis seen in Dent's disease.     

Origin and evolution of Kinesin-like calmodulin-binding protein

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin-14 family, is a C-terminal microtubule motor with three unique domains including a myosin tail homology region 4 (MyTH4), a talin-like domain, and a calmodulin-binding domain (CBD). The MyTH4 and talin-like domains (found in some myosins) are not found in other reported kinesins. A calmodulin-binding kinesin called kinesin-C (SpKinC) isolated from sea urchin (Strongylocentrotus purpuratus) is the only reported kinesin with a CBD. Analysis of the completed genomes of Homo sapiens, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and a red alga (Cyanidioschyzon merolae 10D) did not reveal the presence of a KCBP. This prompted us to look at the origin of KCBP and its relationship to SpKinC. To address this, we isolated KCBP from a gymnosperm, Picea abies, and a green alga, Stichococcus bacillaris. In addition, database searches resulted in identification of KCBP in another green alga, Chlamydomonas reinhardtii, and several flowering plants. Gene tree analysis revealed that the motor domain of KCBPs belongs to a clade within the Kinesin-14 (C-terminal motors) family. Only land plants and green algae have a kinesin with the MyTH4 and talin-like domains of KCBP. Further, our analysis indicates that KCBP is highly conserved in green algae and land plants. SpKinC from sea urchin, which has the motor domain similar to KCBP and contains a CBD, lacks the MyTH4 and talin-like regions. Our analysis indicates that the KCBPs, SpKinC, and a subset of the kinesin-like proteins are all more closely related to one another than they are to any other kinesins, but that either KCBP gained the MyTH4 and talin-like domains or SpKinC lost them.     

Gliding behaviour elicited by lateral looming stimuli in flying locusts

We challenged tethered, flying locusts with visual stimuli looming from the side towards one eye in a way that mimics the approach of a predatory bird. Locusts respond to the lateral approach of a looming object with steering movements and a stereotyped, rapid behaviour in which the wingbeat pattern ceases and the wings are swept into a gliding posture. This gliding behaviour may cause the locust to dive. The gliding posture is maintained for 200 ms or more after which flight is resumed with an increased wingbeat frequency or else the wings are folded. A glide begins with a strong burst of activity in the mesothoracic second tergosternal motor neuron (no. 84) on both sides of the locust. Recordings of descending contralateral movement detector (DCMD) activity in a flying locust show that it responds to small (80-mm diameter) looming stimuli during tethered flight, with a prolonged burst of spikes that tracks stimulus approach and reaches peak instantaneous frequencies as, or after, stimulus motion ceases. There is a close match between the visual stimuli that elicit a gliding behaviour and those that are effective at exciting the DCMD neuron. Wing elevation into the gliding posture occurs during a maintained burst of high frequency DCMD spikes.     

Functional organization of the bovine rumen epithelium

The functional organization of the bovine rumen epithelium has been examined by electron and light microscopy combined with immunocytochemistry to define a transport model for this epithelium. Expression of connexin 43, an integral component of gap junctions, the tight-junction molecules claudin-1 and zonula occludens 1 (ZO-1), and the catalytic alpha-subunit of Na(+)-K(+)-ATPase was demonstrated by SDS-PAGE and Western blotting. From the lumen surface, four cell layers can be distinguished: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Both claudin-1 and ZO-1 immunostaining showed plasma membrane staining, which was present at the stratum granulosum with decreasing intensity through the stratum spinosum to the stratum basale. The stratum corneum was negative for claudin-1 immunostaining. Transmission electron microscopy confirmed that occluding tight junctions were present at the stratum granulosum. Plasma membrane connexin 43 immunostaining was most intense at the stratum granulosum and decreased in intensity through stratum spinosum and stratum basale. There was intense immunostaining of the stratum basale for Na(+)-K(+)-ATPase, with weak staining of the stratum spinosum. Both the stratum granulosum and the stratum corneum were essentially negative. Stratum basale cells also displayed a high mitochondrial density relative to more apical cell layers. We conclude that epithelial barrier function may be attributed to the stratum granulosum and that cell-cell gap junctions allow diffusion to interconnect the barrier cell layer with the stratum basale where Na(+)-K(+)-ATPase is concentrated.     

Functional selectivity, ligand-directed trafficking, conformation-specific agonism: what's in a name?

Research on the design of compounds to selectively affect specific subsets of signals downstream of receptors has burgeoned lately, and several reports discussed at Experimental Biology 2005 indicate progress is being made in the understanding of what makes a drug functionally selective. Different conformations adopted by receptors after associating with specific ligands can determine which intracellular signaling pathways get activated and which do not. The appeal of such specific compounds is enormous when one considers that many disease states might require the subtle manipulation of some (or even one) but not all downstream events stemming from specific receptor activation. Additionally, a better understanding of functional selectivity would likely improve the drug delivery process: if compounds are screened through several functional assays appropriately designed to look for compounds exhibiting a high degree of selectivity, then many potential lead compounds might not be as frequently overlooked.