Dynamic PET imaging of 20 patients with pancreatic ductal adenocarcinoma was used to assess the impact of transport on surrogate metrics of hypoxia: the tumour-to-blood ratio [TBR(t)] at time t post-tracer injection and the trapping rate k3 inferred from a two-tissue compartment model. Transport quantities obtained from this model included the vascular influx and efflux rate coefficients, k1 and k2, and the distribution volume vd≡k1/(k2+k3).The clinical impact of hypoxia in solid tumours is indisputable and yet questions about the sensitivity of hypoxia-PET imaging have impeded its uptake into routine clinical practice. Notably, the binding rate of hypoxia-sensitive PET tracers is slow, comparable to the rate of diffusive equilibration in some tissue types, including mucinous and necrotic tissue. This means that tracer uptake on the scale of a PET imaging voxel—large enough to include such tissue k3 and TBR values were weak to modest: the population average of the Pearson correlation coefficients (r) between voxel-scale k3 and TBR (1 h) [TBR(2 h)] values was 0.10 [0.01] in the 20 patients, while the correlation between tumour-scale k3 and TBR(2 h) values was 0.58. Using Patlak’s formula to correct uptake for the distribution volume, correlations became strong . The distribution volume was substantially below unity for a large fraction of tumours studied, with vd ranging from 0.68 to 1 . Surprisingly, k3 values were strongly correlated with vd in all patients. A model was proposed to explain this in which k3 is a combination of the hypoxia-sensitive tracer binding rate kb and the rate keq of equilibration in slow-equilibrating regions occupying a volume fraction 1−vd of the imaged tissue. This model was used to calculate the proposed hypoxia surrogate marker kb.Correlations between voxel- and whole tumour-scale k3) PET imaging. TBR is reduced by distribution volume effects and k3 is enhanced by slow equilibration. We proposed a novel model to quantify tissue transport properties and hypoxia-sensitive tracer binding in order to improve the sensitivity of hypoxia-PET imaging.Hypoxia-sensitive PET tracers are slow to reach diffusive equilibrium in a substantial fraction of pancreatic tumours, confounding quantification of hypoxia using both static (TBR) and dynamic (The online version of this article (doi:10.1186/s13550-017-0347-3) contains supplementary material, which is available to authorized users. Positron emission tomography imaging of hypoxia is a promising way to detect hypoxia non-invasively in solid tumours , 2. A maAs an example, a typical threshold used to decide whether or not a PET voxel hypoxic is that the voxel-scale tracer concentration exceeds that in blood by 20% after 2 h; i.e.,TBR (2 h) >1.2 –5. This l through the extravascular space of tissue scales as In comparison, the rate at which tracer diffuses across a distance D is the diffusivity of the tracer. For FAZA and similarly sized molecules , D∼10 μm2/s in most tissue  and] andCmodistribution volume: An important tissue transport quantity is the It represents the volume fraction of an imaged ROI in which tracer initially fills; i.e., rapidly equilibrates in. Patlak’s formula , 21, 8\t was used to “correct” TBR for distribution volume effects: for the tumour-to-blood ratio at time Ki≡k3vd is sometimes referred to as the “net trapping rate”. TBR corrected represents the theoretical tumour-to-blood ratio that would have arisen had the distribution volume been unity.In Eq. , Ki≡k3k3, vd, TBR, and TBR corrected, where TBR was calculated as Correlations were analyzed between t=1 and 2 h. Pearson correlation coefficients were calculated to quantify correlations between voxel- and tumour-scale values of these quantities. Voxel-scale coefficients were calculated by fitting the above model to the individual TACs for each voxel, while tumour-scale values were obtained using the average TAC in each tumour. Correlations were reported as the population average (over twenty tumours) of the intra-tumour voxel-scale r values  and as correlations between tumour-scale values .at both k3 and TBR values in each tumour, weak correlations were found at 1 h  and at 2 h . Patient-specific results are shown in Online Resource 1  and moderate correlations were found at 2 h . Although standard imaging protocols call for measurement of TBR at least 2 h after tracer injection, transport coefficient  values obtained using the 1- and 2-h data sets were equivalent to within fit errors to the compartment model. The reduction in correlations is thus a metric for co-registration errors between the 1- and 2-h data sets, as well as the diminished validity of Eq.  but strong correlations with TBRcorrected (mean r = 0.93); see Fig. k3, vd, TBR, and TBRcorrected were identical to the values shown in Table Whole-tumour kinetics are less sensitive to co-registration errors and tumour-scale trapping rate exhibited modest correlations with TBR  but still substantial; see Table In all patients, voxel-scale k3 and vd, we propose a model , and one in which it reaches equilibrium slowly (with concentration Cs): To account for the unexpected correlations between vs represents the voxel volume fraction in which tracer is slow to equilibrate. As noted in the Introduction, tracer will equilibrate slowly in mucinous and necrotic tissue owing to the slow diffusivity and long diffusive distances, respectively.Here, Having defined the above sub-compartments, the distributed-parameter compartment model  that desand vs and vs here ensure detailed balance amongst the compartments. kb is the binding rate due to hypoxia and keq represents the equilibration rate in the regions of slow-equilibration. Recall from the Introduction that we expect it to be on the order of (0.1→1) h −1 when equilibration is driven by diffusion; see Eq.  in Eq. . This isSince 1−1, Eq.  is ident1−1, Eq. , with 1and where we have defined vd defined in Eq. (Equations  and 17)17) are okb and Keq in k3, voxel-scale k3 values were arranged into bins based on distribution volume values. Because there will always be a cohort of normoxic voxels in a tumour for which kb=0 , it is assumed that the lowest M values of k3 in these bins represent equilibration: To distinguish keq values is much smaller than the variance in kb values (so that the two distributions can be distinguished). The choice of M is dictated by their relative sizes: Equation  is stricNb is the total number of values within each bin, σX and X denote the standard deviation and mean values of X=kb or keq. Assuming that the relative variance kb is expected to be large, based on the broad distribution of keq—reflecting that of the size l of the regions in which tracer is slow to equilibrate—is small. This was estimated by calculating the variance in the minimum k3 value in each bin with respect to a vd-dependent average =keq(1−vd)/vd. . Also shown in Fig. k3, keq and kb for the same tumour slices shown in Fig. An example of this algorithm is shown for two patients in Figs. d by Eq.  are plot overlap  and vd from Eq. , keq wik3 and Keq (r=0.57) and kb (r = 0.86) are measures of how much equilibration and binding were found to contribute to the net trapping rate k3. Most of the vd dependence of k3 is contained in Keq, as evidenced by the strong correlations between vd and Keq(r=−0.73) but comparatively weak correlations kb and vd(r=−0.27). Not shown are correlations between these quantities and the vascular influx rate k1 since these were small (|r|<0.15) for all cases.The correlation matrix between derived voxel-scale parameters from our model is shown in Table vd-dependence of k3 in our model is a consequence only of mass conservation and the assumption that there exists a compartment in which tracer is slow to reach diffusive equilibrium. It does not depend on a specific microscopic model for equilibration. We tested the prediction given by Eq. =keq[(1−vd)/vd]γ to determine how close γ was to its predicted value of unity. Averaging over all tumours, we found γ=(0.9±0.4), with the error given by the standard deviation of values across all tumours. This confirms that our model in which tracer equilibrates slowly in a fraction 1−vd of tissue is consistent with our data. The mean equilibration rate derived from these fits was keq=0.44 h −1 , corresponding to an equilibration time of 1/keq∼2.3 h.The n by Eq.  by fitti3] to include diverse cell populations, with widely varying pathology  implied by this result means that unbound tracer will not equilibrate until well-after tracer injection, at times t≫1/keq. At this time, the concentration of tracer in both the slow- and fast-equilibrating regions will approach that in blood and the effect of the distribution volume on TBR will vanish. Ideally, static hypoxia-PET imaging would be carried out when t≫1/keq in order to remove this sensitivity to transport. Unfortunately, the half-life of 18F is short and imaging times are typically restricted to be 3 h or less. The long equilibration time . Although not a sufficient number to be able to definitively attribute the reduced distribution volume to mucous, the patients with the smallest and largest distribution volumes of this four exhibited significant and negligible mucin expression, respectively; see Fig. If slow equilibration were due to necroses, xpressed . Amongsttumour-scale equilibration rates are rapid. The characteristic equilibration rate in the fast-equilibrating regions can be approximated by k1 which, even for the hypo-perfused PDAC tumours studied in this work, was fast compared to kb and keq. The population average of the tumour-scale k1 values was ∼ 0.3 min −1  In recent work, Grkovski et al. discuss the important role of the distribution volume in static PET hypoxia quantification and also report significant negative correlations between k3 and vd [k3 is sensitive both to hypoxia-induced binding as well as diffusive equilibration of un-bound tracer.A key question raised by this work is whether or not the tissue transport effects identified here confound hypoxia quantification using other hypoxia-PET tracers such as FMISO and in other tumour sites. The primary impediment to tracer equilibration is slow diffusivity. FAZA has been estimated to diffuse marginally faster than FMISO , and so  in Ref.  can be u and vd . The prek3 and TBR—are affected by regions where diffusive equilibrium is achieved very slowly, over several hours. We have proposed a scheme to extract the hypoxia-sensitive tracer binding rate as well as the from dynamic PET data and proposed this as a novel hypoxia biomarker. Our results are of relevance for all hypoxia-PET tracers and any tumour site where transport of small-molecular weight agents is challenged.The uptake of hypoxia-sensitive PET tracers in pancreatic tumours depends in a significant way on both tissue transport properties as well as the presence of hypoxia. Both dynamic- and static-PET based hypoxia surrogates— Additional file 1Table S1. (PDF 21 kb)Additional file 2Supplemental information. (PDF 100 kb)


Their minute cross sections minimize tissue displacement upon probe implantation. We functionally validate one probe design in vivo with mice expressing channelrhodopsin-2. Highly local optogenetic neural activation is demonstrated by recording the induced response—both by extracellular electrical recordings in the hippocampus and by two-photon functional imaging in the cortex of mice coexpressing GCaMP6.Optogenetic methods developed over the past decade enable unprecedented optical activation and silencing of specific neuronal cell types. However, light scattering in neural tissue precludes illuminating areas deep within the brain via free-space optics; this has impeded employing optogenetics universally. Here, we report an approach surmounting this significant limitation. We realize implantable, ultranarrow, silicon-based photonic probes enabling the delivery of complex illumination patterns deep within brain tissue. Our approach combines methods from integrated nanophotonics and microelectromechanical systems, to yield photonic probes that are robust, scalable, and readily producible  Ongoing efforts seek to address large functional ensembles of neurons, i.e., “brain circuits,” through realization of tools providing fine enough resolution to interrogate and control each constituent neuron individually and independently. Significant advances in the development of excitatory and inhibitory opsins have been made over the past decade that now permit direct optical control of cellular processes.We identify five critical requirements for realizing widely useful, implantable photonic devices, which we term “visible-wavelength photonic probes”: (i) the probes should provide a multiplicity of microscale illumination sources , each individually controllable and capable of delivering fine illumination, with cellular-scale cross-section dimensions. Ideally, emission from these microscale E-pixels should have minimal spatial overlap, while collectively covering the entire brain volume of interest. (ii) This patterned illumination must be delivered with sufficient intensity to activate optogenetic effectors (actuators/silencers) within the interrogated region. (iii) Associated thermal perturbations of neural tissue at, or adjacent to, the implanted devices must minimally affect neural circuits. Recent studies show that temperature elevation of as small as 1°C can change the neural firing rate and behavior of mice.2,in vitro nor in vivo validation of this particular approach has been reported. Another approach implements modal multiplexing to address several illumination points along an implantable multimode optical fiber.en masse.Various architectures for implantable optical probes have recently been proposed.––,An alternative approach involves the integration of microscale light emitting diodes .The photonic neural probes described herein provide a first proof-of-concept of our paradigm. The prototype devices we report here comprise implantable shanks, initially with nine E-pixels, which are spectrally addressed through “one” single-mode optical fiber. We implement the E-pixels themselves using large diffractive grating couplers that produce beams with low divergence angles, as small as 1.7 deg. This low-light divergence offers beam cross-section dimensions that are comparable to the size of neural cell bodies—even after traversing several hundreds of micrometers. Other recent implementations of implantable probes based on photonic technology3The overall structure of our prototype photonic probes is patterned using standard nanophotonic and MEMS fabrication processes Appendix . The impe-shaped , with tiThe probe head  contains4μ-prism) bridging the external input fiber’s terminus to the on-chip grating coupler. This efficiently couples the light to the photonic waveguides on-chip. Once on-chip, this multispectral light is routed by a single waveguide to an AWG located on the probe head [The visible-wavelength photonic circuitry on the probe is fabricated from an optical multilayer comprising a 200-nm thick silicon nitride . The total IL of these first unoptimized prototypes is about Validation of the capability of our photonic probes to stimulate neural activity, as described in the next section, has been achieved with E-pixel emission power that ranged between 5 to 5Our measurement and simulations results demonstrate that E-pixels emit beams with a propagation direction angle of 2 to 30 deg from the normal to the probe surface . The exaThe beam profile at the surface of the E-pixel is the beam  inset. Wthe beam  and 13, solution , and (iisolution , and 15.Appendix ; Fig. 15 ∼90  μm , is ∼10 n tissue  than in n tissue . However6Our use of WDM makes it possible to independently address on-shank E-pixels by separate temporally modulated multispectral components of the light delivered to the probe. –Our embodiment of the on-chip optical demultiplexer is realized with a visible wavelength AWG . AWGs arc probes . By apprc probes , either c probes . Figure The maximum number of E-pixels addressable by a single AWG is determined by the ratio of the absorption bandwidth of the optogenetic effector to the bandwidth of the individual spectral channels. For example, a typical optogenetic effector such as ChR2x ∼1  nm , thus pe7in vivo, in two separate experimental implementations. In the first, we optogenetically activate neurons in the hippocampus of Thy1:18-ChR2-EYFP transgenic mice,We validate the functional capabilities of our prototype photonic probes ed mouse . In thised mouse  reveals In a second experimental implementation, the functionality of the photonic probes was assessed via simultaneous free-space, two-photon functional imaging of cortical neurons in a mouse coexpressing both ChR2 and GCaMP6s . The phoFor this second set of experiments, 200-ms light pulses with an estimated optical output power of emission .In this second proof-of-concept experiment, an individual neuron was reliably activated by light pulses delivered from an implanted photonic probe. 8To exploit the full potential of optogenetics, it is essential to deliver light with high temporal and spatial resolution at arbitrary locations within the brain. Here, we demonstrate photonic probes operating at visible wavelengths that permit realization of this goal. Our photonic probes leverage technological developments achieved over the past two decades in the field of optical communications at infrared wavelengths, realizing them within the visible range of relevance for present-day optogenetic reporters and effectors operating at visible wavelengths. Specifically, we employ wavelength division demultiplexing using AWG’s, integrated photonic waveguides, and E-pixels realized as diffractive grating couplers. The technology of visible photonics is rapidly advancing, and this makes it feasible to create a spectrum of components for assembling future complex photonic neural probe architectures. Exceptionally promising candidate technologies will enable fast switchingClick here for additional data file.


So-called mild controlled ovarian hyperstimulation (mCOH) has in recent years increased in popularity, claiming to be safer and more patient-friendly, while also improving in vitro fertilization (IVF) outcomes. We here challenge the International Society for Mild Approaches in Assisted Reproduction (ISMAAR) definition of mild stimulation, and especially address four fundamental issues, where our review found conventional COH (cCOH) advantageous over mCOH. They are: prevalence of severe ovarian hyperstimulation syndrome (OHSS), oocyte/embryo quality, pregnancy/live birth rates, and cost. We conclude that an objective review of the literature does not support the routine utilization of mCOH in assisted reproduction. Controlled ovarian hyperstimulation (COH) is essential for in vitro fertilization (IVF) success since it facilitates recruitment of multiple oocytes and, thereby, also often allows for multiple embryo transfers (ETs) , withoutSince investigators used various different definitions and terminologies to describe mild controlled ovarian hyperstimulation (mCOH), the Rotterdam-based International Society for Mild Approaches in Assisted Reproduction (ISMAAR) Consensus Group on Terminology for Ovarian Stimulation for IVF, in 2007, proposed a concise and simplified revised nomenclature for different approaches to ovarian stimulation for IVF .The term natural or unstimulated cycle IVF was proposed when IVF is carried out during a spontaneous menstrual cycle without gonadotropin support, aiming to collect the naturally selected single oocyte of the cycle. mCOH was defined as follicle stimulating hormone (FSH) or human menopausal gonadotropin (HMG) administered at lower doses, and/or for a shorter duration in a GnRH antagonist co-treated cycle, or when oral compounds  are used, either alone or in combination with gonadotropins, aiming for collection of 2–7 oocytes. cCOH was defined by use of GnRH analogues (long/flare agonist or antagonist protocols) with conventional doses of stimulation with FSH or HMG, aiming to collect eight or more oocytes.These definitions can, however, be quite confusing. For example, offering mCOH with a daily gonadotropin dose <150 IU, some patients may respond more vigorously than expected, yielding more than 8 oocytes. Such an oocyte count, however, is already in the cCOH range defined by ISMAAR. How, then, should such a cycle be defined, According to stimulation (mCOH), or patient response (cCOH)?Four other fundamental issues require review:Best IVF is characterized by balance between optimal ovarian stimulation with successful treatment outcomes and minimal rates of severe OHSS and/or  multiple pregnancies. Individualization of treatments, based on patient risk factors, cycle responses, elective single embryo transfer and the option of freezing all embryos have in susceptible cases the potential of reducing the risks and severity of the syndrome . WithholSignificantly reduced clinical pregnancies and increased first trimester pregnancy loss with such treatments , 10, howUtilizing this knowledge, severe OHSS is no longer a complication to fear, as we practically, have eliminated it from our practice for over a decade.A study by Baart et al.  raised cIn 2012, Labarta et al. . Also inLike Labarta et al.  and GleiA recent study  of patieWe, therefore, conclude that there is no reliable evidence to suggest that more aggressive ovarian stimulation increases aneuploidy rates in embryos to significant degrees.Several studies have dealt with pregnancy\live birth rates in mCOH vs cCOH, demonstrating a huge heterogeneity in terms of inclusion criteria, numbers of oocyte retrieved/embryo transferred, or day of the transfer. Verberg et al.  conductep < 0.001, respectively).Distribution of retrieved oocytes was similar for both protocols (range 0–28), with median (25–75 percentile) of 6 –10 and 9Since mCOH cycles more frequently than cCOH cycles fail to reach embryo transfer, outcome analysis with reference cycle start (“intent to treat”) would, likely, demonstrate even more impressive differences in favor of cCOH.More impressive difference was also found comparing stimulated to unstimulated cycles. Sunkara et al.  recentlyAbove noted advantages of cCOH were demonstrated in first fresh-cycle transfers. Those advantages would also become even more obvious if additional frozen-thawed cycle were to be included. Moreover, optimal embryo implantation rates observed with 5 oocytes following mCOH  are realIt was recently also demonstrated , that thGroen et al.  evaluatemCOH has been proposed to provide safer and more patient-friendly IVF, with improving outcomes. Upon careful review, it offers none of these advantages. Regarding occurrence of severe OHSS, oocyte/embryo quality, pregnancy/live birth rates and cost, cCOH is at least comparable or sometime superior over mCOH, discrediting the concept of using mCOH in routine IVF. Further large prospective studies are needed to compare and clarify the role of mCOH vs cCOH in the different subgroups of patients. Moreover, these studies may help fertility specialists in individualization and careful tailoring of the COH protocol for optimizing IVF success.


Enzyme-positive acute coronary syndrome (EPACS) can cause injury to or death of the heart muscle owing to prolonged ischaemia. Recent research has indicated that in addition to liver and brain cells, cardiomyocytes also produce adropin. We hypothesised that adropin is released into the bloodstream during myocardial injury caused by acute coronary syndrome (ACS), so serum and saliva levels rise as the myocytes die. Therefore, it could be useful to investigate how ACS affects the timing and significance of adropin release in human subjectsSamples were taken over three days after admission, from 22 EPACS patients and 24 age- and gendermatched controls. The three major salivary glands  were immunohistochemically screened for adropin production, and serum and saliva adropin levels were measured by an enzyme-linked immunosorbent assay (ELISA). Salivary gland cells produce and secrete adropin locally.Serum adropin, troponin I, CK and CK-MB concentrations in the EPACS group became gradually higher than those in the control group up to six hours (p < 0.05), and troponin I continued to rise up to 12 hours after EPACS. The same relative increase in adropin level was observed in the saliva. Troponin I, CK and CK-MB levels started to decrease after 12 hours, while saliva and serum adropin levels started to decrease at six hours after EPACS. In samples taken four hours after EPACS, when the serum adropin value averaged 4.43 ng/ml, the receiver operating characteristic curve showed that the serum adropin concentration indicated EPACS with 91.7% sensitivity and 50% specificity, while when the cut-off adropin value in saliva was 4.12 ng/ml, the saliva adropin concentration indicated EPACS with 91.7% sensitivity and 57% specificity.In addition to cardiac troponin and CK-MB assays, measurement of adropin level in saliva and serum samples is a potential marker for diagnosing EPACS. Acute coronary syndrome (ACS)  is the dominant cause of death and disability in children and in young,5EPACS is currently diagnosed, according to criteria proposed by the American College of Cardiology (ACC) and European Society of Cardiology (ESC),08Despite this abundance of parameters for diagnosing EPACS, a million patients annually seek care in emergency, cardiology and cardiovascular surgery departments with chest pain or other symptoms suggesting an ACS, although only around 10% are subsequently confirmed to have EPACS.Adropin is a peptide hormone secreted from pancreatic, liver, brain and kidney tissues and from the endocardium, myocardium and epicardium of the heart.10Decreased adropin level is an independent risk factor for endothelial dysfunction, a key early event in atherogenesis, and is integral to the onset of coronary artery disease (CAD) and ACS.18On the basis of these findings, it was hypothesised that the adropin synthesised in the endocardium, myocardium and epicardium20This conflict needs to be resolved. Therefore, the purposes of this study were: (1) to determine the changes in adropin and troponin I concentrations in sera from EPACS patients; (2) to determine whether this hormone is produced by the three major salivary glands, parotid, sublingual and submandibular; and (3) to determine whether saliva contains adropin, because obtaining saliva samples is non-invasive, making it advantageous over blood sampling.All protocols for the human studies in this work accorded with the principles set out  by the Institutional Human Ethics Committee (FUIHC) and with the ethical principles in the most recent version of the Declaration of Helsinki. Written informed consent to participate in the study was individually obtained.A total of 46 subjects (22 EPACS patients and 24 controls) were admitted to the Emergency Department at Elazig Education and Research Hospital due to chest pain or other symptoms (within 30–40 minutes of onset). Our hospital is conveniently located in downtown Elazig so it can be reached from any part of the city within 15 minutes of the first symptoms. The heart team (cardiologists and cardiovascular surgeons) evaluated the patients admitted, as described previously.21A diagnosis of EPACS was made by integrating the history of the presenting illness, an increase in serum troponin I concentration , and associated symptoms of ischaemia, chest pain and/or characteristic ECG signs .The patients were treated as explained elsewhere.Exclusion criteria were: over 75 or under 50 years old, surgery or trauma within two months of the study, known cardiomyopathy, and family history of cardiovascular disease (CVD) .We defined CVD as coronary heart disease, hypertension (hypertension was defined as resting systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg according to WHO–ISH criteria),22 or on current antihypertensive treatment, rheumatic heart disease, known malignant diseases, febrile conditions, acute or chronic inflammatory disease, gastrointestinal diseases, suspected myocarditis or pericarditis, diabetes mellitus of any type, severe heart failure, advanced renal or hepatic disease, alcohol consumption of more than one unit per day, no regular intense exercise (> 15 min of aerobics three times per week), and use of tobacco products (former and current).All the study participants, including the control subjects,underwent a standard clinical examination. Other details relevant to the EPACS studies were described previously.21The first saliva and venous blood samples were collected when patients were admitted to the Emergency Department (within 30–40 minutes of onset) and before angiography. Other samples  were drawn from the antecubital veins of all participants into plain sterile tubes for serum, and into sterile urine cups for whole resting saliva at 08.00 hours in the Department of Cardiology. Saliva and serum were collected simultaneously at each sampling time after thorough rinsing of the mouth with water, as previously described.24Circadian variation in the onset of EPACS has been documented. To avoid this influence, only EPACS patients admitted in the morning were included in this study.Blood samples were divided into two aliquots, one for classical biochemical parameters and the other for measuring adropin levels. The plain sterile tubes for blood and sterile urine cups for saliva contained 500 kIU aprotonin to preclude proteolysis, and after clotting, the samples, were immediately centrifuged at 4 000 rpm and kept frozen (−80°C) pending analysis.All samples were subjected to conventional laboratory analyses using an autoanalyser, including determination of glucose, total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) concentrations. Serum troponin I concentration was measured by chemiluminescence using a Siemens IMMULITE 2000 XPi immunoassay system  and commercial kits .Serum and saliva adropin levels were measured using the same commercial EIA kits (cat no: EK-032-35) and procedures . The saliva adropin assay was validated according to previously published methods.Salivary glands were obtained from the Department of Pathology. They had been removed by surgeons only because of calcification. Adropin was screened immunohistochemically using the Hsu et al. avidin–biotin peroxidase complex (ABC) method, as recently described.Immunostained sections from the parotid, submandibular and sublingual glands were examined with an Olympus BX 50 photomicroscope. Immunohistochemical staining was scored for both intensity and prevalence on a scale of 0 to +3 .All statistical analyses were performed using SPSS for Windows version 21.0 . Differences between groups were analysed with the Kruskal–Wallis test. The Mann–Whitney U-test was used to compare parameters within groups. Comparisons of mean values between groups were expressed as ± 2 SEM.The correlation between serum adropin levels and other clinical characteristics in EPACS patients was measured as the Spearman correlation coefficient, or a chi-squared value when appropriate. Probability values were considered significant at p < 0.05.Immunohistochemical studies revealed no adropin immunoreactivity in the negative controls  for the parotid , sublingValidation of the EIA kit (cat no: EK-032-35) showed it to be as sensitive to saliva adropin as to serum adropin. The lowest detectable adropin concentration in saliva was 0.01 ng/ml, with intra-assay (within day) and inter-assay (between days) variations of less than 10 and 12%, respectively. Assay recovery was between 98 and 106%. The response to salivary adropin was linear over the range 0.50–16.5 ng/ml. Therefore, the sensitivity and specificity of the EIA kit were the same for saliva as for serum adropin concentrations.The serum adropin concentration was slightly (insignificantly) lower in samples taken within 30 minutes (zero time) of patient admission, than the corresponding control value and stable coronary diseases . It rose within two hours post infarct and peaked at six hours; the adropin concentrations at four and six hours post infarct were significantly higher than the controls. At 12 and 24 hours post infarct, the levels were also higher than the corresponding control values but not significantly so .The serum troponin I concentration rose within 30 minutes , peaked at 12 hours post infarct, and remained significantly higher than the controls for up to 72 hours . The timThe saliva adropin concentration was also slightly lower than the controls  in samples taken within 30 minutes (zero time) of hospital admission. Like the serum adropin concentration, saliva adropin then rose within two hours post infarct and peaked at six hours, being significantly higher than the controls at four and six hours, and remaining elevated at 12 and 24 hours post infarct . These rSerum adropin levels were positively correlated with saliva adropin levels  but with neither glucose level nor lipid profiles. There was also a correlation between serum adropin and cTnT levels . Therefore, measuring saliva adropin levels may be an alternative to measuring serum adropin concentrations for diagnosing EPACS or metabolic diseases, for example, diabetes, in which adropin regulates energy homeostasis and insulin resistance.27Serum CK and CK-MB levels were also measured. The initial statistically significant rise in CK-MB above control concentrations occurred within 30–40 minutes (zero time) after the onset of chest pain, peaked at six hours, and returned to baseline at 72 hours. CK levels also started to increase within 30–40 minutes after EPACS (zero time sample: blood taken immediately on admission) and peaked at six hours, thereafter decreasing up to 72 hours. The concentration of CK was significantly higher than the controls from two hours up to 48 hours in the EPACS patients .At four hours after EPACS, the serum adropin concentration measurement had a sensitivity of 91.7% and specificity of 50% at a confidence interval of 95% when the cut-off value was set at 4.12 ng/ml . At six Cardiovascular diseases are the leading cause of death in both developed and developing countries. The World Health Organisation predicts that by 2020, 37% of all deaths worldwide will be from CVDs.In this context, recent animal studies and a human study have suggested that adropin could be useful for diagnosing EPACS in addition to other cardiac biomarkers, but these studies were controversial.Troponin I, CK, CK-MB and adropin concentrations gradually increased in the EPACS group from up to six hours to levels higher than in the controls (p < 0.05), and troponin I and CK-MB continued to increase for up to 12 hours after EPACS. After 12 hours, CK and adropin levels started to decrease for up to 72 hours. These findings confirmed the value of the classical parameters of troponin I, CK and CK-MB for diagnosing EPACS in clinical practice.Saliva adropin concentrations changed in parallel with serum adropin concentrations in ACS. The saliva adropin concentration was generally higher than the serum adropin, possibly because the salivary glands produce adropin (see below). Since adropin is expressed in many tissues, including the heart, all contributing to the serum pool, we had assumed that serum adropin concentration increased after EPACS, as do troponin I or CK-MB, which are released from the myocardium, mainly during EPACS and necrosis following heart injury.9Our clinical results agree with our previous animal experiments, showing that adropin concentration gradually rose above control levels in EPACS patients. This is in contrast to Yu et al., who found that serum adropin levels were significantly lower in EPACS patients than in SAP patients or controls.Yu et al. measured single-time serum adropin levels in EPACS patients, while in our study we measured the time courses of serum and salivary adropin levels in patients and controls.Although the glucose level was within normal physiological limits in the EPACS patients, it was higher than in the controls. This could have been due to the effect of increased epinephrine secretion after EPACS, causing glycogenolysis in the liver and releasing glucose into the blood.Salivary glands are now known to secrete a range of peptides/proteins involved in regulating endocrine metabolism.The adropin levels in saliva were already elevated and increasd further at four and six hours after EPACS. The origin of the high salivary adropin levels is not known but it probably comes from the plasma after saturation, or a larger amount of cardiac adropin is secreted by the salivary glands. Because there is evidence that some of these striated duct proteins are secreted basally, i.e. into the circulation,In this study, receiver operating characteristic (ROC) curves were used to determine the sensitivity and specificity of serum and saliva adropin levels in EPACS patients. At four hours after ACS, serum adropin exhibited 91.7% sensitivity and 50% specificity at a confidence interval of 95% when the cut-off value was 4.43 ng/ml, while serum troponin I exhibited 100% sensitivity and 100% specificity at a confidence interval of 95% when the cut-off value was 0.141 ng/ml. At four hours after ACS, the saliva adropin concentration had a sensitivity of 91.7% and a specificity of 57% at a confidence interval of 95% when the cut-off value was 4.12 ng/ml. At six hours after ACS, the serum adropin exhibited 91.7% sensitivity and 64% specificity at a confidence interval of 95% when the cut-off value was 5.37 ng/ml, while serum troponin I exhibited 100% sensitivity and 100% specificity at a confidence interval of 95% when the cut-off value was 0.226 ng/ml. At six hours after ACS, the saliva adropin concentration had a sensitivity of 91.7% and a specificity of 57% at a confidence interval of 95% when the cut-off value was 4.24 ng/ml. ROC curve analysis indicated that serum troponin I and adropin concentrations diagnosed EPACS with over 90% sensitivity in emergency, cardiology and cardovascular surgery patients.Serum and saliva adropin measurements were not as specific as serum troponin I for diagnosing EPACS. Serum troponin I was still superior to serum or saliva adropin, even though there is the advantage of taking a salivary sample, which can be collected without a venous blood sample. Nevertheless, serum or saliva adropin could still be useful in diagnosing EPACS in the future. First-generation ELISA adropin kits, even from the same company, gave variable results, and there was even more variability among kits from different companies. More reproducible and automated adropin measurements could overcome the current shortfall in specificity, since diagnosis of EPACS by adropin is highly sensitive.Our study had other limitations, especially the small number of patients. Also, this was a single-centre study. The results should be confirmed in a prospective, multicentre study involving more patients. Moreover, adropin is expressed in many tissues, and we did not examine their possible contributions as potential confounders to our measured saliva and serum adropin concentrations. Our previous animal studies revealed that liver and kidney tissue adropin concentrations were considerably changed by isoproterenol-induced EPACS.19The composition and production of saliva is also variable (like urine),36 and this theoretically makes the quantitative determination of any substance unreliable. However, there was a good correlation between the salivary and serum adropin concentrations in this study. This may have been due to the added protease inhibitor (aprotinin) before collection of the biological samples.Despite these limitations, this study provides novel evidence of a connection between increased saliva/serum adropin levels and EPACS. The saliva adropin concentration was higher than the serum adropin level in subjects with and without EPACS. Adropin is synthesised in the intercalated duct, mucous acinus and interlobular cells of human salivary glands, and this could contribute to the high concentrations in saliva. Saliva adropin levels could also be more useful than serum levels for diagnosing some metabolic conditions.Saliva offers advantages over blood, including that collection is non-invasive and therefore stress free for patients, especially children,Overall, four and six hours after EPACS, ROC curve analysis demonstrated that the saliva adropin concentration reflected EPACS with 91.7% (at four hours) and 91.7% (at six hours) sensitivity and 57% (at four hours) and 57% (at six hours) specificity. Therefore, four and six hours after EPACS, the saliva adropin concentration showed the same sensitvity and specifity for diagnosing EPACS as the serum level. All these data promise new possibilities for the diagnosis of EPACS, besides measuring other cardiac enzymes and proteins (troponins).


DZ-1 simplified tumour growth profiling as well, since we were able to correlate NIRF signals with tumour volume and/or tumour-emitting luminescence in mice. Using both orthotopic tumour transplantation and cirrhosis models in parallel, we demonstrated the ability of DZ-1 to differentiate liver tumour from cirrhosis. DZ-1 showed superiority in HCC imaging over indocyanine green by demonstrating significantly enhanced tumour-targeting specificity. At the cellular level, DZ-1 was mainly retained in mitochondria and lysosomes. Additionally, DZ-1 fluorescence spectroscopy has been used for the intraoperative navigation of rabbit liver cancer, to determine surgical margins. We showed that tumor hypoxia and select organic anion-transporting polypeptide genes mediate NIRF dye uptake in HCC, which was supported by clinical evidence. All these findings represent the first evidence that DZ-1 is an effective molecular probe for tumour-specific imaging in HCC, and provide insights into the development of a new generation of imaging agents for intraoperative guidance of cancer surgery.Near-infrared fluorescence (NIRF) dyes have recently emerged as promising tools for non-invasive imaging of different types of cancers. Here, we explored the potential utility of a NIRF DZ-1 dye, with dual imaging and tumour targeting functions, in hepatocellular carcinoma (HCC). We showed the preferential uptake of DZ-1 by HCC cells  Near-infrared fluorescence (NIRF) wavelengths 700-1,000 nm) are attractive for potential biomedical applications due to their increased penetration depth and decreased scattering and absorption in tissues relative to the ultraviolet and visible wavelengths ,000 nm a–4. Therein vitro and in vivo models representing different types of cancers [Recently, a group of NIRF heptamethine carbocyanine dyes have been reported to recognize tumour cells directly without chemical modification, suggesting they are promising agents for non-invasive tumour imaging and targeting , 14. Age cancers , 17, 18. cancers , 8.Hepatocellular carcinoma (HCC) is the fourth most common cancer worldwide . AlthougWe investigated the use of heptamethine carbocyanine dye, DZ-1, for the targeting and visualization of HCC, using preclinical models, cell lines, and clinical samples, and explored the mechanisms of dye uptake. DZ-1 fluorescence imaging was applied for intraoperative navigation in a rabbit liver cancer model as well.in vivo, subcutaneous tumour xenografts of Hep3B-Luc cells were established in the nude mice. We determined that both NIRF signals and BLI increased with the tumour growth, and a significant positive association between the levels of these signals was determined  staining performed using two well-established HCC markers, carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP), which was confirmed by H&E assessment 5CO2H was replaced with (CH2)4SO3-. The novel compound, designated as DZ-1, has an improved hydrophilicity, so that now the same mode as that used for the clinical ICG administration can be used.Our group synthesized a series of heptamethine carbocyanine dyes, including IR-783 and MHI-148 , 24, 26.l groups . The sidin vivo, these dyes did not show cytotoxicity or systemic toxicity at a range of doses appropriate for imaging purposes [In this study, we demonstrated the ability of a group of NIRF heptamethine carbocyanine dyes to specifically recognize HCC cells and tissue samples in multiple preclinical models. Conventional NIRF dyes can identify tumour cells only after they bind to tumour-specific antigens, peptides, enzyme substrates, and tumour markers –30. In cpurposes , 26.in vivo and effectively inhibit HCC cell growth in vitro, suggesting a dual use of NIRG and other similar conjugates as both molecular imaging probes and therapeutic agents for the imaging and treatment of HCC. Furthermore, we demonstrated the superiority of NIRF dyes and their derivatives over ICG for the use in liver tumour imaging. Additionally, NIRF dye was shown to accumulate less in the normal liver than other organs, which facilitates the use of these dyes for HCC imaging specifically, because of the relative reduction in background signal intensity, compared with that observed in other organs.We demonstrated that a significant correlation exists between NIRF dye uptake by orthotopic liver tumour cells and tumour growth, showing the usefulness of NIRF dye for quantitative assessment of HCC growth and progression. Additionally, NIRF dye-drug conjugate, NIRG, was shown to specifically target liver tumours It has been reported that hypoxia mediates the uptake of NIRF dye by different cancer cells via the activation of HIF1α/OATP signalling , 26, whiAdditionally, OATPs are involved in the regulation of NIRF dye uptake by HCC cells and tissues. These molecules belong to a group of cell membrane-bound solute carriers, and were originally thought to mediate cellular transport of amphiphilic compounds, including hormones, drugs, and other exogenous substances . AlteredNIRF dyes do not have strong inherent ligand-labelling abilities, their signal strength (quantum yield) is weaker than that of the fluorophores of shorter wavelength, and they are generally large molecules that do not penetrate well into tissues , since t9. When ICG was injected at the same concentration as DZ-1 into the subcutaneous tumour-bearing mice, no difference in NIRF signal intensity between the tumour site and background was observed, indicating that DZ-1 displays a higher fluorescence quantum-yield than ICG.ICG is the only NIRF dye approved for medical diagnostic use, but it lacks specificity in the recognition of tumour cells. The uptake of ICG by differentiated HCC cells with concomitant biliary excretion disorder was shown to lead to the accumulation of ICG in cancer tissues , which dWe did not detect apparent NIRF signals in HCC orthotopic xenograft mice at 24 h after ICG administration, which may be partially due to the rapid decomposition of ICG, resulting in a rapid quenching of ICG fluorescence signals in blood . Althougin vitro, in subcutaneous and orthotopic xenograft mouse models, and using clinical samples. This dye was shown to be applicable as a NIRF imaging probe for the intraoperative guidance during the surgery of rabbit VX2 liver cancer, and it may serve as a platform allowing drug conjugation, in order to achieve both cancer detection and cancer treatment properties. Additionally, we showed that the underlying mechanisms involve the development of hypoxic conditions in tumours and the activation of selected OATP genes that have not been previously reported as relevant for HCC development or treatment, such as OATP3A1. Although the penetration depth of light in tissues is limited for these dyes, we provided novel insights that will allow further development of this group of dyes for the intraoperative guidance during cancer surgery. Therefore, the results of this study may lead to the clinical development of this dye in the future.In summary, we successfully applied heptamethine carbocyanine DZ-1 dye for the tumour-specific imaging of HCC 2, 5% CO2). ICG dye and bromosulphophthalein (BSP), an OATP inhibitor, were purchased from Sigma-Aldrich . OATP3A1-targeting short hairpin (sh)RNA-carrying lentiviral particles, used for the stable knockdown of these genes, were purchased from Santa Cruz . DAPI was purchased from Tiangen . Heptamethine carbocyanine dyes, DZ-1 and DZ-1 analogue-gemcitabine conjugate (NIRG) were kindly provided by Dr. Leland W.K. Chung  [Human HCC Hep3B2.1-7  and SMMC-7721  cell lines were used . PrimaryCA, USA) . The cheSix-seven-week-old male BALB/c nude mice were purchased from Vital River  and housed in a pathogen-free system at the Laboratory Animal Center of the Fourth Military Medical University . Mice were anesthetized with ketamine  and xylazine  and maintained under isoflurane during surgery and imaging. New Zealand white rabbits (2.5-3 kg) were fed with standard chow and water, and housed in the animal facility. Rabbit VX2 liver tumours were induced by injecting tumour homogenate into the thigh muscle of a rabbit. All animal experiments were approved by the Institutional Animal Care and Use Committee of FMMU (protocol No. 16013). All methods were carried out in accordance with the approved regulations of FMMU.All fresh liver specimens of cancer patients were obtained from the Xijing Hospital of the FMMU, and informed consents were obtained from all patients. The use of these specimens in research was approved by the medical ethics committee of the Xijing Hospital. All methods were performed in accordance with the approved guidelines.6, 3×106, and 9×106) were either inoculated subcutaneously into the right flank of the nude mice or subjected to orthotopic liver transplantation (OLT) to the nude mice according to the following protocol. Briefly, a 100-μL mixture of ketamine and xylazine was intraperitoneally injected into mice. A small cut from the medioventral line with ophthalmic scissors enabled full exposure of the liver, and Hep3B-Luc cells in 50-μL volume were injected into the porta hepatis. After returning the liver to the abdominal cavity, abdominal muscles and skin were sutured. Two weeks later, the operated mice were intravenously injected with DZ-1/NIRG (1 μmol/kg) or ICG (10 μmol/kg) [Hep3B-Luc cells (1×10μmol/kg) . NIRF in3 each were implanted into the hepatic incision. Pressure was applied over the puncture site using gelatine sponge for approximately 2 min. Abdominal muscles and skin were sutured [Rabbits were anesthetized with intravenous ketamine (10 mg/kg) and xylazine (3 mg/kg). Under aseptic condition, a mini-laparotomy was performed in the subxiphoid area, exposing the liver. An incision of 1 cm was made in the liver lobe. Two small rabbit VX2 tumour pieces of 3 mm sutured . Two wee sutured . The rem2 hypoxic conditions or with a prior exposure to BSP . Cells were washed with PBS, and cell nuclei were stained with DAPI after fixation. Control groups were maintained in normoxic conditions or treated with a vehicle.To determine the specific uptake of DZ-1 by HCC cells, GFP-tagged Hep3B cells were co-cultured with normal human hepatic epithelial cells at a ratio of 1:10. Cells were seeded in petri dishes, and DZ-1 (20 μM) was added 24 h after the seeding. Cells were incubated at 37°C for 30 min, followed by PBS wash. Dye uptake was analysed using a NIRF microscope equipped with an ICG filter (excitation/emission: 750-800/820-860 nm). Cell nuclei were stained with DAPI. To determine the organelle-specific cellular localization of NIRF dye, Hep3B cells were seeded on petri dishes, exposed to MitoTracker (200 nM) or LysoTracker (75 nM) 24 h after seeding, and incubated at 37°C for 30 min. DZ-1 (20 μM) was added, and another 30-min incubation was performed at 37°C, followed by the PBS wash. The uptake of probes and dyes was determined by microscopic examination as described. To determine the effects of hypoxia and OATP inhibition on DZ-1 uptake, Hep3B cells were seeded and treated with DZ-1 (20 μM) 24 h after the seeding. Cells were cultured for 24 h under 1% O4)-induced cirrhosis mouse model [4 was dissolved in olive oil (1:5), and 1 μL/μg body weight of CCl4 was injected intraperitoneally into 5-week-old male nude mice twice weekly for 5 weeks. In parallel, orthotopic liver tumour xenografts were established using Hep3B-Luc cells. DZ-1 (1 μmol/kg) was intravenously injected into liver tumour and liver cirrhosis mice. Whole-body fluorescence imaging was used to determine the differential uptake of this dye.We established a carbon tetrachloride  and reverse-transcribed to cDNA using the ReverTra Ace qPCR RT Kit . The expression levels of the OTAP gene family members were determined. PCR analysis was performed using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). All primer sequences used are listed in We investigated the toxicity of DZ-1 in by intravenously injecting the dye. The BALB/c mice (n=10 per group) were divided into four groups. The control group received PBS, while the treatment groups received daily DZ-1 intravenous injections, at the following doses: 1 μmol/kg (imaging dose), 50 μmol/kg, and 100 μmol/kg. The mice were weighed daily, and their physical activities were observed for 1 month after the dye injection. The histopathological morphology of their vital organs, including heart, liver, spleen, lungs, and kidneys were assessed at the time of sacrifice.All values in figures are presented as mean ± standard error of mean (SEM) obtained in at least three independent experiments. Comparisons between the Kaplan-Meier curves were performed using the long-rank test. All other comparisons were performed by using unpaired 2-tailed Student's t test. P≤0.05 was considered significant.


Paris, FranceInformations: Madinina CoxPhone: +33 (0)6 35 31 40 90madinina.cox@mc-com.frE-mail: conference-thoraxparis.com/Site: Paradise Island, BahamasInformations: George Silvaygeorge.silvay@mountsinai.orgE-mail: www.clinicalupdateinanesthesiology.orgSite: Toronto, CanadaInformations: Continuing Professional Developmentwww.cpd.utoronto.ca/cardiactumours/Site:Toronto, CanadaInformations: Amy RicePhone: 416864-9911arice@lungontario.caE-mail: www.betterbreathing.ca/Site: Fort Lauderdale, United StatesInformations: STSPhone: 312-202-5800Fax: 312-202-5801education@sts.orgE-mail: sts.org/annualmeetingSite: Madrid, SpainInformation:www.cursotrasplantepulmonar.com/Site: Windsor, United KingdomInformations: EACTSE-mail: info@eacts.co.ukwww.eacts.orgSite: Houston, United StatesInformations: Rebecca LawPhone: 7607202263Fax: 7607206263rlaw@promedicacme.comE-mail: www.houstonaorticsymposium.com/Site: Paris, FranceInformations: Sharon WilliamsPhone: 7025085200Fax: 7025085200pediatriccardiologists@pediatricsconferences.orgE-mail: pediatriccardiology.conferenceseries.com/europe/Site: Paris, FranceInformations: Cathy Smithheartfailure@cardiologyconference.orgE-mail: heartdiseases.conferenceseries.com/Site: Maastricht, NetherlandsInformations: EACTSinfo@eacts.co.ukE-mail: www.eacts.orgSite:


In a medical context, decision-making is associated with complicated assessment of gains, losses and uncertainty of outcomes. We here provide novel evidence about the brain mechanisms underlying decision-making of analgesic treatment. Thirty-six healthy participants were recruited and completed the Analgesic Decision-making Task (ADT), which quantified individual tendency of risk-taking (RPI), as the frequency of choosing a riskier option to relieve pain. All the participants received resting-state (rs) functional magnetic resonance imaging (MRI) and structural MRI. On rs-functional connectome, degree centrality (DC) of the bilateral anterior insula (aINS) was positively correlated with the RPI. The functional connectivity between the aINS, the nucleus accumbens and multiple brain regions, predominantly the medial frontal cortex, was positively correlated with the RPI. On structural signatures, the RPI was positively correlated with grey matter volume at the right aINS, and such an association was mediated by DC of the left aINS. Regression analyses revealed that both DC of the left aINS and participants’ imagined pain relief, as the utility of pain reduction, could predict the individual RPI. The findings suggest that the functional and structural brain signature of the aINS is associated with the individual differences of risk-taking tendency in the context of analgesic decision-making. When making a medical decision – either for choosing over-the-counter medicines or for shared decision-making between patients and clinicians – one needs to carefully balance between both gains  and losses 135Evidence from functional magnetic resonance imaging (MRI) studies has revealed that when an individual is assessing the gains and losses for a risky financial decision, the anterior insula (aINS) and the nucleus accumbens (NAc), as the core components of the risk-related network, were frequently activated8131417172021We here adopted the ADT for assessing the risk-taking tendency regarding the choice of analgesic treatment, which was quantified as the risk preference index (RPI). We analyzed the structural (GMV) and functional (rs-FC connectome) signatures of a risk-related network composed of 26 brain regions. We hypothesized that at the aINS and the NAc, network degree centrality (DC), FC and GMV, would be correlated with the individual differences in RPI.The current observational study adopted a cross-sectional design. Thirty-six participants  between ages of 21 and 46 years  were recruited in at the university campus . All methods were performed in accordance with the relevant guidelines and regulations. All of the participants provided written informed consent before participating in this study.Previous studies have shown that one’s prior experience of clinical pain and subjective satisfaction  of pain reduction may play a key role in the choice of analgesic treatment24Before performing the ADT, the participants were asked to rate their most intense prior experience of clinical pain, respectively for toothaches, headaches and stomach aches, using a 0–100 scale . Subsequently, we assessed the utility , we calculated the Risk Preference Index (RPI) as follows:ANE), the ADE task (NADE) and the TE task (NTE). The RPI for each task was calculated as a proportion .N is the frequency that a participant chose the riskier option. A higher RPI indicated a stronger tendency that the participant would choose the riskier treatment, i.e., the treatment that has a stronger pain-relieving effect, regardless of the influence of probability, adverse effect or time-course of therapeutic effect. To further investigate risk-taking tendency in each sub-task, we analyzed the frequency that a participant chose the riskier option in the ANE task . The T1-MRI images were acquired with the following parameters: a high-resolution sequence . During rs-fMRI, the participants were instructed to be relaxed, remain awake, and keep their eyes open and fix on a cross symbol on the screen.Resting-state functional MRI (rs-fMRI) and T1-weighted magnetic resonance imaging (T1-MRI) were performed at the 3 T MRI Laboratory of National Yang-Ming University, using a 3 Tesla Siemens MRI scanner . The rs-fMRI images were acquired with the following parameters: gradient echo planar imaging (EPI) with T2* weighted sequence , the difference in imagined pain relief , and the difference in RPI in all three sub-tasks , were respectively investigated, using the Friedman test. Post-hoc pairwise comparisons were performed using the Wilcoxon-signed rank test, with Bonferroni correction for multiple comparison. We adopted the non-parametric methods due to the non-normality of data  of the aINS (DCaINS_L/DCaINS_R) and the NAc (DCNAc_L/DCNAc_R) is associated with the individual RPI. To test the hypothesis, we constructed a risk-related network, primarily based on imaging meta-analysis  as the predictors, and the seed-based functional connectivity as the dependent variable. The bilateral aINS and the NAc were respectively used as the seed. The analyses would reveal the brain cluster which connectivity was positively correlated with RPI. A cluster would be considered statistically significant with a threshold of intensity (uncorrected P [Punc] < 0.005) and a threshold of cluster size (familywise-error corrected P [PFWE] < 0.05).On the aINS/NAc functional connectivity, to understand the pattern of their connections with the other brain regions, we further performed exploratory whole-brain seed-based FC analyses, using the bilateral aINS and the NAc, respectively, as the seeds. We tested a multiple regression model that modeled participants’ sex, age as nuisance regressors and RPI  as the predictors, and grey matter volume (GMV) as the dependent variable. Because we focused on the role of the aINS and the NAc, we performed an ROI-based analysisunc]<0.001) and cluster size .On the structural signature, we first performed a region-of-interest (ROI)-based analysis, focusing on the bilateral aINS and NAc. We tested a multiple regression model that modeled the sex, age, total brain volume, all as nuisance regressors, and RPI  and structural signatures  (see Results). To further clarify the association between these variables, we performed a mediation analysisaINS_R and RPI was mediated by DCaINS_L/DCaINS_R . We assigned GMV as the independent variable, RPI as the dependent variable and DC as the mediator. DC of the right and the left aINS was used, separately, as the mediator. Sobel test was performed to examine the significance of the ‘indirect model’, which represents that a mediational effect of DC.Findings from the previous analyses revealed that RPITOTAL, as the dependent variable, and the following variables as the predictors: (a) imagined pain relief ΔP9 → 6, (b) prior pain experience, and (c) DCaINS_L, which was a significant mediator of RPI, according to the results from the mediational analysis (see Results). The variables ΔP9 → 6 and prior pain experience were log-transformed for normality. We first investigated the covariation between the predictors. The three predictors were not significantly correlated with each other. Secondly, we performed a regression analysis, using the input model, to investigate the predictors which predicted RPITOTAL with a statistically significance. Thirdly, we performed an analysis, using the stepwise method, to investigate the relative effect of prediction from the behavioral and rsFC-connectome predictors.The abovementioned results pointed to the conclusion that functional connectome of the left aINS plays a key role in the individual differences of risk-taking tendency. In addition, imagined pain relief and prior pain experience may also guide one’s analgesic decisions. Therefore, we performed a multiple regression analysis for testing the ad hoc hypothesis that functional connectome of the aINS would predict one’s risk-taking tendency in medical decision-making. We modeled RPITOTAL) . The results confirmed our previous findings that risk-taking tendency is associated with subjective utility of pain reduction  and DCaINS_R , controlled for participants’ sex and age /the right pallidum and putamen/the right aINS /the right inferior frontal gyrus (IFG) /the right IFG/premotor cortex.We found that the individual RPI was significantly positively correlated with the FC between the bilateral aINS/NAc and multiple brain regions . Notablyght aINS  and the us (IFG) , and theus (IFG) . RPITE wTOTAL was positively correlated with the GMV at the right aINS (GMVaINS_R)   was adopted in the analysis. The only above-threshold cluster in the whole-brain analysis is the right prefrontal cortex, which GMV showed positive correlation with RPITE  (We found that RPI volume) . In cont= 0.027) .aINS_R was significantly mediated by the DCaINS_L  . The significant predictors were DC of the left aINS  and imagined pain relief . In contrast, prior experience of pain was not a significant predictor. Thirdly, we performed an analysis using the stepwise method. The results showed that DC of the left aINS was the predominant predictor, which explained 26.1% of the variation of RPITOTAL . Additionally, imagined pain relief explained 12% of the variation .We first investigated the covariation between the predictors. The three predictors were not significantly correlated with each other. Secondly, we performed a regression analysis using the input model. The result showed that all the three predictors explained 38.5% of the variation of RPIaINS_L. Finally, the regression model revealed that both the functional connectome  and psychological utility  can predict risk-taking tendency. Altogether, the findings suggested that the functional and structural brain signatures of the aINS are associated with the individual differences of risk-taking tendency in the context of medical decision-making.In a medical context, decision-making is associated complicated assessment of gains, losses and uncertainty of outcomes514aINS) during the resting status may reflect a heightened salience about the current status, which would direct the individual towards future pain-related experience. Consistently, the seed-based FC analysis showed that the aINS-dACC connectivity, i.e., the main component of the salience networkTOTAL .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Suspensions of gel particles which are pourable or spoonable at room temperature can be created by shearing a gelling biopolymer through its gelation  rather than allowing quiescent cooling – thus the term ‘fluid gel’ may be used to describe the resulting material. As agar gelation is thermoreversible this type of fluid gel is able to be heated again to melt agar gel particles to varying degrees then re-form a network quiescently upon cooling, whose strength depends on the temperature of re-heating, determining the amount of agar solubilised and subsequently able to partake in re-gelation. Using this principle, for the first time fluid gels have been applied to a high viscosity 3D printing process wherein the printing temperature (at the nozzle) is controllable. This allows the use of ambient temperature feedstocks and by altering the nozzle temperature, the internal nature (presence or absence of gel particles) and gel strength of printed droplets differs. If the nozzle prints at different temperatures for each layer a structure with modulated texture could be created. The term fluid gel describes the suspension of microgel particles created when sufficient shear is applied to a gelling biopolymer solution through its gelation, as opposed to allowing the solution to gel quiescently. This has been described in the literature for thermoreversible biopolymer solutions –11, inclA number of studies discuss the possible mechanism of formation of these fluid gel particles, there is general agreement in the literature that fluid gels arise as a result of shear induced phase separation, creating polymer-rich and polymer-poor regions within a solution undergoing gelation due to either spinodal decomposition or a nucleation and growth mechanism. This demixing through gelation in shear constrains gelation and potentially influences the kinetics of network formation inside the gel particle forming a suspension of discrete gel particles in a predominantly aqueous continuous phase. The initially formed gel nuclei grow to an equilibrium particle size, determined by the shear field experienced , 16, 18.Agar is one such biopolymer, extracted from red seaweed, which will undergo thermoreversible gelation. The agar must be dispersed in solvent  then heated and held at high temperature  to ensure polymer chain solubilisation, after which the chains adopt a random coil configuration. Upon cooling, single agar chains form double helices which are then able to aggregate further to create the gel state, as described by the “Domain Model” , 20. Aga2 and removed from the fluid gel supporting medium once gelation had occurred. 3D printing applications using gelling agents as the main material typically utilise this material extrusion method, often referred to as Filament Freeform Fabrication (FFF) or Fused Deposition Modelling (FDM) [Intended application of fluid gels has arisen from their colloidal nature, giving potential as fat replacers in foods or for use in personal care products. There has been one instance of application in 3D printing whereby the interaction between fluid gel particles in suspension has been utilised as a ‘self-healing’ matrix substrate for a technique known as suspended manufacture . In thisng (FDM) . For biong (FDM) . Care mung (FDM) , 27.One study by Gilon et al.  describegel = 35 °C and 1.5% gel solubility in water at 75 °C.Two agars were used to create fluid gels: Luxara-1253  whose composition is available in , 8, and,A 1% stock solution of sodium azide (Sigma Aldrich UK) was diluted to 0.1% in fluid gel formulations.3) for 2% solutions and 15 g water for 3% solutions then 0.4 or 0.45 g, respectfully, of agar was gently added whilst agitating. The vessel was attached to a water bath which was heated to 90 °C and held at this temperature for 30 min to fully solubilise the agar. Whilst stirring at 1000 rpm the water bath cooled the solution to 10 °C at a rate of 1.5°Cmin−1, determined using a temperature probe. For continuity throughout experiments this was the heating and cooling rate carried forward for rheology. Fluid gels were stored at 4 °C until use.A jacketed vessel with a magnetic stirrer was used to create larger volumes of fluid gels. The vessel was filled with 20 g ultrapure water  range to be between 0.01–1% strain (results not shown). Oscillatory temperature sweeps were conducted within the LVE range (0.05%) at angular frequency (ω) = 10rads−1 to heat and cool the fluid gels at 1.5°Cmin−1 to temperatures between 55 and 85 °C whilst measuring the elastic and viscous moduli (G’ and G” respectively). Fluid gels made via the stirred pot method were also subject to amplitude sweeps and the heating and cooling regimes.Creation of fluid gels and rheological tests were conducted on the MCR 301 rheometer  using a cone and plate geometry (50 mm diameter with 50 μm truncation and 2° cone angle) with TruGap functionality and a moisture trap to prevent drying of the sample during heating and cooling stages. Agar solutions were prepared at 2 or 3% via the cold dispersion method described above and a minimum of 30 min stirring at 90 °C was allowed for polymer solubilisation. 1500 μL of hot solution was pipetted into the centre of the plate at 85 °C. Fluid gels were created in the rheometer through application of constant shear at 400 sAn Axiovert 200 M Wide Field Fluorescence Microscope  was used to conduct phase contrast microscopy with ×10, ×40 and ×63 objectives.Fluid gel samples were centrifuged using a Jouan CR3i  at 4100 rpm/2500G for 30 min at a set temperature of 10 °C. The supernatant and pellet portions of each sample were separated and weighed to determine the effective phase volume of fluid gel particles versus the continuous phase.−1. Enthalpies of transition are quoted as Jg−1 of agar.A Setaram MicroDSC III calorimeter  was used to observe ordering and disordering transitions on application of thermal energy. With water as a reference, ≈800 mg of fluid gel sample was loaded into the sample cell at 20 °C. Samples were heated to varying temperatures between 55 and 100 °C and cooled to 10 °C, following this was an immediate reheating step to assess the resulting gel sample; the rate used remained 1°CminA 3-Dimensional stage, controlled by a computer, was used to determine the position of a PICO®Pμlse™ (Nordson EFD) jetting valve throughout printing with overall accuracy of 5 μm; nozzle sizes used were 100 and 150 μm, as the available 50 μm was deemed too close to particle size and risked blockage during printing runs. Cycle time was constant at 10 ms, relating to a frequency of 100 Hz, however stroke, voltage, pulse, open and close times were varied using a PICO Toμch controller and a locking air pressure regulator used to deliver the required pressure.An Eclipse LV100ND (Nikon UK Ltd.) was used to visualise printed structures as soon after printing as possible, to avoid the potential effects of drying.As mentioned in the introduction discrepancies exist in the literature as to the size and shape of fluid gel particles produced, even from agar of the same source at equal concentrations. Figure Here the size and shape of all fluid gel particles was variable throughout the different production processes and concentrations investigated. Aside from the presence or absence of ‘tails’ the particle core ranged from near spherical in shape to anisotropic, often with a high aspect ratio . A trend for the size of particles to decrease as the concentration of agar in solution increased was noted which is consistent with findings in the literature , 16, 18,Samples for phase contrast microscopy were diluted to allow better visualisation of individual particles, therefore little information can be gathered from these regarding particle numbers versus continuous phase volume. After 30 min of centrifugation the supernatant of 2% fluid gels made with Sigma agar was found to be a smaller volume than those made with Luxara agar, thus the pellet collected for Sigma comprised a larger phase volume of the original sample than the Luxara. Based on the weight of separated supernatant and pellet samples collected the Sigma fluid gel particles contributed to 85 ± 6% phase volume whereas Luxara particles only comprised 59 ± 2% phase volume of the total sample. Therefore we can conclude that the effective volume of the tails present will have an effect and result in different behaviour exhibited by the Luxara and Sigma fluid gels.Norton et al.  previousThese observations from the literature on the effect of heating and cooling on the extent of ordering can be used to explain observations presented here based on DSC thermograms.Figure −1 obtained by Cooke et al. [For the purposes of applying fluid gels to a temperature controlled high viscosity print head, in order to use thermal energy input to gain printed gel droplets of varied hardness, it was interesting to observe the extent of re-ordering depicted by the gelation exotherm after an initial heat to a defined temperature (between 55 and 100 °C). Figure e et al.  and Gidle et al. . As the −1) the viscosity was measured throughout and can be plotted against temperature as shown in Fig. For fluid gels created in the rheometer at 400 s00 s−1 thG’ and G” trace versus temperature obtained from one of these sweeps when the fluid gel was heated to 85 °C after production. At temperatures lower than those attributed to the melting endotherm in μDSC the G’ stays relatively constant but begins to decrease rapidly as molecular mobility increases and agar fluid gel particles begin to destabilise and undergo helix to coil transition due to sufficient thermal energy input. The G’ remains low at these elevated temperatures then begins to rise again on cooling at temperatures associated with the μDSC gelation event. Further cooling beyond this point back to 10 °C results in a final G’ much larger than the initial fluid gel sample, closer to that of the quiescent gel. Heating to 85 °C was the highest temperature sweep conducted for all fluid gel samples and was attributed to the highest final G’ values for both types of agar at each concentration. The exception to this is 2% Luxara fluid gels made in the rheometer, where 95–100 °C heating steps were conducted and are presented with μDSC data in Fig. G’ after this temperature sweep was consistent with measurements for quiescent gels. This trend was also seen when samples created in the stirred pot were loaded into the rheometer and subjected to identical temperature sweeps.As mentioned in the materials and methods amplitude sweeps of fluid gels produced were conducted to determine the linear viscoelastic region. Whilst gently oscillating within this region, to monitor viscoelastic moduli, the fluid gels were heated from 10 °C to a value between 55 and 85 °C, held at this point for five minutes then cooled to 10 °C. Figure G’ once re-cooled versus heating temperature for 2 and 3% fluid gels of Luxara agar made both by the stirred pot and rheometer methods. The final G’ achieved between heating to 55 °C versus 85 °C is an increase of almost 2 log for samples made in a stirred pot and 1 log for those made in the rheometer. The higher the heating temperature the more disordering of aggregates and helix to coil transition occurs. In this method of stopping the heating process at a point along the melting endotherm, isothermally holding and then cooling there will be different extents of particle dissolution. At lower temperatures, some aggregates will break and smaller, kinetically trapped helices or chains which may be in the ‘hairy’ regions protruding into the continuous phase can be more easily solubilised than those at the core of the gel particle. This is likely to be due to the formation of tails being at a lower temperature, as discussed earlier, thus they would have a lower melting temperature compared with the particle core. Therefore on cooling the continuous phase is likely to be a weak gel with reinforcing dispersed gelled particles remaining in it. Conversely at higher temperatures most of the aggregates will disorder, enabling double helices to be solubilised in the coil state, thus the continuous phase will have a relatively higher concentration of agar able to take part in gelling but few reinforcing particles. The effect of particle reinforcement may be more pronounced in those fluid gels made via the stirred pot method due to a larger starting particle size, explaining the higher G’ values observed when heating above 70 °C. All temperatures presented in Fig. G’ of samples heated to 85 °C was lower than that for quiescently set agar. Additionally, a five minute isothermal hold is insufficient to allow for full solubilisation of agar molecules but is more representative when considering potential mechanisms in the 3D printing application.Figure G’ achieved on cooling after a temperature sweep. Within this temperature range, after the melting peak maximum, there could be constrained reordering within the fluid gel particle. This would cause a plateau region for both enthalpy on gelation and the measured G’, but after this reordering and heating to temperatures beyond 85 °C melting is able to be completed. At 80 °C 72.5% of the endothermic reaction has taken place with a further 20% occurring between 80 and 85 °C, based on integration of the endothermic transition peak, yet re-heats to these temperatures result in plateau values for G’ and exotherm enthalpy on cooling. Therefore this region begins to indicate heterogeneity between fluid gel particle core and surface upon formation, as there is clearly a significant amount of ordering occurring based on the endotherm integration data, but no uniform increase in gel strength, based on rheology and gelation enthalpy. A further rise in both sets of data upon re-heats to temperatures above 85 °C indicates that this heterogeneity has been overcome, and ordering may continue in a more similar manner to a quiescently set gel. This is exhibited by a G’ consistent with quiescent gel measurements observed upon conducting temperature sweeps to 95 °C and 100 °C and determination of 99.9% of the endothermic reaction being complete at 90 °C for Luxara agar, based on integration.In Fig. Results presented in the sections above can allow for the understanding and development of a model based on heated and re-set thermoreversible fluid gels to be used in practical application. This has been demonstrated for 3D printing using high viscosity ink jet.The main parameters altered to attain good jetting of droplets with the Nordson PicoPulse valve and Pico Touch controlling system were the supplied pressure, pulse , close voltage, stroke percentage and valve open and close times within a cycle time of 10 ms. Preliminary trials have identified suitable operating parameters for fluid gel samples at ambient temperature. For a 2% Sigma Agar fluid gel parameters used were; 3 bar pressure, 0.3 ms pulse, 120 V, 50% stroke, 0.25 ms opening time and 0.2 ms closing time. As heat was applied at the nozzle the solution viscosity decreased therefore pressure was decreased to 2 bar and small adjustments were made to other parameters depending on the printing definition achieved at any given temperature.Printing was initially conducted onto a glass microscope slide however it was discovered that better printed droplet resolution, shape retention and structure removal could be achieved if the glass slide substrates were coated with a thin layer of quiescently set agar, illustrated in Fig. During printing at a variety of temperatures it was possible to create structures of variable height without detriment to the layer underneath. Figure Light microscopy was used to observe fluid gel droplet arrays straight after printing to prevent the samples drying out. Figure G’ after sweeps to and from these temperatures. It was this knowledge which enabled the application of thermoreversible agar fluid gels to high viscosity jetting, creating structures in 3D with varied hardness as a result of the extent of melting of fluid gel particles and forming a fully quiescent set gel or a particle reinforced gel, by controlling the nozzle temperature. When printing fluid gels under ambient conditions, the use of a freezing substrate can cause concentration of agar fluid gel particles and expulsion of some bound water so that they pack closely together to form a structure without the application of heat. This phenomenon should be explored further in addition to conducting more characterisation of all printed structures with the aim of printing more complex geometries. This may also be made possible by printing with ‘mixed’ fluid gels, where relative particle numbers will be determined by both formulation and processing conditions. This addition will allow for further structure control and multiple nozzles, printing different fluid gel feedstocks which provides an exciting future, whose limits are constrained by imagination.We have demonstrated the feasibility and benefit of using fluid gels as a feedstock for 3D printing with application in a high viscosity jetting system. When printing thermoreversible gels the creation of a fluid gel from the polymer of interest prior to printing negates the requirement of keeping the feedstock at elevated temperatures, as has been necessary in previous studies. As demonstrated elsewhere in the literature fluid gel production is not restricted to thermally gelling polymers, it could be useful in the future to look into printing with alternative fluid gels depending on the application. For agar fluid gels there is a clear link between the dependence of particle size, shape and rheological properties on the type of agar used, method of production and concentration. Thermal and rheological studies were concordant in showing a tuneable gelation event after applying a heating and cooling cycle to fluid gels with the extent of gelation dependent on the temperature heated to. The combination of these studies led to the conclusion that the temperature region between 80 and 85 °C, when considering the melting of agar fluid gels, can be attributed to constrained reordering within the particle, determined by a plateau of gelation enthalpy and final


Soft tissue and bone sarcoma represent a broad spectrum of different pathology and genetic variance. Current chemotherapy regimens are derived from randomised trials and represent empirical treatment. Chemosensitivity testing and whole exome sequencing (WES) may offer personalized chemotherapy treatment based on genetic mutations.A pilot, prospective, non-randomised control experimental study was conducted. Twelve patients with metastatic bone or soft tissue sarcoma that had failed first line chemotherapy treatment were enrolled for this study. Human tissue taken at surgical biopsy under general anaesthetic was divided between two arms of the trial. Subsections of the tumour were used for WES and the remainder was implanted subcutaneously in immunodeficient mice (PDX). Results of WES were analysed using a bioinformatics pipeline to identify mutations conferring susceptibility to kinase inhibitors and common chemotherapeutic agents. PDX models exhibiting successful growth underwent WES of the tumour and subsequent chemosensitivity testing.WES was successful in all 12 patients, with successful establishment PDX tumours models in seven patients. WES identified potential actionable therapeutics in all patients. Significant variation in predicted therapeutics was demonstrated between three PDX samples and their matched tumour samples.Analysis of WES of fresh tumour specimens via a bioinformatics pipeline may identify potential actionable chemotherapy agents. Further research into this field may lead to the development of personalized cancer therapy for sarcoma.The online version of this article (10.1186/s13569-018-0090-1) contains supplementary material, which is available to authorized users. The mainstay of systemic cancer therapies has been empirically derived cytotoxic combination protocols based on histological appearance, organ of origin, and tumour staging. This approach has proven effective in lymphomas and other germ cell tumours, but their efficacy has been less in bone and soft-tissue sarcoma due to their heterogeneity . For patKIT, PDGRFα and BRAF have led to the development of selective kinase inhibitors to target them [Recent advances have been made in the utilization of genomic data to facilitate development of targeted therapeutic agents. An example of this is the identification of key genes and signaling pathways in gastrointestinal stromal tumours (GIST). Identification of oncogene mutations in get them –4.9 base pairs, containing coding (exons) and non-coding (introns) regions [The whole human genome contains approximately 3 × 10 regions . Less th regions . It is e regions , 8. As sThe implantation of patient derived tumours into immunodeficient mice is a recently developed technique to provide a more representative microenvironment for measuring tumour response to chemotherapy agents. Reliable mouse models that recapitulate the biological profiles of sarcomas have been developed as a research tool in sarcomas  and indeTyrosine kinases play an important role in the modulation of cell signalling pathways. Their role in oncogenesis of several forms of cancer was recently discovered and arises from genetic mutation that causes dysregulation of these pathways to stimulate a variety of biologic pathways, including angiogenesis and cell growth. Tyrosine kinase inhibitors competitively inhibit ATP binding or impede activation/activity of these kinases, thus leading to pathway deactivation .The use of these drugs in sarcoma has been limited in comparison to other malignancies. In a recent review , it was Herein, we describe a comparison of whole exome analysis of germline, tumour, and murine patient derived xenografts (PDX), with implementation of a bioinformatics pipeline for chemosensitivity prediction in 12 patients with bone and soft tissue sarcoma.A prospective case series study was conducted from March 2016 to March 2017. Institutional approval to conduct the trial was obtained from the Human Ethics Committee. Twelve adult patients with bone or soft tissue sarcoma who had failed to respond to standard chemotherapy, or newly diagnosed local recurrence of bone or soft tissue sarcoma following chemotherapy, were enrolled in the trial. Under-age patients, or those with a cognitive impairment preventing them from providing informed consent were excluded from the trial. Informed consent was obtained by an investigator.Following informed consent, samples of the tumour were taken at the time of surgical biopsy under general or local anaesthesia.3 fragments, mixed with extracellular matrix   and implanted subcutaneously via an 18G needle on the right flank of 6–10 week-old female NSG (NOD.Cg-PrkdcscidIL2Rγtm1Wjl/SzJ) mice . PDX models with successful establishment of tumours were subsequently biopsied with sections of the tumour sent for WES. All PDX tumour biopsies performed for exome sequencing were performed from zero passage mice to minimise the amount of model acquired mutations, which have been demonstrated to increase with each passage [Subsections of the tumour sample were divided between whole exome sequencing (WES) and implantation subcutaneously in immunodeficient mice. Patient blood samples were sent for WES for germline exome identification. DNA was extracted from tumour and blood using a Blood and Cell Culture DNA mini kit according the manufacturer’s instructions . DNA was quantitated using a NanoDrop  and integrity checked by agarose electrophoresis and ethidium bromide staining. Tumour tissue was sliced into approximately 2 mm passage .Samples for WES were prepared using an Agilent SureSelect Target Enrichment Kit and libraries were sequenced via 100 bp PE using an Illumina HiSeq 4000 sequencer . With 100× read depth for germline DNA, and 200× read depth for primary tumour and PDX DNA.Sequence data were then analysed using a modified version of the Integrating Molecular Profiles with Actionable Therapeutics (IMPACT) pipeline developed at the University of Colorado . All anaFurther functional analysis of the gene mutations identified as a potential kinase inhibitor target was performed to investigate whether targeting these kinases had known effects on tumour biology. Known molecular pathways were explored using WebGestalt  and idenThe actionable therapeutics identified for each PDX sample was compared with it’s matched list of tumour derived actionable therapeutics using Fisher’s exact test of independence for assessment of statistical significance.Establishment of tumours in PDX models was successful in seven patients. Patient demographics are outlined in Table Sequencing metrics are outlined in Table Potential kinase inhibitor targets were identified in six of the twelve samples. These samples were of osteosarcoma, Ewing’s sarcoma, undifferentiated pleomorphic sarcoma, alveolar rhabdomyosarcoma, and leiomyosarcoma. The results of this analysis are outlined in Table Analysis using the bioinformatics pipeline successfully identified potential actionable therapeutics in all the twelve samples. An example of the generated list of potential actionable therapeutics is outlined in Table One sample of osteosarcoma demonstrated a mutation of the KIT gene. This gene is a proto-oncogene responsible for the encoding of c-kit, and plays a role in cell survival and proliferation. The mutation identified (E142Q) lies in the extracellular domain and may alter the structure of the receptor. The same mutation of the ALK gene (C928fs) was demonstrated in a sample of Ewing’s sarcoma and leiomyosarcoma. The exact action of this ALK mutation on oncogenesis is still unclear, however it has been implicated in numerous malignancies including non-small cell lung cancer . The ideIn four patients, actionable therapeutics were identified that are already in use as treatment for the corresponding sarcoma: Methotrexate was identified for an osteosarcoma patient as well as a synovial sarcoma patient; Doxorubicin was identified for an Ewing’s sarcoma patient and Vincristine was identified for an alveolar rhabdomysarcoma patient.Establishment of PDX tumour lineages was successful for seven patients. Failure of tumour establishment was defined as lack of observable tumour growth within 24 weeks.All seven PDX tumours underwent WES from which potential actionable therapeutics were identified. Samples from three patients were assessed to be statistically significant different in terms of actionable therapeutics identified in direct tumour and PDX analysis Table . The priThis study outlines a method for the use of WES in combination with the IMPACT bioinformatics pipeline to generate a list of potentially actionable chemotherapeutics in bone and soft tissue sarcoma.KIT), Ewing’s sarcoma (ALK), undifferentiated pleomorphic sarcoma , alveolar rhabdomyosarcoma (NTRK1), and leiomyosarcoma (ALK). Of these targets, two have been described previously in the literature for their respective malignancy. Two separate studies [ALK mutations in Ewing’s sarcoma and have hypothesized that this mutation may be targetable with Crizotinib. Over expression of the KIT gene and resultant increased levels, has previously been identified as a potential therapeutic target in paediatric osteosarcoma [Analysis of the WES data with the bioinformatics pipeline found tyrosine kinase inhibitor targets for half of the patients. Kinase targets found in the analysis were for osteosarcoma ( studies , 29 haveosarcoma .As this is a non-validated method this trial did not aim to alter patient drug treatment. Indeed, due to advance stage of disease in which the biopsies were performed, the majority of patients were deceased at the time of results becoming available. Of note, one patient with osteosarcoma elected to undergo treatment with the predicted tyrosine kinase inhibitor whilst in the terminal stages of their disease. A temporary clinical response was demonstrated with a reduction in size of his recurrent tumour. Whist this is far from conclusive, such a result provides some promise that guided treatment may be feasible.A list of potentially actionable therapeutics was generated for all twelve fresh tumour specimens. Four patients had received chemotherapy agents that were predicted to be actionable, and in none of these were more than one of their current agents predicted to be actionable. The interpretation of this result is difficult, as enrolled patients had already undergone chemotherapy treatment. Darwinian selection pressure in response to chemotherapy exposure may have resulted in the tumours already developing resistance to previous chemotherapy agents. Identifying whether these predicted agents truly are effective is difficult. As this method of drug prediction is not validated we were unable to utilise these results in chemotherapeutic selection. Due to the heterogeneity of sarcoma the data on non-traditional chemotherapy regimes is scarce. Therefore, a larger trial is required with biopsy performed prior to commencement of chemotherapy, and subsequent tracking of chemotherapy response. This would clarify whether there is correlation between predicted drugs and clinical course.Of the genes predicted to be deleterious four were present in more than one sample. A mutation of AQP7 was present seven samples. This gene encodes for Aquaporin 7, and there is some early evidence that it may play a role in tumour cell function . Four deResults of pipeline analysis of PDX samples to identify tyrosine kinase inhibitors indicated varied results in comparison to fresh tumour samples. Of the seven PDX models, five had targetable mutations identified in fresh tumour samples with only three of these remaining present in the PDX analysis. Analysis of all seven PDX samples demonstrated the presence of novel targetable gene mutations in comparison to fresh tumour. The most marked of this was the osteosarcoma sample from patient 4, which demonstrated no actionable kinase inhibitor targets in the tumour WES and 35 novel actionable kinase targets with 227 novel variants in the PDX WES.Actionable chemotherapeutics were successfully predicted in all seven PDX samples. Pipeline analysis identified novel chemotherapeutics in all samples in comparison to fresh tumour results. Samples from three patients were found to be significantly different statistically in terms of actionable therapeutics identified in direct tumour and PDX analysis. These three samples were for osteosarcoma, undifferentiated pleomorphic sarcoma, and leiomyosarcoma.Of the twelve patients enrolled in this trial two had osteosarcoma, two had leiomyosarcoma, and two had undifferentiated pleomorphic sarcoma. The two osteosarcomas demonstrated different mutation, kinase inhibitor, and actionable therapeutic profiles. The leiomyosarcoma samples had different drug profiles but both demonstrated a mutation in AQP7. Likewise, the undifferentiated pleomorphic sarcomas had different drug profiles but a mutation of the gene GXYLT1 was present in both.The results obtained in this study raise several interesting questions. Despite poor earlier evidence for the use of tyrosine kinase inhibitors in sarcoma, the results of the pipeline analysis may suggest that they could play a role if selected individually for patients. It is possible that the genetic mutations that confer a susceptibility to these agents occurs sporadically throughout the sarcoma subtypes. Therefore, careful selection may provide a response in a subset of patients. Secondly, it is interesting to note that very few of the patient’s received chemotherapeutics that were predicted to be actionable by pipeline analysis. An intriguing finding is that pipeline analysis of a chordoma sample indicated the presence of 17 potential actionable therapeutics. Except in rare cases of aggressive chordoma, this sarcoma has traditionally been regarded as having a poor response to chemotherapy .Several limitations are inherent in the design of this study. As this is a pilot investigation, the small number and heterogeneity of the population studied prevents any definitive conclusions to be made. As such, this study is intended to generate hypotheses for further studies and establish a protocol for a larger trial. In comparison to Stebbing et al.  a lower It should be noted that this is a pilot study and further research into this area is required. This is especially important when considering cost and infrastructure required for large scale multi-centre trials. Continued research into the use of WES and drug prediction using bioinformatics pipelines, such as IMPACT, for sarcoma are strongly encouraged. Larger numbers are required to permit meaningful correlation of predicted drug responses and observed clinical response. As this technique is not clinically validated, such a trial would provide support for the development of a multi-centre prospective trial in which chemotherapy selection is guided by chemosensitivity pipeline results.This study provides support for the development of a large-scale trial utilising WES and the IMPACT bioinformatics pipeline to determine potential actionable chemotherapy agents and tyrosine kinase inhibitors. With further advances it is possible that drug prediction via WES may provide clinicians with the ability to deliver personalized therapy to patients with sarcoma and other malignancies.Additional file 1. The full list of potential actionable therapeutics for all twelve patients.