Peptide-based biosensors

Peptides have been used as components in biological analysis and fabrication of novel biosensors for a number of reasons, including mature synthesis protocols, diverse structures and as highly selective substrates for enzymes. Bio-conjugation strategies can provide an efficient way to convert interaction information between peptides and analytes into a measurable signal, which can be used for fabrication of novel peptide-based biosensors. Many sensitive fluorophores can respond rapidly to environmental changes and stimuli manifest as a change in spectral characteristics, hence environmentally-sensitive fluorophores have been widely used as signal markers to conjugate to peptides to construct peptide-based molecular sensors. Additionally, nanoparticles, fluorescent polymers, graphene and near infrared dyes are also used as peptide-conjugated signal markers. On the other hand, peptides may play a generalist role in peptide-based biosensors. Peptides have been utilized as bio-recognition elements to bind various analytes including proteins, nucleic acid, bacteria, metal ions, enzymes and antibodies in biosensors. The selectivity of peptides as an enzymatic substrate has thus been utilized to construct enzyme sensors or enzyme-activity sensors. In addition, progress on immobilization and microarray techniques of peptides has facilitated the progress and commercial application of chip-based peptide biosensors in clinical diagnosis.

General approach for electrochemical functionalization of glassy carbon surface by in situ generation of diazonium ion under acidic and non-acidic condition with a cascade protocol

Immobilization of catechol derivatives on GC electrode surfaces can be performed by in situ generation and reduction of nitrocatechol. We present the oxidative nitration of catechol in the presence of nitrous acid followed by electrochemically reduction of the generated nitro aromatic group to the corresponding amine group and its conversion to diazonium cation at the electrode surface to yield a surface covalently modified with catechol. In this manner, some derivatives of catechol can be immobilized on the electrode surface. Whole of the process is carried out in Triethylammonium acetate ionic liquid as an inert and neutral medium (pH∼7.0). Surface coverage can be easily controlled by the applied potential, time and concentration of catechol. After modification, the electrochemical features of modified surface have been studied. Also modified GC electrode exhibited remarkable catalytic activity in the oxidation of NADH. The catalytic currents were proportional to the concentration of NADH over the range 0.01-0.80 mM. This condition can be used for modification of GC surfaces by various aromatic molecules for different application such as design of sensors and biosensors. © 2014 Elsevier Ltd. All rights reserved.

Taking the sting out of darting : Risks, restraint drugs and procedures for the chemical restraint of Southern Hemisphere otariids

The need to manage otariid populations has necessitated the development of a wide range of capture methods. Chemical restraint by remote drug delivery (i.e., darting) is a highly selective method that can be used to facilitate otariid capture in a range of scenarios, when other methods may be impracticable. However, the risks associated with darting otariids are not widely known and guidelines necessary to promote and refine best practice do not exist. We review the risks associated with darting and in light of our findings, develop darting guidelines to help practitioners assess and minimize risks during capture, anesthesia and recovery. Published studies reveal that mortalities associated with darting predominantly result from complications during anesthetic maintenance (e.g., prolonged respiratory depression, apnea, or hyperthermia), rather than from complications during capture or recovery. In addition to monitoring vital signs and proper intervention, the risk of irreversible complications during anesthesia can be reduced by administering drug doses that are sufficient to enable the capture and masking of animals, after which anesthetic depth can be regulated using gas anesthesia.

Silk powder for regenerative medicine

Two approaches are used for silk particle production: bottom up and top down. In the bottom up approach, different liquid-solid phase transfer techniques are adapted to fabricate particles from silk solution. In the top down approach, silk fibres are milled by various means to prepare ultrafine silk particles. Many important properties of particles such as size, geometry, porosity, stability and biodegradability are dependent on the specific methods of particle production. These properties influence drug loading and release, delivery modes, biocompatibility and their clearance from the body. Particle properties also determine biomechanical properties of particle reinforced composite scaffolds. Thus correlation between preparation, characterisation and application of silk particles for a specific biomedical application is critical. Progress made in this direction and challenges ahead are discussed in this chapter. © 2014 Woodhead Publishing Limited. All rights reserved.

Muscle atrophy, pain, and damage in bed rest reduced by resistive (vibration) exercise

The purpose of this study was to investigate the effectiveness of a short-duration (5-6 min, 3 d·wk) resistive exercise program with (RVE) or without (RE) whole-body vibration in reducing muscle atrophy in the lower limb during prolonged inactivity when compared with that in an inactive control group. METHODS: As part of the second Berlin BedRest Study, 24 male subjects underwent 60 d of head-down tilt bed rest. Using magnetic resonance imaging, muscle volumes of the individual muscles of the lower limb were calculated before and at various intervals during and after bed rest. Pain levels and markers of muscle damage were also evaluated during and after bed rest. Adjustment of P values to guard against false positives was performed via the false discovery rate method. RESULTS: On the "intent-to-treat" analysis, RE reduced atrophy of the medial and lateral gastrocnemius, soleus, vasti, tibialis posterior, flexor hallucis longus, and flexor digitorum longus (P ≤ 0.045 vs control group) and RVE reduced atrophy of the medial and lateral gastrocnemius and tibialis posterior (P ≤ 0.044). Pain intensity reports after bed rest were lower in RE at the foot (P ≤ 0.033) and whole lower limb (P = 0.01) and in RVE at the thigh (P ≤ 0.041), lower leg (P ≤ 0.01), and whole lower limb (P ≤ 0.036). Increases in sarcomere-specific creatine kinase after bed rest were less in RE (P = 0.020) and RVE (P = 0.020). No differences between RE and RVE were observed. CONCLUSIONS: In conclusion, a short-duration RVE or RE can be effective in reducing the effect of prolonged bed rest on lower extremity muscle volume loss during bed rest and muscle damage and pain after bed rest. Copyright © 2014 by the American College of Sports Medicine.

The influence of graphene on the electrical communication through organic layers on graphite and gold electrodes

The influence of graphene on the electrical communication through organic layers fabricated on graphite and gold electrodes is investigated. These layers were prepared by in situ reductive adsorption of 4-aminobenzoic acid in the presence of NaNO2 and HCl to have surface bound carboxylic acid functionalities, followed by covalent attachment of 1-aminopyrene via an amide coupling reaction to have surface bound pyrene groups for graphene immobilization via noncovalent π-π stacking interaction. The coverage of the layers created via reductive adsorption on graphite electrodes was found to be much higher than that on gold electrodes. It was revealed that graphene significantly enhances the electrical communication through the layers on graphite electrodes but on gold electrodes the enhancement effect through the layers was only minor. However, when gold electrodes were modified with a self-assembled monolayer (SAM) of propanethiol the subsequent immobilization of graphene resulted in a significant enhancement of the electrical communication. It is also found that immobilization of graphene could affect the electron transfer between the redox probe, pyrene and the underlying electrodes. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to characterize the graphene sheets. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and X-ray photoelectron spectroscopy (XPS) were also used to characterize the stepwise modified electrodes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of long-term head-down-tilt bed rest and different training regimes on the coagulation system of healthy men

Immobility plus preexisting chronic disease or acute trauma can activate the coagulation system, thus increasing the risk for thromboembolic events. The effects of long-term bed-rest immobility and microgravity on the coagulation system of healthy persons (e.g., during crewed Mars missions) have not yet been studied. The main objective of the second Berlin BedRest Study (BBR2-2) "Coagulation Part" was to investigate adaptations of the hemostatic system during long-term bed rest (60 days) under simulated microgravity (6° head-down-tilt [6°HDT]) and after mobilization in three different volunteer groups (randomly assigned to CTR= inactive control group; RE= resistive exercise only group; and RVE= resistive exercise with whole-body vibration group). In 24 males (aged 21-45 years), before, during, and after long-term bed rest, key parameters of coagulation were measured from venous blood samples: D-dimer (DD), thrombin-antithrombin III complex (TAT), and prothrombin fragment F1 + 2 (PT-F1 + 2). Additionally, modified rotational thrombelastometry (ROTEM (®) ) analysis was performed. Times of exploratory analyses were as follows: baseline data collection 2 days before bed rest (BDC-2); eight different days of 6°HDT bed rest (HDT1-HDT60), and two different days after reambulation (R + 3 and R + 6). We found significant changes in DD, TAT, and PT-F1 + 2 over the total time course, but no consistent effect of physical interventions (RE, RVE) on these parameters. Notably, no parameter reached levels indicative of intravascular thrombin formation. All ROTEM® parameters remained within the normal range and no pathological traces were found. Sixty days of 6°HDT bed rest are not associated with pronounced activation of the coagulation system indicative of intravascular thrombus formation in healthy volunteers independent of the training type during the bed rest.

The control of epidermal growth factor grafted on mesoporous silica nanoparticles for targeted delivery

The performance of biomaterials in a biological environment is largely influenced by the surface properties of the biomaterials. In particular, grafted targeting ligands significantly impact the subsequent cellular interactions. The utilisation of a grafted epidermal growth factor (EGF) is effective for targeted delivery of drugs to tumours, but the amount of these biological attachments cannot be easily quantified as most characterization methods could not detect the extremely low amount of EGF ligands grafted on the surface of nanoparticles. In this study, hollow mesoporous silica nanoparticles (HMSNs) were functionalized with amine groups to conjugate with EGFs via carbodiimide chemistry. Time of flight secondary ion mass spectrometry (ToF-SIMS), a very surface specific technique (penetration depth <1.5 nm), was employed to study the binding efficiency of the EGF to the nanoparticles. Principal component analysis (PCA) was implemented to track the relative surface concentrations of EGFs on HMSNs. It was found that ToF-SIMS combined with the PCA technique is an effective method to evaluate the immobilization efficiency of EGFs. Based on this useful technique, the quantity and density of the EGF attachments that grafted on nanoparticles can be effectively controlled by varying the EGF concentration at grafting stages. Cell experiments demonstrated that the targeting performance of EGFR positive cells was affected by the number of EGFs attached on HMSNs.

Enhanced physiological tremor deteriorates plantar flexor torque steadiness after bed rest

This study evaluated the effectiveness of resistance training to preserve submaximal plantar flexor (PF) torque steadiness following 60 days of bed rest (BR). Twenty-two healthy male subjects underwent either BR only (CTR, n=8), or BR plus resistance training (RT, n=14). The magnitude of torque fluctuations during steady submaximal isometric PF contractions (20%, 40%, 60% and 80% of maximum) were assessed before and after BR. Across contraction intensities, torque fluctuations (coefficient of variation, CV) increased more (P<0.05) after BR for CTR (from 0.31±0.10 to 0.92±0.63; P<0.001), than for RT (from 0.30±0.09 to 0.54±0.27; P<0.01). A shift in the spectral content of torque fluctuations towards increased rhythmic activity between 6.5 and 20Hz was observed in CTR only (P<0.05). H-reflex amplitude (H(max)/M(max) ratio) declined across groups from 0.57±0.18 before BR to 0.44±0.14 following BR (P<0.01) without correlation to CV. The present study showed that increased torque fluctuation after BR resulted from enhanced physiological tremor. Resistance training prevented the spectral shift in isometric PF torque fluctuation and offset ∼50% of the decline in performance associated with long-term BR.

Bone structure and density via HR-pQCT in 60d bed-rest, 2-years recovery with and without countermeasures

We examined the effects of bed-rest, recovery and exercise countermeasures on bone density and structure at the distal tibia and radius as measured via high-resolution peripheral computed tomography. 24 subjects underwent 60-days of head-down tilt bed-rest and performed either resistive vibration exercise (RVE; n = 7), resistive exercise only (RE; n = 8) or no exercise (n = 9; 2nd Berlin BedRest Study; BBR2-2). Measurements were performed regularly during and up to 2-years after 60d bed-rest. At the distal tibia marked reductions in cortical area, cortical thickness and bone density but increases in periosteal perimeter and trabecular area were seen (p all<0.001). Recovery of most parameters occurred within 180d after bed-rest. At the distal radius, persistent increases in cortical area, cortical thickness, cortical density and total density and decreases in trabecular area were seen (p all ≤ 0.005). A significant effect of RVE (p = 0.003), but not RE, was seen on cortical area at the distal tibia, with few effects of the countermeasures observed on the remaining parameters. The current study represents the first implementation of high-resolution peripheral computed tomography in bed-rest in male subjects and helps to understand the patterns of bone remodeling due to bed-rest and recovery.

Heterogeneous atrophy occurs within individual lower limb muscles during 60 days of bed rest

To better understand disuse muscle atrophy, via magnetic resonance imaging, we sequentially measured muscle cross-sectional area along the entire length of all individual muscles from the hip to ankle in nine male subjects participating in 60-day head-down tilt bed rest (2nd Berlin BedRest Study; BBR2-2). We hypothesized that individual muscles would not atrophy uniformly along their length such that different regions of an individual muscle would atrophy to different extents. This hypothesis was confirmed for the adductor magnus, vasti, lateral hamstrings, medial hamstrings, rectus femoris, medial gastrocnemius, lateral gastrocnemius, tibialis posterior, flexor hallucis longus, flexor digitorum longus, peroneals, and tibialis anterior muscles (P ≤ 0.004). In contrast, the hypothesis was not confirmed in the soleus, adductor brevis, gracilis, pectineus, and extensor digitorum longus muscles (P ≥ 0.20). The extent of atrophy only weakly correlated (r = -0.30, P < 0.001) with the location of greatest cross-sectional area. The rate of atrophy during bed rest also differed between muscles (P < 0.0001) and between some synergists. Most muscles recovered to their baseline size between 14 and 90 days after bed rest, but flexor hallucis longus, flexor digitorum longus, and lateral gastrocnemius required longer than 90 days before recovery occurred. On the basis of findings of differential atrophy between muscles and evidence in the literature, we interpret our findings of intramuscular atrophy to reflect differential disuse of functionally different muscle regions. The current work represents the first lower-limb wide survey of intramuscular differences in disuse atrophy. We conclude that intramuscular differential atrophy occurs in most, but not all, of the muscles of the lower limb during prolonged bed rest.

An acetylcholinesterase inhibition biosensor based on a reduced graphene oxide/silver nanocluster/chitosan nanocomposite for detection of organophosphorus pesticides

A sensitive electrochemical acetylcholinesterase (AChE) biosensor based on a reduced graphene oxide (rGO) and silver nanocluster (AgNC) modified glassy carbon electrode (GCE) was developed. rGO and AgNC nanomaterials with excellent conductivity, catalytic activity and biocompatibility offered an extremely hydrophilic surface, which facilitated the immobilization of AChE to fabricate the organophosphorus pesticide biosensor. Carboxylic chitosan (CChit) was used as a cross-linker to immobilize AChE on a rGO and AgNC modified GCE. The AChE biosensor showed favorable affinity to acetylthiocholine chloride (ATCl) and could catalyze the hydrolysis of ATCl. Based on the inhibition effect of organophosphorus pesticides on the AChE activity, using phoxim as a model compound, the inhibition effect of phoxim was proportional to its concentration ranging from 0.2 to 250 nM with a detection limit of 81 pM estimated at a signal-to-noise ratio of 3. The developed biosensor exhibited good sensitivity, stability and reproducibility, thus providing a promising tool for analysis of enzyme inhibitors and direct analysis of practical samples.

Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases, for protein degradation

Enzymatic hydrolysis is a widely used approach to improve the functional, nutritionaland physiological properties of food proteins. In this study, cross-linked enzyme aggre-gates (CLEAs) have been prepared from cell-envelope proteinases (CEPs) of Lactobacillusdelbrueckii subsp. lactis 313 and their proteolytic properties have been evaluated using severalfood proteins. We have optimized cross-linking conditions including ammonium sulphateconcentration, incubation temperatures, agitation speed, glutaraldehyde cross-linker con-centration, reaction time and the addition of proteic feeders. Particularly, the presence ofBSA improves retained activity of cross-linked CEP aggregates (CLCEPAs) from 21.5% to 40.9%.Blocking unreacted cross-linking groups on aggregates is important to enhance recyclabil-ity of CLCEPAs. CLCEPAs had attractive thermal stability at 50◦C and it showed enhancedcatalytic activity over long-term storage after lyophilization. We have demonstrated thatCLCEPAs has proteolytic properties on different food proteins including complex (chickenegg albumin, skimmed-milk protein), fractionated (bovine casein, whey protein isolate), andpurified (bovine serum albumin) proteins. Being the first report of CLEAs from lactobacilliCEPs, this study demonstrates the feasibility of using LDL 313 CLCEPAs for degradation ofvarious food proteins.

Lateral positioning for critically ill adult patients

BACKGROUND: Critically ill patients require regular body position changes to minimize the adverse effects of bed rest, inactivity and immobilization. However, uncertainty surrounds the effectiveness of lateral positioning for improving pulmonary gas exchange, aiding drainage of tracheobronchial secretions and preventing morbidity. In addition, it is unclear whether the perceived risk levied by respiratory and haemodynamic instability upon turning critically ill patients outweighs the respiratory benefits of side-to-side rotation. Thus, lack of certainty may contribute to variation in positioning practice and equivocal patient outcomes. OBJECTIVES: To evaluate effects of the lateral position compared with other body positions on patient outcomes (mortality, morbidity and clinical adverse events) in critically ill adult patients. (Clinical adverse events include hypoxaemia, hypotension, low oxygen delivery and global indicators of impaired tissue oxygenation.) We examined single use of the lateral position (i.e. on the right or left side) and repeat use of the lateral position (i.e. lateral positioning) within a positioning schedule. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2015, Issue 5), MEDLINE (1950 to 23 May 2015), the Cumulative Index to Nursing and Allied Health Literature (CINAHL) (1937 to 23 May 2015), the Allied and Complementary Medicine Database (AMED) (1984 to 23 May 2015), Latin American Caribbean Health Sciences Literature (LILACS) (1901 to 23 May 2015), Web of Science (1945 to 23 May 2015), Index to Theses in Great Britain and Ireland (1950 to 23 May 2015), Trove (2009 to 23 May 2015; previously Australasian Digital Theses Program (1997 to December 2008)) and Proquest Dissertations and Theses (2009 to 23 May 2015; previously Proquest Digital Dissertations (1980 to 23 May 2015)). We handsearched the reference lists of potentially relevant reports and two nursing journals. SELECTION CRITERIA: We included randomized and quasi-randomized trials examining effects of lateral positioning in critically ill adults. We included manual or automated turns but limited eligibility to studies that included duration of body position of 10 minutes or longer. We examined each lateral position versus at least one comparator (opposite lateral position and/or another body position) for single therapy effects, and the lateral positioning schedule (repeated lateral turning) versus other positioning schedules for repetitive therapy effects. DATA COLLECTION AND ANALYSIS: We pre-specified methods to be used for data collection, risk of bias assessment and analysis. Two independent review authors carried out each stage of selection and data extraction and settled differences in opinion by consensus, or by third party adjudication when disagreements remained unresolved. We planned analysis of pair-wise comparisons under composite time intervals with the aim of considering recommendations based on meta-analyses of studies with low risk of bias. MAIN RESULTS: We included 24 studies of critically ill adults. No study reported mortality as an outcome of interest. Two randomized controlled trials (RCTs) examined lateral positioning for pulmonary morbidity outcomes but provided insufficient information for meta-analysis. A total of 22 randomized trials examined effects of lateral positioning (four parallel-group and 18 cross-over designs) by measuring various continuous data outcomes commonly used to detect adverse cardiopulmonary events within critical care areas. However, parallel-group studies were not comparable, and cross-over studies provided limited data as the result of unit of analysis errors. Eight studies provided some data; most of these were single studies with small effects that were imprecise. We pooled partial pressure of arterial oxygen (PaO2) as a measure to detect hypoxaemia from two small studies of participants with unilateral lung disease (n = 19). The mean difference (MD) between lateral positions (bad lung down versus good lung down) was approximately 50 mmHg (MD -49.26 mmHg, 95% confidence interval (CI) -67.33 to -31.18; P value < 0.00001). Despite a lower mean PaO2 for bad lung down, hypoxaemia (mean PaO2 < 60 mmHg) was not consistently reported. Furthermore, pooled data had methodological shortcomings with unclear risk of bias. We had similar doubts regarding internal validity for other studies included in the review. AUTHORS' CONCLUSIONS: Review authors could provide no clinical practice recommendations based on the findings of included studies. Available research could not eliminate the uncertainty surrounding benefits and/or risks associated with lateral positioning of critically ill adult patients. Research gaps include the effectiveness of lateral positioning compared with semi recumbent positioning for mechanically ventilated patients, lateral positioning compared with prone positioning for acute respiratory distress syndrome (ARDS) and less frequent changes in body position. We recommend that future research be undertaken to address whether the routine practice of repositioning patients on their side benefits all, some or few critically ill patients.

Controllable graphene oxide mediated efficient electron transfer pathways across self-assembly monolayers: a new class of graphene based electrodes

In this paper, the influence of chemically reduced graphene oxide sheets (CRGOs) on the electrochemical performance through methyl or carboxylic acid terminated self-assembled monolayers (SAMs) is reported. The gold electrode was initially modified with methyl or carboxylic acid terminated alkanethiols with various carbon chain lengths (n = 4, 6, 8 and 11) and subsequently immobilization of the CRGOs on a SAM surface was achieved via a hydrophobic and electrostatic interaction. By using the potassium ferricyanide as a redox probe, it was observed that CRGOs could effectively enhance the heterogeneous electron transfer (ET) of the SAM due to a tunneling effect. The assemblies based on thiol end groups with methyl head groups were observed to afford more hydrophobic interaction binding with CRGOs with a higher reduction time than the assemblies developed with thiol end groups and a -COOH group which were shown to bind more electrostatically with CRGOs, a lowering reduction time. The Nyquist plots developed show a gradual decrease of the charge transfer resistance (Rct) of [Fe(CN)6]3-/4- redox couple at the CRGOs-SAMs electrode with the controllable adsorption of different CRGO's onto the SAM. Depending on the chain length and terminal functional group the electron transfer rate kinetics were observed to differ considerably.