Our objective was to develop a tool to automate the segmentation of DTI decomposed p and q maps in GBM patients in order to inform construction of radiotherapy target volumes.Glioblastoma multiforme (GBM) is a highly infiltrative primary brain tumour with an aggressive clinical course. Diffusion tensor imaging (DT-MRI or DTI) is a recently developed technique capable of visualising subclinical tumour spread into adjacent brain tissue. Tensor decomposition through p map using the q map as its initial starting point. The reason of choosing this model is because of the robustness of this model on either conventional MRI or only DTI. The method was applied on a data set consisting of 50 patients having their gross tumour volume delineated on their q map and Chan-Vese level set model uses these superimposed masks to incorporate the infiltrative edges.Chan-Vese level set model is applied to segment the q map to p map is clearly visible in all cases and the Dice coefficient (DC) showed a mean similarity of 74% across all 50 patients between the manually segmented ground truth p map and the level set automatic segmentation.The expansion of tumour boundary from q map to p map. We have provided initial validation of this technique against manual contours performed by experienced clinicians.Automated segmentation of the tumour infiltration boundary using DTI and tensor decomposition is possible using Chan-Vese level set methods to expand p maps has the potential to individualise radiation treatment volumes and act as a decision support tool for the treating oncologist.This novel automated technique to generate  Glioblastoma multiforme (GBM) is an aggressive form of primary adult brain tumour, responsible for more average years of life lost than any other common adult malignancy.4,5 Radiation therapy oncology group (RTOG) guidelines converge on a similar 2.5–3 cm margin.6Glioblastoma is characterised by macroscopic and microscopic infiltration into adjacent brain tissue, by contiguous tumour spread, white matter migration, or spread through the subventricular zone. For this reason, generous clinical target volume (CTV) margins are added to the edge of the gross tumour volume (GTV), generally taken as the post-operative resection cavity, at the time of radiotherapy. Current European society for radiotherapy and oncology (ESTRO) consensus guidelines borne out of analysis of patterns of tumour recurrence following chemo-radiation therapy suggest that a 2 cm isotropic CTV margin is adequate to cover over 90% of tumour recurrences.7,8 The DTI abnormality can predict the sites of tumour progression9 and decomposition of the diffusion tensor into isotropic(p) and anisotropic(q) components can provide spatial maps of tumour infiltration zones which correlate to the time of tumour progression.8 The p map is a imaging correlate for the infiltrative edge of the tumour, and therefore a surrogate for the CTV.10 For radiotherapy treatment planning, utilisation of DTI to individualise treatment permits a potential 40% reduction of CTV. Other studies have demonstrated the safety and efficacy of non-individualised reductions in CTV.11 Thus utilisation of DTI data at the time of radiotherapy planning might serve two distinct functions. The first is to reduce the irradiated volume of brain in patients with locally invasive tumours, with consequent reduction in the risk of cerebral radionecrosis and potential reduction in long term neurocognitive deficit. The second is to stratify patients according to risk of tumour infiltration and expected survival. At a practical level, integration of DTI data into conventional treatment planning systems is non-trivial and resource intensive due to the high dimensionality of the image data sets.12Diffusion tensor imaging (DTI) is a technique for imaging of white matter connectivity in the brain. We have previously demonstrated that it can be utilised to image subclinical tumour spread with high degrees of sensitivity and specificity, and patients can be stratified into prognostic phenotypes based on the extent of tumour infiltration as assessed by DTI.In this paper, we demonstrate that DTI decomposition maps by principal component analysis can be combined with automated image segmentation to generated peritumoral tumour infiltration zones. Comparison of these volumes with manually segmented p maps, considered as a surrogate of conventional isotropic CTVs, demonstrates its potential to individualise radiation treatment volumes and act as a decision support tool for the treating oncologist.The framework of the proposed approach is shown in The GTV is manually segmented from DTI q map, this will be the initialisation contour for level set.Segmented GTV is superimposed on DTI p map.Level set automatic segmentation modifies the superimposed GTV to include information from p map, this will incorporate the infiltrative edges.In this paper, Chan-Vese level set segmentation model is applied. The reason of choosing this model is because of the robustness of this model on either conventional MRI or only DTI.b-values  scanned in 13 directions.We utilised image data from a preoperative multimodal imaging study, providing data from 50 patients  with a histologically confirmed diagnosis of Glioblastoma. Written informed consent for research use of imaging was obtained for all patients, and was approved by the local research ethics committee (10/H0308/23). All patients were imaged at 3 T using a Magnetom Trio scanner. , using a standard 8-channel receive head coil and transmission on the body coil. DTI data were acquired using a single-shot echo-planar sequence  with multiple T2 weighted imaging in differentiating the gross tumour mass from the tumour in ltration.8 These derived quantities are the isotropic and anisotropic components of the diffusion tensor, respectively called p and q, and de ned respectively as follows. As the tensor flow is written as a 3 × 3 matrix for each voxel of the image, with eigenvalues DTI images is an imaging technique aiming to the mobility of water within the brain. Furthermore, derived parameters from the tensor reconstruction have shown to be more sensitive than D is the mean diffusivity where 13 The manual ROIs were independently contoured by the three observers: a neurosurgeon with >8 years of experience (CL), a neurosurgeon with >9 years of experience (JLY), a researcher with >4 years of brain tumour image analysis experience (NRB). The manual segmentation was blinded to the model construction and validation phases. The inter-rater reliability testing was performed using similarity coefficient scores to assure consistency among the observers.12,14Gold-standard manual segmentation of the preoperative GTV and CTV, together with p and q maps were performed by expert clinicians for all patients in the data set using the three-dimensional (3D) slicer application.15 to improve the shortcomings of the Snake technique introduced by Kass in 1987.16 Level set methods are a form of active contour which has the freedom of movement inside an image until it converges to the desired boundary/region. The level set needs to be initialized as a boundary. It was initially introduced as a Eulerian formulation of a propagating front, which moves with the speed F perpendicular to the curve. Level set’s front , propagates implicitly as the zero-crossings of a level set function Level set methods was developed in 1988 by Osher-SethianIt adjusts the height of the z function which vanishes the topological problem. Such a level set is a growing or shrinking contour based on curvature-dependent speed for propagating fronts. It uses Hamilton-Jacobi equations to reconstruct complex shapes.18 as two-phase level set method without edges.19 The main concept of this model was the improvement of energy minimization problem which is based on the mean intensity values in each region of the level set (inside or outside in two-phase), Chan-Vese proposed based on Mumford-Shah functionalH is the Heaviside function,At each iteration, the values of The Chan-Vese level set evolution equation is as follow where represents a one-dimensional Dirac function.p and q images have the volume of 240 × 330 × 23 pixels and a pixel size of 0.977 x 0.977 mm in nifti format. The manual segmentation/ground truth which refers to the GTV on q-map was used to initialise level set and tries to segment p maps which refer to CTV. In order to evaluate the reliability of the proposed model, we seek to demonstrate that the expansion of tumour volume on p-mask can be used to measure CTV automatically. In 20Concerning conforming indices, the best agreement was determined with a mean score of 74% across all 50 patients’ average DC with the standard deviation of 7%. When the initial contour was done manually, further analysis revealed that the average CTV contouring time of proposed model was 0.5 s which is less than the overall average time in planning practice done by clinicians. The consistency in the proposed model’s segmentation among all different patients shown in this figure is a great indication to reduce the inter- and intraobserver variability in GTV and CTV contour generation.Segmenting GBM and its recurrence boundaries is a very challenging area as the image modalities are restricted to conventional MRI before treatment. Although the outcome of treatment in GBM demonstrates the lack of information on used MR images, the proposed approach in analysing GBM tumours based on DTI p and q-maps images would help the clinicians to better estimate the location and size of the tumour as well as recurrence area of tumour which is not really visible to the conventional MR images. The standard treatment for GBM patients is still surgery. Surgery is also of use, because it provides tissue specimens to evaluate, so that an appropriate treatment can be defined based on the accurate evaluation of the tumour, biologically. A better individualisation of treatment approaches requires a more precise understanding of the recurrence regions of GBM. However, there are some real limitations to surgery. The proposed model would give an indication to the clinicians about the potential areas of tumour using radiotherapy. The great possibility in generating accurate CTV using DTI and specificity using p and q maps which are completely different, would lead into better merging the isotropic and anisotropic information of the brain white tracts. The proposed CTV modelling improves using the p and q maps segmentation.21 As suggested by Price et al,22 isotropic p component appears to increase with tumour infiltration due to the increased tissue water from vasogenic oedema. This is a very common finding in GBM imaging and automation of part of the analysis for this oedematous, infiltrated region may have further research and clinical applications.Our algorithm can reliably generate DTI sequence derived p maps from manually segmented q maps. Automatic segmentation of p and q abnormalities can be performed robustly. As we have previously demonstrated that the p map represents the infiltrative margin of the tumour, it acts as a biological surrogate for the CTV concept of microscopic disease extent used in radiotherapy planning. The volumes derived from our automated tool are highly comparable to the manual CTV contours derived by experts using a geometric expansion round the tumour of 2 cm in the case of the ESTRO ACROP (Advisory Committee on Radiation Oncology Practice) guidelines or the 2.5 cm in the case of the RTOG guidelines.Due to limitations on scanning time, DTI acquisitions are noisy when compared to clinical T1 and T2 sequences. The Chan-Vese model performs robustly in the presence of this noise due to its ability to utilise global properties such as regional image intensity. Also, the level set algorithm considers the interrelation of p and q (defined by the equation), since it initializes from the q region to derive an accurate segmentation of p. The robust results of the algorithm, comparable to manual segmentation by experienced clinicians, suggests that this iterative level set method may perform well in the GBM population with further parameter tuning.T1 weighted sequences with contrast and FLAIR sequences. Use of these without reference to the information available from DTI-derived sequences can considerably underestimate the true tumour volume. By automating the conversion of a q region to a p region, this additional information may also assist in guiding the treatment plans of clinicians considering resective surgery or radiotherapy, e.g. to establish whether or not total macroscopic excision of the tumour is feasible.Whilst manual segmentation of image maps by expert observers is considered the gold-standard, it is well appreciated that manual segmentations are subject to significant inter- and intraobserver variability. Current clinical management of GBM is very firmly based on the convention of using In radiotherapy, the GTV contours are manually delineated way for each patient. In this work, we have not used user-input to find the location of the GTV. Using the proposed technique would provide personalised CTV solutions for each patient at the same time. This would influence the resulting radiation dose calculation and final treatment toward the desired direction of harming less healthy cells and radiating more cancerous cells. Also, this method would significantly reduce the CTV contouring task complication time. Thus, the use of such automatic contouring tools can be encouraged in radiotherapy planning software.e.g., the adjacent subventricular zone) and careful consideration would need to be given to the handling of these regions in an individualised margin protocol.We know that the central concept of the CTV is to encompass subclinical tumour infiltration. Given that our previous work demonstrates the validity of the p-map as a surrogate for tumour infiltration, we consider that the automatically generated CTV has value as the basis of personalising the CTV. Clearly, personalisation of the CTV that differs significantly from conventional margins would require validation in a prospective clinical study but existing data on patterns of treatment failure would support such an approach. Furthermore, there may be other anatomical zones at risk of tumour infiltration that are not adequately visualised using DTI . The second is for radiotherapy target volume definition. The technique offers the opportunity to generate an automated yet individualised CTV for treatment planning, rather than the current CTV which is a simple geometric expansion that is driven by an assessment of tumour infiltration across a population of patients. Such a CTV might be used as the basis of an individualised dose intensity-modulated radiation therapy dose painting strategy. Further cross-validation of the segmentation technique is required to confirm its utility.


Advanced fluid models relating viscosity and density to resonance frequency and quality factor of vibrating structures immersed in fluids are presented. The numerous established models which are ultimately all based on the same approximation are refined, such that the measurement range for viscosity can be extended. Based on the simple case of a vibrating cylinder and dimensional analysis, general models for arbitrary order of approximation are derived. Furthermore, methods for model parameter calibration and the inversion of the models to determine viscosity and/or density from measured resonance parameters are shown. One of the two presented fluid models is a viscosity-only model, where the parameters of it can be calibrated without knowledge of the fluid density. The models are demonstrated for a tuning fork-based commercial instrument, where maximum deviations between measured and reference viscosities of approximately ±0.5% in the viscosity range from 1.3 to 243 mPas could be achieved. It is demonstrated that these results show a clear improvement over the existing models. When electromechanical transducers are immersed in a fluid, the mechanical properties of the fluid influence the resonance spectrum measured at the electrical terminals . Based orameters . Althougrameters ,8), suchQ-factor. It is therefore possible to establish a relationship between the physical fluid properties and the measured resonance parameters. The task of the sensor designer is to guarantee that the resonance parameters can be correctly measured and that a fluid model is available which can be transformed to yield unique and accurate fluid parameters from the resonance parameters. As the exact hydrodynamic function is only known for some special cases, the conventionally used fluid models derived, e.g., by [Piezoelectric cantilevers ,11,12,13sections ,19. A nusections ,21, respe.g., by ,24,25,26e.g., by ,29,30,31e.g., by ,27. It eThe remainder of the paper is structured as follows: In The closed form solution for the vibrating cylinder is chosen as a vehicle to reveal functional dependencies which apply also for more complex cross-sections, but for which no closed forms are known.R, v, and j is the imaginary unit hydrodynamic function. We demand that the vibration amplitudes are small enough that the relation between force and velocity amplitudes remain in the linear range. This means that the convective part in the Navier–Stokes equations causing non-linearity and fluid instability is neglected. Although not generally permissible, this assumption holds for many vibrators, especially for QCMs [Reynolds number [non-dimensional frequency [The complex valued fluid force amplitude per length given by ,23(1)F′ial flow , where tfor QCMs , QTFs anfor QCMs . It is ts number , or non-requency . Furtherr models ,25,26, t3+H.O.T,Γ_≈1+42β−3+H.O.T,Γ_≈1+42β−In general, hydrodynamic functions for different cross-sections must be determined numerically. For the numerical modeling of complex geometries the characteristic variables are not easily recognized in the modeling approach. By means of a dimensional analysis, with details given in the tube see , for insEquation .The fluid forces In the vicinity of a resonance, a vibrating structure can be approximated by an equivalent spring-mass-damper system with the respective parameters Q-factor by Y.  The resonance parameters are given byThe Nyquist plots of such admittances resemble circles as is shown in Equation  in real Q-factor and angular resonance frequency in the fluid and in vacuum, respectively. A). Consequently, the model from Heinisch et al., yields lower deviations between the fluid reference values and the measured values, as is also outlined in [Here, cηρk for , C1,C2,C3,C4 for ,25, ML,a1,b2 for  or A,B,C,B,C for  differ. ,B,C for  where thg et al. . FurtherEquation , the samg et al.  =gbgaA procedure for model calibration similar to that in In this section, it was shown that extending the usual fluid models to higher polynomial order is straightforward. Given Equations  and A17A17, it iSynthetic data, generated using the closed form solution for the cylinder, as well as experimental data obtained with quartz tuning fork sensors, are analyzed. For the latter, the hydrodynamic function is estimated from measured data and compared to theoretical predictions. The deviations between the reference values of certified standard fluids and estimates using different model orders, are analyzed in detail. It will be shown that with increasing order, the systematic model deviations reduce to a point where measurement errors and presumably deviations between actual and nominal fluid parameters or unnoticed fluid degradations become dominant.Q-factor of Equations  and 13)13) were Equation  was usedThe measurement results were obtained with the VDC100 measurement cell and the MFA200 resonance analyzer, both provided by MicroResonant . DetailsThe set of four fluids # 2, 13, 19, and 23 in n m3/kg)a0=2.9983g is calculated from the resonance parameters in g and the solid lines are the models for polynomial orders ranging from 1 to 5. (All fluids were used for these parameter fits.) The intersections of dashed and solid lines represent The function W and thickness H of one prong of the QTF are 610 As was mentioned in Equation  derived c moduli ,38.). Ththe ends . In addithe ends . It can Fluid models providing a higher degree of approximation of the hydrodynamic function than the conventionally used ones were derived and applied to measurement results obtained with a quartz tuning fork sensor setup. These models demonstrate a clear advantage in terms of reduced model deviations for this particular problem, but it is assumed that they can be applied to many other types of vibrating sensors as well. Increased orders, however, do not significantly increase the complexity of the model calibration method and the viscosity and density calculation from measured resonance parameters.


Background: Pregnancy-related acute kidney injury (PRAKI) is still a common serious problem in developing countries. Insulin-like growth factor-binding protein 7 (IGFBP7) and tissue inhibitor metalloproteinases-2 (TIMP-2) can identify critically ill patients at risk for the development of severe AKI. Aim: To identify main causes and timing of PRAKI and to study the G1 cell cycle arrest biomarkers in cases diagnosed with (PRAKI) as a diagnostic tool. Methods: 80 pregnant women diagnosed with PRAKI were recruited from a single hospital as well as 30 age-matched pregnant women with normal pregnancy participated in the present study. A urine specimen was collected from all study participants with established AKI within 24 h of ICU admission to measure [TIMP-2]*[IGFBP7]. Results: The incidence of PRAKI was 1.1%. The most common cause of PRAKI is pre-eclampsia/eclampsia spectrum (61%). Most of the cases occur in the third trimester (60%) and postpartum period (23%). At a cutoff 0.33 ng/ml, the estimated sensitivity and specificity of urinary [TIMP-2]*[IGFBP7] in predicting PRAKI is 100% (95% CI) with NPV and PPV are 100%. Conclusion: Urinary [TIMP-2]*[IGFBP7] serves as a sensitive and specific biomarker in the diagnosis of PRAKI. Pregnancy-related acute kidney injury (PRAKI) is still a common issue in developing countries with serious consequences on the mother and fetus . ConsensAcute kidney injury in pregnancy is defined as a serum creatinine level of >1.1 mg/dl or a doubling of the serum creatinine concentration in the absence of other renal disease . FormulaPRAKI occurs mainly during the third trimester and postpartum period. The etiology of PRAKI is highly variable depending on the country and trimester of pregnancy. Hypertensive complications of pregnancy, namely pre-eclampsia and eclampsia, are the leading cause of PRAKI in both developed and developing countries. Pregnancy and its complications play a predominant primary role in the genesis of PRAKI in most cases, or in few cases act as a trigger in genetically susceptible patients .The biomarker [TIMP-2]*[IGFBP7] panel has been shown to predict future development of AKI within the next 12 h . Then, tThe study was reviewed and approved by the local institutional ethics and review committee of Minia University Hospital (MU1562017) and was conducted in accordance with the Helsinki Declaration. Informed consent was obtained from all participants. The present study was an observational study to study the cause of PRAKI admitted to the hospital and to assess the sensitivity and specificity of cell cycle arrest urinary biomarkers; tissue inhibitor of metalloproteinases-2 (TIMP-2), and insulin-like growth factor-binding protein 7 (IGFBP7) in the diagnosis of PRAKI, with the participants recruited from the emergency room and critical care unit of Minia University Hospital over the period from June 2017 to December 2018. The study enrolled 80 pregnant patients with PRAKI out of 7253 deliveries during this period; as well as 30 age- and sex-matched healthy pregnant controls. The inclusion criteria were all pregnant and postpartum patients who develop PRAKI. Exclusion criteria were pregnant patients with pre-existing renal disease. A urine specimen was collected from each participant within 24 h of admission to ICU and kept frozen at −80° until further analysis for IGFBP7 and TIMP-2 then the results multiplied ([TIMP-2]*[IGFBP7]) expressed in (ng/ml) 2/1000.The diagnosis of AKI in pregnancy was based on the following criteria (any one of three): Serum creatinine > 1.1 mg/dl, doubling of serum creatinine from baseline, oliguria/anuria > 12 h duration, or the need for dialysis .Urine samples were drawn within 24 h of ICU admission from each of the study participants with established AKI. Measurements of urine IGFBP7 concentrations were performed using a two-site second-generation Enzyme-Linked Immunosorbent Assay (ELISA) Kit  with reference range is 0.05–20 ng/ml, intra-assay precision is CV<8%, and inter-assay precision is CV<10%. The microtiter plate was coated with monoclonal anti-IGFBP7 antibody. About 50 μl of standards or samples are added to the appropriate microtiter plate wells, and incubate for 60 min at 37°C. Remove the liquid of each well; add 50 μl of a biotin-conjugated polyclonal anti-IGFBP7 antibody to each well and incubate for 1 h at 37°C. Aspirate each well and wash with wash buffer, repeating the process three times for a total of three washes, followed by the addition of 50 μl of Avidin conjugated to horseradish peroxidase (HRP) to each microplate well and incubated for 30 min at 37°C. aspirate and wash five times. Color development was achieved using a 50 μl TMB substrate solution is added to each well and incubated for 10 min at 37°C in the dark. Only those wells that contain, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme–substrate reaction is terminated by the addition of 50 μl sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Serial dilutions of recombinant human IGFBP7 were used to establish a standard curve.Urine samples were drawn within 24 h of ICU admission from each of the study participants with established AKI. Measurements of urine TIMP-2 concentrations were performed using a two-site second-generation Enzyme-Linked Immunosorbent Assay (ELISA) Kit  with reference range is 0.05–200 ng/ml, intra-assay precision is CV<8%, and inter-assay precision is CV<10%. The microtiter plate was coated with monoclonal anti-TIMP-2 antibody. About 50 μl of standards or samples are added to the appropriate microtiter plate wells, and incubate for 60 min at 37°C. Remove the liquid of each well; add 50 μl of a biotin-conjugated polyclonal anti-TIMP-2 antibody to each well and incubate for 1 h at 37°C. Aspirate each well and wash with wash buffer, repeating the process three times for a total of three washes, followed by the addition of 50 μl of Avidin conjugated to horseradish peroxidase (HRP) to each microplate well and incubated for 30 min at 37°C. aspirate and wash five times. Color development was achieved using a 50 μl TMB substrate solution is added to each well and incubated for 10 min at 37°C in the dark. Only those wells that contain, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme–substrate reaction is terminated by the addition of 50 μl sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. Serial dilutions of recombinant human TIMP-2 were used to establish a standard curve.Demographic data with maternal history including gestational age, parity, gravidity, and maternal and fetal complications as well as physical examination were obtained at enrollment. Complete blood cell count (CBC), prothrombin time and concentration, INR, APTT, C-reactive protein (CRP), renal function tests, liver function tests were measured using stored ethylenediaminetetraacetic acid (EDTA) plasma samples using standard techniques. Complete urine analysis, spot urine protein/creatinine ratio and abdominal ultrasound were performed in all patients. Serological investigations such as ANA, anti-dsDNA, C3, C4, LDH, anticardiolipin antibodies, lupus anticoagulant assay and β2 glycoprotein were performed in selected patients. Kidney biopsy was performed only in patients with anuria and/or oligo-anuria of >4 weeks duration and in those with partial recovery of renal function after 3 months of the diagnosis of AKI.T test for parametric quantitative data between the two groups, Mann–Whitney test for nonparametric quantitative data (expressed as median) between the two groups and Fisher’s exact test for qualitative data between the two groups. For all tests P-values #0.05 were considered significant. To analyze the predictive power of these biomarkers receiver operating characteristic curves (ROC) were calculated and the area under the ROC curve (AUC) was determined. 95% confidence intervals (CI) were reported.For the primary analysis, and to compare the difference between the study groups independent samples P<0.001), white blood cells count , platelets count . With respect liver function tests, PRAKI showed statistically significant higher INR , AST , ALT , total bilirubin , and direct bilirubin . Importantly, renal function showed statistically significant higher blood urea  and serum creatinine  in PRAKI group as compared with control group.Most of the PRAKI occurs in the third trimester (48 patients = 60%) as compared with postpartum period (18 patients = 23%) followed by first trimester (9 patients = 11%), and finally second trimester (5 patients = 6%) .The most common obstetric complication that leads to PRAKI is hypertensive disorders of pregnancy, namely pre-eclampsia and eclampsia that contribute collectively to 61% of the cases. The second common cause was sepsis attributed to intrauterine fetal death (IUFD), premature rupture of membranes and postoperative sepsis. This is followed by postpartum hemorrhage with subsequent ischemic acute tubular necrosis. Other causes contribute to only 14% of cases which include hyperemesis gravidarum, abruptio placenta, thrombotic microangiopathy, lupus nephritis and postrenal obstruction .P<0.001) with the median is  as shown in Analysis of [TIMP-2]*[IGFBP7] as a diagnostic biomarker of PRAKI showed that [TIMP-2]*[IGFBP7] is statistically significant higher in PRAKI cases than controls  as shown in Sub-analysis of PRAKI cases according to categories of AKI showed that the median [TIMP-2]*[IGFBP7] is higher in prerenal and postrenal causes as compared with intrinsic renal causes but without statistical significance (14.4(5.6–27.2), 15.9(15.9–15.9) and 7.5(4.9–12.5), respectively; .ROC curve for prediction of PRAKI using [TIMP-2]*[IGFBP7] is shown in . AKIN and RIFLE criteria used to define AKI in the general population are not well validated in pregnancy because they depend on changes in serum creatinine *[IGFBP7] in obstetric patients. This may be attributed to higher incidence of sepsis (33%) and shock (16%) in this critically ill small cohort with some patients had more than one co-morbidity [We test the diagnostic performance of [TIMP-2]*[IGFBP7] in PRAKI. We found that [TIMP-2]*[IGFBP7] to be higher in cases of PRAKI as compared with controls, with higher levels noticed in prerenal and postrenal cases. We used a cutoff point of 0.33 ng/ml for the prediction of PRAKI with both sensitivity 100% and specificity 100% (95% confidence interval: 0.967–1.0, orbidity .Our study has some limitations. First, it is a single center study in small number of patients especially in the prerenal and postrenal categories of PRAKI which might affect the statistical power; but this is attributed to the low incidence of these categories in pregnancy. Second, in the intrinsic renal category of PRAKI, larger number of patients with different causes need to be recruited to investigate the possible role of [TIMP-2]*[IGFBP7] in the differentiation between them. Third, to complete the validation as a diagnostic marker, further research is needed to compare AKI patients with a larger number of patients who experience the same complications but without development of AKI.In conclusion, we report that PRAKI is still a common problem in developing countries with the third trimester and postpartum period are the most vulnerable period, pre-eclampsia/eclampsia spectrum is the most common cause of PRAKI, and most importantly [TIMP-2]*[IGFBP7] is a valuable tool in the diagnosis of PRAKI that needs further research on a large scale of patients to determine the ability to differentiate between different causes of PRAKI and to study its predictive value in the diagnosis of PRAKI through serial measurements in high risk pregnant patients.


The use of Twitter hashtags at medical conferences has revolutionized the way healthcare professionals interact and advance their education. We aim to investigate the scope of the Academic Surgical Congress's online reach and engagement through the use of Twitter hashtags #ASC from 2015 to 2019, by analyzing the number of impressions and tweets and retweets.A cross sectional study of Twitter data through Symplur with the following conference hashtags for the Academic Surgical Congress annual meetings for years 2015–2019: #ASC2015, #ASC2016, #ASC2017, #ASC2018, and #ASC2019. Data on tweets, retweets, users, and impressions was reviewed along with information on the top 10 influencers and the most frequently tweeted links. Symplur Signals software was utilized to extract and assimilate data. Statistical Significance was defined as p < 0.05.Twitter engagement metrics significantly increased from 11,400 to 32,100 from 2015 to 2017 (p < 0.05). However, from 2017 to 2019, there was a significant decline in engagement metrics from 32,100 to 26,100 (p < 0.05). Impressions increased significantly from 13,100 in 2015 to 71,800 impressions in 2019 (p < 0.05). Users grew significantly from 1500 in 2015 to peak at 4600 in 2017 before dropping back to 3300 in 2019 (p < 0.05). The most influential organizations during these years were the organizers of the conference: Association for Academic Surgery and the Society of University Surgeons. Conference attendance progressively increased from approximately 1700 in 2016 to about 2100 in 2019 (p < 0.05).Twitter engagement metrics at the Academic Surgical Congress 2015–2019 has fluctuated, while impressions significantly increased through the years indicating the consistent dissemination of conference content. •Cross sectional review of the Academic Surgical Congress's online reach and engagement through the use of Twitter hashtags #ASC 2015–2019.•Twitter engagement metrics at the Academic Surgical Congress 2015–2019 has fluctuated, while impressions significantly increased through the years.•Using Twitter to maximize the dissemination of scientific surgical research and evidence-based practices is highly beneficial to medical professionals. Instant dissemination of new ideas and online interactions through the use of Twitter hashtags has greatly magnified the reach of medical conferences materials. Twitter is uniquely suited to improve communication and spread of ideas at conferences because of its simple, free access and interactive format. Users publish tweets with a 280-character limit that can include identifying hashtags, that other users can “like,” retweet and respond to. In addition to the text, users can include pictures, videos and links in their tweets.Twitter provides the unique opportunity for physicians to build an online brand or personality. This has improved opportunities for connections and even mentorship with medical trainees or other professionals . The samMany studies have shown that Twitter improved medical conferences online participation from year to year by documenting increasing numbers of tweets, users and impressions . One stuUsing conference hashtags to increase a conference's online presence benefits not only those in attendance but those following the conference proceedings remotely. Through a targeted emphasis on the online impact of medical conferences, we can help ensure those who are unable to attend conferences due to work or time constraints are still able to learn from the novel ideas presented.In this study, we aim to investigate the scope of the Academic Surgical Congress's (ASCs) online reach and engagement through the use of Twitter hashtags #ASC from 2015 to 2019. We measure this by analyzing the number of impressions (reachability metrics measured in millions) and engagement (tweets & retweets metrics measured in thousands). The ASC is a combined meeting of the two leading associations for academic surgery – The Association for Academic Surgery (AAS) and The Society of University Surgeons (SUS). As a prominent international surgical conference, which is greatly attended, the potential for impact is more significant than many other conferences that have been previously analyzed. Additionally, there have been no studies investigating the reach of the ASCs Twitter use over the period of multiple years. We hypothesize that increased Twitter impressions and engagements will result in wide dissemination of knowledge/conference material and increase the conference attendance rate.2A cross sectional review of Twitter data at the ASC from 2015 to 2019. Symplur Signals software , designed specifically for hashtags related to healthcare topics was used to review and analyze studied Twitter metrics. We analyzed the following conference hashtags: #ASC2015, #ASC2016, #ASC2017, #ASC2018 and #ASC2019. From this analysis, we obtained information on the number of engagements, impressions, and users who participated by using the conference hashtags. Only publicly available twitter data metrics were utilized for analysis. This study was reviewed by our Institutional Review Board (IRB) and the Western Institutional Review Board (WIRB) and deemed exempt.Data obtained from Symplur was analyzed to obtain a more thorough understanding of the breadth of Twitter use at the ASC from 2015 to 2019. The term “impression” is defined as the number of people who only viewed a tweet containing the conference hashtag, but did not engage. In contrast, “engagement” is defined as action on conference content through user tweets containing the conference hashtag or “likes”, retweets or comments on a Tweet containing the conference hashtag. “Twitter influencers”, who are accounts of either organizations or individuals, are able to gather a significant amount of impressions and tweet engagement due to their large number of followers. Therefore, we investigated the role of twitter influencers on hashtag engagement. These influencers are defined through Symplur as having high conference hashtag utilization. This study was reported in line with the Strengthening the Reporting of Cohort Studies in Surgery (STROCSS) 2019 guidelines [3Impressions continued to significantly grow during the time period of 2015–2019, from 13,100 to 39,900 to 63,100 to 65,900 to peak at 71,800 impressions in 2019, respectively. From 2015 to 2017 there was a consistent increase in engagement from 11,400 to 27,700 to 32,100, respectively. However, from 2017 to 2019, there was a significant decline in engagement from 32,100 to 26,100. Data regarding tweets, retweets, users, impressions and Top 3 Links tweeted are summarized in The term “users” refers to Twitter accounts that participated in meeting conversation by tweeting the conference hashtag. Users grew from 1500 in 2015 and 3800 in 2016, to peak at 4600 in 2017 before dipping back to 3700 in 2018 and 3300 in 2019.Symplur signals provides data on the Most Tweeted Links containing the meeting hashtag. The Most Tweeted Links represent articles that were presented at the conference, however there were a significant amount of outdated links that have since been deleted as our study refers back to posts dating as far back as 2015.The Top 10 Twitter influencers by year were primarily occupied by the Association for Academic Surgery , followed by the Society of University Surgeons . Other contributing organizations included the Association of Women Surgeons . The remaining influencers were individual physicians at varying academic levels and from a myriad of surgical specialties as summarized in Understanding where the majority of the conference tweets originate is crucial to the discussion on engagement. In this study, engagement is defined as the number of “likes”, tweets, retweets, or comments; however, these numbers may not reflect continuous conversation or involvement as active as it may appear. For example, in 2017 when engagement was the highest, 71% of users only used the conference hashtag once, which does not indicate active participation or conversation. These numbers remained largely the same, even in 2019, when only 13.5% of users tweeted more than 5 times.4Through an analysis of hashtag data usage and interaction for the ASC from the years of 2015–2019, we aimed to investigate relationships between online impact and attendance levels. Our study found that though engagement peaked in 2017 and declined subsequently in 2018–2019, impressions continued to increase. This exhibits that while Twitter interaction may have decreased in more recent years, users were still viewing conference content. One phenomenon to explain the increasing impression but decreased engagement is social media overload and fatigue . Social Engagement significantly decreased from the years of 2017–2019 by nearly 6000. There are several possible explanations for this phenomenon. First, an insufficient level of twitter participation or interest in utilization of conference hashtags in more recent years may have influenced engagement numbers during this time. As we discovered, attendance did not grow between 2017 and 2018 and minimally grew in 2019, which may also affect Twitter engagements. In addition to the social media overload and fatigue, overall Twitter usage patterns over the same time interval. According to Twitter data AI experts, Twitter usage and membership began to stagnate in 2018, prior to which the social media site was gaining users and engagement [As Twitter has the potential to include participants nationally and globally, it has a particularly unique role in enhancing engagement in an international conference such as the ASC. In a study of the Canadian Geriatrics Conference, tweets were separated by content such as conference sessions, networking, resource sharing, and conference promotion. This study also found that 60% of Twitter participants were not Canadian, indicating an international reach . Up to 2A study of 14 Anesthesiology conferences showed no relationship between meeting attendance, registration, total tweets, impressions and retweets. However, a positive correlation was demonstrated between the number of Twitter users participating using the conference hashtags and the Twitter metrics of tweets, retweets, impressions and comments . AdditioWe recommend that scientific medical meetings use Twitter to increase their online participation of those attending and those following along remotely in order to increase knowledge dissemination. Ensuring that the hashtag is advertised on Twitter and on the association's website can boost hashtag use before and during the conference . DissemiThis study has several limitations. Data regarding conference attendance in 2015 was not available. In addition, information may have been tweeted regarding the conference where the hashtag was not used. Conversely, hashtags may have been utilized in tweets not containing relevant conference information. Symplur Signals is not an exhaustive software, and certain information could not be obtained from our analysis, such as the occupation of each individual user who interacted with meeting content. Therefore we were unable to ascertain which tweets are from medical professionals versus the general public. Filters created by Symplur signals were utilized to provide data only on Tweets published in English and in the North American time zone. Importantly, Twitter engagement relates to the previous years’ attendance. Finally, there may be a proportion of meeting participants and attendees who do not utilize social media or Twitter for academic discussions.The recent COVID-19 pandemic has clearly shown that such sudden unanticipated disruptions that lead to health and safety concerns result in the cancellation of upcoming medical meetings, conferences and training courses. This has led to renewed interest for consideration of developing a virtual web-based platform to minimize the impact of not being able to attend these educational symposia in person. Twitter and other social media platforms may serve as an important alternative to continuing to disseminate the educational component of these meetings. We theorize that these internet-based services will continue to grow in importance and may provide a resilient solution to any future global health crises and/or inclement climate issues, while potentially minimizing missed educational opportunities. They will continue to provide an easily accessible network to allowing meeting content to be available for remote audiences. We also suspect that engagement will increase during these times as the transition to remote conferences will encourage active participation by conference attendees. Developing these innovative alternative options is crucial to continue the spread of medical research knowledge and improved clinical care despite impediments.Twitter serves as a free of cost online forum to increase the interactions and engagement between surgeons, facilitate the spread of new research ideas and advance the scientific learning process. As social media is constantly growing in the evolution of our digital society, we anticipate that the role of Twitter will continue to increase in conferences in the future.5Twitter engagement metrics and number of users at the ASC has fluctuated through the years, however the consistent increase in numbers of Twitter impressions show conference content can still be widely disseminated. Twitter hashtag usage may have contributed to the increased in attendance rates throughout the years. Using Twitter to maximize the dissemination of scientific surgical research and evidence-based practices and guidelines is highly beneficial to all medical professionals, particularly surgeon scientists, leading to not only improving their scientific knowledge and research skills but also ultimately translating into improved patient care.This study was conducted in compliance with ethical standards, reviewed by our institutional review board and the Western Institutional Review Board and deemed exempt.None.AE, KS, MM– Conception of study, acquisition of data, analysis and interpretation of dataKS, AE, AS, EM, DB, MM – drafting of manuscript, critical revision of manuscript.KS, EM, AS, AE, DB, MM – Approval of the final version for submission.researchregistry5961.Mark McKenney.Not commissioned, externally peer reviewed.Authors declare no competing interests.


Fluorescence probes with aggregation-induced emission (AIE) characteristics are of great importance in biomedical imaging with superior spatial and temporal resolution. However, the lack of toxicity studies and deep tissue imaging in nonhuman primates hinders their clinical translation. Here, we report the blood chemistry and histological analysis in nonhuman primates treated with AIE probes over tenfold of an intravenous dose of clinically used indocyanine green (ICG) during a study period of 36 days to demonstrate AIE probes are nontoxic. Furthermore, through bright and nontoxic AIE probes and fluorescence imaging in the second window , we achieve an unprecedented 1.5-centimeter-deep vascular imaging in nonhuman primates, breaking the current limitation of millimeter-deep NIR-II fluorescence imaging. Our important findings, i.e., nontoxic features of AIE probes and centimeter-deep NIR-II vascular imaging in nonhuman primates, may facilitate successful translation of AIE probes in clinical trials. Fluorescence imaging with organic molecules as probes has become an essential tool for bench researchers and clinicians interested in understanding disease mechanism or improving human health due to excellent spatial and temporal resolution –3. Howevin vivo imaging application in the NIR-II window using these probes. Nonhuman primate studies are known to facilitate clinical trials because of the evolutionary relationship between nonhuman primates and humans  calculated for C30H34NS 440.2406; found: 440.24050.A solution of 3-octylthiophene 1  in THF (40 mL) was dropwise added with 2.5 Mn-BuLi  at -78°C. The mixture was stirred for 3 h under nitrogen protection. Then, SnBu3Cl  and stirred overnight at room temperature. The mixture was quenched with water and washed with ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4 and concentrated. The residual was solved in toluene as 1 M solution. The 1 M solution  was added with 4,7-dibromobenzobis BBTD  and beta-4-platinum  and dissolved in toluene (5 mL) under nitrogen protection. After refluxing for 24 h and then cooling to room temperature, the mixture was quenched with 1 M KF aqueous solution and washed with ethyl acetate. The combined organic phase was dried over anhydrous Na2SO4 and evaporated. The crude product was subjected to column chromatography on silica gel with PE/EA 10 : 1 to TTB as a black green solid . 1H NMR  δ 7.57 , 7.36 , 7.30-7.26 , 7.15-7.03 , 2.58 , 1.63-1.61 , 1.34-1.26 , 1.18-1.10 , 0.81 . 13C NMR  δ 153.24, 147.60, 147.45, 147.00, 145.68, 129.36, 128.18, 126.78, 124.80, 124.67, 123.39, 123.22, 116.08, 31.79, 30.51, 30.35, 29.73, 29.46, 29.31, 29.16, 22.64, 14.12. HRMS (ESI): m/z [M+] calculated for C66H64N6S4 1068.40686; found: 1068.40698.A solution of 2  in THF (20 mL) was dropwise added with Mn-BuLi  at -78°C. The mixture was stirred for 3 h under nitrogen protection. Then, the reaction mixture was added with SnBusample is the QY of AIE probes, QYref is the QY of IR-26 (0.5%), and nsample and nref are the refractive indices of AIE probes and IR-26.The NIR-II fluorescence QY of the TTB-based AIE probes was measured according to the previous reports . General2. Human umbilical vein endothelial cells (1.0 × 104 cells per well) were seeded in 96-well plates and incubated with various concentrations of AIE probes  for 24 h. The whole blood of monkey was collected, centrifuged  to separate the red blood cells, and washed  with PBS three times. 10% RBC  was incubated with AIE probes  at 37°C for 3 h, respectively. After centrifugation, the supernatant of the suspensions was collected and analyzed by a UV-vis spectrometer at 541 nm.Human umbilical vein endothelial cells were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin with a humidified environment containing 5% CO−1) dispersed in 0.9% NaCl were filter-sterilized before injection. Blood samples for the analysis of clinical chemistry and clinical hematology analyses were collected on days 0, 1, 2, 4, and 7 of each injection cycle. Body mass, temperature, appearance, and exploratory behavior were recorded at the same time. The clinical chemistry and clinical hematology analyses were carried out on the pocH-100ivD fully automated hematology analyzer .Four cynomolgus monkeys  were obtained from the Huazhen Laboratory Animal Breeding Centre . All animal experiment procedures were performed in compliance with the Institutional Animal Care and Use Committee of Huazhen Laboratory Animal Breeding Centre, China (no. HZ-AEC-TORM-051). The cynomolgus monkeys were housed individually in stainless steel cages  and fed a commercial monkey diet. Fresh fruits were also supplemented, and sufficient water was available. Research staff inspected the monkeys three times each day. Cynomolgus monkeys remained healthy and active, and food intake and body weight were normal. After collecting fasting blood samples, AIE probes . Tissue sections were examined by two independent clinical pathologists.−2, 1250 nm long-pass filter, and 200 ms exposure time using a 640 × 512 pixel two-dimensional InGaAs/SWIR camera , with an intravenous dose of 2 mg kg−1.NIR-II fluorescence imaging was performed using NIR-OPTICS Series III 900/1700 small animal imaging system. The fluorescence imaging parameters were 808 nm laser with a power density of 30 mW cm


Advances in imaging have enabled the identification of prostate cancer foci with an initial application to focal dose escalation, with subvolumes created with image intensity thresholds. Through quantitative imaging techniques, correlations between image parameters and tumour characteristics have been identified. Mathematical functions are typically used to relate image parameters to prescription dose to improve the clinical relevance of the resulting dose distribution. However, these relationships have remained speculative or invalidated. In contrast, the use of radiobiological models during treatment planning optimisation, termed biological optimisation, has the advantage of directly considering the biological effect of the resulting dose distribution. This has led to an increased interest in the accurate derivation of radiobiological parameters from quantitative imaging to inform the models. This article reviews the progress in treatment planning using image-informed tumour biology, from focal dose escalation to the current trend of individualised biological treatment planning using image-derived radiobiological parameters, with the focus on prostate intensity-modulated radiotherapy (IMRT). It is well-established that prostate cancer (PCa) exhibits high multifocality and heterogeneity ,2,3 and “Dose painting” was first proposed by Ling et al.  to targeDPbN requires demonstration of correlation between imaging features and specific tumour biology characteristics ,6,7 and This article aims to review the progress in treatment planning using image-informed tumour biology, from focal dose escalation to the current trend of individualised biological treatment planning using voxel-level, image-derived radiobiological parameters to achieve optimal treatment outcomes, with the focus on prostate IMRT.Follow-up studies on treating PCa with a uniform-dose distribution have shown that local recurrences typically occur at the site of the primary tumour ,9,10,11,The FLAME trial investigated the treatment efficacy of focal dose escalation, the largest and the first randomised trial of its kind . The FLAA major limitation of escalating the dose in only the DIL delineated on imaging is the risk of missing other biologically significant tumours as PCa is a multifocal disease. Furthermore, a large inter-observer variation in DIL delineation on multimodality imaging has been observed ,21,22,23lowI was not chosen as the lowest intensity found in the image to minimise the effect of noise. Instead, the dose was escalated between 25% and 100% of the 95th percentile PET voxel intensity values. DPbN essentially establishes a mathematical link between imaging parameters and dose prescriptions that optimise chosen clinical endpoints ,5. The mnth order polynomial function or a sigmoidal function  model appeared in the late 1940s ,38 and bIf it is assumed that any surviving clonogenic cell may repopulate a tumour, then the probability of tumour control is the same as the probability that all clonogenic cells are killed, given by:For fractionated EBRT with The initial number of clonogenic cells in voxel Then, the overall TCP of a given dose distribution within the target structure is calculated as the product of all voxel TCPs:Taking account of the tumour growth dynamics where the combined influence of cell doubling, the proportion of proliferating cells, and accelerated clonogen proliferation are considered, the TCP can then be described by:eatments . α and then utilising a normalisation with a weighted sum:As radiosensitivity varies among individuals in a population, inter-patient radiosensitivity can be taken into account by applying a population distribution to the radiosensitivity parameter Hypoxic tumour cells are radioresistant and hence the presence of hypoxia alters the radiosensitivity parameters. The amount which the tumour radiosensitivity changes is described by the oxygen enhancement ratio (OER):Early attempts in incorporating TCP in biological optimisation often involved maximisation of subvolume TCP with limited modeling of intra-tumour heterogeneity from quantitative imaging. Kim and Tomé  used a pEach subvolume was assigned different values of the radiobiological parameters depending on the risk of recurrence. All voxels within a subvolume were assumed to possess identical tumour characteristics. n et al. . A varien et al. . For all−1 and −1, introducing inter-tumour heterogeneity. Biological optimisation was performed using research software and the resulting plan was replanned on clinical software using the dose-volume (DV) metrics. Uzan et al. reported the potential of biological optimisation in yielding high tumour control with promising toxicity profile. Only two patients experienced grade 2 urinary toxicity and two patients had ≤ grade 2 late rectal toxicity over a median follow-up of 36 months.Uzan et al.  maximiseMore sophisticated intra-tumour biology was modeled for Betts et al. and Haworth et al. ,56,57, wWhile population-based parameters can be extracted from large trial datasets, spatial voxel-level tumour characteristics are ideal for a patient-specific DPbN plan. Deriving an accurate, quantitative relationship between image features and radiobiological parameters is challenging, leading to very few treatment planning studies incorporating image-derived PCa parameters so far. Dircherl et al.  used proGrönlund et al.  used a pWhile improved radiobiological models have been used in biological optimisation, the derivation of the model parameters from imaging has largely been limited to invalidated functions of image intensity. The combination of radiomics and machine learning methods appears to be a potential solution for accurate image-derived radiobiological parameters. Radiomics aims to extract quantitative features from images. These features can then be used to develop predictive models using machine learning methods when “ground-truth” voxel-level data is available. Many investigators have explored and yielded promising results from using radiomics and machine learning of mpMRI for PCa detection ,66,67,68One such approach proposed by Haworth et al. is called the biofocused radiotherapy (BiRT) ,75. The The future of prostate radiotherapy treatment planning is promising with work underway to improve various aspects of biological optimisation. However, it is still far from clinical deployment, largely owing to uncertainties in radiobiological models and their parameters. In addition to methods of incorporating these uncertainties in treatment planning optimisation, hypofractionation and adaptive therapy are of interest in PCa radiotherapy. It is expected that future research addressing these issues, some of which are outlined below, will lead to the realisation of patient-specific, biologically-optimised radiotherapy as the standard of care.There are several significant limitations in current TCP models that need to be overcome. Firstly, these models are usually mechanistic in nature and lack the ability to incorporate important nondosimetric clinical factors such as age, interactions with other treatment responses. Secondly, the TCP model described above fundamentally assumes that clonogens do not communicate with each other, that cell killing events are independent and that clonogens only respond to local dose deposition. These assumptions ignore the bystander effect where cytotoxic effects are seen in nonirradiated cells. The indirect effects of ionising radiation in tumour control is another aspect of tumour biology that has not been incorporated in TCP modeling. One such effect is the vascular effect where high local doses create an unfavourable tumour microenvironment through vascular damage. In studies with experimental tumours, a dose/fraction size greater than 10 Gy has demonstrated the ability to induce severe vascular damage such as endothelial apoptosis that leads to tumour cell killing through the reduced blood supply, thus starving the tumour of oxygen and other nutrients ,81,82,83The reporting of sensitivity and specificity of quantitative imaging in detecting tumour biology information is necessary for a complete understanding of the accuracy of a biological optimization approach in treatment planning. Prostate mpMRI has primarily been investigated in tumour detection and localisation demonstrating high sensitivity and specificity ,88. TherFor biological optimisation where a radiobiological model is informed by imaging via predictive models, quantitative imaging accuracy is combined with other model uncertainties as overall predictive model accuracy. Factors that influence model accuracy include reconstruction methods, machine learning methods, and size of the training dataset. The effect of the overall uncertainty in the predicted parameter value on the resultant plan should be performed on a case-by-case basis.The standard approach to incorporate treatment delivery uncertainty in radiotherapy has been the use of a margin surrounding the CTV to generate a PTV . It is aWith a consensus that the han 5 Gy ,102,103 f 5-8 Gy ,105, hypf 5-8 Gy ,112,113.f 5-8 Gy ,115 due f 5-8 Gy . As suchα/β ratio allows extreme hypofractionation with less than five fractions. The optimal imaging frequency and timing remains unknown. Standard radiotherapy assumes that the tumour response will be consistent throughout treatment. However, cancer is known to adapt to therapies and its biological characteristics are often temporally and spatially heterogeneous. To address the time-dependent changes in tumour characteristics, an adaptive therapeutic strategy has been proposed . AdaptivAdvances in quantitative imaging of PCa allow better targeting of the tumour using radiobiological information derived from the images. Clinical studies that focused on translating image parameters to the desired dose demonstrated promising results in improving tumour control with minimal normal tissue toxicity. However, the direct consideration of the desired endpoint during treatment planning optimisation appears to be the most logical approach for clinical implementation of personalised medicine in prostate radiotherapy. It is expected that the accumulation of PCa radiobiology information and their associated uncertainties will lead to the realisation of patient-specific, biologically-optimised radiotherapy as the standard of care.


In preclinical studies serotonin stimulates and dopamine inhibits tumour growth and angiogenesis. Information regarding serotonin and dopamine receptor (5-HTR and DRD) expression in human cancers is limited. Therefore, we screened a large tumour set for receptor mRNA overexpression using functional genomic mRNA (FGmRNA) profiling, and we analysed protein expression and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 with immunohistochemistry in different tumour types. With FGmRNA profiling 11,756 samples representing 43 tumour types were compared to 3,520 normal tissue samples to analyse receptor overexpression. 5-HTR2B overexpression was present in many tumour types, most frequently in uveal melanomas (56%). Receptor overexpression in rare cancers included 5-HTR1B in nasopharyngeal carcinoma (17%), DRD1 in ependymoma (30%) and synovial sarcoma (21%), and DRD2 in astrocytoma (13%). Immunohistochemistry demonstrated high 5-HTR2B protein expression on melanoma and gastro-intestinal stromal tumour cells and endothelial cells of colon, ovarian, breast, renal and pancreatic tumours. 5-HTR1B expression was predominantly low. High DRD2 protein expression on tumour cells was observed in 48% of pheochromocytomas, and DRD1 expression ranged from 14% in melanoma to 57% in renal cell carcinoma. In conclusion, 5-HTR1B, 5-HTR2B, DRD1, and DRD2 show mRNA overexpression in a broad spectrum of common and rare cancers. 5-HTR2B protein is frequently highly expressed in human cancers, especially on endothelial cells. These findings support further investigation of especially 5HTR2B as a potential treatment target.The online version of this article (10.1007/s12253-019-00734-w) contains supplementary material, which is available to authorized users. Serotonin and dopamine are biogenic amines, which are produced in the central nervous system and gastrointestinal tract. Throughout the body, they are transported by platelets. Serotonin and dopamine play a role in vascular tone, gastrointestinal motility, limb movement control and other physiological processes , 2. PrecAngiogenesis is one of the hallmarks of cancer. It is a prerequisite for tumour growth as it secures oxygen and nutrient supply and removal of break-down products from the tumour microenvironment . SerotonSerotonin and dopamine receptor agonists and antagonists are prescribed for the treatment of Parkinson’s disease, psychosis, nausea, and migraine , 13, 14,RNA microarray expression data of 11,756 human tumour samples were collected from Gene Expression Omnibus (GEO), a large publicly available data set . From thTo determine a threshold for overexpression, FGmRNA profiling of 3,520 normal human tissue samples was performed, and the 97.5th percentile for 5-HTR1B, 5-HTR2B, DRD1 and DRD2 was calculated  –8, 12 orn = 12), ovarian cancer (n = 12), breast cancer (n = 12), renal cell carcinoma (n = 14), and pancreatic cancer (n = 12) were used. A minimum of 12 tumours were selected per tumour type, because if 0/12 tumour samples show expression, the chance that it is relevant in >10% of patients is low. Furthermore, 3 tissue microarrays (TMA) were used, namely a melanoma TMA containing 36 tumour samples, a pheochromocytoma TMA containing 63 tumour samples, and a GIST TMA containing 76 tumour samples. The TMAs contained 3 cores with a diameter of 0.6 mm per patient.Formalin-fixed paraffin-embedded primary tumour tissues of colon cancer ; Vector Laboratories, Brunschwig Chemie, Amsterdam, The Netherlands) and human serum blocks were performed. The sections were incubated with the primary antibody for 1 h at room temperature for 5-HTR2B, DRD1, and CD31 or overnight at 4 °C for 5-HTR1B and DRD2. Primary antibodies used were mouse monoclonal 5-HTR1B 1:100 , mouse monoclonal 5-HTR2B 1:1000 , mouse monoclonal DRD2 1:100 , rat monoclonal DRD1 1:75 , and mouse monoclonal CD31 1:50 . Subsequently, tissue sections were incubated with secondary antibodies . Between steps, slides were washed with PBS or in case of DRD2 with PBS plus 0.1% Tween-20. Staining was visualized using 3,3′-diaminobenzidine (DAB) and hematoxylin for counterstaining. Positive and negative controls (including immunoglobulin class-matched control sera) were included for each staining. Colon, testis, and prostate tissue served to validate the stainings as positive and negative controls. In addition standard hematoxylin & eosin (H&E) stainings were performed to evaluate tissue morphology.Formalin-fixed, 4 μm thick paraffin-embedded sections and TMAs were deparaffinized and rehydrated. Antigen retrieval was performed using heated citrate buffer (pH 6.0) for 5-HTR1B, 5-HTR2B, DRD1, and DRD2 or tris/EDTA buffer (pH 9.0) for CD31 for 15 min. For DRD2, slides were blocked with phosphate-buffered saline  plus 0.1% Tween-20 for 20 min at room temperature. Endogen peroxidase was blocked in all slides with 1% HAfter immunohistochemical staining, slides were digitally scanned using the NanoZoomer  and were scored using accompanied NanoZoomer Digital Pathology (NDP) software. All slides were scored by two independent investigators (M.P. and C.M. or H.H.) and compared to assure a minimal inter-observer difference. As an extra control, random slides were also evaluated by a pathologist (H.H.).Tissue morphology was evaluated using H&E-stained slides. Staining intensity of the tumour cells and percentage of positive tumour cells was scored. Staining intensity of tumour cells was scored as negative (0), low (1), moderate (2), or high (3). The percentage of positive tumour cells was scored as no positive cells (0), 1–4% positive cells (1), 5–24% positive cells (2), 25–49% positive cells (3), 50–74% positive cells (4), and 75–100% positive cells (5). Receptor expression was scored using an immunoreactive score (IRS) to account for the heterogeneous staining in the section slides . The IRSReceptor expression on tumour-associated blood vessels was only assessed on the whole tissue sections, as blood vessels were only in limited numbers or not at all present in the TMA samples due to their small diameter. Serotonin and dopamine receptor expression was scored by staining intensity (ranging from 0 to 3). Receptor expression was considered negative if intensity was 0, low if intensity was 1, and high if intensity was 2 or 3, CD31 staining was performed to confirm localization of tumour vessels.For the frequency of overexpression of serotonin and dopamine receptors per tumour type, see Table 5-HTR1B was overexpressed in a low percentage of tumour samples per tumour type. The highest percentage was found in nasopharyngeal carcinoma: in 7 of 42 (17%) samples.Of the four receptors, 5-HTR2B was most frequently overexpressed, especially in uveal melanomas (59 of 106 (56%) samples). Also in certain brain tumours and sarcomas, and in hepatocellular carcinoma and non-small cell lung cancer, a relatively high proportion of tumour samples showed overexpression of 5-HTR2B: 6 out of 24 astrocytomas (25%), 15 out of 76 liposarcomas (20%), 4 out of 26 osteosarcomas (15%), 71 out of 346 hepatocellular carcinomas (20%) and 16 out of 103 small cell lung cancers (16%).Overexpression of DRD1 was most frequently found in ependymoma and synovial sarcoma with 46 out of 156 (30%) respectively 7 out of 34 (21%) of the samples.DRD2 was most frequently overexpressed in adrenal neuroblastoma (24 out of 96 (25%) samples) and astrocytoma (3 out of 24 (13%) samples).n = 12), also small foci of strongly positive tumour cells were observed within a field of negative tumour cells. DRD1 was highly expressed on tumour cells of approximately 50% of colon cancers, ovarian cancers, breast cancers, renal cell carcinomas and GIST samples. For melanoma and pheochromocytoma, this was the case in less than 25% of samples. High DRD2 expression by tumour cells was most frequently observed in pheochromocytomas (30 out of 63 samples), ovarian cancer (6 out of 12 samples) and pancreatic cancer (6 out of 12 samples) . Expression of DRD1 on endothelial cells varied per tumour type. In breast cancer, DRD1 was highly expressed on endothelial cells of 7 out of 12 samples. For the other tumour types, high expression of DRD1 on endothelial cells was only observed in 4 out of 14 renal cell carcinomas and 1 pancreatic cancer sample. DRD2 expression was low or absent on endothelial cells expression in a broad range of tumour types with both FGmRNA profiling and immunohistochemistry. We found that 5-HTR2B is more frequently overexpressed compared to 5-HTR1B, DRD1, and DRD2. All four receptors, however, are expressed across tumour types including rare cancers. 5-HTR2B is highly expressed on tumour cells in all melanomas and on tumour endothelial cells of colon, ovarian, breast, renal, and pancreatic cancer samples.One of the great advantages of FGmRNA is that it enabled us to screen overexpression of serotonin and dopamine receptors in a large number of samples, leading to interesting findings in rare cancers like brain tumours and sarcomas. FGmRNA profiling identified mRNA overexpression based on a strictly chosen cut-off level, being receptor expression higher than the 97.5th percentile of expression in normal tissue (16). This may have led to the relatively low overexpression percentages found with FGmRNA profiling compared with the number of samples with high expression found with immunohistochemistry.FGmRNA profiling and immunohistochemistry both demonstrated high 5-HTR2B expression in  melanoma. Two in vitro studies evaluated the effect of serotonin on melanoma growth with opposite results. Serotonin inhibited proliferation of 5-HTR2B expressing human melanoma cell line IPC298 but it did not affect B16F0 murine melanoma cell proliferation, in which however, receptor expression was not assessed . On the 5-HTR2B was highly expressed on endothelial cells of five tumour types evaluated using immunohistochemistry . This was in concordance with studies from a research group from Malmö, Sweden, which demonstrated 5-HTR2B protein expression by endothelial cells in 29 ovarian cancer and 102 breast cancer samples , 22. In Preclinical research demonstrated that dopamine inhibits tumour angiogenesis via activation of DRD2. To our knowledge, this is the first study evaluating DRD2 expression on vasculature of human tumours. Previous studies demonstrated DRD2 protein expression on vessels of mouse ears and HUVEC . In our In conclusion, serotonin and dopamine receptors are differentially (over)expressed in various tumour types by tumour and endothelial cells. Of these, 5-HTR2B is expressed most frequently. This study demonstrates that selection of patients with tumours of different backgrounds but with similar receptor expression profiles is possible. This could offer interesting future possibilities for basket studies. Basket studies include different tumour types and select patients with the same tumour characteristic for targeted treatment . This alESM 1(DOCX 14 kb)


These data revealed that protamine/HAp (less than 1000 μg·mL−1) had both antimicrobial activity and biocompatibility and can be applied for bone substitutes in orthopedic fields.Bacterial infection of biomaterials is a serious problem in the field of medical devices. It is urgently necessary to develop new biomaterials with bactericidal activity. Antimicrobial peptides and proteins (AMPs), alternative antibacterial agents, are expected to overcome the bacterial resistance. The aim of this study was to develop a new intelligent material in bone tissue engineering based on protamine-loaded hydroxyapatite (protamine/HAp) that uses AMPs rather than antibiotics. It was found that the adsorption of protamine to HAp followed the Langmuir adsorption model and was due to electrostatic and/or hydrophobic interactions. In vitro bacterial adhesion and growth on protamine/HAp was inhibited in a protamine dose-dependent manner. Adherent bacteria exhibited an aberrant morphology for high dosages of protamine/HAp, resulting in the formation of large aggregates and disintegration of the membrane. The released protamine from protamine/HAp also prevented the growth of planktonic bacteria in vitro. However, a high dosage of protamine from powders at loading concentrations over 1000 μg·mL Implants have been used to improve loss of function, replace lost tissue, or optimize appearance. Various medical devices with different raw materials and shapes have been developed. Hydroxyapatite (HAp) is the principal inorganic constituent of hard tissues (bone and teeth) and has been widely applied in tissue engineering as an orthopedic and dental material due to its biocompatibility and osteoconductivity. Furthermore, HAp can form very tight bonds with living bone, resulting in the promotion of new bone formation. In many researches, HAp has been employed as bioactive coatings onto titanium  and polyStaphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., and Escherichia coli in surgical site infections (SSIs), which were defined as infections by organisms occurring up to 30 days after surgery [Causative pathogens are reported to be  surgery ,11. TherAntimicrobial peptides and proteins (AMPs) have attracted much attention as a promising alternative to antibiotics. AMPs are the host defense peptides and are key elements of innate immunity . TargetiProtamine, which is an arginine-rich protein extracted from fish milt, is a promising potential AMP. Protamine is a cationic AMP that inhibits or kills many microorganisms, including bacteria and fungi. The electrostatic interaction between (positively charged) protamine and (negatively charged) bacterial membrane is suggested to be the driving force for the expression of the protamine bactericidal activity. However, the precise mechanism of action of protamine at the molecular level remains unclear. Additionally, protamine, biomedical application, has been focused in the field of tissue engineering and regenerative medicine. Basic protamine molecules complexed with acidic molecules such as heparin, flagmin, and DNA form complexes via ionic interaction. Nano/microparticles were generated by electrostatic interaction and can function as immobilizers or attractants of various cytokines. Those nano/microparticles have been applied as biomaterials such as cell carriers, protein carriers, and growth factor carriers ,18.In this study, to develop a new bone substitute with antimicrobial activity, protamine was applied to biomaterials. We evaluated the bactericidal properties of protamine-loaded HAp (protamine/HAp) and demonstrated its suitability for use in bone tissue engineering.−1 protamine. The adsorption of protamine to HAp followed the Langmuir adsorption isotherm.To prepare the protamine/HAp powders, HAp powders were mixed with various concentrations of a protamine solution for 48 h and were adsorbed by the batch method. The protamine adsorption to HAp powders increased with increasing the concentration of protamine in a dose-dependent manner  and reacAn examination of the XRD patterns of each protamine/HAp powder showed no obvious changes due to protamine adsorption with only HAp single phase peaks observed (data not shown).−1 protamine. As shown in Next, to investigate the properties of the protamine/HAp powders, the zeta potential was measured. The zeta potential of the protamine/HAp powders also increased with increasing the concentration of protamine a and reaEscherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were seeded on different types of protamine/HAp discs and cultured in Luria-Broth (LB) medium for 24 h. At 24 h after plating, the ability of protamine/HAp discs to affect the viability of E. coli and S. aureus was tested using the BacLight LIVE/DEAD staining kit (E. coli and S. aureus on the HAp disc showed that the surface of the disc was covered by adhered living bacteria (green), and these bacteria started the formation of a biofilm  on the protamine/HAp discs was observed  appeared as normal rod-like shapes (E. coli on the protamine/HAp discs (doses of 250 and 500 μg·mL−1) showed elongated shapes (−1) caused the formation of large aggregates  induced cell aggregation and led to an increase in the number of bacteria. By contrast, a high dosage of protamine (over 500 μg·mL−1) induced cell death. These data indicate that the bactericidal ability of protamine/HAp discs was mainly due to the release of protamine from HAp. Interestingly, the release of protamine from discs only occurred in the ion-rich media such as the LB medium and simulated body fluid. Analysis of the ion concentration in the LB medium showed that the concentration of sodium ions in LB decreased due to the release of protamine (data not shown). These results suggest that the adsorbed protamine was released through ion exchange.Next, to clarify the mechanism of antimicrobial properties of protamine/HAp on E. coli, the concentration of the protamine released into the LB medium was measured. It was found that the concentration of protamine in the LB that was released from the discs increased with increasing amounts of adsorbed protamine  for 24 and 72 h. As shown in −1. However, the viability of the cell culture exposed to a high dosage of protamine/HAp (over 1000 μg·mL−1) induced cell death. These data indicated that released protamine from protamine/HAp powders (over 1000 μg·mL−1) was toxic for osteoblasts. On the other hand, extracts at dilutions of 50% and 25% (50 and 25 μg·mL−1) did not negatively affect the viability at 24 and 72 h (data not shown).Generally, antibacterial ability conflicts with biocompatibility. Therefore, to evaluate the biocompatibility of protamine/HAp, in vitro cytotoxicity tests were carried out according to ISO 10993-5:2009 “Tests for Cytotoxicity—In vitro Methods” [−1) in vivo, the specimens  were implanted into the tibiae of Japanese rabbits for eight weeks (−1) were both osteoconductive and biocompatible in vitro and in vivo.Furthermore, to evaluate the biocompatibility of the protamine/HAp . For the intermediate levels of protamine (500–1000 μg·mL−1), we observed elongated E. coli that could not divide  that was higher than the saturated adsorption level. This phenomenon was also observed in our previous study [As for the release profile of protamine, the amount of the released protamine increased with the increase in the adsorbed protamine concentration . Protamius study . These r−1) were dead due to the disruption of the membrane by the attached protamine. Additionally, we observed the differences of susceptibility to protamine between E. coli and S. aureus, which might be caused by the differences of the bacterial membrane structures. In the present study, S. aureus were more susceptible to protamine, and a low concentration of protamine (100 μg·mL−1) could disrupt the membrane, leading to cell death. In contrast to S. aureus, protamine would be able to past the cell wall without causing damage and bind to DNA in E. coli [E. coli changed their morphology without achieving cell division. However, the precise mechanism of action of protamine remains unknown, and further studies are needed to clarify other mechanisms.As previously reported ,14,28, A E. coli . The inh−1 [With regards to the biocompatibility of AMPs, it was previously found that AMPs can selectively bind to the outer surface of the negatively charged bacterial cell membranes without interacting with the outer surface of the neutral eukaryotic cell membranes . In our −1 . Therefo−1) were diluted by phosphate-buffered saline (PBS). HAp powder  were added into each protamine solution (45 mL) and incubated for 48 h at room temperature. The samples were centrifuged for 15 min at 8.000 rpm, and the supernatant was assayed for the protein using the Bio-Rad  protein assay following the manufacturer’s instructions. Powders were washed in PBS 5 times and freeze-dried. To fabricate the discs , the resulting powders (0.30 g) were uniaxially compressed at 50 MPa.Various concentrations of protamine sulfate solutions  in a 10 mM NaCl solution at room temperature. The zeta potential was calculated from the measured electrophoretic mobility. The complete experiment was repeated for a total of three separate assays. The morphological observation of the sample powders was performed at an accelerating voltage of 15 kV by scanning electron microscopy .E. coli, ATCC 27325) and Staphylococcus aureus  were used in this study. Bacteria were grown in a culture broth  and culture agar  at 37 °C in an incubator.To evaluate the antibacterial activity, Escherichia coli  was solubilized in PBS to a final concentration of 100 mg∙mL−1 and sterilized by filter with a 0.22-μm pore size. Protamine solutions then were diluted to yield final concentrations of 0.5, 1.0, 2.0, and 4.0 mg mL−1. Dilutions were dispensed (0.02 mL/well) in the wells of a polypropylene microtiter plate already containing 0.16 mL/well of the prepared 1 × 106 cfu mL−1 bacteria inoculum and 0.02 mL/well of Alamar blue . Final protamine concentrations of 50, 100, 200, and 400 μg∙mL−1 were tested. Each concentration was tested in triplicate. Samples were incubated for 18 h at 37 °C without shaking. The bacterial growth in suspension was monitored at 570 nm and 600 nm. The relative cell viability (%) was calculated by a ratio of optical density (OD)sample/ODcontrol × 100 for each of the values. The experiment was repeated for a total of three separate assays.Using the microtiter broth dilution method , the ant−1 using 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution (pH 7.4), containing 150 mM NaCl. Protamine stock dilution (0.1 mL) was mixed with 0.9 mL of the bacterial suspension. The bacterial suspensions were dispensed into microtubes and incubated for 15 min at room temperature. The zeta potential was calculated from the electrophoretic mobility. The complete experiment was repeated for a total of three separate assays.The zeta potential analyses were carried out using an ELSZ-1000  at room temperature. Protamine solutions were prepared at the concentrations ranging from 50 to 400 μg·mL® 9 and propidium iodide (PI) were mixed in microtube and then the dye mixture solution was added to PBS. Following resuspension of each sample in 1 mL of the staining solution, samples (bacteria on the discs or in the supernatants) were incubated for 15 min in the dark at room temperature. After washing with PBS, samples then were imaged using fluorescence microscopy . Each concentration was tested in triplicate.Bacteria cultured on various concentrations of protamine/HAp discs were examined for cell viability using a BacLight LIVE/DEAD staining kit . Equal volumes of SYTO8 CFU·mL−1).Optical density (OD), measured in a spectrophotometer , can be used as a measure of the concentration of bacteria in a suspension. Bacterial concentrations were determined by measuring the OD at 600 nm. The OD values of the inoculum were standardized to OD = 1.0 at 600 nm , which were filled with bacterial suspension at 37 °C. After 24 h culture, concentrations of protamine released from discs were then measured using the Bio-Rad protein assay.® CRL-1427TM . MG-63 cells were cultured in Eagle’s minimum essential medium  supplemented with 10% heat-inactivated fetal bovine serum  and 0.1% antibiotics  in a humidified atmosphere containing 5% CO2 at 37 °C. According to ISO 10993-5:2009 “Tests for Cytotoxicity—In vitro Methods” [−1 of the culture medium for 24 h at 37 °C). For cytotoxicity test, cells were seeded at 5 × 103 cells·well–1 on 96-well plates and cultured for 24 h at 37 °C/5% CO2. Each extract (0.1 mL) was then applied to cultured cells. After 24 h and 72h of incubation, culture medium was replaced with a fresh medium. Subsequently, 5 mg·mL−1 of MTT solution (10 μL) was added, and cells were cultured for 4 h at 37 °C/5% CO2. The MTT solution was then removed, and formed formazan crystals were solubilized by DMSO (100 μL) per well. After that, the absorbances at 570 nm (measurement wavelength) and 650 nm (reference wavelength) of each well were quantified by a microplate reader . The relative cell viability (%) was calculated by a ratio of ODsample/ODcontrol (HAp at 24 h) × 100 for each of the values.Human osteoblasts, MG-63 cells, were obtained from ATCCTo assess the biocompatibility and osteoconductivity of protamine/HAp discs in vivo, 16-week-old male rabbits (average weight: 3 kg) were used in this study. The specimens  were sterilized by ethylene oxide gas. The tibia of a rabbit was exposed, and cylindrical defects (4.2 mm in diameter) were drilled in the epiphysis of the tibia. The HAp or protamine/HAp discs were then implanted into the defect for 8 weeks. After implantation, the rabbit was sacrificed using sodium pentobarbital, and the tibia was removed. For undecalcified histological analysis, harvested implants were fixed 70% ethanol, dehydrated in an alcohol series, defatted, embedded in methylmethacrylate, and cut into 7 μm sections using a microtome. After staining with Villanueva bone stain, these specimens were observed under light microscopy . All experiments were approved by the Keio University Institutional Animal Care and Use Committee (09067-(10)).We developed a novel biomaterial with bactericidal coatings using protamine in this study. Protamine, a cationic peptide, is adsorbed to HAp through electrostatic and/or hydrophobic attraction. Both the protamine immobilized on HAP and the protamine released from HAp inhibited the adhesion and early growth at the material’s surface. These protamines are likely to disintegrate the bacterial membrane and bind to DNA, resulting in the inhibition of DNA synthesis. The findings reported in the present study suggest that protamine can be effectively used for antimicrobial materials. Protamine-loaded calcium phosphates such as HAp and tricalcium phosphate are believed to have great potential for use as antimicrobial biomaterials in bone tissue engineering.