Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P <0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P <0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

Shear Strength Model for FRP-Strengthened RC Beams with Adverse FRP-Steel Interaction

RC beams shear strengthened with externally bonded fiber-reinforced polymer (FRP) U strips or side strips usually fail owing to debonding of the bonded FRP shear reinforcement. Because such debonding usually occurs in a brittle manner at relatively small shear crack widths, some of the internal steel stirrups intersected by the critical shear crack may not have reached yielding at beam shear failure. Consequently, the yield stress of internal steel stirrups in such a strengthened RC beam cannot be fully utilized. This adverse shear interaction between the internal steel shear reinforcement and the external FRP shear reinforcement may significantly reduce the benefit of the shear strengthening FRP but has not been considered explicitly by any of the shear strength models in the existing design guidelines. This paper presents a new shear strength model considering this adverse shear interaction through the introduction of a shear interaction factor. A comprehensive evaluation of the proposed model, as well as three other shear strength models, is conducted using a large test database. It is shown that the proposed shear strength model performs the best among the models compared, and the performance of the other shear strength models can be significantly improved by including the proposed shear interaction factor. Finally, a design recommendation is presented.

Development and validation of the first high performance-lateral flow immunoassay (HP-LFIA) for the rapid screening of domoic acid from shellfish extracts

A lateral flow immunoassay (LFIA) has been developed and fully validated to detect the primary amnesic shellfish poisoning (ASP) toxin, domoic acid (DA). The performance characteristics of two versions of the test were investigated using spiked and naturally contaminated shellfish (mussels, scallops, oysters, clams, and cockles). The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits. The new rapid assay (LFIA version 2) was designed to overcome the performance limitations identified in the first version of the assay. The improved test uses an electronic reader to remove the subjective nature of the generated results, and the positive cut-off for screening of DA in shellfish was increased from 10 ppm (version 1) to 17.5 ppm (version 2). A simple extraction and test procedure was employed, which required minimal equipment and materials; results were available 15 min after sample preparation. Stability of the aqueous extracts at room temperature (22 C) at four time points (up to 245 min after extraction) and across a range of DA concentrations was 100.3±1.3% and 98.8±2.4% for pre- and post-buffered extracts, respectively. The assay can be used both within laboratory settings and in remote locations. The accuracy of the new assay, to indicate negative results at or below 10 ppm DA, and positive results at or above 17.5 ppm, was 99.5% (n=216 tests). Validation data were obtained from a 2-day, randomised, blind study consisting of multiple LFIA lots (n=3), readers (n=3) and operators (n=3), carrying out multiple extractions of mussel tissue (n=3) at each concentration (0, 10, 17.5, and 20 ppm). No matrix effects were observed on the performance of the assay with different species (mussels, scallops, oysters, clams, and cockles). There was no impact on accuracy or interference from other phycotoxins, glutamic acid or glutamine with various strip incubations (8, 10, and 12 min). The accuracy of the assay, using naturally contaminated samples to indicate negative results at or below 12.5 ppm and positive results at or above 17.5 ppm, was 100%. Variability between three LFIA lots across a range of DA concentrations, expressed as coefficient of variation (% CV), was 1.1±0.4% (n=2 days) based on quantitative readings from the electronic reader. During an 8 week stability study, accuracy of the method with test strips stored at various temperatures (6, 22, 37 and 50 C) was 100%. Validation for both versions included comparisons with results obtained using reference LC-UV methods. © 2013 Elsevier B.V.

Near Field Enhancement and Subwavelength Imaging Using Resonantly Loaded Apertures

It is demonstrated that the electromagnetic (EM) transmission through a subwavelength or non-resonant aperture in a conductive screen can be dramatically enhanced by loading it with folded metallic strips exhibiting resonant properties. When illuminated by an EM plane wave these loaded apertures enable very tight, subwavelength, collimation of the EM power in the near field zone. We propose planar and quasi-planar resonant insertion geometries that should allow, for the first time, two-dimensional dual-polarization subwavelength field confinement along with ability to focus both electric and magnetic fields. The proposed technique for resonance transmission enhancement and near field confinement forms a basis for a new class of microwave near field imaging probe with subwavelength resolution capable of operating over a wide range of imaging distances (0.05–$0.25lambda$). Measurement results demonstrate the possibility of high contrast (more than 3 dB in amplitude and 40 degrees in phase) near field subwavelength imaging of 2D and 3D resonant and non-resonant metallic and dielectric targets in free space and in moderately lossy layered media.

Mechanical properties of structures 3D printed with cementitious powders

The three dimensional (3D) printing technology has undergone rapid development in the last few years and it is now possible to print engineering structures. This paper presents a study of the mechanical behavior of 3D printed structures using cementitious powder. Microscopic observation reveals that the 3D printed products have a layered orthotropic microstructure, in which each layer consists of parallel strips. Compression and flexural tests were conducted to determine the mechanical properties and failure characteristics of such materials. The test results confirmed that the 3D printed structures are laminated with apparent orthotropy. Based on the experimental results, a stress-strain relationship and a failure criterion based on the maximum stress criterion for orthotropic materials are proposed for the structures of 3D printed material. Finally, a finite element analysis was conducted for a 3D printed shell structure, which shows that the printing direction has a significant influence on the load bearing capacity of the structure.

抗剪加固FRP与混凝土界面粘结性能的试验研究

In this study, 21 small beam specimens with a pre-set through-crack were tested with FRP strips bonded to the sides to study the bond behavior between shear-strengthening FRP and concrete (referred to as "the interface" for simplicity hereafter). The test parameters included the bond length, width, thickness of FRP strip and the angle between fiber orientation and crack opening direction (referred to as "fiber tensile angle" θ hereafter) on the bond behavior of the interface. Test results showed that: 1) the small beam test setup with a pre-set crack is suitable for studying the bond behavior between FRP and concrete; 2) the bond length, width, and thickness of the FRP as well as the fiber angle have significant effects on the bond strength; 3) the distribution of FRP strains along the direction perpendicular to the fiber orientation (FRP width direction) is non-uniform; this is mainly attributed to the progressive debonding of the FRP strips in the width direction, with the FRP at the location of larger crack width debonding earlier.

Strengthening timber beams with near surface mounted carbon fiber reinforced polymer rods

Many timber structures may require strengthening due to either decay and aging or an increase of load. This paper presents an experimental study in which eleven timber beams were tested, including three unstrengthened reference beams and eight beams strengthened with NSM CFRP bars. The test parameters include the position of NSM (tensile face or the bottom of the sides), the number of CFRP bars (1 or 2), and additional anchorage of NSM CFRP bars (steel wire U anchors or CFRP U strips). The test results show that the ultimate flexural strength of the timber beams were increased by 14%∼85% with an average of 47% due to NSM CFRP bar strengthening. Their deflection corresponding to the peak load was increased by 33% in average.

Improved shear strength model for FRP-strengthened RC beams accounting for adverse FRP-steel interaction

RC beams shear-strengthened with externally-bonded FRP side strips or U-strips usually fail by debonding. As such debonding occurs in a brittle manner at relatively small shear crack widths, some of the internal steel stirrups may not have reached yielding at beam shear failure. Consequently, the internal steel stirrups cannot be fully utilized. This adverse shear interaction between internal steel stirrups and external FRP strips may significantly reduce the benefit of shear-strengthening FRP but has not been considered by any of the existing FRP strengthening design guidelines. In this paper, an improved shear strength model capable of accounting for the effect of the above shear interaction is first presented, in which the unfavorable effect of shear interaction is reflected through a reduction factor (i.e. shear interaction factor). Using a large test database established in the present study, the performance of the proposed model as well as that of three other shear strength models is then assessed. This assessment shows that the proposed shear strength model performs better than the three existing models. The assessment also shows that the inclusion of the proposed shear interaction factor in the existing models can significantly improve their performance.

Resonantly loaded apertures for high-resolution near field surface imaging

A novel type of microwave probes based on the loaded aperture geometry has been proposed and experimentally evaluated for dielectrics characterisation and high-resolution near-field imaging. Experimental results demonstrate the possibility of very accurate microwave spectroscopic characterisation of thin lossy dielectric samples and biological materials containing water. High-resolution images of the subwavelength lossy dielectric strips and wet and dry leaves have been obtained with amplitude contrast around 10-20 dB and spatial resolution better than one-tenth of a wavelength in the near-field zone. A microwave imaging scenario for the early-stage skin cancer identification based on the artificial dielectric model has also been explored. This model study shows that the typical resolution of an artificial malignant tumour with a characteristic size of one-tenth of a wavelength can be discriminated with at least 6 dB amplitude and 50° phase contrast from the artificial healthy skin and with more than 3 dB contrast from a benign lesion of the same size. It has also been demonstrated that the proposed device can efficiently deliver microwave energy to very small, subwavelength, focal areas which is highly sought in the microwave hyperthermia applications.

Recent developments in bridge weigh-in-motion (B-WIM).

Bridge Weigh in Motion (B-WIM) uses accurate sensing systems to transform an existing bridge into a mechanism to determine actual traffic loading. This information on traffic loading can enable efficient and economical management of transport networks and is becoming a valuable tool for bridge safety assessment. B-WIM can provide site specific traffic loading on deteriorating bridges, which can be used to determine if the reduced capacity is still sufficient to allow the structure to remain operational and minimise unnecessary replacement or rehabilitation costs and prevent disruption to traffic. There have been numerous reports on the accuracy classifications of existing B-WIM installations and some common issues have emerged. This paper details some of the recent developments in B-WIM which were aimed at overcoming these issues. A new system has been developed at Queens University Belfast using fibre optic sensors to provide accurate axle detection and improved accuracy overall. The results presented in this paper show that the fibre optic system provided much more accurate results than conventional WIM systems, as the FOS provide clearer signals at high scanning rates which require less filtering and less post processing. A major disadvantage of existing B-WIM systems is the inability to deal with more than one vehicle on the bridge at the same time; sensor strips have been proposed to overcome this issue. A bridge can be considered safe if the probability that load exceeds resistance is acceptably low, hence B-WIM information from advanced sensors can provide confidence in our ageing structures.

MMP-8 Activity Profiling In GCF Samples

Background: In healthy tissues a family of enzymes known as matrix metalloproteinases (MMPs) play an important role in regulating turnover and metabolism of connective tissue collagen. MMPs have been implicated in a wide variety of pathological conditions including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using enzyme-linked immunosorbent assay (ELISA). Although ELISA quantifies the presence of the MMP-8 protein, it is not possible to determine enzyme activity using this method. Furthermore, since members of the MMP family have poor substrate sequence specificity, a peptide substrate alone cannot differentiate the activity of MMP-8 from other MMPs that may be present in biological samples. Objectives: In the present study, a method to specifically measure MMP-8 activity in gingival crevicular fluid (GCF) samples was developed. Methods: GCF was collected from healthy patients and those with periodontal disease using Perio paper strips. Samples were stored frozen until required for analysis. A specific MMP-8 antibody was used to coat 96 well microtitre plates to selectively remove MMP-8 from the GCF samples. Following a washing step, the activity of bound MMP-8 was measured over 70 minutes using a fluorogenic (FRET) substrate. Results: GCF from healthy subjects exhibited basal MMP-8 activity but in diseased samples MMP-8 activity was significantly higher. Minimal binding of other recombinant MMPs to the specific MMP-8 antibody was observed in cross-reactivity studies. Conclusion: We show for the first time that MMP-8 activity was significantly increased in GCF from periodontitis sites compared with activity levels in healthy sites. Further studies of MMP-8 activity in GCF samples should improve our understanding of its destructive role in periodontal disease.

Before the “Comics:” On the Seriality of Graphic Narratives during the Nineteenth Century

While the connection between seriality and comics in the twentieth century has frequently been a subject of study, far less attention has been paid to the role of serialisation in the previous century, when the language of comics gradually developed in the illustrated and satirical press. This article discusses a heterogeneous group of graphic narratives published in various European countries between the 1830s and the 1880s, before a new generation of comic magazines influenced by American newspaper strips transformed this emerging field into the autonomous medium of comics. Serial works flourished during this period and included diverse modes, such as series of “graphic novels,” the use of recurring characters, the serialisation of picture stories in humour periodicals, and the use of graphic narratives as a regular feature in the illustrated news magazines. By providing a panoramic survey of various types of serial texts, the article suggests that the hybrid nature of these graphic narratives and the publishing strategies applied to them can be better understood if considered in relation to the larger context of nineteenth-century print culture, rather than in comparison with the future of the medium.