Artificial neural network study of whole-cell bacterial bioreporter response determined using fluorescence flow cytometry.

Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

Whole-cell bioprocessing of human fetal cells for tissue engineering of skin.

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity (simple culture conditions), maintenance of differentiated phenotype for primary human cell culture and transmission of communicable diseases. Cultured primary fetal cells from one organ donation could possibly meet the exigent and stringent technical aspects for development of therapeutic products. Master and working cell banks from one fetal organ donation (skin) can be developed in short periods of time and safety tests can be performed at all stages of cell banking. For therapeutic use, fetal cells can be used up to two thirds of their life-span in an out-scaling process and consistency for several biological properties includes protein concentration, gene expression and biological activity. As it is the intention that banked primary fetal cells can profit from the prospected treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability and safety of the process including donor tissue selection, cell banking, cell testing and growth of cells in out-scaling for the preparation of whole-cell tissue-engineering products.

Electrochemical As(III) whole-cell based biochip sensor.

The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy.

Analysis of bioavailable arsenic in rice with whole cell living bioreporter bacteria.

A multiwell plate bioassay was developed using genetically modified bacteria (bioreporter cells) to detect inorganic arsenic extracted from rice. The bacterial cells expressed luciferase upon exposure to arsenite, the activity of which was detected by measurement of cellular bioluminescence. The bioreporter cells detected arsenic in all rice varieties tested, with averages of 0.02-0.15 microg of arsenite equivalent per gram of dry weight and a method detection limit of 6 ng of arsenite per gram of dry rice. This amounted to between approximately 20 and 90% of the total As content reported by chemical methods for the same sample and suggested that a major proportion of arsenic in rice is in the inorganic form. Calibrations of the bioassay with pure inorganic and organic arsenic forms showed that the bacterial cells react to arsenite with highest affinity, followed by arsenate (with 25% response relative to an equivalent arsenite concentration) and trimethylarsine oxide (at 10% relative response). A method for biocompatible arsenic extraction was elaborated, which most optimally consisted of (i) grinding rice to powder, (ii) mixing with an aqueous solution containing pancreatic enzymes, (iii) mechanical shearing, (iv) extraction in mild acid conditions and moderate heat, and (v) centrifugation and pH neutralization. Detection of mainly inorganic arsenic by the bacterial cells may have important advantages for toxicity assessment of rice consumption and would form a good complement to total chemical arsenic determination.

Whole-cell living biosensors--are they ready for environmental application?

Since the development of the first whole-cell living biosensor or bioreporter about 15 years ago, construction and testing of new genetically modified microorganisms for environmental sensing and reporting has proceeded at an ever increasing rate. One and a half decades appear as a reasonable time span for a new technology to reach the maturity needed for application and commercial success. It seems, however, that the research into cellular biosensors is still mostly in a proof-of-principle or demonstration phase and not close to extensive or commercial use outside of academia. In this review, we consider the motivations for bioreporter developments and discuss the suitability of extant bioreporters for the proposed applications to stimulate complementary research and to help researchers to develop realistic objectives. This includes the identification of some popular misconceptions about the qualities and shortcomings of bioreporters.

Comparison of naphthalene bioavailability determined by whole-cell biosensing and availability determined by extraction with Tenax

A rapid biological method for the determination of the bioavailability of naphthalene was developed and its value as an alternative to extraction-based chemical approaches demonstrated. Genetically engineered whole-cell biosensors are used to determine bioavailable naphthalene and their responses compared with results from Tenax extraction and chemical analysis. Results show a 1:1 correlation between biosensor results and chemical analyses for naphthalene-contaminated model materials and sediments, but the biosensor assay is much faster. This work demonstrates that biosensor technology can perform as well as standard chemical methods, though with some advantages including the inherent biological relevance of the response, rapid response time, and potential for field deployment. A survey of results from this work and the literature shows that bioavailability under non-equilibrium conditions nonetheless correlates well with K(oc) or K(d). A rationale is provided wherein chemical resistance is speculated to be operative.

Novel use of a whole cell E. coli bioreporter as a urinary exposure biomarker.

Bacterial bioreporters have substantial potential for contaminant assessment but their real world application is currently impaired by a lack of sensitivity. Here, we exploit the bioconcentration of chemicals in the urine of animals to facilitate pollutant detection. The shore crab Carcinus maenas was exposed to the organic contaminant 2-hydroxybiphenyl, and urine was screened using an Escherichia coli-based luciferase gene (luxAB) reporter assay specific to this compound. Bioassay measurements differentiated between the original contaminant and its metabolites, quantifying bioconcentration factors of up to one hundred-fold in crab urine. Our results reveal the substantial potential of using bacterial bioreporter assays in real-time monitoring of biological matricesto determine exposure histories, with wide ranging potential for the in situ measurement of xenobiotics in risk assessments and epidemiology.

Red blood cell structure and dynamics explored with digital holographic microspcopy

Digital holographic microscopy (DHM) is a technique that allows obtaining, from a single recorded hologram, quantitative phase image of living cell with interferometric accuracy. Specifically the optical phase shift induced by the specimen on the transmitted wave front can be regarded as a powerful endogenous contrast agent, depending on both the thickness and the refractive index of the sample. Thanks to a decoupling procedure cell thickness and intracellular refractive index can be measured separately. Consequently, Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), two highly relevant clinical parameters, have been measured non-invasively at a single cell level. The DHM nanometric axial and microsecond temporal sensitivities have permitted to measure the red blood cell membrane fluctuations (CMF) on the whole cell surface. ©2009 COPYRIGHT SPIE--The International Society for Optical Engineering.

Biologicals and fetal cell therapy for wound and scar management.

Few biopharmaceutical preparations developed from biologicals are available for tissue regeneration and scar management. When developing biological treatments with cellular therapy, selection of cell types and establishment of consistent cell banks are crucial steps in whole-cell bioprocessing. Various cell types have been used in treatment of wounds to reduce scar to date including autolog and allogenic skin cells, platelets, placenta, and amniotic extracts. Experience with fetal cells show that they may provide an interesting cell choice due to facility of outscaling and known properties for wound healing without scar. Differential gene profiling has helped to point to potential indicators of repair which include cell adhesion, extracellular matrix, cytokines, growth factors, and development. Safety has been evidenced in Phase I and II clinical fetal cell use for burn and wound treatments with different cell delivery systems. We present herein that fetal cells present technical and therapeutic advantages compared to other cell types for effective cell-based therapy for wound and scar management.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells.

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1.

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Membrane potential dynamics of neocortical projection neurons driving target-specific signals.

Primary sensory cortex discriminates incoming sensory information and generates multiple processing streams toward other cortical areas. However, the underlying cellular mechanisms remain unknown. Here, by making whole-cell recordings in primary somatosensory barrel cortex (S1) of behaving mice, we show that S1 neurons projecting to primary motor cortex (M1) and those projecting to secondary somatosensory cortex (S2) have distinct intrinsic membrane properties and exhibit markedly different membrane potential dynamics during behavior. Passive tactile stimulation evoked faster and larger postsynaptic potentials (PSPs) in M1-projecting neurons, rapidly driving phasic action potential firing, well-suited for stimulus detection. Repetitive active touch evoked strongly depressing PSPs and only transient firing in M1-projecting neurons. In contrast, PSP summation allowed S2-projecting neurons to robustly signal sensory information accumulated during repetitive touch, useful for encoding object features. Thus, target-specific transformation of sensory-evoked synaptic potentials by S1 projection neurons generates functionally distinct output signals for sensorimotor coordination and sensory perception.