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Simple Summary: The instability of viral RNA, which is susceptible to ubiquitous RNases, poses a significant challenge for its transport and detection in diagnosis. To address this challenge, this study aimed to evaluate the stability and detection limits of various RNA viruses, including the avian influenza virus, Newcastle disease virus, and African horse sickness virus, on Flinders Technology Associates cards. This investigation provides empirical evidence supporting the efficacy of Flinders Technology Associates cards for sample collection and subsequent viral RNA recovery, highlighting their suitability for use in molecular diagnostics. Consequently, based on the demonstrated effectiveness, stability, and safety implications observed in this study, Flinders Technology Associates cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens. The Flinders Technology Associates (FTA) card, a cotton-based cellulose membrane impregnated with a chaotropic agent, effectively inactivates infectious microorganisms, lyses cellular material, and fixes nucleic acid. The aim of this study is to assess the stability and detection limit of various RNA viruses, especially the avian influenza virus (AIV), Newcastle disease virus (NDV), and African horse sickness virus (AHSV), on the FTA card, which could significantly impact virus storage and transport practices. To achieve this, each virus dilution was inoculated onto an FTA card and stored at room temperature in plastic bags for durations ranging from 1 week to 6 months. Following storage, the target genome was detected using conventional reverse transcription polymerase chain reaction. The present study demonstrated that the detection limit of AIV ranged from 1.17 to 6.17 EID50 values over durations ranging from 1 week to 5 months, while for NDV, it ranged from 2.83 to 5.83 ELD50 over the same duration. Additionally, the detection limit of AHSV was determined as 4.01 PFU for both 1 and 2 weeks, respectively. Based on the demonstrated effectiveness, stability, and safety implications observed in the study, FTA cards are recommended for virus storage and transport, thus facilitating the molecular detection and identification of RNA viral pathogens. [ABSTRACT FROM AUTHOR]

Functional gastrointestinal disorders (FGIDs) have prominent sex differences in incidence, symptoms, and treatment response that are not well understood. Androgens are steroid hormones present at much higher levels in males than females and could be involved in these differences. In adults with irritable bowel syndrome (IBS), a FGID that affects 5% to 10% of the population worldwide, we found that free testosterone levels were lower than those in healthy controls and inversely correlated with symptom severity. To determine how this diminished androgen signaling could contribute to bowel dysfunction, we depleted gonadal androgens in adult mice and found that this caused a profound deficit in gastrointestinal transit. Restoring a single androgen hormone was sufficient to rescue this deficit, suggesting that circulating androgens are essential for normal bowel motility in vivo. To determine the site of action, we probed androgen receptor expression in the intestine and discovered, unexpectedly, that a large subset of enteric neurons became androgen-responsive upon puberty. Androgen signaling to these neurons was required for normal colonic motility in adult mice. Taken together, these observations establish a role for gonadal androgens in the neural regulation of bowel function and link altered androgen levels with a common digestive disorder., Introduction Almost all disorders of the gut-brain axis, from inflammatory bowel disease to neurological disorders with GI comorbidities, exhibit sex differences (1-3). Irritable bowel syndrome (IBS) is one of the [...]

Introduction γ-amino butyric acid (GABA) is not only the major inhibitory neurotransmitter in the central nervous system (CNS), but it also plays an important role in the lung, mediating airway smooth muscle relaxation and mucus production. As kinases such as protein kinase A (PKA) are known to regulate the release and reuptake of GABA in the CNS by GABA transporters, we hypothesized that β-agonists would affect GABA release from airway epithelial cells through activation of PKA. Methods C57/BL6 mice received a pretreatment of a β-agonist or vehicle (PBS), followed by methacholine or PBS. Bronchoalveolar lavage (BAL) was collected and the amount of GABA was quantified using HPLC mass spectrometry. For in vitro studies, cultured BEAS-2B human airway epithelial cells were loaded with .sup.3H-GABA. .sup.3H-GABA released was measured during activation and inhibition of PKA and tyrosine kinase signaling pathways. Results β-agonist pretreatment prior to methacholine challenge attenuated in vivo GABA release in mouse BAL and .sup.3H-GABA release from depolarized BEAS-2B cells. GABA release was also decreased in BEAS-2B cells by increases in cAMP but not by Epac or tyrosine kinase activation. Conclusion β-agonists decrease GABA release from airway epithelium through the activation of cAMP and PKA. This has important therapeutic implications as β-agonists and GABA are important mediators of both mucus production and airway smooth muscle tone., Author(s): Jennifer Danielsson[sup.1] , Sarah Zaidi[sup.2] , Benjamin Kim[sup.3] , Hiromi Funayama[sup.1] [sup.4] , Peter D. Yim[sup.1] , Dingbang Xu[sup.1] , Tilla S. Worgall[sup.3] , George Gallos[sup.1] , Charles W. [...]

Tumor necrosis factor (TNF)-[alpha] is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-[alpha] has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-[alpha] receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-[alpha] in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or Gi proteins. TNF-[alpha] caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-[alpha] also increased phosphorylation of [Ser.sup.338] on raf-1 kinase, indicative of activation. IL-1[beta] and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-[alpha] transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase. GW5074; mitogen-activated protein kinase; phosphorylation; immunoblot; cell culture doi:10.1152/ajplung.00123.2006.

Vascular disease produces changes in lumenal shape evident in magnetic resonance angiography (MRA). However, quantification of vascular shape from MRA is problematic due to image artifacts. Prior deformable models for vascular surface reconstruction primarily resolve problems of initialization of the surface mesh. However, initialization can be obtained in a trivial manner for MRA using isosurfaces. We propose a methodology for deforming the isosurface to conform to the boundaries of objects in the image with minimal a priori assumptions of object shape. As in conventional methods, external forces attract the surface toward edges in the image. However, smoothing is produced by a moment that aligns the normals of adjacent surface triangles. Notably, the moment produces no translational motion of surface triangles. The deformable isosurface was applied to a digital phantom of a stenotic artery, to MRA of three renal arteries with atherosclerotic disease and MRA of one carotid artery with atherosclerotic disease. Results of the surface reconstruction from the deformable model were compared with conventional X-ray angiography for the renal arteries. Measurement of the degree of stenosis of the renal arteries was within 12% [+ or -] 6%. The deformable model provided improvements over the isosurface in all cases in terms of measurement of the degree of stenosis or improving the surface smoothness. Index Terms--Carotid artery, deformable model, magnetic resonance angiography, renal artery.

Three-dimensional (3-D) angiographic methods are gaining acceptance for evaluation of atherosclerotic disease. However, measurement of vessel stenosis from 3-D angiographic methods can be problematic due to limited image resolution and contrast. We present a method for reconstructing vessel surfaces from 3-D angiographic methods that allows for objective measurement of vessel stenosis. The method is a deformable model that employs a tubular coordinate system. Vertex merging is incorporated into the coordinate system to maintain even vertex spacing and to avoid problems of self-intersection of the surface. The deformable model was evaluated on clinical magnetic resonance (MR) images of the carotid (n = 6) and renal (n = 2) arteries, on an MR image of a physical vascular phantom and on a digital vascular phantom. Only one gross error occurred for all clinical images. All reconstructed surfaces had a realistic, smooth appearance. For all segments of the physical vascular phantom, vessel radii from the surface reconstruction had an error of less than 0.2 of the average voxel dimension. Variability of manual initialization of the deformable model had negligible effect on the measurement of the degree of stenosis of the digital vascular phantom. Index Terms--Atherosclerosis, deformable model, magnetic resonance angiography, surface reconstruction.

Recently, we characterized blue light--mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic β2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2. [ABSTRACT FROM AUTHOR]

The clinical administration of GABAergic medications leads to hypotension which has classically been attributed to the modulation of neuronal activity in the central and peripheral nervous systems. However, certain types of peripheral smooth muscle cells have been shown to express GABAA receptors, which modulate smooth muscle tone, by the activation of these chloride channels on smooth muscle cell plasma membranes. Limited prior studies demonstrate that non-human large-caliber capacitance blood vessels mounted on a wire myograph are responsive to GABAA ligands. We questioned whether GABAA receptors are expressed in human resistance arteries and whether they modulate myogenic tone. We demonstrate the novel expression of GABAA subunits on vascular smooth muscle from small-caliber human omental and mouse tail resistance arteries. We show that GABAA receptors modulate both plasma membrane potential and calcium responses in primary cultured cells from human resistance arteries. Lastly, we demonstrate functional physiologic modulation of myogenic tone via GABAA receptor activation in human and mouse arteries. Together, these studies demonstrate a previously unrecognized role for GABAA receptors in the modulation of myogenic tone in mouse and human resistance arteries. [ABSTRACT FROM AUTHOR]

The protein actin can polymerize from monomeric globular G-actin to polymeric filamentary F-actin, under the regulation of thermodynamic variables such as temperature, pressure, and compositions of G-actin and salts. We present here new measurements of the extent of polymerization ([lowercase_phi_synonym]) of actin under pressure (P), for rabbit skeletal muscle actin in H2O buffer in the presence of adenosine triposphate and calcium ions and at low (5–15 mM) KCl concentrations. We measured [lowercase_phi_synonym] using pyrene-labeled actin, as a function of time (t) and temperature (T), for samples of fixed concentrations of initial G-actin and KCl and at fixed pressure. The [lowercase_phi_synonym](T,P) measurements at equilibrium have the same form as reported previously at 1 atm: low levels of polymerization at low temperatures, representing dimerization of the actin; an increase in [lowercase_phi_synonym] at the polymerization temperature (Tp); a maximum in [lowercase_phi_synonym](T) above Tp with a decrease in [lowercase_phi_synonym](T) beyond the maximum, indicating a depolymerization at higher T. From [lowercase_phi_synonym](T,P) at temperatures below Tp, we estimate the change in volume for the dimerization of actin, ΔVdim, to be -307±10 ml/mol at 279 K. The change of Tp with pressure dTp/dP=(0.3015±0.0009) K/MPa=(30.15±0.09) mK/atm. The [lowercase_phi_synonym](T,P) data at higher T indicate the change in volume on propagation, ΔVprop, to be +401±48 ml/mol at 301 K. The [lowercase_phi_synonym](t) measurements yield initial relaxation times rp(T) that reflect the behavior of [lowercase_phi_synonym](T) and support the presence of a depolymerization temperature. We also measured the density of polymerizing actin with a vibrating tube density meter, the results of which confirm that the data from this instrument are affected by viscosity changes and can be erroneous. [ABSTRACT FROM AUTHOR]

Studies of the dependence of actin polymerization on thermodynamic parameters are important for understanding processes in living systems, where actin polymerization and depolymerization are crucial to cell structure and movement. We report measurements of the extent of polymerization, F, of rabbit muscle actin as a function of temperature [T=(0 - 35)°C], initial G-actin concentration [[G[SUB0]]=(1 - 3) mg/ml], and initiating salt concentration [[KCl]=(5-15) mmol/l with bound Ca[SUP2+]], in H[SUB2]O and D[SUB2]O buffers and in the presence of adenosine triphosphate (ATP). A preliminary account of the data and analysis for H[SUB2]O buffers has appeared previously [P. S. Niranjan, J. G. Forbes, S. C. Greer, J. Dudowicz, K. F. Freed, and J. F. Douglas, J. Chem. Phys. 114, 10573 (2001)]. We describe the details of the studies for H[SUB2]O buffers, together with new data and analysis for D[SUB2]O buffers. The measurements show a maximum in φ(T) for H[SUB2]O buffers and D[SUB2]O buffers. For H[SUB2]O buffers, T[SUBp] decreases as either [G[SUB0]] or [KCl] increases. For D[SUB2]O buffers, T[SUBp] decreases as [KCl] increases, but T[SUBp] is not monotonic in [G[SUB0]]. The measurements are interpreted in terms of a Flory-Huggins-type lattice model that includes the essential steps: monomer activation, dimerization of activated species, and propagation of trimers to higher order polymers. The competition between monomer activation and chain propagation leads to the observed nonmonotonic variation of φ(T). The actin polymerization in D[SUB2]O buffer differs considerably from that in the H[SUB2]O buffer and underscores the significant deuterium effect on hydrophobic interactions and hydrogen bonding in the polymerization process. [ABSTRACT FROM AUTHOR]

Interpretation of magnetic resonance angiography (MRA) is problematic due to complexities of vascular shape and to artifacts such as the partial volume effect. We present new methods to assist in the interpretation of MRA. These include methods for detection of vessel paths and for determination of branching patterns of vascular trees. They are based on the ordered region growing (ORG) algorithm that represents the image as an acyclic graph, which can be reduced to a skeleton by specifying vessel endpoints or by a pruning process. Ambiguities in the vessel branching due to vessel overlap are effectively resolved by heuristic methods that incorporate a priori knowledge of bifurcation spacing. Vessel paths are detected at interactive speeds on a 500-MHz processor using vessel endpoints. These methods apply best to smaller vessels where the image intensity peaks at the center of the lumen which, for the abdominal MRA, includes vessels whose diameter is less than 1 cm. Index Terms--Magnetic resonance angiography, skeletonization, visualization.