Critical review of dog detection and the influences of physiology, training, and analytical methodologies.

Detection dogs serve a plethora of roles within modern society, and are relied upon to identify threats such as explosives and narcotics. Despite their importance, research and training regarding detection dogs has involved ambiguity. This is partially due to the fact that the assessment of effectiveness regarding detection dogs continues to be entrenched within a traditional, non-scientific understanding. Furthermore, the capabilities of detection dogs are also based on their olfactory physiology and training methodologies, both of which are hampered by knowledge gaps. Additionally, the future of detection dogs is strongly influenced by welfare and social implications. Most importantly however, is the emergence of progressively inexpensive and efficacious analytical methodologies including gas chromatography related techniques, "e-noses", and capillary electrophoresis. These analytical methodologies provide both an alternative and assistor for the detection dog industry, however the interrelationship between these two detection paradigms requires clarification. These factors, when considering their relative contributions, illustrate a need to address research gaps, formalise the detection dog industry and research process, as well as take into consideration analytical methodologies and their influence on the future status of detection dogs. This review offers an integrated assessment of the factors involved in order to determine the current and future status of detection dogs.

Establishing the volatile profile of pig carcasses as analogues for human decomposition during the early postmortem period.

Following a mass disaster, it is important that victims are rapidly located as the chances of survival decrease greatly after approximately 48 h. Urban search and rescue (USAR) teams may use a range of tools to assist their efforts but detector dogs still remain one of the most effective search tools to locate victims of mass disasters. USAR teams can choose to deploy human scent dogs (trained to locate living victims) or human remains detection (HRD) dogs (trained to locate deceased victims). However, little is known about the variation between live human scent and postmortem human remains scent and the timeframe during which one type of scent transitions to the other. The aim of the current study was to measure the change in the scent profile of human decomposition analogues during the first 72 h postmortem by measuring the volatile organic compounds (VOCs) that comprise the odour. Three pig carcasses (Sus scrofa domesticus L.) were placed on a soil surface and allowed to decompose under natural conditions. Decomposition odour was sampled frequently up to 75 h postmortem and analysed using comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GC×GC-TOFMS). A total of 105 postmortem VOCs were identified during the early postmortem period. The VOC profile during the early postmortem period was highly dynamic, changing both hourly and daily. A transition period was observed after 43 h postmortem, where the VOC profile appeared to shift from a distinct antemortem odour to a more generalised postmortem odour. These findings are important in informing USAR teams and their use of detector dogs for disaster victim recovery.

In vitro volatile organic compound profiling using GC×GC-TOFMS to differentiate bacteria associated with lung infections: a proof-of-concept study.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Profiling the decomposition odour at the grave surface before and after probing.

Human remains detection (HRD) dogs are recognised as a valuable and non-invasive search method for remains concealed in many different environments, including clandestine graves. However, the search for buried remains can be a challenging task as minimal odour may be available at the grave surface for detection by the dogs. Handlers often use a soil probe during these searches in an attempt to increase the amount of odour available for detection, but soil probing is considered an invasive search technique. The aim of this study was to determine whether the soil probe assists with increasing the abundance of volatile organic compounds (VOCs) available at the grave surface. A proof-of-concept method was developed using porcine remains to collect VOCs within the grave without disturbing the burial environment, and to compare their abundance at the grave surface before and after probing. Detection and identification of the VOC profiles required the use of comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) due to its superior sensitivity and selectivity for decomposition odour profiling. The abundance of decomposition VOCs was consistently higher within the grave environment compared to the grave surface, except when the grave surface had been disturbed, confirming the reduced availability of odour at the grave surface. Although probing appeared to increase the abundance of VOCs at the grave surface on many of the sampling days, there were no clear trends identified across the study and no direct relationships with the environmental variables measured. Typically, the decomposition VOCs that were most prevalent in the grave soil were the same VOCs detected at the grave surface, whereas the trace VOCs detected in these environments varied throughout the post-burial period. This study highlighted that probing the soil can assist with releasing decomposition VOCs but is likely correlated to environmental and burial variables which require further study. The use of a soil probe to assist HRD dogs should not be disregarded but should only follow the use of non-invasive methods if deemed appropriate.

The effect of soil texture on the degradation of textiles associated with buried bodies.

There are many factors which affect the rate of decomposition in a grave site including; the depth of burial, climatic conditions, physical conditions of the soil (e.g. texture, pH, moisture), and method of burial (e.g. clothing, wrappings). Clothing is often studied as a factor that can slow the rate of soft tissue decomposition. In contrast, the effect of soft tissue decomposition on the rate of textile degradation is usually reported as anecdotal evidence rather than being studied under controlled conditions. The majority of studies in this area have focused on the degradation of textiles buried directly in soil. The purpose of this study was to investigate the effect of soil texture on the degradation and/or preservation of textile materials associated with buried bodies. The study involved the burial of clothed domestic pig carcasses and control clothing in contrasting soil textures (silty clay loam, fine sand and fine sandy loam) at three field sites in southern Ontario, Canada. Graves were exhumed after 2, 12 and 14 months burial to observe the degree of degradation for both natural and synthetic textiles. Recovered textile samples were chemically analyzed using infrared (IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) to investigate the lipid decomposition by-products retained in the textiles. The findings of this study demonstrate that natural textile in contact with a buried decomposing body will be preserved for longer periods of time when compared to the same textile buried directly in soil and not in contact with a body. The soil texture did not visually impact the degree of degradation or preservation. Furthermore, the natural-synthetic textile blend was resistant to degradation, regardless of soil texture, contact with the body or time since deposition. Chemical analysis of the textiles using GC-MS correctly identified a lipid degradation profile consistent with the degree of soft tissue decomposition. Such information may be important for estimating time since deposition in instances where only grave goods and associated materials are recovered from a burial site.

Analytical separations of mammalian decomposition products for forensic science: a review.

The study of mammalian soft tissue decomposition is an emerging area in forensic science, with a major focus of the research being the use of various chemical and biological methods to study the fate of human remains in the environment. Decomposition of mammalian soft tissue is a postmortem process that, depending on environmental conditions and physiological factors, will proceed until complete disintegration of the tissue. The major stages of decomposition involve complex reactions which result in the chemical breakdown of the body's main constituents; lipids, proteins, and carbohydrates. The first step to understanding this chemistry is identifying the compounds present in decomposition fluids and determining when they are produced. This paper provides an overview of decomposition chemistry and reviews recent advances in this area utilising analytical separation science.

A capillary electrophoresis method for the determination of selected biogenic amines and amino acids in mammalian decomposition fluid.

A simple capillary zone electrophoresis method for the determination of selected biogenic amines (tyramine and tryptamine) and amino acids (tryptophan, phenylalanine and tyrosine) in mammalian decomposition fluids is presented. Separations were carried out in a fused silica capillary (75microm i.d., total length 65cm, effective length 56cm) with detection by ultraviolet absorbance spectrophotometry at 200nm. In order to improve resolution and total analysis time, the method was subjected to optimisation utilising a chemometric approach. A screening design was carried out followed by a central composite design (CCD), using peak resolution and total analysis time as response factors. The influences of four experimental variables (pH, background electrolyte concentration, percentage of organic modifier (methanol) and applied voltage) were investigated. Optimum separation conditions were determined to be; a background electrolyte of boric acid (70mM) adjusted to pH 9.5 with 0.1M sodium hydroxide with 32% methanol (v/v). Applied voltage was 30kV, with the resulting current being less than 26microA. Under these conditions the analytes were separated within 12min. Tryptamine, tyramine, tryptophan, tyrosine and phenylalanine were identified by migration time and spiking in porcine decomposition fluids.

Studies of adipocere using attenuated total reflectance infrared spectroscopy.

Attenuated total reflectance (ATR) infrared spectroscopy has been used to characterize pig adipocere formation. The compositions of adipocere samples obtained by burial of pig adipose tissue in soil and in mock coffins were compared with that of the original adipose tissue using this technique. The ART spectra show that bands resulting from triglyceride and fatty acid C=O stretching are particularly useful for monitoring the changes in adipocere formation. The technique is able to be used to investigate how the burial environment affects the rate of adipocere formation and supports the results of earlier gas chromatograph and mass spectrometer and diffuse reflectance infrared studies. ATR spectroscopy has also been demonstrated to have the advantage of convenient sample preparation.

Lacrosse stick entrapment injury to the thumb.

A case of injury to the left thumb following an errant stick check, and subsequent entrapment of the digit in the open sidewall of a lacrosse stick, is presented. A circumferential laceration, severe swelling, and bruising to the proximal phalanx resulted. This case report emphasises the need to limit the dimensions of openings in the sidewalls of lacrosse sticks to prevent the occurrence of this and other preventable injuries.

The identification of adipocere in grave soils.

Soil samples recovered from grave exhumations have been analysed in an attempt to identify and characterise adipocere contained in the samples. The soil samples were collected from different environments, including samples recovered from forensic grave sites. Gas chromatography-mass spectrometry (GC-MS) was employed to identify adipocere and characterise the fatty acid composition. X-ray diffraction was used to characterise the soil environments.

Targeted transgenic expression of beta(2)-adrenergic receptors to type II cells increases alveolar fluid clearance.

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.

Polymorphic deletion of three intracellular acidic residues of the alpha 2B-adrenergic receptor decreases G protein-coupled receptor kinase-mediated phosphorylation and desensitization.

A polymorphic variant of the human alpha(2B)-adrenergic receptor (alpha(2B)AR), which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301-303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the alpha(2B)ARs and GRK2 revealed that the Del301-303 receptor displayed approximately 56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301-303 alpha(2B)AR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.

An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling.

alpha(2A)-Adrenergic receptors (alpha(2A)AR) are presynaptic autoinhibitory receptors of noradrenergic neurons in the central and peripheral sympathetic nervous systems, which act to dynamically regulate neurotransmitter release. Signaling through the G(i)/G(o) family of G-proteins, the receptor subserves numerous homeostatic and central nervous system functions. A single nucleotide polymorphism of this receptor, which results in an Asn to Lys substitution at amino acid 251 of the third intracellular loop, was identified in the human population. The frequency of Lys-251 was 10-fold greater in African-Americans than in Caucasians, but was not associated with essential hypertension. To determine the consequences of this substitution, wild-type and Lys-251 receptors were expressed in CHO and COS-7 cells. Expression, ligand binding, and basal receptor function were unaffected by the substitution. However, agonist-promoted [(35)S]GTPgammaS binding was approximately 40% greater with the Lys-251 receptor. This enhanced agonist function was observed with catecholamines, azepines, and imidazolines albeit to different degrees. In studies of agonist-promoted functional coupling to G(i), the polymorphic receptor displayed enhanced inhibition of adenylyl cyclase (60 +/- 4. 4 versus 46 +/- 4.1% inhibition) and markedly enhanced stimulation of MAP kinase (57 +/- 9 versus 15- +/- 2-fold increase over basal) compared with wild-type alpha(2A)AR. The potency of epinephrine in stimulating inositol phosphate accumulation was increased approximately 4 fold with the Lys-251 receptor. Unlike previously described variants of G-protein-coupled receptors, where the minor species causes either a loss of function or increased non-agonist function, Lys-251 alpha(2A)AR represents a new class of polymorphism whose phenotype is a gain of agonist-promoted function.

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction.

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.

A four amino acid deletion polymorphism in the third intracellular loop of the human alpha 2C-adrenergic receptor confers impaired coupling to multiple effectors.

The alpha(2)-adrenergic receptors (alpha(2)ARs) play a critical role in modulating neurotransmitter release in the central and peripheral sympathetic nervous systems. A polymorphism of the alpha(2)AR subtype localized to human chromosome 4 (the pharmacologic alpha(2C)AR subtype) within an intracellular domain has been identified in normal individuals. The polymorphism (denoted Del322-325) is because of an in-frame 12-nucleic acid deletion encoding a receptor lacking Gly-Ala-Gly-Pro in the third intracellular loop. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were permanently transfected with constructs encoding wild-type human alpha(2C)AR and the polymorphic receptor. The Del322-325 variant had decreased high affinity agonist binding (K(H) = 7.3 +/- 0.95 versus 3.7 +/- 0.43 nm; %R(H) = 31 +/- 4 versus 49 +/- 4) compared with wild-type indicating impaired formation of the agonist-receptor-G protein complex. The polymorphic receptor displayed markedly depressed epinephrine-promoted coupling to G(i), inhibiting adenylyl cyclase by 10 +/- 4.3% compared with 73 +/- 2.4% for wild-type alpha(2C)AR. This also was so for the endogenous ligand norepinephrine and full and partial synthetic agonists. Depressed agonist-promoted coupling to the stimulation of MAP kinase ( approximately 71% impaired) and inositol phosphate production ( approximately 60% impaired) was also found with the polymorphic receptor. The Del322-325 receptor was approximately 10 times more frequent in African-Americans compared with Caucasians (allele frequencies 0.381 versus 0.040). Given this significant loss of function phenotype in several signal transduction cascades and the skewed ethnic prevalence, Del322-325 represents a pharmacoethnogenetic locus and may also be the basis for interindividual variation in cardiovascular or central nervous system pathophysiology.

Fusion of beta 2-adrenergic receptor to G alpha s in mammalian cells: identification of a specific signal transduction species not characteristic of constitutive activation or precoupling.

The forward and antegrade interactions that comprise the agonist receptor-G protein complex were studied in Chinese hamster fibroblasts transfected to express the beta(2)-adrenergic receptor (beta(2)AR), the beta(2)AR and the alpha-subunit of its cognate G protein (G(s)), and a protein consisting of the beta(2)AR fused at its carboxy terminus with G(alpha)(s) (beta(2)AR-G(s)). Expression levels were matched at approximately 600 fmol/mg. Basal adenylyl cyclase activities were increased with the fusion receptor membranes compared to coexpressed receptor plus G(alpha)(s), and to wild-type beta(2)AR (20.5 +/- 1.8 vs 9.0 +/- 0.88 vs 8.7 +/- 0.93 pmol min(-)(1) mg(-)(1)), confirming in mammalian cells that the fusion of beta(2)AR and G(alpha)(s) results in a state not attained by expression of unfused components. However, agonist-stimulated activities were not increased proportionally, such that the stimulation over basal of the beta(2)AR-G(s) fusion protein (1. 5-fold) was less than wild-type beta(2)AR (2.1-fold). Agonist competition studies performed in the absence of guanine nucleotide exhibited high-affinity binding sites with a lower K(H) (1.75 vs 8. 47 nM) and greater %R(H) (51% vs 44%) for beta(2)AR-G(s), but GppNHp failed to convert most of these to the low-affinity state. Functional studies with the inverse agonist ICI 118551 did not show enhanced efficacy or potency with the fusion protein. Adenylyl cyclase studies with three partial agonists with diverse structures (dobutamine, ritodrine, and phenylephrine) showed no enhancement of efficacy with beta(2)AR-G(s) and a minor trend toward enhanced potency. Taken together, these results indicate that the tethering of G(alpha)(s) to the beta(2)AR causes a conformational change in the receptor that stabilizes a species "trapped" between the non-guanine nucleotide-bound state and the GTP-bound form. Functionally the receptor is not characterized by a consistent pattern of properties ascribed to other states such as constitutive activation or precoupling, but rather represents a unique state in the transition from high- to low-affinity forms.

Transgenic overexpression of beta(2)-adrenergic receptors in airway smooth muscle alters myocyte function and ablates bronchial hyperreactivity.

beta(2)-Adrenergic receptors (beta(2)AR) act to relax airway smooth muscle and can serve to counteract hyperresponsiveness, although the effect may not be ablative even in the presence of exogenous agonist. Within this signaling cascade that ultimately transduces smooth muscle relaxation, a significant "spare receptor" pool has been hypothesized to be present in the airway. In order to modify the relationship between beta(2)AR and downstream effectors, transgenic mice (TG) were created overexpressing beta(2)AR approximately 75-fold in airway smooth muscle using a mouse smooth muscle alpha-actin promoter. While >90% of these receptors were expressed on the smooth muscle cell surface, the percentage of receptors able to form the agonist-promoted high affinity complex was less than that found with nontransgenic (NTG) cells (R(H) = 18 versus 36%). Nevertheless, beta(2)AR signaling was found to be enhanced. Intact airway smooth muscle cells from TG had basal cAMP levels that were greater than NTG cells. A marked increase in agonist-stimulated cAMP levels was found in the TG ( approximately 200% stimulation over basal) compared with NTG ( approximately 50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC(50) for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to beta-agonist, with a 60-fold decrease in the ED(50), indicating that the enhanced signaling imposed by overexpression results in an increase in the coordinated function of the intact airway cells. In vivo studies showed a significantly blunted airway resistance response to the inhaled bronchoconstrictor methacholine in the TG mice. Indeed, with beta-agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with beta(2)AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the beta(2)AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an anti-asthma phenotype.

Polymorphisms of the 5' leader cistron of the human beta2-adrenergic receptor regulate receptor expression.

Cellular expression of the beta2-adrenergic receptor (beta2AR) is controlled in part by a 19-amino acid peptide that regulates mRNA translation. This peptide is encoded by a short open reading frame, termed the 5' leader cistron (5'LC), which is 102 bp upstream of the beta2AR coding block. In 176 normal subjects we found a single nucleotide polymorphism resulting in either Arg (previously denoted wild-type) or Cys at position 19 of this peptide. Allele frequencies were 0.37 for Arg and 0.63 for Cys. To determine if these variants altered beta2AR expression, COS-7 cells were transfected with polymorphic constructs consisting of 1,989 bp encompassing the 5'LC and the beta2AR coding block exactly as found in the human gene. beta2AR density, as determined by [125I]CYP radioligand binding, was 72% higher in cells transfected with the 5'LC-Cys19 construct as compared with those transfected with the 5'LC-Arg19 construct and 110% higher when a cotransfection technique with a luciferase construct was used to control for transfection efficiency. Levels of the two mRNA transcripts were not different, confirming in vitro studies that the upstream peptide regulates receptor expression at the translational level. In human airway smooth muscle cells that natively express beta2AR, receptor expression was approximately twofold higher in those bearing the Cys versus the Arg polymorphism, confirming the phenotype in a relevant cell type. Linkage disequilibrium was observed between the 5'LC-Cys polymorphism and the beta2AR coding block polymorphisms Arg16 and Gln27 (P < 0.0001), although several different haplotypes were identified. Thus, beta2AR expression in the human population is controlled by a common polymorphism of this 5'LC, and may be responsible for interindividual variation in betaAR responsiveness.

Eye injuries in women's lacrosse: strict rule enforcement and mandatory eyewear required.

OBJECTIVE: To inform practitioners of the risk of ocular injury in women's lacrosse and to advocate the mandatory use of protective eyewear. DESIGN, MATERIALS AND METHODS: Athletes reported retrospectively, via a questionnaire, on the nature and circumstances leading to their injuries. MEASUREMENTS AND MAIN RESULTS: Four case reports of injury to ocular and adjacent nasal structures are presented. None of the athletes was wearing protective equipment at the time that her injuries were incurred. CONCLUSIONS: Mandatory eye protection should be introduced into the game of women's field lacrosse. Eyewear may have prevented the injuries reported in this article.