Strength and endurance training reduces the loss of eccentric hamstring torque observed after soccer specific fatigue

Objectives: To investigate the effect of two hamstring training protocols on eccentric peak torque before and after soccer specific fatigue. Participants: Twenty-two university male soccer players. Design: Isokinetic strength tests were performed at 60°/s pre and post fatigue, before and after 2 different training interventions. A 45-minute soccer specific fatigue modified BEAST protocol (M-BEAST) was used to induce fatigue. Players were randomly assigned to a 4 week hamstrings conditioning intervention with either a maximum strength (STR) or a muscle endurance (END) emphasis. Main outcome measures: The following parameters were evaluated:– Eccentric peak torque (EccPT), angle of peak torque (APT), and angle specific torques at knee joint angles of 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80° and 90°. Results: There was a significant effect of the M-BEAST on the Eccentric torque angle profile before training as well as significant improvements in post-fatigue torque angle profile following the effects of both strength and muscle endurance interventions. Conclusions: Forty-five minutes of simulated soccer activity leads to reduced eccentric hamstring torque at longer muscle lengths. Short-term conditioning programs (4-weeks) with either a maximum strength or a muscular endurance emphasis can equally reduce fatigue induced loss of strength over this time period

Reducing driving forces for pressed-in piles

Pile jacking techniques use static loading to install sheet piles with minimal environmental effects, such as noise and ground vibrations, which are often associated with other methods of driven piling. This paper describes a series of 20g centrifuge tests conducted to investigate two methods of reducing the large driving forces that can be associated with jacked sheet piles in stiff clay. In practice, pre-auguring at the clutch positions and water jetting techniques are routinely carried out prior to the installation of sheet piling in order to reduce the driving forces. However, these methods are known to contribute to ground movements and can detract from the advantages of jacked sheet piles. The tests involved driving model sheet piles, which were modified either by installing driving shoes to the base of the pile, or by ribbing the profile of the pile shaft. The driving forces of the modified piles were compared with those of a plain pile. The tests showed that the use of driving shoes and ribs can lead to the reduction of driving forces at greater depth

Antibody mimetic receptor proteins for label-free biosensors

The development of high sensitivity biosensors, for example for clinical diagnostics, requires the identification of suitable receptor molecules which offer high stability, specificity and affinity, even when embedded into solid-state biosensor transducers. Here, we present an electrochemical biosensor employing small synthetic receptor proteins (Mw < 15 kDa) which emulate antibodies but with improved stability, sensitivity and molecular recognition properties, in particular when immobilized on a solid sensor surface. The synthetic receptor protein is a non-antibody-based protein scaffold with variable peptide regions inserted to provide the specific binding, and was designed to bind anti-myc tag antibody (Mw � 150 kDa), as a proof-of-principle exemplar. Both the scaffold and the selected receptor protein were found to have high thermostability with melting temperatures of 101 �C and 85 �C, respectively. Furthermore, the secondary structures of the receptor protein were found to be very similar to that of the original native scaffold, despite the insertion of variable peptide loops that create the binding sites. A label-free electrochemical sensor was fabricated by functionalising a microfabricated gold electrode with the receptor protein. A change in the phase of the electrochemical impedance was observed when the biosensor was subjected to anti-myc tag antibodies at concentrations between 6.7 pM and 6.7 nM. These findings demonstrate that these non-antibody receptor proteins are excellent candidates for recognition molecules in label-free biosensors

Rapid cell separation with minimal manipulation for autologous cell therapies

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of > 98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities

Perceptions About the Present and Future of Surgical Simulation: A National Study of Mixed Qualitative and Quantitative Methodology.

Objectives: Assess expert opinion on the current and future role of simulation in surgical education. Design: Expert opinion was sought through an externally validated questionnaire that was disseminated electronically. Participants: Heads of Schools of Surgery (HoS) (and deputies) and Training Program Directors (TPD) (and deputies). Results: Simulation was considered a good training tool (HoS: 15/15, TPD: 21/21). The concept that simulation is useful mostly to novices and for basic skills acquisition was rejected (HoS: 15/15, TPDs: 21/21; HoS: 13/15, TPDs: 18/21). Further, simulation is considered suitable for teaching nontechnical skills (HoS: 13/15, TPDs: 20/21) and re-enacting stressful situations (HoS: 14/15, TPDs: 15/21). Most respondents also felt that education centers should be formally accredited (HoS: 12/15, TPDs: 16/21) and that consultant mentors should be appointed by every trust (HoS: 12/15, TPDs: 19/21). In contrast, there were mixed views on its use for trainee assessment (HoS: 6/15, TPDs: 14/21) and whether it should be compulsory (HoS: 8/15, TPDs: 11/21). Conclusion: The use of simulation for the acquirement of both technical and nontechnical skills is strongly supported while views on other applications (e.g., assessment) are conflicting. Further, the need for center accreditation and supervised, consultant-led teaching is highlighted

Selection and characterisation of Affimers specific for CEA recognition

Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications

Reagentless Affimer- and antibody-based impedimetric biosensors for CEA-detection using a novel non-conducting polymer

Polyoctopamine (POct), an amine-functionalised non-conducting polymer, as the transducer layer in an electrochemical biosensor, is presented. This polymer offers versatile covalent coupling either through thiol linker conjugation, carboxyl or aldehyde functional groups without the requirement of pre- or post-surface activation. The colorectal cancer biomarker carcinoembryonic antigen (CEA) was selected as the target analyte, whilst an antibody and a synthetic binding protein, an Affimer, were used as distinct bioreceptors to demonstrate the versatility of polyoctopamine as a transducer polymer layer for oriented immobilisation of the bioreceptors. The electrodeposited polymer layer was characterised using cyclic voltammetry, electrochemical impedance spectroscopy, and on-sensor chemiluminescent blotting. The performance of optimised POct-based biosensors were tested in spiked human serum. Results showed that the electropolymerisation of octopamine on screen printed gold electrode generates a thin polymer film with low resistance. Close proximity of the immobilised bioreceptors to the transducer layer greatly enhanced the sensitivity detection. The sensitivity of the smaller monomeric bioreceptor (Affimer, 12.6 kDa) to detect CEA was comparable to the dimeric antibody (150 kDa) with limit of detection at 11.76 fM which is significantly lower than the basal clinical levels of 25 pM. However, the Affimer-based sensor had a narrower dynamic range compared to the immunosensor (1–100 fM vs. 1 fM – 100 nM, respectively). All electrochemical measurements were done in less than 5 min with small sample volumes (10 μl). Hence, polyoctopamine features a simple fabrication of impedimetric biosensors using amine-functionalisation technique, provides rapid response time with enhanced sensitivity and label-free detection

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4–5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers

A database of microRNA expression patterns in Xenopus laevis

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase

Glucocorticoids regulate AKR1D1 activity in human liver in vitro and in vivo

Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids