The plant hormone auxin regulates various developmental processes including root formation, vascular development, and gravitropism. Mutations within the AUX1 gene confer an auxin-resistant root growth phenotype and abolish root gravitropic curvature. Polypeptide sequence similarity to amino acid permeases suggests that AUX1 mediates the transport of an amino acid-like signaling molecule. Indole-3-acetic acid, the major form of auxin in higher plants, is structurally similar to tryptophan and is a likely substrate for the AUX1 gene product. The cloned AUX1 gene can restore the auxin-responsiveness of transgenic aux1 roots. Spatially, AUX1 is expressed in root apical tissues that regulate root gravitropic curvature.

SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost.

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

Biosensors are ideally portable, low-cost tools for the rapid detection of pathogens, proteins, and other analytes. The global biosensor market is currently worth over 10 billion dollars annually and is a burgeoning field of interdisciplinary research that is hailed as a potential revolution in consumer, healthcare, and industrial testing. A key barrier to the widespread adoption of biosensors, however, is their cost. Although many systems have been validated in the laboratory setting and biosensors for a range of analytes are proven at the concept level, many have yet to make a strong commercial case for their acceptance. Though it is true with the development of cheaper electrodes, circuits, and components that there is a downward pressure on costs, there is also an emerging trend toward the development of multianalyte biosensors that is pushing in the other direction. One way to reduce the cost that is suitable for certain systems is to enable their reuse, thus reducing the cost per test. Regenerating biosensors is a technique that can often be used in conjunction with existing systems in order to reduce costs and accelerate the commercialization process. This article discusses the merits and drawbacks of regeneration schemes that have been proven in various biosensor systems and indicates parameters for successful regeneration based on a systematic review of the literature. It also outlines some of the difficulties encountered when considering the role of regeneration at the point of use. A brief meta-analysis has been included in this review to develop a working definition for biosensor regeneration, and using this analysis only ∼60% of the reported studies analyzed were deemed a success. This highlights the variation within the field and the need to normalize regeneration as a standard process across the field by establishing a consensus term.

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Transduction of the auxin imulus in plants Is thought to entail binding of the hormone to a soluble auxin-binding protein (ABP) outside the cell and subsequent interaction between this auxin-protein complex and an integral membrane receptor ("docking") [Ca2+] with EGTA had no effect on IK,.l response to the peptide. However, virtually complete and reversible block of the response was achieved when cytoplasmic pH (pH) was brought under experimental control using the weak acid butyrate. Parallel measurements of pH, using the fluorescent dye 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein (BCECF) and dual-wavelength laser-scanning confocal microscopy demonstrated that the C-terminal peptide evoked rapid and reversible cytoplasmic alkalnizations of 0.4 ± 0.1 pH, unit and confirmed the antagonism of the pH, response in the presence of butyrate. These, and comparable results with the auxins indole acetic acid and 1-naphthyleneacetic acid, implicate the C-terminal domain of ABPzml in auxin-ABP coupling to pH and an associated intracellular signaling cascade. stimulus by associating with an integral membrane "docking" protein at the plasma membrane (1,9).Some support for this scheme has come from immunochemical studies. Microelectrode impalements of mesophyil protoplasts have yielded evidence of auxin-evoked and anti-ABP-sensitive membrane hyperpolarizations that are thought to reflect the activity of the plasma membrane H+-ATPase (10, 11). Furthermore, auxin-agonist activity has been found with polyclonal antibodies to the putative auxinbinding domain of ABPzml, the dominant ABP from Zea (12). Yet, while otherwise compelling, these studies have only confirmed an antigenic similarity between the ABP and the auxin receptor in vivo. Evidence for the postulated docking protein is still lacking.As an alternative approach, we have used synthetic peptides corresponding to surface domains ofthe protein (13, 14) to probe ABP structure directly, assaying for function in modulating ionic second messengers and ion transport across the guard cell plasma membrane. In guard cells, which regulate gas exchange through the stomatal pores of higherplant leaves, auxin coordinates the activities of [Ca2+]i-and pHi-sensitive K+ channels and of anion channels to effect stomatal opening and closing (1,15). Here we report that biological activity of ABPzml is associated with the C-terminal region of the protein and that its activity in controlling guard cell K+ channels is mediated through cytoplasmic alkalinization.MATERIALS AND METHODS Cells and Protocol. Viciafaba L. was grown and epidermal peels containing the guard cells were prepared as described (16,17). Peels were bathed in fast-flowing solutions containing 5 mM Ca2+ Mes (pH 6.1) ([Ca2+] 1 mM), and KCI and sodium butyrate (pH 6.1) were added as required. Peptides were added under stopped-flow conditions by complete exchange with 5x the chamber volume. Control experiments showed no measurable effect of stopped flow over the periods of these exposures.Electrophysiology and ...

Current diagnostic tests often fail to diagnose anastomotic leak at an early stage that enables timely intervention and minimizes serious morbidity and mortality. Emerging technologies, based on detection of local biomarkers, have achieved proof of concept status but require further evaluation to determine whether they translate into improved patient outcomes. Further research is needed to address this important, yet relatively unrecognized, area of unmet clinical need.