Fuzi  is such an herb with its toxic ingredient, aconites. Aconitine, mesaconitine, and hypaconitine are the main toxic components of Fuzi, which are hydrolyzed into non-toxic derivatives by water decoction. Therefore, long-time decoction was commonly applied as a traditional way to detoxify Fuzi before use. Nevertheless, recent clinical trials presorted on adverse events induced by long-time decocted Fuzi, putting some doubt on the safety of Fuzi after the traditional detoxification procedure. To thoroughly determine whether or not long-time decocted Fuzi was safe, we conducted in vivo acute toxicity assays using both rodent and zebrafish models and performed chemoprofile analyses using HPLC and UPLC-MS. The HPLC analysis showed that toxic aconitine components were hydrolyzed into benzoyl derivatives with increasing time of decoction. These aconitines were undetected by HPLC in Fuzi after 2 h-decoction (FZ-120), indicating seemingly non-toxicity of FZ-120. Unlike the non-decocted Fuzi (FZ-0) and 60 min-decocted Fuzi (FZ-60) with lethal toxicity, FZ-120 at 130 g/kg did not cause any deaths or side effects in mice regarding body weight and biochemical parameters. This seems to confirm safety of Fuzi after long-time decoction. However, histopathological observations revealed an abnormal liver phenotype and a significant decrease of the liver index following FZ-120 treatment, indicating a potential hepatoxicity of FZ-120. By using a zebrafish model, we observed that FZ-120 at a dose range from 288 to 896 μg/ml caused considerable adverse events including arrhythmia, liver degeneration, yolk sac absorption delay, length decrease, and swim bladder loss, which clearly speak for acute toxicity on cardiovascular, digestive, development, and respiratory systems. The dose range of FZ-120 was lower than that used for clinical application in human beings. Moreover, UPLC-MS revealed that FZ-120 still contained toxic aconitines that were not detectable by HPLC, which might explain its acute toxicity in zebrafish. We concluded that Fuzi is not sufficiently safe even after long-time decoction. The zebrafish model combined with UPLC-MS assay may represent an appropriate test system to unravel aconitine-related acute toxicity.Many herbs of traditional Chinese medicine (TCM) possess not only therapeutic efficacy, but also toxicity towards normal tissues. The herbal toxicities occasionally cause serious adverse events or even fatal poisoning due to the erroneous use of TCM herbs.  Aconitum species. From 1989 to 2006, over 45 aconite poisoning cases were reported in China, among which three cases were fatal  gains increasingly popularity around the world. Adverse effects of herbal TCM were less frequently reported than that of conventional Western medicine . The caal., 1990; Ono et al., 1990).Fuzi, the lateral root of Aconitum carmichaeli Debx. (Ranunculaceae), is a distinguished TCM herb originally grown in the district of Jiangyou in the Sichuan province, China. It was first recorded in the earliest Chinese Pharmacopeia, ''Shennongʹs Materia Medica'' (Shengnong Ben Cao Jing), in the Eastern Han Dynasty of China (24−220 AD). Another Chinese medical classic, ''Treatise on Febrile Diseases'' (Shang Han Lun), elucidated its clinical effects. There are more than 20 herbal formulas containing Fuzi as a main ingredient, such as 'Si Ni Tang', 'Fu Zi Tang' and 'Zhen Wu Tang' etc. . As oneal., 1991). Howevehan, 2009). The dihan, 2009; Lin et han, 2009). Water-han, 2009; Tong ethan, 2009). This iang, 2007). Fuzi still caused adverse events in patients during the treatment of osteoarthritis . This iAconitum carmichaeli (Fuzi) was obtained from Sichuan Jiangyou Zhongba Aconiti Technology Development CO., LTD  and authenticated by the authors (voucher specimen number: JY160301). The materials were powdered and soaked in 10-fold stilled water for 30 min, followed by boiling and decoction for 60 min and 120 min, respectively. Then, the water extract was collected after filtration, and the residue was boiled and decocted with water for another 60 min and 120 min, respectively. After filtration, the second water extract was collected and mixed with the first extract, followed by concentration to dryness. The extract from 60 min decoction and 120 min decoction were labeled as FZ-60 and FZ-120, respectively. Water suspension of the powdered material of Fuzi was used and labeled as FZ-0 (0 min decoction).The clinically used decoction pieces of the lateral root of The reference standards of aconitine (Batch number: 0720-9406), mesaconitine (Batch number: 0799-9203), and hypaconitine (Batch number: 0798-9202) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . The purities of the three chemicals were all above 99.8 %. Methanol, triethylamine, chloroform, and dichloromethane were of HPLC grade and commercially obtained. The distilled water was purified by Smart2Pure 6 UV/UF .ad libitum during the testing periods. All experimental procedures were in strict accordance with the China legislation on the use and care of laboratory animals.ICR mice with both sexes weighing 18−22 g were purchased from the Shanghai Laboratory Animal Center of Chinese Academy of Sciences . The mice were kept in a controlled breeding room  for one week acclimatization and fed rodent laboratory chow with tap water 3).Wide-type AB strain of Zebrafish was purchased from China Zebrafish Resource Center (CZRC), Institute of Hydrobiology, CAS  and bred by Hunter Biotechnology, Inc. . All fishes were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAA LAC) International (SYXK2012-0171). After natural pair-mating and reproduction, larval zebrafish (2 dpf) were generated and housed in a light-controlled aquaculture facility with a standard 14:10 h day/night photoperiod and fed with live brine shrimp twice a day and dry flake once a day. The temperature of fish water was maintained at 28 °C , FZ-60 (130 g/kg) and FZ-120 (130 g/kg), respectively, for 7 days. The normal group was daily treated with an equal volume of water for 7 days. Toxicity-related parameters were analyzed, including body weight, survival rate, weight index of main organs, histopathology of main organs, and serum biochemical . Equal amount of male and female ICR mice were randomly divided into four groups with each 16 animals, including normal group, FZ-0 group, FZ-60 group, and FZ-120 group. All mice were fasted for 12 h with water 10 and MNLC of FZ-120, 180 larval zebrafish (2 dpf) were grouped and distributed into 6-well plates (30 fishes per well) with 3 ml water for each well. FZ-120 was mixed into the water at doses of 600, 700, 800, 900, 1000 μg/ml for oral administration for three days. Untreated well was used as normal control. During the treatment, mortality was daily recorded to generate dose-mortality response curve by using Origin 8.0 . MNLC and LC10 were determined by logistic regression calculation. To determine the LC10 of FZ-120, respectively for 3 days. After treatment, the heart, brain, eyes, liver, intestine, spine, and behaviors of each fish were observed under the microscope. The occurrence of edema, hemorrhage, and thrombosis were also observed in the animals. All abnormal phenotypes were statistically recorded by double-blind evaluation.To evaluate the acute toxicity on target organs, 120 larval zebrafish (2 dpf) were orally treated with 1/10 MNLC, 1/3 MNLC, MNLC, and LC18 column   at 30 °C. The mobile phase consisted of methyl alcohol, water, chloroform, and triethylamine (100 : 50 : 3 : 0.15) with flow rate of 1.0 ml/min. The sample injection volume was 2 μl for FZ-0, FZ-60, and FZ-120, and the detection wavelength was 234 nm. The data was analyzed to determine the contents of aconitine, mesaconitine, and hypaconitine in all samples.HPLC analysis was performed on an Agilent 1260 Infinity HPLC system . Chromatographic separation was achieved on a Hypersil BDS-C18 column  maintained at 40 °C.The UPLC-MS analysis was performed on an Acquity UPLC system  equipped with a Xevo TQS triple quadrupoleelectrospray ionization (ESI) MS  operated in positive ESI-mode. Chromatographic separation was carried out on an Acquity BEH CAll tests were replicated until the experimental condition was optimized. Data were expressed as mean ± SD and subjected to one-way ANOVA, followed by Fisherʹs LSD comparison, using DPS software .The HPLC chromatographic comparison of aconitine components among FZ-0, FZ-60, and FZ-120 is shown in Figure 1P<0.05) . However, FZ-120 did not change the kidney index, but significantly decreased the liver index (P < 0.01). The liver abnormality induced by both FZ-60 and FZ-120 was verified through histopathological observation, in which cytoplasmic degeneration (CD) and fatty degeneration (FD) were found , mesaconitine (0.13 ± 0.001 mg/g), and hypaconitine (1.40 ± 0.013 mg/g) as determined by UPLC-MS analysis Figure 5. These tin vivo mammalian screening approaches prior to approval by the authorities in accordance with the OECD guidelines  and although in vivo animal systems exhibit high similarities with the human physiology, sometimes it remains difficult to comprehensively predict drug toxicity due to the disadvantages of low throughput, expensive and time consuming test procedures  are such a well-established animal model, which may be well suited for toxicity assessment. It offers several advantages compared to traditional in vivo and in vitro models, including: (1) optically large and transparent body for real-time visible observation of organ response; (2) high sensitivity to toxic insults; (3) similar cellular and molecular processes as human beings; (4) high-throughput application and less experimental time due to high fecundity and rapid development; and (5) low overall costs . Althouung, 2009). In ordal., 2014). Therefal., 2005; McGrathal., 2005; Vliegenal., 2005). Opticaal., 2005; Liang eal., 2005; Gorgulhal., 2005), demons003. Althal., 2005).Fuzi has to be pre-decocted for an extra 30 min before the conventional decoction with other herbs in one prescription , but not from the commonly used type (Hei Shun Pian with peel). Secondly, toxic aconitines (mesaconitine and hypaconitine) still existed after long-time decoction. Thirdly, the applied body weight and death rate of mice were not adequate as parameters for the acute toxicity test. Therefore, safety of the long-time decocted Fuzi cannot be reliably determined, especially if it caused adverse events in the clinic  can be completely detoxified by long-time decoction. HPLC analysis showed that toxic aconitine components were hydrolyzed into benzoyl derivatives with increasing time of decoction, and aconitine, mesaconitine, and hypaconitine were not detected in FZ-120 (2 h decocted Fuzi)  the use of Fuzi after long-time decoction is not sufficiently safe and urgently deserves more attention by clinicians and patients; and (2) zebrafish model combined with UPLC-MS assay may represent an appropriate test system to unravel aconitine-related acute toxicity.Decoction for longer time represents a traditional way to detoxify toxic TCM herbs. For example, tee, 2015). It meaal., 2013). Three al., 2016). The pr Figure 1. Acute t Figure 1. These d Figure 1, which ial., 2013). Appareal., 2013), which al., 2013). MoreovFuzi after long-time decoction, suggesting that the current practice of long-time decoction as only preventive measure is not adequate for aconite-related detoxification. New and more efficient approaches are needed to completely remove the toxicity of Fuzi. For example, co-decoction with Radix Glycyrrhizae (Gancao) can reduce the toxicity of Fuzi by complexation between aconitine in Fuzi and liquiritin in Gancao. Adding an excess of Gancao or free liquiritin makes the prescription much safer  and Semen Strychni (seed of Strychnos nuxvomica). More toxicity we can find, more safety patients have. Further, not only the toxicity, but the efficacy of herbs should also be considered. In this study, toxic aconitine amounts were reduced with increased contents of benzoyl derivatives during decoction . The co Figure 1. The benWan Sun, Bo Yan and Rongrong Wang contributed equally as first authors.Jiawei Huang, Peijian Tong  and Letian Shan  contributed equally as corresponding authors.This study was funded by the Zhejiang Provincial Natural Science Foundation of China , the National Natural Science Foundation of China (Grant No: 81774331 and 81673997), the Zhejiang Provincial Major Science and Technology Project of Medical and Health of China (Grant No: 201487674), and the Zhejiang Provincial Science and Technology Project of Traditional Chinese Medicine of China (Grant No: 2013ZQ007 and 2016ZZ011).


There is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury.To investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of β-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the β-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth.The novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson’s disease. There is currently no effective treatment for promoting regeneration of injured nerves in patients following brain trauma or spinal cord injury (SCI). The principal cause of disability that results from such injuries is the regenerative failure of mammalian CNS axons. This is due in part to upregulation of growth-inhibitory chondroitin sulphate proteoglycans (CSPGs) in the region of injury , 2,3.P. Vulgaris, promotes axon regeneration following CNS injury. It functions by removing growth-inhibitory CSPGs at the lesion site and this also promotes neural plasticity by dissolution of perineuronal nets [Chondroitinase ABC (ChABC), an enzyme isolated from the bacterium nal nets , 5,6. ThThe robustness of efficacy of ChABC in experimental SCI has been demonstrated in many injury models and in several mammalian species , 5, 7. Cin vitro model of neurite outgrowth to study the effects of targeting ChABC to the axonal compartment of neurons and to investigate the signalling pathways involved. SH-SY5Y cells are used as a model of neurons and chondroitin-4-sulphate (CSA) as a substrate inhibitory to neurite outgrowth. SH-SY5Y cells are a human neuronal cell line, derived from the sympathetic nervous system. They differentiate into neurons following treatment with retinoic acid [We also unveil some of the previously unidentified mechanisms underlying these effects. We used an oic acid . CriticaThe eukaryotic expression plasmid, pcDNA 3.1-(Invitrogen), encoding mammalian compatible ChABC  and contOligonucleotide sequences.                    N CCPGCC5’AATTC CTG AAT TGC TGC CCC GGC TGC TGC ATG GAG CCC ATG    EcoRI          Tetracysteine tag    Y  E  N  N  P  T  Y  K  F  F  E  Q  M  Q  NGGA TAT GAG AAT CCA ACT TAC AAG TTC TTT GAG CAA ATG CAG AAC                                Axon targeting signal TAG G 3’  stop BamHIGATCC CTA GTT CTG CAT TTG CTC AAA GAA CTT GTA AGT TGG ATT  5’  BamH1G 3’  CTC ATA TCC CAT GGG CTC CAT GCA GCA GCC GGG GCA GCA ATT CAG EcoR1                                                                                TM). These were propagated in DMEM, 10% FCS 100 units/ml penicillin and 100μg/ml streptomycin. Cells were passaged 24h prior to transfection and plated to achieve 60% confluency on the day of transfection. Dorsal Root Ganglion neurons (DRGs): Adult Sprague-Dawley rats, 250-400g, were supplied by Charles River Laboratories, Margate, UK. They were housed under a 12h light/dark cycle with ad libitum access to food and water. Euthanasia was carried out by decapitation. This is considered a regulated procedure under the Animals Scientific Procedures Act 1986 and as such was authorised under a project License  which was ethically reviewed by the University of Cambridge Animal Welfare &Ethical Review Body (AWERB) prior to submission and subsequent approval by the Secretary of State. The University of Cambridge holds an Establishment License  and is committed to animal welfare, with all animal facilities designated under the above Act. DRGs were dissected from adult rats, dissociated with 2% collagenase (Sigma) and 0.1% trypsin (Invitrogen) for 15 min at 37°C, then washed Hanks balanced salt solution (HBSS) prior to plating. Cultures were grown in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen), glutamic acid, penicillin and streptomycin (Sigma).SH-SY5Y Neurons: , according to the manufacturer’s instructions. GFP-transfected cells or non-transfected cells served as controls. A plasmid encoding mcherry or FIAsH-staining was used to assess transfection efficiency in the groups transfected with the ChABC plasmids. After 5h, the medium was replaced with medium containing serum and the cells incubated overnight. 24h post-transfection they were plated onto 4 well slides at a density of 1.7x10 neurons . For the5 cells suspended in electroporation buffer (invitrogen). These were then electroporated at 1200V, 20ms, 2pulses, then plated onto laminin/CSA coated coverslips at 2.0–4.0x106 cells/cm2 in serum free medium . Neurite lengths were measured 72h post-plating.DRGs were transfected by microporation using a neon kit (Invitrogen), according to the manufacturer’s instructions. Briefly, 0.5μg of plasmid (encoding GFP (control), non-targeted ChABC or axon-targeted ChABC) was added to~1x104 well slides (Millipore), were coated with 100μg/ml poly-l-lysine and incubated at 37°C overnight. The poly-l-lysine was then removed and the slides washed with PBS and left to dry. The poly-l-lysine-coated slides were then coated with 10ug/ml laminin in DMEM. This provides a substrate permissive for neurite outgrowth. Chondroitin-4-sulphate (CSA), is known to be inhibitory to axon outgrowth . CSA froCoverslips were coated with laminin 1μg/ml or laminin and CSA, 25μg/ml, a concentration previously shown to be inhibitory to neurite outgrowth of these neurons .Aequorea Victoria, was used to stain GFP-transfected cells, and mouse monoclonal anti-β1-integrin  clone, 102DF5, immunogen: tissue extract from human myometrium, was used to stain SH-SY5Ycells or rabbit polyclonal anti-pFAK  immunogen: synthetic peptide, that contains tyrosines 579&580, (which are conserved in rat and human), was used to stain DRGs. Cells stained for β1-integrin, were not permeabilised, in order to restrict detection to surface β-integrin expression. Staining for β1-integrin with DRGs was found to be weak, therefore these cells were stained for the presence and activation (phosphorylation) of focal adhesion kinase (pFAK) instead. This allows a direct assessment of integrin activation. The activation of integrin triggers a signalling cascade within the cell  and integrin clustering results in a rapid auto-phosphorylation of FAK [Following fixation with 4% paraformaldehyde, cells were permeabilised in 0.2% Triton X-100 for 5 minutes and washed 3 times in phosphate buffered saline (PBS). They were then blocked: first with Image-iT FX Signal Enhancer (Invitrogen) for 30 minutes in a humid environment; then with block buffer  for 2 hours. They were then stained with primary antibody, in block buffer overnight at 4°C. Cells were then washed 4 times, 5minutes each in PBS, before a 1h incubation in the secondary antibodies. After three 5minute washes in PBS, they were mounted with Prolong Gold anti-fade reagent (Invitrogen) and coverslipped. The primary antibodies used were: Mouse monoclonal anti-β tubulin III antibody, clone 2G10, , immunogen: synthetic peptide corresponding to amino acids 436–450 of human neuronal specific β-tubulin III, was used to visualize the neurites. Staining with anti-β tubulin III also confirmed that the SH-SY5Y cells had successfully differentiated into mature neuron-like cells, as β-tubulin is almost exclusively expressed in neurons . Rabbit n of FAK . IntraceP.Vulgaris ChABC (Sigma) was used as a standard. The reaction was incubated at 37°C for 20min, then stopped by boiling for 1min. Potassium borate solution  was added and the mixture boiled for 7min. It was then chilled on ice and centrifuged in a microfuge at 13,000rpm for 10 mins. 1ml of glacial acetic acid was added to the supernatant and mixed before centrifugation for a further 20min. To 1ml of supernatant, 0.4ml of Morgan-Elson Reagent  was added and incubated at 37°C for 20min. Product was measured by absorbance at 550nm.This enzyme assay measures ChABC activity by the N-acetylation of product disaccharides and subsequent reaction to give a coloured product. The reaction contains 100μl of 40mM sodium acetate, 40mM Tris-Cl pH8.0, 10mg/ml chondroitin-6-sulphate (Sigma), mixed with 20μl of enzyme sample. Images were captured on a Zeiss Axioplan microscope, under red or green fluorescent light (depending on the antibodies used) using a digital camera (QImaging) and QCapture Pro6.1 imaging software. This allows regulation of the acquisition exposures. The exposure, once optimized for photography for any set of photographs, was kept constant for all the photographs in that set.All image analysis was performed using ImageJ (NIH). A segmented line was used to measure neurite lengths. Measured lengths were converted to their actual size using a multiplication factor, determined from a photograph of a scale bar at 40x power. The average neurite length was then calculated from all neurons on each coverslip, to give a final measurement for each condition. Fluorescent measurements were carried out using a formula adapted by Gavet &Pines , as follWhole cell signal is the sum of the intensity of the pixels from one cell.Axon signal is the sum of intensity of the pixels for the cells' axon.Background signal is the average signal per pixel for a region selected just beside the cell.Whole cell or axon signal corrected is the whole cell signal or axon signal with the background signal subtracted.Cell body signal corrected is the whole cell signal corrected minus the axon signal corrected.Data was statistically analysed using IBM SPSS statistics software, .Neurite length and number analysis: The Shapiro-Wilk test was used to test whether the data followed a normal distribution. The data were found not follow a normal distribution, P<0.001. This was the case for both neurite length and number. Therefore, the non-parametric Mann Whitney-U-test (MWU), was used to compare neurite length and number between cells transfected with different constructs and plated onto the different substrates. It was also used to compare cell fluorescence intensities and gene expression analysis. A P value <0.05 was considered significant. Where more comparisons between groups were made, a Holm adjustment was performed. This method enables multiple comparisons to be made between groups statistically analysed by a Mann Whitney-U test. It uses a stepwise approach to compute the significance levels depending on the P value based rank of hypotheses . In the Real time (qPCR) was used to measure intracellular levels of PTEN in the different experimental groups. Differentiated SH-SY5Y cells were resuspended in RNA later (Life Technologies). RNA was extracted using the High Pure RNA Isolation Kit, (Roche), its integrity and concentration was assessed using agarose gel electrophoresis and nanodrop respectively. 1μg RNA was then reverse transcribed into cDNA using Nanoscript Reverse-Transcription Kit (Primer Design), following the manufacturers protocols. Intron-spanning primers were designed, specific for PTEN or ACTB, primer sequences shown in Real-time quantification was carried out using GoTaq® Probe qPCR Master Mix  according to manufacturer’s instructions, in a final volume of 10μl. The Light Cycler 480  was used for amplification and data acquisition, using the following cycling conditions; 95°C for 10 min, then 40 cycles at 95°C for 15s and 60°C for 1 min. Relative gene expression levels were calculated using the standard curve method, and gene expression was normalised using beta-actin (ACTB) housekeeping gene.Dot blot: Serum-free conditioned medium (DMEM) from SH-SY5Y cells, cultured for 72h, was spotted onto nitrocellulose membrane using a dot blot apparatus. CSPG mix (Millipore) diluted with DMEM were used as a positive control. Half the samples from each group were treated with ChABC for 3h, prior to incubation of the membrane with antibody 2B6  1:500, Seikagaku. This detects a stub epitope exposed by ChABC digestion of CSPGs. The membrane was then washed and incubated with HRP anti-mouse IgG 1:10,000. The signal was detected using chemiluminescence (Luminata) and visualised using hyperfilm (Amersham).CSPG assay: A Blyscan sulphated glycosaminoglycan assay (Biocolor) , was use-6M retinoic acid to induce differentiation into mature neurons [SH-SY5Y neurons were transduced with a lentivirus encoding ChABC (LVC) described in Zhao et al. [ neurons . After 4The axon-targeted ChABC construct was introduced into DRGs via microporation, then plated onto coverslips and stained for Rho A. The presence of intracellular RhoA was detected, using an AlexaFluor 488 secondary antibody.-6M retinoic acid to induce differentiation into mature neurons [One group of SH-SY5Y cells were transduced with a lentivirus encoding ChABC (LVC) , 100μMPic and ChABC on PTEN gene expression, cells from each group were plated into six, 25cm2+ and Mg2+. Cells were washed x6 with HBSS, then fixed with 4% paraformaldehyde. Cultures were then washed twice with HBSS, permeabilised with 0.2% Triton-X100 in PBS for 5mins, then washed x3 with HBSS. The FIAsH reagent was added (0.5ml/well) and the cultures incubated at RT for 30min, protected from light. BAL buffer was then used to wash the cells . Once stained, slides were mounted with Prolong Gold anti-fade reagent (Invitrogen), and examined within 48h to avoid fading.FIAsH staining was adapted from the manufacturer’s instructions (ThermoFischerscientific). The optimal working concentration was determined to be 1.25μg/ml in HBSS with CaCultures of SH-SY5Y cells transfected with targeted ChABC and plated onto CSA, had significantly longer neurites when compared to cells transfected with GFP, plated onto CSA (MWU-test: P<0.001) .Of particular note, are the remarkably long neurites seen on some neurons expressing targeted ChABC, which are almost 20 times the length of those of the GFP-transfected cells . These cin vivo [in vivo study.In this study, there was no significant difference in the number of neurites per cell between controls and neurons transfected with the non-targeted construct. This contrasts with the results observed in vivo  and may We also conducted an additional experiment to determine if the results obtained with our neuronal cell line, are recapitulated in cultures of primary neurons. To this end we analysed the effect of targeted ChABC on neurite outgrowth of dissociated dorsal root ganglion cells plated onto CSA. We extended our studies to investigate the mechanism underlying the enhancement of neurite outgrowth by axon-targeted ChABC on laminin. We show that SH-SY5Y cells produce and shed CSPGs. The Blyscan assay is a sensitive quantitative dye binding method for detecting sulphated glycosaminoglycans and a measure of total sulphated glycosaminoglycan content. The dye, 1,9-dimethlmethylene blue is a specific label for the sulphated polysaccharide component of proteoglycans. Using this assay we show that CSPGs were also present in the conditioned medium of SH-SY5Y cells at ~4.0μg/ml. These results confirm that SH-SY5Y neurons produce CSPGs, and are consistent with the hypothesis that ChABC-axon enhances neurite outgrowth on laminin by digesting the CSPGs produced by these neurons. The magnitude of the effect on axon length suggest that endogenously produced CSPGs provide a significant block to neurite outgrowth.Further investigation of the mechanisms responsible for the enhancement of neurite outgrowth showed that the cell adhesion molecule β1-integrin, is up-regulated at the surface of neurons into which the ChABC gene is introduced. To determine whether the enhancement of neurite outgrowth observed in primary cultures of DRGs transfected with targeted ChABC was also associated with integrin-mediated mechanism we measured integrin levels in transfected DRG neurons. The antibody used to stain the SH-SY5Y cells functioned poorly with the DRGs, so integrin expression was measured indirectly by staining for phosphorilated focal adhesion kinase (pFAK).Integrins can exist in both activated and inactivated forms. When integrins are activated, focal adhesion kinase (FAK) becomes phosphorylated (pFAK). Therefore, importantly, we have demonstrated the presence of pFAK in primary cultures of neurons transfected with the targeted version of ChABC, RhoA is a potent inhibitor of axon regeneration . InteresMoreover, when ChABC expression is directed to the axon, RhoA staining is additionally reduced in the axonal compartment, This suggests that ChABC has to be targeted to the axon in order to decrease RhoA expression in this compartment. These findings identify an additional mechanism for ChABC-mediated promotion of axon outgrowth, and again expression of the enzyme in the axonal compartment would be predicted to give optimal performance.SH-SY5Y cells were transduced with a lentivirus encoding non-targeted ChABC . A thirdThe increase neurite length observed in ChABC expressing neurons is similar to that produced by the PTEN inhibitor VO(OH)PIC . Moreovein vivo [in vivo, is a result of ChABC-mediated dissolution of perineuronal nets, structures not present in our SH-SY5Y cultures. The observation that targeting ChABC to the axon enhances the sprouting potential of ChABC is an important finding, as the ability to promote plasticity is a key mechanism of promoting repair following SCI [Addition of the peptide containing the YENPTY motif targets proteins with the tag to the axonal compartment of a neuron . This wain vivo  but coulwing SCI . Using twing SCI . Howeverwing SCI , targetiWe show that SH-SY5Y neurons produce CSPGs which are shed into the medium. Therefore, it is likely that the increase in neurite length of the cultures transfected with targeted ChABC, compared to the GFP transfected cells, seen on a laminin substrate, which is normally permissive to neurite outgrowth, is due in part to ChABC digestion of neuron-derived CSPGs. The difference in neurite length between GFP-transfected cells and those transfected with targeted ChABC plated on laminin is significant, suggesting that neuron-derived CSPGs may provide an additional block to neurite outgrowth. This finding has direct relevance to the regeneration of cortical neurons, as they also produce CSPGs, which are located on the cell surface and shed into the extracellular matrix . This prWe show that ChABC upregulates β-integrin at the cell surface of SH-SY5Y neurons. Moreover, β-integrin expression is further up-regulated on axons when neurons are transfected with the targeted form of the enzyme. This is consistent with the targeted form of ChABC being more effective at promoting axon outgrowth than the non-targeted version.β-integrins can exist in both activated and inactivated forms. Therefore, importantly, we have demonstrated that the enhanced staining for β1-integrin is accompanied by an increase in staining for pFAK, consistent with the β-integrins being in the activated state. They would therefore be able to promote neurite outgrowth via an increase in cell adhesion. CSPGs are known to down-regulate cell surface integrin expression  and alsoWe have demonstrated a role for PTEN in ChABC mediated neurite outgrowth.PTEN mRNA and protein expression were significantly reduced in cells expressing ChABC, and this was accompanied by the increase neurite length. A similar increase in neurite length was produced by the PTEN inhibitor VO-OHpic . This isAlthough the enhancement of neurite outgrowth associated with PTEN inhibition is modest, it is the first report of ChABC enhancing neurite outgrowth via an intrinsic mechanism. This finding, taken together with the upregulation of cell-surface β1-integrin, adds to the list of the enzymes’ known benefits and strengthens the case for including ChABC as an essential component of any combination treatment for SCI.in vivo study to reveal its true potential.The observation that targeting ChABC to the axon, enhanced neurite length compared to non-targeted ChABC, suggests that this may be an even more powerful way of enhancing axon regeneration. Importantly, the finding that ChABC down-regulates PTEN in neurons, and promotes neurite outgrowth of neurons expressing surface CSPGs on laminin, has important implications for promoting long-distance regeneration of the corticospinal tract. This is still a major challenge that remains to be overcome, before a successful treatment for SCI is attained. Indeed, the combination of PTEN knockdown, with CSPG removal has been predicted to be a promising strategy to promote extensive plasticity in adult mammals . In summS1 Table(DOCX)Click here for additional data file.S1 FigNeurons were stained with anti-β-tubulin-III to visualise the neurites. The MWU-test showed a significantly lower median neurite length on a substrate containing laminin+ CSA 75μg/ml compared to laminin alone, P<0.001, n = 230, indicating that this concentration of CSA is inhibitory to neurite outgrowth. L = laminin, C4S = chondroitin-4-sulfate (CSA).(TIF)Click here for additional data file.S2 FigThe neurite indicated by the arrow is ~ 194μm. The colour represents pixel intensity, as shown in the scale to the left of the figure, where white is maximum intensity and black is minimum.(TIF)Click here for additional data file.S3 FigThe Neurites of DRG’s transfected with targeted ChABC and plated onto CSA, mean neurite length, 283.6μm, are significantly longer than those of DRGs transfected with non-targeted ChABC, mean length 255.0μm, plated on CSA, Z = 16.13, P<0.0001, and the GFP controls, mean length 209.78μm, plated on CSA, z = 17.3, P<0.0001. The neurites of DRGs transfected with non-targeted ChABC and plated onto CSA are also significantly longer than the GFP controls, z = 17.3, P<0.0001. Values are mean+/-SEM. MWU test with Holm adjustment. n = 200.(TIF)Click here for additional data file.S4 FigLong neurites are associated with enhanced staining for β-integrin.(TIF)Click here for additional data file.S5 FigDRGs plated onto CSA and transfected with targeted chABC  or GFP  and stained for β-tubulin (Left hand panels) or pFAK (Right hand panels). Left hand panel (Top) shows staining for β-tubulin III is present in both the cell body and the axons of controls, (A) and targeted ChABC transfected DRGs (bottom panel), B. Right hand panel (Top), A shows weak staining for pFAK in the cell body of GFP-transfected DRGs and no staining is observed in the axonal compartment. In contrast, targeted ChABC transfected DRGs show bright staining for pFAK in the cell bodies and diffuse staining in the axonal compartment, indicating β-integrin activation, .(TIF)Click here for additional data file.S6 FigRho A-staining of DRGs plated onto CSA and transfected with targeted chABC (bottom panel) or GFP (Top panel). Left hand panels show staining for β-tubulin III is present in both the cell body and the axons of controls (Top) and DRGs transfected with targeted ChABC(bottom). Right hand panels show strong staining for RhoA in both the cell body and axons of control neurons (Top). Neurons transfected with targeted ChABC show staining for RhoA in the cell body, but weak staining of the axonal compartment (bottom).(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Elastic nails made of the nickel-titanium shape memory alloy (Nitinol) have been reported to control bone modeling in animal studies. However, the mechanical stability of the Nitinol nail in the fixation of long bone fractures remains unclear. This study compared mechanical stability among nails made of three materials, namely Nitinol, titanium, and stainless steel, in the fixation of long bone fractures. These three materials had identical shapes (arc length: π/2 and radius: 260 mm). A cylindrical sawbone with a 10-mm gap and fixed with two C-shaped elastic nails was used to examine the stability of the nails. A finite element (FE) model was developed based on the sawbone model. The end cap for elastic nails was not used in the sawbone test but was considered based on a constraint equation in FE simulation. The results of stability tests appeared to depend on the presence or absence of the end cap. In the sawbone test, the titanium nail yielded a higher ultimate force against the applied load than did the stainless steel and Nitinol nails before the gap completely closed; the difference in linear stiffness between the nails was nonsignificant. In FE simulation, the titanium nail produced smaller gap shortening than did stainless steel and Nitinol nails without the end cap; the difference in gap shortening between the nails was minor with the end cap. The titanium elastic nail should be a better choice in managing diaphyseal long bone fractures when the end cap is not used. For Nitinol and stainless steel nails, the end cap should be used to stop the nail from dropping out and to stabilize the fractured bone. The elastic stable intramedullary nail (ESIN) is used for the fixation of diaphyseal long bone fractures in adolescents, children, and small people ,2,3. TheNitinol  is a type of SMA and has been widely used in many implant types, such as vascular stents and memory staples ,14, as wThe Nitinol nail has been demonstrated to increase bone density and promote bone healing in animal models ,11. FurtTo investigate the effect of various nail materials, namely titanium, stainless steel, and Nitinol, on the stability of the fractured long bone fixed with two C-shaped nails, an experimental test and FE simulation were used.A cylindrical sawbone  with a length of 260 mm, an outer diameter of 35 mm, and a wall thickness of 11 mm was used, and a 10-mm transverse gap was created at the middle of the sawbone. Subsequently, two prebent, C-shaped elastic nails were used to fix the fractured sawbone. The outer diameters of the nails was 3 mm [A material property test system  was used to apply axial compression force to the fractured sawbone fixed with two C-shaped elastic nails a. The diBased on the present sawbone nail model, a solid model was developed using SolidWorks 2014  to examine the internal responses of the nail and bone under loading. The outer diameter and prebending degrees of the nail were the same as those used in the experimental setting b. The naThe elastic modulus and Poisson’s ratio of the bone were 12 GPa and 0.3 (according to the manufacturer), respectively. Titanium and stainless steel were set as plastic behavior, and the bilinear hardening effect was then adopted for these two metals. Material parameters were used from the engineering database in ANSYS Workbench . The supTwo types of loading conditions based on our previous study were used to examine the stability of the fractured bone fixed with nails fabricated from different materials . To simuIn the experimental compression test, the loading with the gap completely closed was recorded as “ultimate load,” and the relationship between force and displacement at the initial linear stage was referred to as “linear stiffness” for comparison . The valExperimental results indicated that compared with stainless steel and Nitinol nails, the titanium nail yielded a significantly higher ultimate load before the gap completely closed (P < 0.05); the difference in the ultimate load between stainless steel and Nitinol nails was not significant . FurtherFE simulation results indicated that the titanium nail could sustain the applied loading (150 N) without gap closing, either under axial compression or bending a,b, in tIn this study, the mechanical stability of the currently used titanium and stainless steel nails was compared with that of the Nitinol nail in the fixation of diaphyseal long bone fractures by using the sawbone test and FE simulation. The results of the sawbone test and FE simulation both indicated that the titanium nail provided the highest stability for the bone fracture, particularly when the end cap was not used. The Nitinol nail demonstrated the lowest stability. Although the Nitinol nail has facilitated a favorable outcome for bone union in animal studies ,11, it pThe titanium nail resulted in a higher ultimate load (sawbone test) and stability (FE simulation) than did stainless steel and Nitinol nails, because the yield stress of titanium (800 MPa) is considerably higher than that of stainless steel (210 MPa). Although the elastic modulus of titanium in the linear phase is only half of that of stainless steel, the linear elastic zone (before the yield stress) of titanium is much wider than that of stainless steel. Compared with the stainless steel nail, the titanium nail developed a higher contact force, thereby generating a higher frictional force against the applied load. The superelastic property of Nitinol could supply a consistent force when phase transformation was triggered, and the force did not increase with strain. Although the linear zone  of Nitinol is slightly smaller than that of titanium, the elastic modulus of Nitinol is considerably lower than that of titanium. Compared with the titanium nail, the Nitinol nail developed a lower contact force between the nail and canal; consequently the Nitinol nail’s ultimate force was also lower.Titanium and stainless steel are commonly used metals for preparing ESINs. Nails composed of stainless steel and titanium may exhibit different responses under loading. Whether the titanium nail provides higher stability than the stainless steel nail remains controversial ,30. In tIn previous FE simulation studies, the end cap was reported to be crucial to the stability of the titanium nail when the contact force between the nail and canal was not adequate to stop the nail from sliding against the inner aspect of the canal, particularly in nails with low prebending degrees ,23. In tAlthough the Nitinol nail demonstrated relatively low stability in this study, it remains a potential biomaterial for many medical devices because of its unique shape memory effect and superelastic material properties. Furthermore, compared with the elastic moduli of titanium (110 GPa) and stainless steel (210 GPa), the elastic modulus of Nitinol (30–70 GPa) is much closer to that of the cortical bone. Hence, the stress shielding effect caused by a material with a higher elastic modulus than that of the bone can be decreased by reducing the elastic moduli of implants. The shape memory effect enables the implant to be delivered into the bone through a small tunnel, and then recovery to the original complex shape for supporting, such as kyphoplasty ,32. In aThe elastic modulus of Nitinol used in this study was only approximately 30 GPa, which was lower than that reported in the literatures ,37,38. IThis study involved some limitations that should be addressed. First, the simulation results are approximate solutions, and some small errors likely occurred during the transformation of the complex geometry into simple elements. Second, the FE bone model was simplified as a cylinder for comparison with the sawbone. The real contact between the nail and canal is more complex than that in the present model. Third, the effect of temperature on the material properties of Nitinol was not considered in the FE simulation. Fourth, the solid model of the end cap was not modeled, and the contribution of the end cap was replaced by a constraint equation in the simulation. Fifth, the difference of frictional coefficient between the materials was not considered in the simulation. Sixth, the Nitinol with a higher elastic modulus is unavailable, hence the stability may be underestimated. We have substituted the elastic modulus of the Nitinol with a higher value in the simulation and it indeed increased the stability.Nitinol has received substantial attention in recent years, particularly for its applications in orthopedic implants. Thus, the differences in mechanical behavior between Nitinol and conventional metals should be clarified before this material is further applied clinically, particularly in weight-bearing limbs. The present study is the first to demonstrate the mechanical stability of titanium, stainless steel, and Nitinol nails in the fixation of diaphyseal long bone fractures. According to the results of this study, the C-shaped titanium elastic nail should be the top choice for the management of diaphyseal long bone fractures when the end cap is not used. For Nitinol and stainless steel nails, use of the end cap is suggested to prevent the nail from dropping out and to stabilize the fractured bone. Additionally, loading with the gap completely closed (the ultimate load proposed in this study) is a key index—along with structural stiffness and gap deformation—for judging the stability of elastic stable intramedullary nails fabricated from various materials.


To evaluate the material flows associated with construction and demolition in different countries it is necessary to have a consistent set of data. However, data collected by regulators and governments differ and this study used concrete as a case in point. Concrete is a significant man-made material in construction whose use reflects socio-economic variation between countries. Flows of natural components, cement and aggregates, are investigated from extraction to final disposal following demolition . The housing sector dominates the use of concrete in urbanized areas and greatly reflects socio-economic and resource extraction issues. To compare concrete stock, use and policies of contrasting countries the data from Thailand and Great Britain (GB) are considered, but as reported they differ for each country. We present here the results of the calculations required to generate an internally consistent database for Great Britain and for Thailand that enables an informed materials flow analysis to be undertaken on materials consumed and generated during construction and demolition of concrete structures. The research methodology and calculations for national cement and concrete production  and the resulting datasets help to make projections that shape policy requirements for Thailand and other emerging economies as reported in . The rawSecondly, inputs required to make concrete are provided by either using data from referenced sources, or calculations based on technical proportions. Finally, quantities of concrete stocks in housing and other construction types are calculated .Table 9P2We utilize knowledge of the chemical reactions involved in calcination Table 32 emissions are calculated based on weight loss after mineral calcination and fuel combustion (COmbustion . Clinkermbustion . Table 6A brief comparison of national datasets such as population, economy, and urbanisation, building lifespan, policy and regulation


The readiness of academics to engage in the service-learning (SL) institutionalisation process is not accentuated in research on SL institutionalisation in South Africa. The argument has been advanced that SL scholarship and willingness of key stakeholders are crucial for SL institutionalisation at the academic programme level.The research focus of the study being reported here was on readiness of respondents to embed SL in the curricula of the nursing programme.This study used a quantitative, exploratory and descriptive design. A self-administered structured questionnaire was used to collect data from a stratified sample comprising 34 respondents. The data were analysed for descriptive statistics using SPSS 19.The demographic profile of the respondents indicated that 31 (66%) were between 31 and 50 years old; 36 (75.16%) had a minimum of 10 years’ nursing experience; 19 (39.6%) had a master's degree, two (4.2%) had a doctorate; and 29 (60.4%) had been employed by the school for a maximum of five years. The results indicated that the nurse educators were in need of SL capacity-building because 9 (18.8%) had limited or no knowledge of SL and 24 (50%) confused SL with other forms of community engagement activities. However, only 15 (33%) of the clinical supervisors and 13 (27%) of the lecturers indicated a willingness to participate in such a programme.The school was not ready to embed SL in the academic programme because of a lack of SL scholarship and willingness to remediate the identified theory–practice gaps. Although research on service-learning (SL) institutionalisation has proliferated both internationally and, lately, in South Africa, the researcher agrees with Bender :138 in tThe research focus of the study being reported here was on readiness of respondents to embed SL in the curricula of the nursing programme.et al.  is conceptualised as an engaged pedagogy:that integrates theory with relevant community service. Through assignments and class discussions, students reflect on their service in order to increase their understanding of module … content, gain a broader appreciation of a discipline, and enhance their sense of social responsibility. HEQC :32SL institutionalisation refers to the process through which SL is perceived and supported as being an essential component of the education process and thus embedded in the culture and organisation of the HEI. Tangible institutional commitment to mainstream SL is demonstrated by making SL an integral part of the infrastructure and everyday operations of the HEI's academic programmes, ensuring that SL is part of each student's academic experience , clinical supervisors and senior academic officers had to be in the employ of the School of Nursing for at least four months prior to the research taking place in order to be eligible for inclusion in the study. The assumption was that, at that stage, they would have been fully oriented regarding the teaching and learning approaches of the undergraduate nursing programme and the teaching and learning policy of the university.Key stakeholders of the academic programme were involved to ensure cooperation, support and ownership , version 19.0 . The data were entered into a Microsoft® Excel spreadsheet and imported into SPSS for descriptive statistical analysis. The data are presented as frequencies and percentages in the form of tables and graphs.The school is the largest residential nursing school in South Africa and has been offering the Bachelor of Nursing (B Nursing) degree as its core undergraduate programme since 2004 :2. The sThis article reports data related to the demographics and the challenges to service-learning institutionalisation pertaining to the SL scholarship and capacity-building needs of the respondents.The demographic profile included gender, age group, employment category, highest nursing qualification, total number of years’ nursing experience and years employed at the current institution. See n = 31; 66%) were between 31 and 50 years old, whilst 9 (19.1%) were older than 51 years.The findings indicate that the workforce of the school is mature in years because only 7 (14.9%) of the sample were 20–30 years old. The majority of respondents (It can be concluded that the School of Nursing had an experienced workforce because 36 (75.16%) of the respondents had a minimum of 10 years’ nursing experience compared to only 12 (25%) who had less than 10 years’ nursing experience.The findings indicate that 10 (20.8%) of the respondents had a diploma, which is lower than the prescribed minimum qualification to teach on the bachelor's nursing programme. The findings also indicate that nursing scholarship may be compromised because only 19 (39.6%) of the respondents held a master's degree, whilst 2 (4.2%) had a doctorate at the time of data collection.n = 29; 60.4%) had been employed in the school for a maximum of 5 years, with only 14 (29.2%) having worked at the School of Nursing for between 6 and 11 years. The findings hint at a high turnover rate of employees at the School of Nursing.The majority of participants (The representation of lecturers (academics) and clinical supervisors was almost equal, with 22 (45.9%) lecturers and 23 (47.9%) clinical supervisors, respectively. Hence, the sample reflected the perspectives of both the theoretical and clinical components of the nursing programmes.Since institutional change is strongly associated with transformational shifts that occur within individuals, the transformational mindset regarding SL within the School of Nursing was determined by the following question: What is the proportion of respondents who identified a need for SL capacity-building as determined by personal commitment to develop SL scholarship? The SL scholarship needs of the respondents were determined by their previous exposure to SL, their request for foundational SL material and their willingness to participate in SL capacity-building. The assumption was that awareness of SL theory–practice gaps would be the catalyst that moves individuals to the commitment described by Bender .The SL capacity-building needs were based on the level of SL scholarship at the time of the research within the school as assessed by previous SL exposure, understanding of SL and self-identified SL training needs (requesting information) of the respondents. Overall willingness to participate in capacity-building activities was also determined.Respondents were asked about attendance at SL training sessions, awareness of HEQC policy guidelines and assessment criteria and SL discussions in their communities of practice. Respondents were asked to describe their understanding of SL. These responses were coded according to the following four categories: correct understanding of SL; confusing SL with other forms of community engagement activities; limited or no knowledge of SL; and no response. Four (8.3%) of the respondents had a correct understanding, 9 (18.8%) had limited or no knowledge of SL, 11 (22.9%) did not indicate any response and 24 (50%) confused SL with other forms of community-engagement activities.Respondents were also asked to indicate whether they needed training in the philosophy, theoretical foundations and development of SL modules and information on the national SL policy guidelines. The responses are summarised in Twenty-six (54.2%) of the respondents indicated their willingness to attend SL training sessions. Overall willingness, assessed by combining training needs and willingness to participate in SL capacity development, was indicated by 31 of the respondents (64.6%). The clinical supervisors were the most willing group, with 15 (33%) indicating willingness, followed by academics with 13 (27%) and academic officers with 2 (4%).The respondents were required to give written consent before the research project commenced Creswell :89. In aThis issue refers to the researcher's commitment not to violate the individuals’ privacy or to breach confidentiality by unwanted exposure of information. In order to prevent the researcher from linking them to the data entered on the survey, the respondents were not expected to write any personal details on the questionnaire. No personal details that might identify a respondent will be included in the findings or any subsequent research reports and publications :64–65. ANone of the respondents were coerced to participate in the study – they were informed about the proposed study and that the choice to participate was voluntarily. In addition, they were informed about their right to withdraw from the study at any time without any penalty or being required to give a reason :279.Since the researcher was ethically bound to protect the respondents against any known physical and emotional harm, the information letter stated explicitly that no harm was associated with this study :280.All the information collected and the communications related to the study were kept confidential because only those who were directly involved with the research had access to it. The instruments and the data will be kept in a safe place for at least five years after the results had been published, after which it would be destroyed in accordance with the institution's policy :278.Cronbach's alpha coefficient was used to test the internal consistency amongst scale items Creswell :149. Theet al. Validity refers to the degree to which an instrument measures what it is supposed to be measuring  of participants were unfamiliar with the national SL policy guidelines, untrained in SL methodology and had no SL discourse in their respective communities of practice. This finding is a concern for various reasons. From 2003–2005, the School of Nursing participated in the national Community – Higher Education – Service Partnerships (CHESP) initiative in that the Gender-Based Violence SL module was developed as part of the Monitoring, Evaluation and Research Programme of CHESP. This contributed to the drafts of the HEQC's Policy Guidelines for community engagement and SL (Julie et al. The findings in et al. A surprise finding reflected in The low proportion of academics indicating willingness is a concern if we concede that ‘curricular community engagement’ is a scholarly endeavour that ‘moves beyond the service experience’ Bender :128. SinThe findings related to the conceptual understanding of SL revealed that conceptual confusion was prevalent amongst both academics and clinical supervisors at the School of Nursing because only 8.3% provided a correct understanding of SL. Although this finding reflects a national trend, as stated by Bender :81, it iThe findings confirmed the readiness of academics in terms of SL scholarship as well as willingness, which is crucial to SL institutionalisation. The self-assessment tool for SL institutionalisation in higher education developed by Furco  should tThe management of the School of Nursing should address the existing conceptual confusion by developing an operational definition for SL in order to differentiate it from other forms of community engagement.SL champions should be identified across the four year-levels of the programme and provide the necessary enabling environment in order to motivate academics to participate in SL capacity-building.This study was confined to one school that had a relatively high staff turnover. Hence, the study should be extended to other faculties in the university.This article reported on the individual's readiness as a crucial component for SL institutionalisation at the programme implementation level. In terms of ‘cracking the nut’, the results revealed that the respondents were not ready for SL institutionalisation because of a lack of SL scholarship and willingness to remediate the self-identified theory-practice gaps.


An effective sterilisation technique that maintains structure integrity, mechanical properties, and biocompatibility is essential for the translation of new biomaterials to the clinical setting. We aimed to establish an effective sterilisation technique for a biodegradable (POSS-PCL) and nonbiodegradable (POSS-PCU) nanocomposite scaffold that maintains stem cell biocompatibility. Scaffolds were sterilised using 70% ethanol, ultraviolet radiation, bleach, antibiotic/antimycotic, ethylene oxide, gamma irradiation, argon plasma, or autoclaving. Samples were immersed in tryptone soya broth and thioglycollate medium and inspected for signs of microbial growth. Scaffold surface and mechanical and molecular weight properties were investigated. AlamarBlue viability assay of adipose derived stem cells (ADSC) seeded on scaffolds was performed to investigate metabolic activity. Confocal imaging of rhodamine phalloidin and DAPI stained ADSCs was performed to evaluate morphology. Ethylene oxide, gamma irradiation, argon plasma, autoclaving, 70% ethanol, and bleach were effective in sterilising the scaffolds. Autoclaving, gamma irradiation, and ethylene oxide led to a significant change in the molecular weight distribution of POSS-PCL and gamma irradiation and ethylene oxide to that of POSS-PCU (p<0.05). UV, ethanol, gamma irradiation, and ethylene oxide caused significant changes in the mechanical properties of POSS-PCL (p<0.05). Argon was associated with significantly higher surface wettability and ADSC metabolic activity (p<0.05). In this study, argon plasma was an effective sterilisation technique for both nonbiodegradable and biodegradable nanocomposite scaffolds. Argon plasma should be further investigated as a potential sterilisation technique for medical devices. Synthetic biomaterials are being used to replace the extracellular matrix to restore damaged and failing tissues and organs . AmongstOur group have developed and patented two families of nanocomposite polymers for the development of organs and tissues –9. The nAn increasingly popular method of modifying the surface functionality to enhance cell behaviour on scaffolds is plasma modification , 11. PlaThis study compared ethylene oxide, argon plasma, bleach, antibiotic/antimycotic, ethanol, ultraviolet radiation, autoclaving, and gamma irradiation sterilisation procedures for morphological alteration, chemical damage, effects on polymer degradation, and biocompatibility. The viability of ADSCs was assessed following the sterilisation of POSS-PCU and POSS-PCL scaffolds to assess biocompatibility of the sterilisation techniques. trans-cyclohexanechloroydrinisobutyl-silsesquioxane (Hybrid Plastics Inc.) was heated to 135°C and then cooled to 70°C. Flakes of 4,4′-methylenebis (phenyl isocyanate) (MDI) were then added to the mixture, at 75–85°C for 90 minutes to form the prepolymer. Then N,N-dimethylacetamide (DMAc) was to form a solution. Further chain extension was completed by the drop-wise addition of ethylenediamine and diethylamine in DMAc. This then created the POSS-modified polycarbonate urea-urethane in DMAc. All chemicals and reagents were purchased from Aldrich Limited, Gillingham, UK.The nanocomposite scaffolds were manufactured as previously described , 13. In  trans-cyclohexanechlorohydrinisobutyl-polyhedral oligomeric silsesquioxane (POSS) were mixed and heated to 135°C. Then 9.4 g of 4,4′-methylenebis(cyclohexylisocyanate) was added to form a prepolymer. Following this, 100 g of DMAC was added to the prepolymer. Chain extension was performed by drop-wise addition of 1 g of ethylenediamine in 80 g of dry DMAC. Following this, 2 g of 1-butanol in 5 g of DMAC was added to form the nanocomposite. All chemicals and reagents were purchased from Aldrich Limited, Gillingham, UK.POSS-PCL nanocomposites solution was manufactured as described previously . In brieμm) was dissolved in POSS-PCL and POSS-PCU in DMAc containing Tween-20 surfactant (1:1 weight ratio). The solution was dispersed and degassed in a Thinky AER 250 mixer . The polymer mixture was spread onto steel moulds. The moulds were then washed in deionised water to dissolve the solvent and DMAC for 7 days. Following washing, polymer sheets with 700-800 μm thickness were manufactured. For cell culture analysis, 16 mm polymer disks were cut from the sheets using a steel manual shape cutter.Polymers were fabricated as 3D scaffolds using the phase separation/particulate-leaching technique as described previously . Firstly60Co gamma-ray source . Scaffolds were exposed on a continuous path for 10 hours as described previously [Scaffolds were irradiated with a dose of 25 kGy at room temperature, using a eviously .The scaffolds were exposed to steam at 121°C for 15 minutes at pressures of 115 kPa as previously described .Polymer scaffolds were submerged in 70% (v/v) ethanol on a roller mixer for 30 minutes as previously described . Followi in vitro analysis to prevent hydrophobic recovery of the scaffolds.Scaffolds are placed in 24-well plates for treatment by LF (radio frequency) argon plasma generator operating at 40 KHz at 100 W. Scaffolds then enter the chamber of the electrode-less, glow discharge apparatus, which is purged 3 times with argon gas  for 2 minutes. The chamber is then evacuated to 1.0 Torr. Plasma is then ignited by a radio frequency excitation source and was maintained at 100 W for 5 minutes. The scaffolds were treated with plasma and immediately seeded with cells forThe ethylene oxide sterilisation is initiated with a preconditioning of the samples which is carried out at 41°C for 13 hours at 42% humidity. The sterilisation step is then performed by 100% ethylene oxide atmosphere at 49°C for 2 1/2 hours. Scaffold are then in air for 9 hours at 43°C.2) for 3 hours as previously described [Scaffolds were UV irradiated by placement in a UV decontamination device  antibiotic antimycotic solution ) for 24 hours at 4°C. Following sterilisation, the scaffolds were washed five times with sterile deionised water on a roller for 15 minutes each time.The scaffolds were submersed in 1000 ppm slow chlorine releasing compound sodium dichloroisocyanurate dihydrate (SDIC) at room temperature for 20 minutes as described previously . The scan = 6) using an Instron 5565 . Young's modulus of elasticity at the 0-25% portion of the curve, maximum tensile strength, and elongation at break were calculated. Statistical differences in the tensile properties between sterilisation techniques were evaluated using two-way ANOVA with post hoc Turkey test.Tensiometry to evaluate the mechanical properties of the scaffolds following sterilisation was performed as described previously . In brieFTIR spectrophotometer was used to analyse changes in the surface chemistry of the scaffolds treated with the different sterilisation techniques as previously described . Chemicaμm nylon filter. GPC analysis was conducted on the Agilent 1260 infinity system using 2 PLgel 5 μm mixed-D columns (300 × 7.5 mm), a PLgel 5 mm guard column (50 × 7.5 mm), a differential refractive index (DRI), and variable wavelength detector (VWD). Statistical differences in the GPC analysis between sterilisation techniques were evaluated using two-way ANOVA with post hoc Turkey test.Molecular weight averages and polymer dispersity were determined by GPC as previously described  (n=6). Iμl volume of deionised water was used in all experiments. Measurement of a single drop was performed on six independent scaffolds (n=6). The average contact angle was calculated using the KRUSS drop shape software (version 1.90.0.14).The static water contact angle of the scaffolds was performed as described previously . In brieThe surface of the scaffolds treated with the different sterilisation techniques was analysed using SEM as described previously  (n=6). S3. The tissue was then further digested in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F12) containing 300 U/mL crude collagenase I  for 30 minutes in an incubator . Following filtration through 70 μm Cell Strainers  the samples underwent centrifugation . Then the ADSC-rich cell preparation formed a pellet at the bottom of the tube. The ADSC cells were cultured for up to 2 passages. When the ADSCs reached approximately 80% confluence, subculture was performed through trypsinisation. For cell cultures analysis, 1.5 X 104 ADSCs at passage 2 were seeded on each polymer disk following sterilisation. Written consent was taken from all patient donors in the study and was approved by the North Scotland ethical review board, reference number 10/S0802/20.ADSCs were isolated from adipose tissue according to the method described by Zuk et al. with modifications as previously described , 14, 15.4 ADSC per well. At 1, 3, 7, and 14 days medium was removed and 10% AlamarBlue prepared in fresh media was added for 3 hours. Following incubation, AlamarBlue fluorescence was quantified at the respective excitation and emission wavelength of 540 and 595 nm. The mean fluorescent units for the six replicate cultures of three individual experiments were calculated for each exposure treatment and the mean blank value was subtracted from these.AlamarBlue, viability assay, was performed as described previously , 13. BriTo study adhesion and morphology of the ADSC onto the scaffolds, immunocytochemistry morphology staining was performed as described previously , 13. At n = 9).All samples were tested for the effectiveness of sterilisation as previously described . In briep value of ≤ 0.05 was defined as significant. The exact statistical analysis performed for each dataset is described in the figure legend.All statistical analyses were performed using Prism software . Means and standard deviations were calculated from numerical data. In figures, bar graphs represent means, whereas error bars represent 1 standard deviation (SD). A All samples withstood treatment with UV, antibiotic/antimycotic, bleach, and plasma treatment. Although the POSS-PCU samples were unaffected by the autoclaving process, the POSS-PCL samples were destroyed; therefore, it was not possible to examine the autoclaved POSS-PCL samples further. Both the POSS-PCU and POSS-PCL samples held up well against gamma irradiation with slight discolouring/yellowing of the POSS-PCU samples being observed. Ethylene oxide gas caused slight yellow discolouring and ethanol visible deformation of POSS-PCL samples.Quantitative values of mechanical properties of POSS-PCL and POSS-PCU samples subjected to ethanol, bleach (SDIC), plasma, ethylene oxide gas, UV radiation, antibiotic/antibiotic treatment, gamma irradiation, autoclaving (only POSS-PCU), and unsterilised controls for each sterilisation method are presented in The difference in surface hydrophilicity of POSS-PCL and POSS-PCU samples after each sterilisation technique was assessed by measuring the water contact angle . Plasma −1 and 1557 cm−1 and an increase in the peak at 1520 cm−1. POSS-PCL FTIR spectra were not significantly affected by the other sterilisation techniques. FTIR spectra of POSS-PCU samples were unaffected by different sterilisation techniques.Mw) of 91200 g/mol and a number average molecular weight (Mn) of 47700 g/mol, whereas POSS-PCL had an Mw of 361100 and Mn of 141000 g/mol. After ethanol, bleach, UV radiation, and antibiotic treatments there was a negligible impact on Mw, for any of the samples. However, solely amongst POSS-PCL samples, ethanol caused a decrease of 21% in Mn, whilst UV increased Mn by 10% in comparison to unsterilised POSS-PCL. No major changes in molecular weight distributions were detected in autoclaved samples of POSS-PCU. However, autoclaved POSS-PCL samples showed a 52% decrease in Mw and 38% decrease in Mn. Exposure to gamma irradiation had a significant impact on all of the samples. Mn of POSS-PCU decreased significantly by 16%, whereas Mw increased by 48%. Meanwhile, gamma irradiation decreased both Mn and Mw of POSS-PCL by 68% and 58%, respectively. Ethylene oxide increased Mw of POSS-PCU by 23% and decreased Mn of POSS-PCU by 28%. Concurrently, it decreased Mw and Mn of POSS-PCL by 23% and 31%, respectively. Plasma had no significant impact on the molecular weight distribution of POSS-PCU or POSS-PCL polymers.Gel permeation chromatography (GPC) results are summarised in SEM images of POSS-PCL and POSS-PCU after different sterilisation techniques are shown in The results of the alamarBlue viability assay after 1, 3, 7, 10, and 14 days of incubation are presented in Confocal laser scanning microscopy images indicated that ADSC developed different morphologies when grown on differently sterilised surfaces . Cells gThe polymeric materials were incubated in TSB and THY for 7 days to test the efficiency of sterilisation with the resultant level of bacterial growth reported in Polymer degradation after sterilisation techniques can be assessed using (i) macroscopic characterisation methods, providing information on “bulk” properties such as mechanical performance, (ii) microscopic characterisation, looking at molecular weight and its dispersity, (iii) characterisation of the molecular structure and composition, such as FTIR analysis, and (iv) surface characterisation, i.e., scanning electron microscopy and surface wettability . AccordiThe three leading sterilisation methodologies with FDA approved for medical devices are gamma irradiation, autoclaving, and ethylene oxide. Autoclaving has been the earliest method for the sterilisation of biomaterials. Sterilisation with moist heat in an autoclave is usually performed at temperatures equal to or higher than 121°C; dry heat sterilisation requires considerably higher temperatures to effectively inactivate bacterial spores. The suitability of steam sterilisation has been questioned for polyurethanes, as the high temperature may soften the polymer and deform the material . In thisGamma sterilisation involves ionising radiation from either cobalt 60 isotope or accelerated electrons. Gamma irradiation can generate free radicals in the polymer, causing surface oxidation and subsequent degradation due to chain scission and cross-linking with increasing dosages of radiation . HoweverGamma irradiation has been shown to cause the release of free radicals in polymers, causing surface oxidation and subsequent degradation of the polymer. Surface oxidation of the polyurethanes is indicated by a strong yellowing of samples, which was also found in this study with some POSS-PCL scaffolds. Despite the degradation and potential surface oxidation of gamma on PCL scaffolds, the water contact of POSS-PCL did not change after gamma sterilisation compared to control. There are reports in the literature that show no change in water contact angle of biomaterials following gamma sterilisation , 24. ThiGamma irradiation showed no changes in material properties including mechanical, surface chemistry, or molecular number for POSS-PCU scaffolds. However, SEM did show some surface changes after gamma sterilisation on POSS-PCU. Although suitable for POSS-PCU, POSS-PCL was unsuitable for gamma sterilisation with significant changes in molecular weight and number.EtO is the final sterilisation technique, which has been approved for medical implants. EtO is a liquid below 11° so in contrast to steam sterilisation it is a low temperature method . In addi in vitro and in vivo examination. The use of SDIC prevented contamination of the samples and was not associated with any significant changes in mechanical properties or any visual deformation of both nanocomposite polymers. Although longstanding polymer exposure to bleach has been associated with increased hydrophobicity [Although not considered sterilisation techniques for medical devices, we also evaluated the effect of different disinfectant methods. Techniques such as bleach, using antibiotics, and UV radiation are used in the laboratory setting to sterilise scaffolds forhobicity , we did hobicity . In thisEthanol and UV are all useful sterilisation techniques for laboratory analysis and provided sterility of the POSS-PCU and POSS-PCL nanocomposite scaffolds. For POSS-PCU scaffolds, UV and ethanol did cause some changes to the surface as shown by SEM although bulk mechanical properties were not affected. UV decreased the contact angle of POSS-PCU, which is consistent with other reports of UV creating hydrophilic surfaces –29. UV sPOSS-PCL scaffolds also maintained their bulk mechanical properties after ethanol and UV modification. However, reports in the literature show that whilst Gram-positive, Gram-negative, acid-fast bacteria, and lipophilic viruses show high susceptibility to concentrations of ethanol in water ranging from 60 to 80%, hydrophilic viruses and bacterial spores are resistant to the microbial effects of ethanol . Antibio in vitro compatibility of the sterilised scaffolds was performed to evaluate the release of cytotoxic molecular weight products from the sterilisation. Viability data demonstrated that significantly more cells were able to adhere to POSS-PCU and POSS-PCL after argon plasma surface modification compared to other sterilisation techniques by Day 7 (Plasma technology is defined as neutral ionised gas and includes photons, electrons, positive, and negative ions, atoms, free radicals, and nonexcited molecules . Severalby Day 7 . SeveralMenashi et al. first reported the use of argon for sterilisation in 1968 after sterilising the surface of vials contaminated by bacterial spores . Since tThere were limitations to this work. During the plasma treatment the scaffolds were exposed to the unsterile environment whilst moving them from the plasma chamber. To further optimise the plasma treatment the apparatus will be placed in a sterile laminar flow cabinet in the future.In summary, argon plasma maintained the properties of the nanocomposite scaffolds in addition to improving cell adhesion . Plasma In conclusion, the FDA approved sterilisation technique of autoclaving maintained POSS-PCU characteristics but was unsuitable for biodegradable POSS-PCL scaffolds. Taking all results into account, we argue that plasma surface modification using argon gas may effectively sterilise polyurethane biomaterials as well as improve cytocompatibility of tissue engineering scaffolds by modification in surface wettability and topography. Argon plasma should be explored and optimised for the sterilisation of further biomaterials.


A survey of presences and absences of specific species across multiple biogeographic units (or bioregions) are used in a broad area of biological studies from ecology to microbiology. Using binary presence-absence data, we evaluate species co-occurrences that help elucidate relationships among organisms and environments. To summarize similarity between occurrences of species, we routinely use the Jaccard/Tanimoto coefficient, which is the ratio of their intersection to their union. It is natural, then, to identify statistically significant Jaccard/Tanimoto coefficients, which suggest non-random co-occurrences of species. However, statistical hypothesis testing using this similarity coefficient has been seldom used or studied.p-values and false discovery rates. The proposed estimation methods are orders of magnitude faster than the exact solution, particularly with an increasing dimensionality. We showcase their applications in evaluating co-occurrences of bird species in 28 islands of Vanuatu and fish species in 3347 freshwater habitats in France. The proposed methods are implemented in an open source R package called jaccard (https://cran.r-project.org/package=jaccard).We introduce a hypothesis test for similarity for biological presence-absence data, using the Jaccard/Tanimoto coefficient. Several key improvements are presented including unbiased estimation of expectation and centered Jaccard/Tanimoto coefficients, that account for occurrence probabilities. The exact and asymptotic solutions are derived. To overcome a computational burden due to high-dimensionality, we propose the bootstrap and measurement concentration algorithms to efficiently estimate statistical significance of binary similarity. Comprehensive simulation studies demonstrate that our proposed methods produce accurate We introduce a suite of statistical methods for the Jaccard/Tanimoto similarity coefficient for binary data, that enable straightforward incorporation of probabilistic measures in analysis for species co-occurrences. Due to their generality, the proposed methods and implementations are applicable to a wide range of binary data arising from genomics, biochemistry, and other areas of science. Specifically, for k=1,…,m,yi,k∼i.i.d.Bernoulli(pi) and yj,k∼i.i.d.Bernoulli(pj). Because this conventional definition is undefined if both binary vectors contain only zeros such that yi∪yj=0, we refine the definition of Jaccard/Tanimoto coefficientUnder the null model of independence, m trials . Four categories arising from presence-absence data are N1=yi∩yj,N2=yi∩(1−yj),N3=(1−yi)∩yj and N4=m−N1−N2−N3. From pi and pj, probabilities of those four categories are pipj,pi(1−pj),(1−pi)pj and (1−pi)(1−pj), respectively. Putting them together, N= is distributed according to a multinomial distribution, Multi,(1−pi)pj,(1−pi)(1−pj)).Following the definition of Jaccard/Tanimoto similarity coefficient in Eq. , we deriyi and yj are independent, thenIf N1+N2+N3. We observe that N1|N1+N2+N3 follows N1+N2+N3>0, we haveN1+N2+N3=0, we haveFirst, we compute conditional expectation given Therefore,This allows us to define the centered Jaccard/Tanimoto coefficient asThis accounts for expected values, naturally distinguishing negative and positive associations. Generally, we would like to measure the deviation of an observed coefficient from an expected value, instead of simply looking at a magnitude of an observed statistics. Furthermore, this centered coefficient may be scaled by variance in order to span a pre-defined range.yi and yj are independent, a following statistical hypothesis testing is performed:To evaluate whether H0 is that the centered Jaccard/Tanimoto coefficient equals zero. Note that this is equivalent to that the conventional (uncentered) Jaccard/Tanimoto coefficient equals an expected value under independence. Therefore, although we propose and use the centered coefficient, this hypothesis testing is attributed to both uncentered and centered versions. Then, a p-value indicates a probability of observing a coefficient equal to or more extreme than an observed coefficient under the null hypothesis.The null hypothesis p-value, we derive the distribution of Jaccard/Tanimoto coefficient under the null hypothesis. In terms of N=, the Jaccard/Tanimoto coefficient can be expressed asTo obtain its pi and pj are known, the p-value is given by When pi and pj are usually unknown. Therefore, we define the centered Jaccard/Tanimoto coefficient by pi and pj respectively. Plug-in estimates of pi and pj for each configuration  separately.However, in practice, probabilities into Eq.  will resSo in this case, the critical region is defined as followswhere Results for comparison), we introduce an asymptotic approximation when m→∞. It may be useful when dealing with very large binary data, where computational power is a bottleneck. Denote by q1=pipj the probability that both yi and yj have ones, and by q2=pi+pj−2pipj the probability that only one of two vectors has one. Similarly, m→∞, we can estimate the variance:Because the exact distribution is computationally expensive  with N1+N2+N3+N4=m, such that Iε, we have following boundsThis can be quickly and accurately estimated by the measure concentration algorithm (MCA) with a known error bound . For eveIn addition, N= and N∼N′, if N′ can be obtained from N by moving one element to a different class. We construct the set Iε as follows.The idea behind the algorithm is that a multinomial distribution concentrates around its mode. Two possible states Iε contains only the mode of multinomial distribution. We find the mode by a simple hill climbing algorithm, which starts with a state close to the mean of the multinomial distribution and follows the direction of increasing probability until the maximum is reached. Because of unimodality, it is indeed a global maximum. In the next steps, we add the neighbors of states which were previously visited. The procedure is repeated until the total probability of set Iε reaches the desired value 1−ε. The details of the above method can be found in  re reAdditiAdditional file 3 Hypergeometric p-values of similarity among independent presence-absence vectors of m=200 with p=.5. We used a hypergeometric distribution [gt and plt can be “interpreted and reported as p-values”. The dashed red lines indicate theoretically correct Uniform distributions.ribution  to obtaiAdditional file 4 Scatterplot of marginal occurrences of 53 bird species and Jaccard/Tanimoto coefficients. As expected, we observe high correlation (Pearson correlation =0.43) between marginal occurrences and Jaccard/Tanimoto coefficients.Additional file 5 Histograms of conventional and centered Jaccard/Tanimoto similarity coefficients. The conventional (uncentered) Jaccard/Tanimoto coefficients are centered by their expected values under the independence assumption.Additional file 6 Comparison of p-values from the bootstrap and measure concentration algorithm (MCA). Both algorithms were applied on 1378 co-occurrences of bird species. The difference between estimated p-values from two methods is minimal with a mean squared deviation of 1.15×10−4. The diagonal red line indicates the identity.