Quantitation of human Fab in periplasmic extracts was performed by Surface Plasmon Resonance (SPR) on a PARP signaling Biacore A4000 or Biacore 2000 instrument (GE Healthcare, NJ). A standard curve was generated by diluting human Fab (Jackson Immunoresearch) in two-fold serial dilutions into assay running buffer and used for the estimation of Fab concentrations (Supplementary methods, tables and figures). Fab standard and unknowns were injected

over a goat-anti-human IgG [specific for F(ab′)2] surface (Jackson Immunoresearch) immobilized at a high density on a Biacore CM5 Sensor chip (GE Healthcare). Data analysis was performed using the BIAevaluation software (GE Healthcare). For the first round of phage panning using a naïve scFv library (Schwimmer et al., 2013), 4.7 × 1013 cfu of phage particles from a scFv kappa library or 1.6 × 1013 cfu of phage particles from a Fab lambda library combined with 2.2 × 1013 cfu of phage particles from a Fab kappa library were blocked for 1 h at RT in

5% non-fat dry milk (Marvel Premier Foods, UK) in PBS buffer with gentle rotation. Blocked phage was twice deselected for 45 min against streptavidin-coated magnetic Dynabeads® M-280 (Invitrogen Dynal AS, Oslo, Norway). The kinase antigen (R&D Systems, MN) that GSK126 mouse was used for the scFv panning, and Tie-2 antigen (R&D Systems) that was used for the Fab panning, were biotinylated with Sulfo-NHS-LC-Biotin (Pierce) using the manufacturer’s protocol in 20-fold molar excess of biotin reagent and confirmed by ELISA. The biotinylated products were then incubated with blocked streptavidin-coated magnetic Dynabeads® M-280 for 1 h with gentle Glycogen branching enzyme rotation in order to immobilize biotinylated antigen and remove unbiotinylated material. Antigen-captured beads were then washed twice with PBS. For the first, second and third rounds of selection, 100, 50 and 10 pmol of biotinylated kinase or Tie-2 were used, respectively. For the first round, the deselected phage was divided into two aliquots: one was used to infect TG1 cells, and the other was used to infect TG1 cells harboring pAR3-cytFkpA. The rescued, deselected phage was used to perform parallel first round pannings by incubation with

biotinylated kinase streptavidin beads for 90 min at room temperature. The input phage for rounds two and three was generated with separate rescues from either the round one TG1 infection or the round one TG1 with pAR3-cytFkpA. For the first round of panning, beads were washed quickly (i.e., beads were pulled out of solution using a magnet and resuspended in 1 ml wash buffer) three times with PBS—0.05% TWEEN®-20, followed by three times with PBS. For the second round of panning, beads were washed for five times with PBS—0.05% Tween followed by one 5-minute wash. Similar washes were performed with PBS. For the third round of panning, beads were washed quickly for 4 times with PBS—0.05% TWEEN®-20 followed by four 5-minute washes with PBS—0.05% TWEEN®-20.

The mean values of contents in calibration Selleckchem MLN0128 set and validation set were approximately equal with similar ranges in variation ( Table 3). The PLS regression statistics of cross-validation and test set validation are shown in Table 4. The model for the ground powder protein had the highest coefficient

of correlation (r2 = 0.97) followed by starch (r2 = 0.93). The protein model also had the highest RPD of 4.09 in the cross validation and 4.05 in the external validation, which indicated extremely on good prediction. The starch model of the milled powder, with a coefficient of correlation of 0.93 and RPDs of 2.64 and 2.95 in cross validation and external validation, demonstrated a good predictive capacity. The RPDs over 2.00 and below 2.50 showed that the predictive

capability for total polyphenol in the milled powder and for protein and starch in whole seeds could be used for rough estimation of their content. The oil NIR models could not be used find more for practical germplasm analysis. The optimal model for ground powder with lower values of rank was better than for seeds ( Table 4). Fig. 3 and Fig. 4 represent the optimized regression lines of PLS models in the cross validation of the constituents. As determined by automatic selection which was based on the values of BIC (Bayesian Information Criterions) across different clustering solutions, the optimized number of clustering was three. The clustering features covered constructors (sample number and producing area) and seed composition characteristics. The three groupings consisted of 91 samples in Group 1 (46.7%), 62 samples in Group 2 (31.8%) as well as other 42 samples in Group 3 (21.5%, Table 5). Group 1 was characterized by low content of starch (40.96 ± 1.49%) Selleck 5 FU and total polyphenol (3.52 ± 0.79 mg g− 1) with a high content of oil (1.30 ± 0.32%). Group 2 had high content of protein (28.12 ± 1.39%). Group 3 was in low content of protein (26.56 ± 1.12%) and oil (0.93 ± 0.24%) but was high in starch (44.04 ± 1.05%) and total polyphenol (5.06 ± 0.98 mg g− 1). These

results showed the typical features of groupings clustered by a two-step cluster analysis. Canonical discriminant analysis demonstrated that the concentration of protein (Wilk’s Lambda = 0.825, F = 20.302, P = 0.000), starch (Wilk’s Lambda = 0.615, F = 60.129, P = 0.000), oil (Wilk’s Lambda = 0.785, F = 26.232, P = 0.000) and total polyphenol (Wilk’s Lambda = 0.671, F = 46.999, P = 0.000) were all significantly important in the determination of the three groups. The correction ratio of validation was high (79.5%), which indicated agreement with the results of the calibration set. The outliers of discrimination included that thirteen varieties in Group 1 were predicted to Group 2 and one to Group 3; eighteen in Group 2 were assigned to Group 1 and five to Group 3; one in Group 3 was placed in Group 2 and two in Group 1. Group 2 was clustered into two subgroups (Table 5).

Transgenic enhancer assays also enable the activity of a human ncHAR sequence to be compared to its ortholog from chimpanzee or other mammals. Of 26 ncHAR enhancers that have been tested using both human and non-human primate sequences, seven drive human-specific expression patterns in mouse embryos at day 11.5. The tissues with differential expression

buy CAL-101 are limb (HAR2, 2xHAR114), eye (HAR25), forebrain (2xHAR142, 2xHAR238), and the midbrain–hindbrain boundary (2xHAR164, 2xHAR170). The functional implications of these expression differences remain to be discovered, but it is tempting to speculate that changes in the development of these tissues could influence human anatomy and traits such as fine motor skills, spoken language, and cognition. The past few years have seen a shift in HAR research from sequence based studies

to functional validations, including the discovery of several human-specific enhancers, suggesting that developmental gene regulatory changes played a significant role in human evolution. However, there are still many hurdles to linking genetic changes to divergent traits. One caveat of using transgenic mice or fish to assay HAR activity is that the trans environment is not identical to either human or chimpanzee. Indeed, trans regulatory factors have played a significant role in human evolution (Nowick, in this issue). However, a study that tested http://www.selleckchem.com/products/gkt137831.html orthologous human and zebrafish enhancers in both zebrafish and mouse found almost no trans effects [49]. Another problem is that only a small number of candidate enhancers can be screened with these relatively costly and time-intensive techniques. New genomic

technologies, such as massively parallel reporter assays [50 and 51] and genome editing [52], are opening the door to high-throughput screens of many HARs in model systems as well as human and non-human primate cells. These approaches will enable the validation and comparative analysis of HARs in more cell types and a larger range of developmental stages, which is critical for discovery of HARs with divergent enhancer activity. They may also lead to ways to easily test non-enhancer Racecadotril functions, such as insulators and repressors for which there are currently no straightforward assays. Without a doubt, we are likely to learn the molecular functions of many more HARs in the coming decade. But the critical downstream functional studies needed to link molecular changes to traits remain low-throughput and challenging for the foreseeable future. Perhaps as more humans are sequenced we will be able to study the traits of individuals with ancestral or mutant versions of HARs to discover functional effects at the population level. It will be particularly interesting to discover disease associations in HARs and to eventually unravel the roles HARs played in the evolution of human disease. In conclusion, it is important to remember that accelerated regions are not a human-specific trait.

The perfused livers were dispersed in 50 mL solution A, and the isolated hepatocytes were filtered through a 180 μm nylon filter and centrifuged at 500 rpm for 10 min. After repeating the washing step, the cells were resuspended in Modified Eagle Medium (MEM) Ca2+, 1.8 mM, (Gibco®) and supplemented with 5% fetal bovine serum, 26.2 mM NaHCO3, 1 mM

pyruvate, 0.2 mM aspartic acid and 0.2 mM l-serine. After trypan blue staining, viable hepatocytes were counted by haemocytometry, and 2.5 × 105 or 2.5 × 106 find more cells were plated on 60 mm (for genotoxicity assays) or 90 mm (for mRNA quantitation) collagen-coated dishes, respectively. Hepatocytes were allowed to attach for 3 h and viability was found to range from 85 to 90%. After attachment, the medium was removed and replaced with fresh MEM (Ca2+, 1.8 mM). After replacing the MEM (1.8 mM, Ca2+), PB (CAS 50-06-6) (prepared in 0.9% NaCl) was added directly to the cultures in a final concentration of 1 mM. After 16 h, rat hepatocyte cultures

were incubated with NDEA (CAS 55-18-5) at concentrations ranging from 0.21 to 105 μg/mL (corresponding Pexidartinib ic50 to 0.05 to 25 mM final concentrations) for 3 h. The cells were subsequently washed once with MEM, 0.4 mM, Ca2+, and re-incubated with MEM, 0.4 mM, Ca2+, supplemented with 40 ng/mL EGF (Sigma) and 0.1 μM insulin (Sigma) for 48 h. RNA was extracted after 6 h and cytogenetic assays were terminated after 48 h of NDEA treatment. As a positive control for the cytogenetic assays, the cells were treated with 0.5 μM N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Cytogenetic studies were performed in triplicate as described by Eckl and Riegler (1997) with the following modifications. For determination of the mitotic index (the percentage of total cells in some stage of mitosis) and the number of micronucleated cells, MEM (0.4 mM, Ca2+) was replaced with cold fixative methanol–glacial acetic acid (3:1). The cells were incubated for 15 min on the petri dish, rinsed with distilled water for 2 min and

air dried. The fixed cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) using a solution of 0.2 μg/mL dissolved in McIlvaine buffer (0.1 M citric acid, 0.2 M Na2HPO4, pH 7.0) for 40 min. After washing with McIlvaine buffer tuclazepam for 2 min, the cells were briefly rinsed with distilled water and mounted in glycerol. To determine the mitotic index and number of cells with micronuclei, 1000 cells per petri dish (2000 cells per animal/group concentration) were analyzed under the fluorescence microscope (Reichert Univar) at an excitation wavelength of 350 nm. The micronucleus results are presented as a percentage of cells containing micronuclei in 2000 total cells/group concentration analyzed. The presence of glowing bright and homogenous nuclei in cells was considered the normal phenotype morphology. Apoptotic nuclei were identified by condensed chromatin gathering at the periphery of the nuclear membrane or by fragmented nuclear body morphology.

Thus, it is also not dependent on the accumulated MS dose. However, the slope of the MS concentration–response relationship for 18 months selleck products inhalation is approximately 3-fold steeper than that of the 5 + 4-month schedule (Fig. 5 and Fig. 7), which could erroneously be interpreted as an increased response due to a higher accumulated dose. A more detailed analysis of tumor multiplicity data obtained in the previous Study 1 (Stinn et al., 2012), though, revealed that there is an increase in the slope of the MS concentration–response relationship with the duration of the post-inhalation

period or, rather, with the age of the mice upon final dissection (Fig. 9A), while there is no increase in this slope

upon prolonged MS inhalation periods to the same final study duration or age (Fig. 9B). An age-dependent impact on the slope GSK2118436 chemical structure of the concentration–response relationship can indeed be expected in view of the supra-linear age-dependence of the spontaneous tumor development (Fig. 8). Considering a similar development of spontaneous and induced tumors, steeper dose–response relationships should be expected in long-term rather than in shorter-term chronic studies. Comparing various exposure scenarios, the factor between induced and control tumor multiplicities seems to be the most robust measure of tumorigenesis in this model. Within a given study design with specified inhalation and post-inhalation periods, though, the concentration–response relationship should still be the main parameter to be evaluated CHIR-99021 clinical trial in studies with the objective to compare MS generated from different cigarette types. For this purpose, the current dataset on MDDs provides information that can be used to determine group sizes to fit a certain study design and to provide the required discriminatory

power. The MDDs that can be obtained for the MS tumorigenesis model – if used according to OECD guidelines (Organisation for Economic Co-operation and Development, 2009) in terms of dose levels and group size, are compatible to the discriminatory power that was determined for chemical, in vitro, and subchronic inhalation studies with MS (Oldham et al., 2012). Pulmonary tumorigenesis in the A/J mouse is dependent on the presence and transcriptional activity of a mutated Kras oncogene ( Chen et al., 1994 and To et al., 2006). Previous studies in mouse lung tumorigenesis models showed a lack of mutational effects on Kras by smoke inhalation, which has been interpreted as being indicative of a tumor promoting rather than an initiating activity of smoke ( Wang et al., 2005 and Witschi et al., 2002). This would be consistent with the results of applications of the two-stage carcinogenesis model to epidemiological data ( Hazelton et al., 2005).

Variables with positively skewed distributions were transformed to natural logarithms before further statistical analysis. Regression analysis of data from the LC children was used to assess the relationships between age (as a continuous variable) and sex with each variable (anthropometric, biochemical or dietary). Sex was not a significant Selleckchem DZNeP factor in predicting any of the variables with the exception of creatinine, and therefore was not included in the models presented in this paper. However, 25OHD, iCa, P, FGF23, 1,25(OH)2D, PTH, Cys C, Cr and albumin were influenced by age. Age-adjustments were

therefore included for these variables. To adjust for age in linear regression, age was added as an independent variable in all models. Standard deviation scores (SDS) were calculated for

all variables to enable age-adjusted comparisons to be made between RFU and LC children. As the data from RFU and LC children were collected at the similar time of year, the SDS were, by definition, adjusted for season. SDS http://www.selleckchem.com/products/Dasatinib.html was calculated in the following way: [(value RFU − meanLC) / SDLC] within the specific age bands as indicated in Local community children (LC children). Group differences between RFU and LC children were determined by 2-sample Student’s t-tests using SDS values. This method allowed for the small sample size of LC children in each age band and therefore was a more conservative estimate of the significance of group differences than considering the significance of the deviation of the SDS of RFU children from zero. The sample size of 35 RFU and 30 LC children, meant that the study was able to detect significant group differences in SDS of approximately 0.66 SD (two thirds of

a standard deviation) or greater, at p ≤ 0.05 with 80% power. TCa was corrected for albumin (corr-Ca) by normalising to an albumin concentration of 36 g/l using a correction factor of 0.016 mmol TCa/g albumin. This correction factor was calculated from the slope of the relationship between TCa and albumin in LC children [12]. Urinary excretion and clearance data were corrected for age-appropriate body surface area (BSAage). BSA was calculated using Resminostat the Mosteller formula BSA = √((ht (cm) × wt (kg)) / 3600) m2[13] and then corrected to the age-appropriate mean BSA for each LC AG (AG1: 0.81 (0.12) m2, AG2: 1.16 (0.17) m2, AG3: 1.38 (0.16) m2). As no difference was found between BSAage when calculated with standing height or sitting height, standing height was used for all BSAage adjustments. Estimated glomerular filtration rate (eGFR ml/min), was derived in four ways from equations which use plasma Cys C and/or plasma Cr as markers. The Cys C based equations include: 1) Cys C-eGFR = [74.835 / (Cys C(mg/l)1/0.75)] ml/min [14] and 2) Counahn–Barret ( C-B-eGFR) = [39.1 [ht (m) / Cr (mg/dl)]0.516 × [1.8 / Cys C (mg/l)]0.294[30 / urea (mg/dl)]0.169 × [1.099]male [ht (m) / 1.4]0.188] [15].

5). We observed that trovafloxacin and APAP induced increased dose- and time-dependent cytotoxicity and decreased cell viability at physiologically relevant drug concentrations close to the Cmax (Table 1) after chronic exposure (8 or 15 days) of human 3D liver cultures, but not after acute treatment (2 days) of human 2D

hepatocyte monolayers (Figs. 5A and B). Almost no toxicity was detected with levofloxacin and AMAP in 3D liver cells and 2D hepatocytes (Figs. 5A and B). As described above, troglitazone elicited cytotoxicity and cell viability INK 128 in vivo decrease in human but not in rat 3D liver cells (Fig. 4B). To further characterize this effect, we measured additional endpoints after repeated drug-treatments (up to 8 days) including cell apoptosis (caspase 3/7 activity), cell cytotoxicity (dead-cell protease activity and ALT-release) and cell viability (live-cell protease activity). In human 3D liver cells, troglitazone elicited

cytotoxicity (LDH release) already after 1 day of treatment, demonstrating an acute drug effect (Fig. 6A). After 8 days of treatment, trogitazone caused a dose-dependent increase in caspase 3/7 and dead-cell protease activity, as well as decrease in live-cell protease activity in human 3D liver cells (Fig. 6A). In contrast, pioglitazone applied during 8 days to human 3D liver cells Astemizole did not cause any significant changes in TGF-beta inhibitor any of these parameters (Fig. 6B). In agreement with this result, we observed a dose-dependent increase of ALT release in troglitazone, but not in pioglitazone treated human 3D liver cells (Figs. 6A and B). In this work, we characterized a 3D liver culture model of rat and human for detection of single or repeated drug-treatment induced toxicities. We compared the 3D liver co-culture model with standard monolayer hepatocytes grown on collagen type I, since this

model is one of the most frequently used model in pharmacological industry for drug toxicity screening and mechanistic studies. Other culture models expose hepatocytes between two layers of Matrigel and/or collagen-I gels that allow better retention of cell cyto-architecture, CYP activity and prolonged functional lifespan for up to 14 days compared with cultures on rigid collagen (Dunn et al., 1989, Guguen-Guillouzo and Guillouzo, 2010, Hewitt et al., 2007, LeCluyse, 2001, Lecluyse et al., 2012 and Mingoia et al., 2007). Recent data have shown that heterotypic cell–cell interactions between parenchymal and nonparenchymal cells induce higher levels of phenotypic functions in human hepatocytes than extracellular matrix configuration or composition (Griffith and Swartz, 2006, Guillouzo, 1998 and LeCluyse, 2001).

The concentration of chlorophyll a was determined using several methods: in situ using a Pump Probe immersion fluorometer (PrimProd-EcoMonitor, selleck chemical Russia, in accordance with the methodology developed by Falkowski and Kiefer, 1985 and Falkowski et al., 1986; see also Ostrowska, 2001 and Matorin

et al., 2004); in samples of lake water using HPLC ( Stoń-Egiert & Kosakowska 2005), and the standard spectrophotometric technique (e.g. Jeffrey & Humphrey 1975) (for details, see Ficek 2012). 235 sets of data points obtained from simultaneous measurements of the reflectance spectra Rrs(λ), chlorophyll a concentrations Ca, suspended particulate matter concentrations CSPM, and absorption spectra aCDOM(λ) were used in the analysis and interpretation of the remote sensing reflectance spectra Rrs(λ) described in Ficek et al. (2011) and in the present paper. The waters of the Pomeranian lakes investigated in this study differ widely in their contents of optically active components (OAC); consequently, ERK inhibitor order their spectral optical properties

are also different. As in most inland and coastal sea waters, the OAC they contain consist of suspended particulate matter (SPM) and coloured dissolved organic matter (CDOM), usually in large concentrations. On the basis of numerous empirical investigations (to be presented below), these waters can be conventionally classified into three types differing in their optical properties, although this distinction is not a sharp one – waters with properties intermediate between these types have also been recorded. In waters of Type I OAC concentrations are relatively low: SPM (including phytoplankton1) is dominant and the concentration of CDOM2 is relatively low (Table 2). The optical properties of these waters are similar to those

of Baltic waters (see e.g. Kowalczuk et al., 1999 and Ficek et al., 2011). The waters Y-27632 chemical structure we designated as Type II (humic lakes3) have a very high CDOM concentration, so high that the light attenuation coefficient and other properties of such waters are completely dominated by the light absorption aCDOM in practically the whole spectral range of visible light. Our Type III lake waters are supereutrophic, in which the OAC is dominated by phytoplankton; for practical purposes the absorption/scattering properties of this phytoplankton determine the optical properties of such waters (see Table 2). Figure 1, Figure 2, Figure 3 and Figure 4 illustrate light absorption spectra in the surface waters of the lakes investigated. Figures 1a and b emphasize above all the very great differentiation in the absorption properties of these waters due to the large differences in OAC concentrations in them.

,

2003, Traynor et al., 2006, Cunningham et al., 2007 and Konat et al., 2009). TLR3 stimulation induces a much more robust anti-viral response than TLR4 stimulation (Doyle et al., 2003) and this is characterised by high expression of type I interferons. In the current study, we hypothesized that the neurodegenerating brain is primed with respect to stimulation by systemic anti-viral mimetics. Thus, we predicted that ME7 prion-diseased animals would show similar systemic cytokine responses but amplified CNS inflammatory and sickness behavioural responses to systemic poly I:C stimulation, with respect to normal animals given the same stimulus. We have examined the CNS inflammatory profile and in particular, have focussed on type I interferons VE-822 in vivo and downstream pathways. We Sorafenib mouse also predicted that poly I:C would accelerate disease progression but have no lasting consequences for

normal animals. Female C57BL/6 mice (Harlan, Bicester, UK), were housed in groups of five and given access to food and water ad libitum. We used females in order to avoid fighting and injury, which has significant effects on behaviour. Animals were kept in a temperature-controlled room (21 °C) with a 12:12 h light–dark cycle. The mice were anaesthetised intraperitoneally (i.p.) with Avertin (2,2,2-tribromoethanol) and positioned in a stereotaxic frame. Two small holes were drilled in the skull either side of the midline to allow for bilateral injection of 1 μl of a 10% w/v ME7-infected C57BL/6 brain Thymidylate synthase homogenate made in sterile PBS. Injections were made into the dorsal hippocampus (co-ordinates from bregma: anteroposterior, – 2.0 mm; lateral, – 1.6 mm; depth,

– 1.7 mm) using a microsyringe (Hamilton, Reno, Nevada) with a 26 gauge needle. Control animals were injected with a 10% w/v normal brain homogenate (NBH) in PBS, derived from a naive C57BL/6 mouse. All procedures were performed in accordance with United Kingdom Home Office and Republic of Ireland Department of Health & Children licenses and all efforts were made to minimise both the suffering and number of animals used. Poly I:C was obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). It was prepared for injection by resuspending in sterile saline, heating to 50 °C at a concentration of 2 mg/ml to ensure complete solubilisation and then allowing to cool naturally to room temperature to ensure proper annealing of double-stranded RNA. Poly I:C was stored at −20 °C until use. Experimental groups at 18 weeks post-inoculation with ME7 or NBH were challenged intraperitoneally (i.p.) with either poly I:C (12 mg/kg) or sterile saline to examine systemic and CNS inflammatory responses to systemic poly I:C.

Only he would recognise, in a room full of students, the important meaning of a patient drumming his fingers on the couch. Geoff was a visionary and an innovator. In the preface Crizotinib to the first edition of Vertebral Manipulation [1964] he recognises “The practical approach to the use of manipulation is to relate treatment to the patient’s symptoms and signs rather than to diagnosis” and that “…it is often impossible

to know what the true pathology is…symptoms and signs [of a disc lesion] may vary widely and require different treatments His vision was instrumental in giving us what are now established competencies, including, “Patient-Centred Care”, the use of mobilisation for pain modulation, and an awareness of “the nature of the person” and its impact on treatment. He highlighted the need for deep and broad theoretical knowledge to support and inform clinical practice.

He advocated the discipline of evaluating everything we do to prove our worth and with this came the use of patient reported and orientated outcome measures [subjective and functional asterisks] and the demand for accurate recording of treatment and its effects. Geoff was also at the forefront of research by Physiotherapists for Physiotherapists at a time when it was seen as the role of the Doctor to report on Physiotherapy and decide which Physiotherapy modalities should be prescribed. Geoff wrote extensively for the Australian Journal of Physiotherapy as well as for other medical and Physiotherapy journals world-wide. He wrote, for example, about 5-FU solubility dmso “Some observations

on Sciatic Scoliosis” in 1961, “The hypotheses of adding compression when examining and treating synovial joints” in 1980 and “Movement of pain-sensitive structures in the vertebral canal in a group of physiotherapy students” also in 1980. Look in any respectable physiotherapy or manual therapy journal and you will see G.D. Maitland cited frequently. Researchers in manual therapy are still referring back to Geoff’s models for practice and using Glycogen branching enzyme his descriptions of examination and treatment techniques as their methodological standards. Geoff was a great believer in quality education for Physiotherapists. In 1965 he ran the first 3-month course on “Manipulation of the Spine” based at the South Australian Institute of Technology in Adelaide. In 1974 this course developed into the one-year post-graduate diploma in Manipulative Physiotherapy and subsequently became a Master’s Degree course currently under the auspices of Geoff’s closest colleagues, Mr Mark Jones and Dr Mary Magarey. Geoff always led from the front. As well as being active on various Physiotherapy Committees and Boards in Australia, he was well aware of the much bigger, International, picture and in 1974 was involved in the foundation of the International Federation of Orthopaedic Manipulative Therapy [IFOMPT], a branch of the WCPT.