The pursuit of mimicking complex multiscale systems has been a tireless effort with many successes but a daunting task ahead. A new perspective to engineer complex cross-linked meshes and branched/tree-like structures at different scales is presented here. Control over Saffman-Taylor instability which otherwise randomly rearranges viscous fluid in a ‘lifted Hele-Shaw cell’ is proposed for the same. The proposed control employs multiple-ports or source-holes in this cell, to spontaneously shape a stretched fluid film into a network of well defined webs/meshes and ordered multiscale tree-like patterns. Use of multiple ports enables exercising strong control to fabricate such structures, in a robust and repeated fashion, which otherwise are completely non-characteristic to viscous fingering process. The proposed technique is capable of fabricating spontaneously families of wide variety of structures over micro and very large scale in a period of few seconds. Thus the proposed method forms a solid foundation to new pathways for engineering multiscale structures for several scientific applications including efficient gas exchange, heat transport, tissue engineering, organ-on-chip, and so on. Proposal of multi-port Hele-Shaw cell also opens new avenues for investigation of complex multiple finger interactions resulting in interesting fluid patterns. Tree-like structure with integrated source and sink segments have been demonstrated to be mechanically strong and at the same time energy efficient in flow and mass transport than any other design configuration14. Vascular systems also exhibit tree-like patterns at higher scales with extremely branched constructs that transform them into a web of interconnected micro capillaries16. These micro-capillaries are less in the form of tree-like and more as complex web of interconnected networks. In vitro models of such micro networks are of tremendous help to the field of artificial organ growth, response of tissues to various in-development treatment drug, curing diseases, cell sorting and many other tissue engineering applications17.Plentiful existence of branched tree-like patterns in nature is an outcome of natural iterative process, over the millennia, towards the perfection of such designs. These branched structures therefore have been of interest to be used for various applications. Some examples include, microfluidic mixers and networks18, well-defined engineered micro arrays/channel-networks20 have shown momentous advancement in bio-applications23. Fabrication techniques presented in the literature are limited either in scalability23 or in time efficiency or controllability24. We propose a non-conventional, spontaneous, time-efficient, lithography-less method of shaping fluid into well-defined meshes and tree-like structures with source and sink segments by exercising control over Saffman-Taylor instability in a lifted Hele-Shaw cell. In Hele-Shaw cell25 a thin viscous fluid film, sandwitched between two parallel plates, when displaced by a less viscous fluid  develops Saffman-Taylor fingering instability26. In a “lifted Hele-Shaw cell” a variant of the same interface instability is induced by lifting apart the cell plates, sucking-in low viscous air as long air-columns/fingers from its peripheries27. Lifting of cell plates, done either angularly28 or parallely30, leaves a random and normally short-lived network of high-viscous fluid branches on both the cell plates. The pattern layout could substantially be altered by micro-patterning anisotropies on the cell slides33. We recently proposed use of yield-stress fluids in a lifted Hele-Shaw cell to retain the structures permanently34 and also the use of pits as anisotropies to control the Saffman-Taylor instability for shaping/fabricating fractal-like structures31.Among many techniques of fabricating such complex capillary-networks31 demonstrated controlled shaping of fluid into branched patterns using control over fluid-air interface offered by blind pits that cross the interface. Additional control was exercised by manipulating depth of these pits. In contrast, in this paper, we use fundamentally different scientific principle of using multiple source-holes on the cell plate (forming what we term as multiport Hele-Shaw cell) inside stretched fluid interface. Thus the proposed control in this case is exercised by relative placement of these source-holes as against the limited control offered by depth of pits only at boundary in the previous case. Controlled branched patterns obtained in this paper, hence show distinct difference in terms of connected branch ends as against free branch ends in our previous work31. For example, we demonstrate in this paper shaping of fluid into ordered mesh patterns at two different scales and a replica of leaf venation. Thus, the proposed philosophy of using source-holes establishes solid foundation for shaping fluid into families of desired patterns at multiple scales. The shaping/fabrication process, irrespective of the structure scale, completes in a period of few seconds and the structures thus obtained are solidified followed by casting them into hollow channel-networks using an elastomer or a hydrogel as per the application requirement.In this paper, we first characterize the effect of a single hole (termed as source-hole) in lifted Hele-Shaw cell. We further propose to use multiple such sources-holes/ports as controlling units, for the first time to the best of our knowledge, to shape fluid in the desired patterns mentioned above. Our previous workShaping of fluid into desired structures involve exercising control over highly noise sensitive viscous fingering process in a Hele-Shaw cell. Section-1.1 introduces fundamental concepts behind the proposed shaping of fluid by controlling viscous fingering and identifies process parameters. Section-1.2 discusses development of recipe for the desired shaping by using the results of characterization of process parameters. Structure fabrication/shaping, based on the control recipe thus evolved, is demonstrated in Section 1.3. Discussion in Section-2 summarizes the work, followed by methods in Section-3 explaining materials and experimental procedures used.35, is what patterns the fluid into a highly disordered/random structure, graphically shown in Fig. 36 whereas their size in length and width continues to increase37.Random pattern evolution in a lifted Hele-Shaw cell Fig.  is a twoσ and viscosity τ0 is a function of lifting velocity V and cell parameters bo and R. In what follow in Section 1.2, a modified non-dimensional number involving these parameters is proposed to carry out the characterization of finger splitting event. Although the pattern evolution process is explained here for uniformly spaced port arrays, it can be seen easily that the concepts can be extended to predict pattern evolution with non-uniform arrays of ports. Note here that because of multiple ports pressure inside film gets redistributed as against parabolic increase, from periphery towards cell center, in a cell without ports.We propose shaping of fluid using multiple-ports or source-holes in lifted Hele Shaw cell (LHSC) now termed as multiport lifted Hele Shaw cell (MLHSC). Introduction of multiple-ports/source-holes in a cell plate allows the controlled initiation of air fingers into the stretched film at many desired points. Such initiation of fingers is explored to completely transform the process for shaping fluid into well-defined mesh patterns as against random branched patterns formed in lifted Hele-Shaw cell. For obtaining well-defined mesh patterns, for example, source-holes are placed at the centroid of each cell-unit or each square as schematically shown in Fig. Rn) from the film center. Non-uniform interaction implies the unequal growth of fingers for shaping the fluid into an ordered tree-like pattern. Controlled non-uniform interaction via initiation-manipulation is again possible only for fingers which propagate while maintaining their stability i.e., which do not undergo tip-splitting mechanism27. To ensure this the lifting velocity, is always maintained below a threshold above which random tip-splitting events are observed.As another example to demonstrated effectiveness of proposed control method, we consider shaping of fluid into tree-like patterns. For controlled shaping of fluid into tree-like pattern source-holes are arranged along the circumferential region, just inside the stretched film interface as shown in Fig. μV/σ against cell parameters b0/R and a curve is fitted to separate region with unstable evolution from that with stable. Validation of fitted curve is confirmed by studying the destabilization for two fluids (Fluid 1 and Fluid 2) with a contrasting difference in σ and τ0. The fluids used are shear-thinning fluids which follow Herschel-Bulkley model  is very very low (<1 mm/s) as shown in Supplementary Fig. V  the number of branches formed (tip-splitting events) is less frequent and decreases as b0 increases. For evolution without destabilization the fluid gets collected into a circular ring as can be seen in the last image of each row in Fig. V) it is found that both the outer and inner interface stabilizes at lesser b0. All the arrayed patterns could be fabricated with interface-stability dependable parameters laying on or to the right of curve in Fig. Towards development of recipe for shaping fluid into desired arrayed structures, parameters influencing the process are identified and characterized first. As mentioned in Section 1.1 fabrication involves uniform interaction of fingers with no destabilization, the characterization of parametric dependencies of the finger destabilization process is thus presented in Fig. Given arrayed pattern, determine the centroids of each unit cell as locations for source-hole anisotropies.For multiport Hele Shaw cell with anisotropies determined in step 1 use parameters in stable region of Fig. Having mentioned characterization results and fundamental concepts in proposed control, the following simple recipe is proposed for fabrication of arrayed structures:35 demonstrated shielding of a finger by using numerically advanced initial positions to fingers adjacent to it. Further widening of these side fingers in a continuously decreasing area of spread, while progressing towards the center, completely stops/shields the central fingers to a certain length. Inspired by this and considering simultaneous finger initiation (as mentioned earlier) from all ports in our case, we propose relative placement of ports to affect shielding process. Specifically, we place ports for initiation of two outer fingers at a smaller radius R0 relative to radius R1 where port for initiation of central finger is placed  between two adjacent source-holes for different 35 the normalized finger length r1/R follow a linear relation against θ given by r1/R = Cθ + I, where C is slope and I the intercept. Graph in Fig. δ1, the value of C remains constant (=1.74) where as I decreases linearly. With r1/R showing a reliance on both θ and δ1 (represented by changing I), its variation against δ1 is separately plotted in Supplementary Fig. rn is measured as a distance traveled by that finger beyond the circumferential-line passing through source-holes placed at R0 as shown in Figs For shaping of fluid into desired tree-like pattern, as shown schematically in Fig. see Fig.  against δ1 can further be used to conceive recipe for several desired tree-like patterns. As an example we consider a Cayley-tree type structure38 with co-ordination number 3 and upto three generations as shown in Fig. R3 so that fingers initiate from them travel smallest length r1. Source-hole 3 and 7, which give rise to 2nd generation fingers, travel a distance of r2 while shielding all 1st generation fingers are thus placed at R2 such that R2 < R3. Finger from source-hole 5 terminates at r3 before shielding 1st and 2nd generation fingers along its way thus setting a position for source-hole 5 at R1 such that R1 < R2. Finally finger 1 and 9 have to be ahead of all other fingers so that they can supersede all other fingers before shielding finger 5 at r3 and are therefore places at a minimum distance of R0. The recipe can be generalized as: In order to obtain an nth order Cayley tree with coordination number 3, source-holes generating fingers The characterization of single mm and 900 μm and Cayley tree structures (with co-ordination number 3) upto three generations, the control strategy can be extended to fabricate even higher generations and over much larger areas. Multiport Hele-Shaw cell is used to fabricate mesh’s with different linkage configurations viz. square, triangular and hexagonal. Micro structures with branch dimensions in micro domain and on other hand same structures with much bigger branch dimensions were considered for fabrication. To fabricate a structure, say square mesh shown schematically in Fig. μm where as in macro structures  is 8 and 0.5 mm respectively. The proposed method can not only fabricate arrayed symmetric structures  and all the 9 source-holes form Fig. Similar to the first generation -holes 1– or 5–9 aThis paper presented simple yet elegant control over Saffman-Taylor instability leading towards development of time and cost effective recipes that shape viscous fluids into patterns widely observed in the nature at multiple scales. Specifically two distinct classes of patterns were demonstrated: a mesh patterns with desired layouts and an ordered branched pattern with smoothly varying branch thicknesses  in the present paper, investigation using proposed ideas with different fluids (for example viscoelastic) can further open up interesting fluid dynamics and applications.A setups to work as parallely lifted Hele-Shaw cell, shown in Supplementry Fig. A fluid with a small yield stress property is prepared from a monomer HDDA  added with alumina , 4wt.% Benzoin ethyl ether  and 2.5 wt.% Phosphate ester . Alumina powder is incrementally added while the ceria stabilized zirconia balls were used to homogenize the fluid on a pot mill. The prepared fluid is a photo-resist ceramic suspension with viscosity, surface tension, and yield stress values dependent on the volume fraction of alumina added. Modular compact rheometer (Anton paar Physica MCR301) and Wilhelmy plate method was used to measure the fluid viscosity-profile and surface tension, respectively.mm for all the experiments.Using a CNC-micro drilling machine source-holes were drilled on one of the cell plate for fabricating the structures. Diameter of source-holes in the present study is kept equal to 0.5 b0) and radius (R). Source-holes, machined using the CNC machine, are sealed in advance form one side to prevent the penetration of fluid into them while squeezing the fluid. Before the separation process begins the sealing is removed to let the source-holes act as sources of low-viscous fluid. For tree-like pattern fabrication, compressed fluid film is allowed to spread radially beyond the source-holes by a maximum distance of around 1.5 mm. A delay of 30 seconds is introduced to stabilize the fluid film, followed by plate separation at constant velocity of 4 μm/s. Constant separation velocities in mesh structure fabrication case varies from 2.5 to 12.5 μm/s for different experiments. Complete experimental process is recorded by a camera (18 MP NIKON S9400) at 30 fps. Structure features like the finger lengths A measured quantity of the fluid at the center cell plate is first compressed to required thickness (Supplementary MovieSupplementary File


The design of a patient-specific virtual tumour is an important step towards Personalized Medicine. However this requires to capture the description of many key events of tumour development, including angiogenesis, matrix remodelling, hypoxia, and cell state heterogeneity that will all influence the tumour growth kinetics and degree of tumour invasiveness. To that end, an integrated hybrid and multiscale approach has been developed based on data acquired on a preclinical mouse model as a proof of concept. Fluorescence imaging is exploited to build case-specific virtual tumours. Numerical simulations show that the virtual tumour matches the characteristics and spatiotemporal evolution of its real counterpart. We achieved this by combining image analysis and physiological modelling to accurately described the evolution of different tumour cases over a month. The development of such models is essential since a dedicated virtual tumour would be the perfect tool to identify the optimum therapeutic strategies that would make Personalized Medicine truly reachable and achievable. Precision Medicine Initiative (https://www.whitehouse.gov/precision-medicine) with cancer and diabetes as the main targets [ Personalized Medicine, is thus based on the identification of common characteristics in patient subpopulations. That means that treatment is adapted to specific subclasses but cannot be, per se, individualized as Personalized Medicine aims it to be [At the State of the Union address 2015, President Obama launched theit to be –6. Althoit to be . First, it to be , 9. Secoit to be –14.i.e., tumour size, shape, density, and vascular configuration) to be able to define, in a near future, truly personalized and optimized treatments.To that end, we developed a computational model that integrates key pathophysiological mechanisms to describe the growth of real tumours observed in a mouse pinna, so as to build an avatar or virtual tumour for each observed case. In order to build an accurate virtual clone, the experimental mouse model was chosen in a way to provide sufficiently detailed microscopic information on the tumour evolution and on its vascular environment. We chose to use immunodeficient nude mice to ensure the growth of the tumour and also in order to neglect the interplay with the immune system. This allows us to simplify the elements to integrate in the computational model in this first stage towards the development of a mouse-specific virtual tumour. As proof of the concept, the virtual tumour evolution should mimic its real life evolution. The growth of seven different tumour cases were virtually described with a good accuracy over a month. Moreover the model was able to capture a characteristic event in the experimental growth process corresponding to the well-known angiogenic switch  and thatnu mice were used. Mice were housed in ventilated cages with food and water ad libitum in a 12 h light/dark cycle at 26 ± 1°C. For in vivo imaging or injections, mice were anaesthetized using isoflurane (5% for induction and 2% during the experiment) in a 80% air and 20% O2 gas mixture.We used a mouse ear tumour model which consists in the injection of tumour cells in the dermis of mice pinna . This miμg/mL). The cells were kept in standard culture conditions . The culture medium was changed twice weekly.The U87-MG cell line (primary human glioma) was obtained from the American Type Culture Collection (ATCC HTB-14) where cell line authentication and species identification was conducted. The cells were transfected with the Green Fluorescence Protein (GFP). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated foetal bovine serum, 2% L-glutamine, penicillin (100 IU/mL), and streptomycin  in mouse ear dermis. During the whole injection procedure, cells and cells/matrigel solution were kept on ice. Prior to injection, anaesthesia was performed and ears were taped to a conical tube for easy injection of the tumour cells using a 26-gauge custom needle mounted on a RN-701 Hamilton syringe. Immediately after injection the presence of tumour cells was controlled by fluorescence microscopy.Tumour was generated by injecting a 2 The ear was gently placed in a custom-built ear holder and immobilized under a coverslip with ultrasonic gel in between the ear and the coverslip. Body temperature was monitored with a rectal probe and maintained at 36°C during the whole imaging session using a heating pad with feedback. Acquisitions were performed only when body temperature reached 36°C in order to avoid hypothermia and vascular constriction during anaesthesia.μL of a 20 mg/mL solution of Rhodamine B isothiocyanate-dextran (Sigma Aldrich) in the vein of the mouse tail on days 7 and 14.Tumour imaging was performed twice a week during one month using a Nikon AZ100 multizoom microscope , equipped with 1x (0.1 NA), 2x (0.2 NA), and 4x (0.4 NA) objectives. Fluorescence and bright field imaging was performed for each tumour  with NISImage analysis was performed using ImageJ (version 1.47). To monitor apparent tumour growth, a grey-level threshold was applied on the GFP-images. Yen's thresholding method was used  and manuX) and the tumour is identified as left (L) or right (R). The different tumours are thus coded MX-L/R (with X from 1 to 4).The results presented correspond to experiments made on 4 mice bearing two tumours, one on each ear. Each mouse (M) is assigned a number  level of oxygen is the proliferative state. If the level of oxygen decreases below a threshold and the cell is in an oxygen-sensitive phase, which is assumed to be restricted to the G1-phase of its cycle, then the cell becomes quiescent [A cellular automaton, under the form of a square grid, is used to describe tumour growth and the evolution of the tumour cells state. Full details are available in , 24. Trauiescent . It can uiescent .μ) of the cycle of the mother cell with standard deviation σ = 0.2 hours. Only the duration of the G1-phase is assumed to be altered since this phase is known to vary the most between cells coming from a same clone [The duration of the cycle of a cell after division (daughter cell) is assumed to be slightly different from this of the dividing cell (mother cell). It is determined using a truncated Gaussian distribution centred on the duration  where E0 corresponds to the normal density of capillaries which ensures a physiological ground level of oxygen. In the model, it can be locally degraded  by proteases produced by proliferating tumour cells [Lb = Δx, if the vessel is on the edge (border) of the element and i, j) the vessels weight Wi,j can be calculated to evaluate the contribution of the neovessels v in providing oxygen or in consuming growth factors:Based on the results from a previous study , the capur cells , 30. Shoed as in , 24, 31. via the proteases produced by the migrating and proliferating endothelial cells [It evolves when the tumour develops and can play an important role to stabilize the tumour or at the opposite to favour tumour invasion . Here weal cells .V) mainly produced by hypoxic tumour cells trigger vascular growth through angiogenesis and reciprocally the new vessels provide oxygen (O) that fuels the growth of the tumour through cell proliferation. The other diffusive molecules involved in the model are the proteases produced by the proliferating tumour cells (p) and by the migrating endothelial cells (m) that degrade the capillary network and extracellular matrix fibres, respectively. The equations that rule the spatiotemporal dynamics of the concentrations for all diffusive species {V, O, p, m} are given byDV, DO, Dp, and Dm are diffusion coefficients, αV, αp, and αm are production rates, and νV, νp, and νm are decay rates for the related species; λV is the consumption rate of growth factors by endothelial cells and by unit length of vessels; γv is the permeability coefficient of the vessels; Ov is the intravascular concentration of oxygen, taken as a constant; Vmax is the maximum uptake of growth factors when all cell receptors are saturated. The coefficient ki,j is the uptake rate of oxygen which depends on the cell state in element : if  ∈ P, ki,j = kP (proliferative cells), if  ∈ Q, ki,j = kQ (quiescent cells), and if  ∈ A or N (apoptotic or necrotic dead cells), ki,j = 0, else ki,j = k0  with kP ≥ k0 ≥ kQ. ni,j represents an angiogenic sprout  located on one edge of the grid element . The cells forming the sprouts have an intense proteolytic activity, via the production of proteases that degrade the matrix to ease cell migration. All the parameter values are given in Such growth factors and oxygen influence the relationship between tumour growth and vascular growth in a reciprocal way. Growth factors . The GFP fluorescence image is used to extract the tumour cells repartition and the information on cell density is captured by the level of fluorescence intensity. Three intensity thresholds for the fluorescence are applied successively on the original image  to diffeμm are segmented and their coordinates are transposed in the model. Very often, venules and arterioles are parallel to one another. Since we do not distinguish them in our computational framework, only one of the two is represented in that case. Only the vessels next to the tumour are taken into account. The segmented image is cropped and resized to the size of the computation grid (200 × 200 pixels) , they almost immediately exhaust the oxygen resource by consuming oxygen and by degrading the underlying capillary network field , 30, 34  that is, heterogeneous which allows us to differentiate dense regions corresponding to higher fluorescence intensity from low density regions with weaker signal intensity. With time, the image texture becomes smoother, which reveals that cell proliferation and movements homogenize the cells spatial distribution.Similarly in vivo, we observe that tumours become more compact with time. Compaction can be qualitatively assessed from the texture of the GFP fluorescence images  and the The changes in the texture on the tumour fluorescence images from day 3 to day 28 are quantified and characterized from the distribution curves of fluorescence intensity. To that end, the fluorescence intensity and the intensity range have been normalized to make the comparison possible since the intensity levels are different from one image to another depending on how the image has been tuned to avoid pixel saturation. The resulting curve corresponding to the previously presented tumour is displayed in In the meantime, the tumour size which is estimated from its apparent area is multiplied in average by 1.67 ± 0.36 in 28 days . The apparent area taken alone is however not sufficient to assess correctly the tumour growth in the two-dimensional plane, that is, the increase of the number of tumour cells in this plane. Correlation with the granularity is necessary to obtain a better estimation related to tumour cells density. A qualitative good match is attained between the real tumour and the virtual one based on three criteria: the shape, size, and texture.Comparisons of the real and virtual (simulated) tumour growth are realised from the evaluation of the tumour area at discrete time points. Experimentally the tumour area is measured from the fluorescence images and depends on the filter used to segment the image . For the virtual tumour, the area can be directly calculated from the number of cells since each cell occupies one element of the cellular automaton. This gives us the effective tumour area. However we also estimate the apparent tumour area which is obtained by delineating the tumour edge. The different curves for the evolution of the tumour area with time are plotted on a same graph in t = 0. The virtual tumour areas are smaller than the experimental ones. This is due to the strategy we employ to define the initial virtual tumour. Only the bulk of the tumour is taken into account for the estimation of the tumour area. The scattered cells are not taken into account  (k = 1 to 7) have been normalized by their integral value, that is, Experimental and simulated growth curves of all the tumour cases are presented in Figures  bottleneck that more accurately describes the observe phenomena by which slow growing tumours (slower than the average) have a higher angiogenic potential since they possess a higher proportion of hypoxic cells (producing growth factors), whereas faster growing tumours (faster than the average) produce a lower angiogenic response. As a result, the growth curves coincide at a specific time period due to a progressive and adapted angiogenic regulation which starts to develop after about a week and reaches its full capacity about 10 days later, leading to the emerging convergence of the normalized growth curves on day 17 (with the approximation of time sampling for image acquisition).This corresponds to the well-known angiogenic switch  that we i.e., hypoxic cells) in the simulated tumours is highlighted in The proportion of quiescent cells  tumour growth over this specific time period for all the tumour cases. We note that this phenomenon appeared in the simulations as an emerging property of the physiological model. It shows that the model, although simple, is able to catch a major characteristic of the experimental model. Specifically, the basic mechanisms for cell growth activation and inhibition through the regulation of the oxygen level and the mediation of growth factors are sufficient to reproduce the characteristics of tumour development in terms of shape, size, and density. The vascular structure of the microenvironment influences the cell shape and the tumour heterogeneity (active proliferating zones versus dormant quiescent zones) through its angiogenic potential stimulated by the tumour itself.In this study we developed a model for tumour growth and angiogenesis that has been applied to build up a virtual clone of a real tumour. The model successfully describes the development of seven different tumour cases over a period of about a month, without requiring any changes or adjustments in the model parameters from one tumour to another. This shows that the model with the default set of parameters , mostly d day 17 . It can Although successful in catching key aspects of reality, the physiological model should be further improved to become reliable on a longer time scale. First the computational model can be generalized in 3D to exploit the potential of two-photon imaging that gives access to a tissue depth of a few hundreds microns for the reconstruction of a large tumour volume. Second, cell compaction that has been overlooked in this version of the model can be easily integrated in the computational framework by allowing more than one cell in a grid element. The consequences of the increase mechanical pressure on the modulation of the cell proliferation rate  and on vi.e., by defining the drug dose, administration duration, and frequency). This is even more helpful in combined therapy protocols that may influence each other like the use of antivascular and cytotoxic drugs. This should help to identify the optimum therapeutic strategy for the real tumour. It is expected that such a personalized treatment, which takes into account all the tissue specificities of the patient (tumour shape and density and vascular configuration), would considerably increase its efficacy. Although precision medicine has been recently promoted and advertised through the rise of big data [With this study, we have been able to show that a biological object, as complex as a tumour, can be transposed into a virtual clone to predict its overall behaviour. But the main interest in detaining such a reliable tool is its potential to investigate and predict the effects of therapies. Its main object is to use it as a virtual substitute to test a panel of therapeutic protocols (big data –6, we rePersonalized Medicine is pursued as a major goal to fight cancer and requires the assistance of biologically based theoretical models. However deriving such highly informed and dedicated models is not easy. In this study we developed a virtual tumour based on basic mechanisms for cell growth activation and inhibition through the regulation of the oxygen level and the mediation of growth factors. Those mechanisms appeared to be sufficient to reproduce the characteristics of tumour development in terms of size and shape over a month. Moreover, the key angiogenic transition in the growth process was very well captured by the model, by spontaneously emerging in the simulations as a consequence of these simple physiological rules.Simulation of tumour growth over 28 days corresponding to the experimental cas M4-L displayed in figure 4.


We sought to test whether non-invasive evaluation of tissue stiffness is indicative of pathological tumour changes and can be used to monitor therapeutic efficacy. US-SWE was performed in tumour xenografts in 15 NCr nude immunodeficient mice, which were treated with either the cytotoxic drug, Irinotecan, or saline as control. Ten tumours were imaged 48 hours post-treatment and five tumours were imaged for up to five times after treatment. All tumours were harvested for histological analysis and comparison with elasticity measurements. Elastic (Young's) modulus prior to treatment was correlated with tumour volume . Irinotecan administration caused significant delay in the tumour growth (p = 0.02) when compared to control, but no significant difference in elastic modulus was detected. Histological analysis revealed a significant correlation between tumour necrosis and elastic modulus . SWE measurement provided complimentary information to other imaging modalities and could indicate potential changes in the mechanical properties of tumours, which in turn could be related to the stages of tumour development.The objective of this study was to evaluate the potential value of ultrasound (US) shear wave elastography (SWE) in assessing the relative change in elastic modulus in colorectal adenocarcinoma xenograft models  In addition, it has been used to detect treatment response to drugs in tumour models3 and in patients4, especially in oncology, as the change in the elastic ‘phenotype’ is considered a manifestation of mechanical processes determined on a cellular level such as desmoplastic stroma and inflammation which may precede macroscopic changes5.Elastography, the imaging of elastic properties of soft tissues, is a range of imaging techniques that provide useful information about various pathological processes such as cancer, fibrosis and atherosclerotic plaques. The importance of clinical elasticity imaging lies in its ability to provide complementary information to other imaging techniques. Elastography can detect lesions, invisible or barely visible by ultrasound (US) and Magnetic Resonance Imaging (MRI)6, mechanical force probing7 or Atomic Force Microscopy8 were the main methods to assess tissue elasticity in biopsy samples. However these methods, which cannot be performed in real time, require the acquisition of an invasive biopsy, and only account for a small part of the whole tissue (the biopsy site). This is clinically relevant given the high degree of heterogeneity in solid tumours. Removing tissue from its original environment might also cause alterations in its mechanical properties which affect the obtained results.Until recently, rheometryin vivo quantitative application has only recently emerged. The supersonic shear wave imaging (SSI) is a quantitative ultrasound imaging method that allows assessing the tissue elasticity in vivo9. Recently, Chamming’s et al. used this method to successfully assess the stiffness of tumours formed from human breast cancer cells implanted first into the murine fat pad10 and then subcutaneously11. Using MR elastography (MRE) Jugé et al. reported a correlation between stiffness, vessel density and cellularity of colon cancer tumours subcutaneously implanted in mice12. In another MRE study, Li et al. assessed the stiffness of tumours before and after treatment with a vascular disrupting agent3.Although ultrasound has always been a major tool for elastography application, an However, all these studies used anti-angiogenic or vascular disrupting drugs. In this pilot study our purpose was to evaluate the potential of SWE to monitor the changes in tumour elastic properties as a response to cytotoxic drugs, and correlate this with histological analysis to investigate the longitudinal dependence between necrosis and elastic modulus.13 and the ARRIVE guidelines14. All experimental protocols in the license were approved by the UK Home Office, and the individual protocols were prepared according to the licensed experimental protocols. 5 × 106 SW620 human colorectal adenocarcinoma cells  in 100 µl of serum free culture medium were injected subcutaneously into the right flank of 15 seven-week-old female NCr nude immunodeficient mice (Charles River Ltd. UK). After the tumour was palpable and measured 2 mm by 2 mm, its volume was measured by callipers and calculated using the ellipsoid volume formula, V = π/6 × L × W × H, where L, W and H correspond to length, width and height of the tumour respectively15. To estimate the error, each tumour measurement was performed eight times. The potential bias was removed by making the measurer blind to the records of the calliper.All procedures involving animals were performed in accordance with the local ethical review panel, the UK Home Office Animals (Scientific Procedures) Act 1986, the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer researchThe mice were scanned by a commercial US scanner with an integrated SWE measurement property, Aixplorer , every second day from day 10 after implantation, and were treated after the third scan.The design of experiments was such to determine the optimal analysis which could be performed thereby reducing and optimising the number and size of groups using observations from previously published work. Overall, the aim was to perform experiments where a measurable effect could be determined using a minimal number of animals. Using 80% power and 95% confidence, a predicted 25% practical difference and 15% coefficient of variation, group sizes of 5 animals are sufficient, where 1 tumour was implanted per animal.16. Mice in cohort I and cohort II were culled by cervical dislocation after the fourth scan, 48 h after the treatment. We hypothesized that drug-treated tumours would become more necrotic with time. Thus, to examine longitudinal dependence between necrosis and elastic modulus, mice in cohort III were left until their tumours reached ethical limits (six to ten days after drug treatment). Figure The mice were randomly divided into three cohorts. Cohort I (four mice) was treated with saline solution (control); cohort II (six mice) and cohort III (five mice) were treated intraperitoneally with a single dose 100 mg/kg) of the topoisomerase inhibitor Irinotecan00 mg/kg 2) for approximately 20 minutes. The US probe  was mounted on a 3D scanning motor to allow tumour positioning and eliminate artefacts during acquisition. The US probe was partially immersed in a nylon pocket water bath and degassed US gel  was applied to the scanning area for better coupling. Two B-mode and elasticity images, one in the transverse and one in the sagittal plane, at the centre of each tumour were obtained in each acquisition. A total of 144 ultrasound shear wave elastography measurements were performed. An example of an image is shown in Fig. During image acquisition, mice were placed on a table  heated to 37 °C to maintain animal core temperature under isoflurane anaesthesia  to allow free hand drawing of the region of interest (ROI) and therefore produced the result only within a chosen area. The program defines the colour scale, scans the image and converts it to kPa using colourbar. Mean values of elastic modulus and standard deviation were recorded. An ROI including the whole of each tumour and excluding skin was drawn, and the mean value of the elastic modulus from sagittal and transverse planes was calculated Fig. .Figure 317.Tumours were excised at the end of the experiments. The tumours were fixed in 10% formalin solution  for 24 hours and then into 70% alcohol solution where they were stored until being embedded into paraffin. The paraffin embedding was performed using a Leica EG 1160 embedder (Germany). Formalin fixed paraffin embedded tumours were sectioned using a microtome into 4 µm sections and stained with haematoxylin and eosin (H&E) for cellular visualisation, the endothelial cell marker CD31 and the connective tissue marker Masson’s trichrome. Stained sections were visualised using Cell^P  installed on an Olympus Bx51 microscope  with an attached ProScan II motorized scanning stage system . The percentage of stained area for CD31 and Masson’s trichrome included the whole area of the tumour was calculated using an in-house written Matlab script which detects the pre-defined colour in the image. With CD31 staining, the vascular areas appear in brown on a purple background, while collagen appears blue or green on the red-purple background. The script identifies brown (for CD31) or green (for Masson’s) pixels and gives their percentage. A surface area in viable regions was calculated from the H&E stained sections using ImageJTwo-way ANOVA was used to compare volume and elastic modulus changes between control and drug-treated groups. The p-values less than 5% were taken to be statistically significant. Pearson’s correlation was used to compare between tumour volume and tumour elasticity.First, the Pearson’s correlation between the tumour volume and the tumour elastic modulus for all 15 mice prior to drug treatment (i.e. after and including the third scan) was calculated and a moderate positive correlation between elastic modulus and tumour volume  was detected. One mouse from a second cohort developed a large scab and a hole in a tumour by the fourth scan and therefore was withdrawn from further results.2, E2) and last pre-treatment data  was calculated , no significant difference was observed in the change of the elastic modulus suggesting volume is independent of elastic modulus in this model . The elastic modulus did not change in the control group; however in drug-treated mice we observed approximately 25% reduction in elastic modulus, which whilst not statistically significant, highlights a response to the drug. Table Next, the changes in tumour volume (V) and elastic modulus (E) after treatment were assessed. Here cohort II and cohort III were considered together as one drug-treated group since all conditions were identical up to this point. Figure ted Fig. . Despitent data V, E1 was Irinotecan is a topoisomerase I inhibitor which induces DNA damage and leads to cytotoxicity with ensuing apoptosis leading to necrosis within tumours. Given the observed decrease in elasticity we detected using SWE, along with the moderate increase in tumour volume, we reasoned that Irinotecan may be causing tumour necrosis leading to the decrease in tumour elasticity. A significant negative correlation between the degree of necrosis (percentage of tumour) and elastic modulus was observed when the respective data from cohort I and cohort II were analysed . A greater negative correlation  was also observed in cohort III at the later time point indicating that as necrosis increases following Irinotecan treatment, tumour elasticity also decreases.Figure In addition, we found that cohort I had a 16% lower degree of necrosis  than it’s matched cohort (II) (p = 0.04).There was no statistically significant difference in vessel density within the viable areas of the tumour among the groups, suggesting that a loss of vessel density is unlikely to be the cause of tumour necrosis. However, in the control group the vessel density was found to be highly proportional to the tumour volume . Only a very small amount of collagen, less than 1%, was detected in the histological analysis, and the staining had inadequate contrast to permit automatic analysis in most of the sections.Figure 5. Elastography is therefore an important tool for tracking pathological processes.It has been noticed since the dawn of medicine that some diseases are associated with altered tissue stiffness. Among those are several types of cancer, such as breast and prostate, as well as fibrosis of the liver11, in the present model the variability appeared too high to robustly draw a conclusion. Prior to Irinotecan treatment, the elastic modulus of tumours increased in all mice concurrently with the tumour volume, which is in line with previously published results10, although in the aforementioned study a maximum diameter was used instead of tumour volume.In this study we have shown that US SWE can track changes in elastic modulus in response to cytotoxic therapy. However, we note that the changes in the values of elastic modulus between the drug-treated and the control group are small and did not reach significance in this study. While previous reports in different models with other drugs have observed detectable differenceset al., who used MRE to detect changes in elastic modulus during anti-vascular treatment of Balb-C mice with colon tumours12. Although in that work another cell line and another class of drug were used, the common outcome was that necrotic tumours exhibited a lower elastic modulus. This initial increase in elastic modulus is generally attributed to collagen deposition and further post-transitional modifications of extracellular matrix components18, along with rapid cellular expansion. In the present study, only a very small amount of collagen was detected. This is, again, in accordance with Jugé et al. who also observed a low collagen presence12.After the treatment, either with the drug or with saline, tumours continued to grow but the growth rate was slower in the Irinotecan treated group. With regard to the elasticity, the trend was reversed and in all cohorts a decrease in the elastic modulus was observed over time. This is most probably attributed the development of a necrotic core as the necrotic area was found to lie mostly in the centre of the tumour. Thus, while the outer part continued to grow, the core became necrotic. In control cohorts, the lower level of necrosis likely owed to the development of hypoxia (cohort I) indicating a baseline level of tumour necrosis which occurs in developing tumours. However, in Irinotecan treated mice, the increase in necrosis is likely a combination of hypoxia and drug-induced necrosis (cohorts II and III). The same pattern of behaviour was observed by Jugé In the present study, a statistically significant negative correlation between elastic modulus and necrosis was observed. We show that the percentage of necrosis was significantly higher in both drug-treated cohorts compared to control. This indicates that in addition to the developing hypoxia the drug used in this study causes additional cell death and a breakdown of tumour tissue leading to a decreased elasticity.11, but most have reported softening20. In this study, we also observed a decrease in elastic modulus, albeit small; and although we could not differentiate between drug-treated and control groups based on elasticity measurement alone, we could detect pathological changes, as increased necrosis correlated with tissue softening.Extant literature does not provide a definite answer to the direction of elastic modulus change upon drug treatment. Some studies have reported stiffening11 only circular ROI provided by the SSI software have been used, while our in-house written software allows free hand drawing of the ROI, thus calculating the elastic modulus only within a chosen area. This improves the accuracy of the calculation as we computed the elastic modulus within the rim of the tumour, regardless of its shape, not necessarily relying on the default circular ROI of the Aixplorer. With improved resolution images and using our method, it would be pertinent to look at elasticity in different regions of the tumour to study heterogeneity in elastic modulus.We employed an improved method of ROI calculation in the Aixplorer. In the previous studies5.We observed a decrease in vascularity, but without statistical significance, and the decrease in CD31 signal is probably related to a smaller viable area within drug-treated cohorts. Whilst our data do not indicate a direct correlation between vascularity and tumour stiffness, further studies would be required to interrogate this. Despite the lack of significant difference in vessel density, in the control cohort it is highly proportional to the tumour volume. This is not surprising, since larger tumours typically require more vascularizationet al. in their comparison of six commercially available elastography systems acquired ten repeated measurements21. To further study the changes in elastic modulus and necrosis one may add another control group that would be comparable to cohort III.For future studies a larger number of mice, with tumours excised at different time points, would have allowed a better insight into variation of elastic modulus over time. This is particularly important since measurements in some mice can be difficult owing to highly necrotic tumours, scabs or unresponsive tumours. Secondly, more than two repeated measurements per every scan result might be taken to provide more reliable data. Milkowski This study improves our understanding of the dynamics of tumourigenesis with regard to the tumour elasticity. Whether necrosis was caused by drug-treatment or by hypoxia it can be assessed non-invasively by measuring tumour elastic modulus. Elasticity measurements are only one of many available techniques for cancer identification and monitoring of drug efficacy; but as we show here, it provides complimentary information to biopsy and could increase the reliability and accuracy of the results.


The development of nanoscale molecular probes capable of diagnosis, characterization, and clinical treatment of disease is leading to a new generation of imaging technologies. Such probes are particularly relevant to inflammation, where the detection of subclinical, early disease states could facilitate speedier detection that could yield enhanced, tailored therapies. Nanoparticles offer robust platforms capable of sensitive detection, and early research has indicated their suitability for the detection of vascular activation and cellular recruitment at subclinical levels. This suggests that nanoparticle techniques may provide excellent biomarkers for the diagnosis and progression of inflammatory diseases with magnetic resonance imaging (MRI), fluorescent quantum dots (QDs), and surface enhanced Raman scattering (SERS) probes being just some of the new methodologies employed. Development of these techniques could lead to a range of sensitive probes capable of ultrasensitive, localized detection of inflammation. This article will discuss the merits of each approach, with a general overview to their applicability in inflammatory diseases.


The thoracic fluid content (TFC) and its percent change compared to the baseline (TFCd0%) derived from a bioreactance technique using a noninvasive cardiac output monitoring (NICOM) device correlate well with the amount of fluid removal in patients undergoing hemodialysis and with intraoperative fluid balance in pediatric patients undergoing cardiac surgery. We hypothesized that TFC or TFCd0% would also be a useful indicator allowing fluid management in pediatric patients undergoing a Fontan procedure.T0) and just before transfer of the patient from the operating room to the ICU (T1). The analyzed variables were hemodynamic parameters, TFC, TFCd0%, stroke volume variation, body weight gain, change in the central venous pressure, and difference in the TFC (ΔTFC). The medical records of patients who underwent an elective Fontan procedure were reviewed retrospectively. The intraoperative variables recorded at two time points were used in the analysis: when the NICOM data obtained just after anesthesia induction (p = 0.01); between TFCd0% and body weight gain% 0.572 (p = 0.007); and between TFCd0% and intraoperative fluid balance 0.554 (p = 0.009). The coefficient of determination derived from a linear regression analysis of TFCd0% versus body weight gain was 0.30 (p = 0.01); between TFCd0% and body weight gain% 0.33 (p = 0.007); and between TFCd0% and intraoperative fluid balance 0.31 (p = 0.009). The correlation coefficient between TFCd0% and body weight gain was 0.546 ( TFCd0% correlated well with body weight gain, body weight gain%, and intraoperative fluid balance. It is a useful indicator in the intraoperative fluid management of pediatric patients undergoing a Fontan procedure.KCT0002062. This trial is registered with Clinical Research Information Service  During pediatric cardiac surgery, an accurate determination of the patient's volume status, and the choice of an appropriate fluid management strategy are important factors leading to a stable hemodynamic status and thus a better clinical outcome. These considerations are even more important than in adult cardiac surgery, due to the potential for large fluid shifts compared with the pediatric patient's small body surface area –3. In adIn adult patients, volume status is traditionally evaluated by measuring the central venous pressure (CVP) or pulmonary capillary wedge pressure (PCWP) in the past. It is now widely demonstrated that cardiac filling pressures could not reliably predict fluid responsiveness. CVP also has been widely used to estimate the intravascular volume status of pediatric patients because of any other suitable alternative parameter for fluid status. However, as an indicator of exact volume status its use in this population is inappropriate, due to various factors that affect its accuracy, such as venous capacitance, cardiac chamber compliance, cardiac valve competence, and pulmonary artery pressure –7. MeasuIn pediatric patients undergoing a Fontan procedure, the exact volume status cannot be determined based on the CVP value, because the latter is actually the postoperative pulmonary arterial pressure following bidirectional cavopulmonary shunt (BCPS). The development of a good indicator of the volume status of pediatric patients will allow fluid management in those undergoing a Fontan procedure.The thoracic fluid content (TFC) and its percent change compared to the baseline (TFCd0%) are derived from the application of a bioreactance technique performed using a noninvasive cardiac output monitoring (NICOM) device. The values obtained with this method correlate well with the amount of fluid removal in patients undergoing hemodialysis  and withhttp://cris.nih.go.kr (KCT0002062). The medical records, including the anesthetic and operative records, of patients with a congenital functional single ventricle who underwent an elective Fontan procedure at Konkuk University Medical Center from April 2011 to December 2015, were reviewed retrospectively. Demographic, diagnostic, procedural, and hemodynamic information were obtained and analyzed.This study was approved by the Institutional Review Board of Konkuk University Medical Center, Seoul, South Korea , and subsequently registered at Anesthesia induction and maintenance were performed according to the institutional standard regimen for pediatric cardiac surgery: sufentanil-based anesthesia with sevoflurane. Anesthesia induction was followed by invasive arterial pressure monitoring at radial artery and central venous catheterization for CVP monitoring at internal jugular vein. Mean arterial pressure (MAP) and CVP were measured using pressure transducers , which were placed on the patient at the level of the mid-axillary line under the guidance of a laser leveler  and then fixed to the operation table. Two NICOM electrode strips (right and left) were then placed on each side of the chest of the patient and connected to a NICOM controller . Each electrode strip consisted of upper and lower contact points. The upper thoracic electrode strips were placed at the mid-subclavian region and the lower electrode strips in the middle region of the lower costal margin. After an initial calibration of the electrodes, hemodynamic variables, including cardiac output (CO), cardiac index (CI), stroke volume (SV), and stroke volume variation (SVV), were monitored continuously and recorded every 10 min until just before the patient was transported to the intensive care unit (ICU). After surgical procedure was over, the patients were transported to the ICU with intubation status.Ф) between input and output signals, which reflects changes in blood volume in the aorta. SV determined by NICOM can be estimated using SV = C · VET · dФ/dtmax, where C is a constant of proportionality, VET is ventricular ejection time, and dФ/dtmax is the peak rate of change of Ф [C has been optimised in prior studies, accounting for patient age, gender, and body size [The system's signal processing unit determines the relative phase shift , moving in the opposite direction to the water content. TFC provides a relative measure of changes in thoracic fluid. Specifically, higher TFC values compared with baseline indicate that the water content of the patient's chest is increased and vice versa. TFCd0% is the percentage change in TFC compared with the baseline TFC value at the measured point, and it changes with variations in TFC. Parameters measured by the NICOM device were average values during 1 min, with a 1 min interval between measurements.The water content of the body, whether in the blood (intravascular) or outside the blood (extravascular), contains high concentrations of various electrolytes, such as sodium, chloride, potassium, and calcium. These electrolytes are good conductors of electricity. The more fluid a patient has in his/her chest, the more electrolytes are available for electrical conductance and vice versa. Thoracic fluid content (TFC) is calculated as TFC = 1/T0) and just before transfer of the patient from the operating room to the ICU (T1). The analyzed variables were MAP (mmHg), heart rate , CVP (mmHg), CO (L/min), CI (L/min/m2), TFC (1/Ω), TFCd0% (%), and SVV (%). The body weight gain of the patient, the change in the CVP, and the difference in the TFC (ΔTFC) were calculated as the difference between the values at T1 and T0. The intraoperative fluid balance (intake/output) was estimated based on intraoperative fluid and blood product administration, urinary output, and the fluid balance during CPB.Among the intraoperative variables, those recorded at two time points were used in the analysis: the NICOM data obtained just after anesthesia induction  using the same method. The percent gain in body weight was calculated as follows: body weight gain% = 100 × (body weight at T1 − body weight at T0)/body weight at T0.The patient's body weight was measured twice, according to the standard protocol for pediatric cardiac surgery at our institution. After anesthesia induction with routine invasive  and noninvasive patient  monitoring, a central venous catheter and a urinary catheter were inserted and the electrode strips of the NICOM were attached. The patient's body weight was then measured using portable scales . This was the t-test or Wilcoxon's signed-rank test, to compare the values obtained at the two measurement points. Student's t-test or the Mann–Whitney rank sum test was used to compare values between groups. Categorical variables were analyzed using a χ2 test. Correlations between variables were determined using the Pearson product moment correlation analysis and linear regression analysis. The data are expressed as the mean ± standard deviation or as the median (25th to 75th percentile) and the number of patients. A p value < 0.05 was considered to indicate statistical significance.Statistical analyses were conducted using SigmaStat software . Continuous variables were analyzed using the paired From April 2011 to December 2015, data from the medical records of 22 patients were collected. One patient was excluded for incomplete medical records. The preoperative demographic profiles of the 21 patients included in the study are summarized in T1 versus T0, whereas HR, CVP, TFC, SVV, and body weight were significantly higher at T1 ; between TFCd0% and body weight gain% 0.572 (p = 0.007); and between TFCd0% and intraoperative fluid balance 0.554 (p = 0.009). The correlation coefficient between ΔTFC and body weight gain was 0.449 (p = 0.41); between ΔTFC and body weight gain% 0.479 (p = 0.028); and between ΔTFC and intraoperative fluid balance 0.518 (p = 0.016). The correlation coefficient between the change in the CVP value and body weight gain was 0.494 (p = 0.023); between the change in the CVP value and body weight gain% 0.510 (p = 0.018); and between the change in the CVP value and intraoperative fluid balance 0.482 (p = 0.027). The correlation coefficient between body weight gain and intraoperative fluid balance was 0.821 (p < 0.001).The correlation coefficient between TFCd0% and body weight gain was 0.546 (R2) derived from a linear regression analysis of TFCd0% versus body weight gain was 0.30 ; between TFCd0% and body weight gain% 0.33 ; and between TFCd0% and intraoperative fluid balance 0.31  ; between ΔTFC and body weight gain% 0.23 ; and between ΔTFC and intraoperative fluid balance 0.27  ; between the change in the CVP value and body weight gain% 0.26 ; and between the change in the CVP value and intraoperative fluid balance 0.23   (The coefficient of determination (= 0.009) . The R2 = 0.016) . The R2 = 0.027) . The R2 < 0.001) .In the present study, the values of the correlation coefficients derived from the correlation analyses were acceptable, with the strongest correlations between TFCd0% and body weight gain and between body weight gain% and intraoperative fluid balance. The coefficients of determination derived in the linear regression analysis were also acceptable, with the highest being those between TFCd0% and body weight gain and between body weight gain% and intraoperative fluid balance. Because the intraoperative fluid balance is almost the only factor that affects patients' body weight and the result derived from linear regression analysis between body weight gain and intraoperative fluid balance showed high correlation coefficient, these findings suggest that TFCd0% is an appropriate indicator for intraoperative fluid management in pediatric patients undergoing a Fontan procedure.In pediatric patients with congenital heart disease who are undergoing surgery, CVP is an inappropriate parameter for estimating the exact volume status. This can be explained by factors such as intracardiac shunt (left to right or right to left), altered pulmonary vascular resistance, altered left ventricular and right ventricular compliance, and the complex anatomy that often occurs in congenital heart disease . MoreoveT0 and T1, the values of HR, CVP, TFC, and SVV were higher at T1 than at T0. The higher HR and SVV values may have been related to postoperative conditions, such as those resulting from the use of inotropes, a lack of effective circulating blood volume, and the inflammatory reaction induced by CPB. The higher TFC and body weight may have been due to a positive intraoperative fluid balance. There were various possible reasons for the higher CVP at T1 than at T0. It may have been related to more blood volume crossing the pulmonary vascular bed, higher PVR from exposure to cardiopulmonary resistance, and elevated end diastolic pressure due to myocardial edema. However, it is not clear what was the definite reason because the methods for their appropriate measurement of pulmonary blood volume or PVR are lacking.In comparisons of the variables between Prior to undergoing a Fontan procedure, most patients have already been treated surgically and have a postoperative status of BCPS. This was the case in the present study, in which all of the patients had previously undergone a BCPS procedure. Therefore, the CVP value was instead a measurement of the pulmonary artery pressure (PAP), both before and after surgery. In the determination of PAP, pulmonary vascular resistance is the important factor; however, it is also affected by other factors and, thus, does not reflect the exact volume status of the patient.T1. This allowed an evaluation of the relationship between TFCd0% and other variables. Comparisons between the low and high TFCd0% groups with respect to body weight gain, intraoperative fluid balance, CVP, and the changes in the TFC showed that only the absolute value and the change in CVP did not differ between groups. All other variables were higher in the high TFCd0% group. This demonstrates that the absolute CVP value and the change in CVP do not reflect either the change in the patient's body weight or the intraoperative fluid balance. Instead, both can be determined using the TFCd0%, which may also be an appropriate indicator in intraoperative fluid management.The patients were divided into two groups based on the value of TFCd0% 30% at In a previous study, TFCd0% correlated well with intraoperative fluid balance and with body weight gain, based on correlation coefficients of ~0.7 . AlthougAmong the limitations of the present study was its retrospective design. A prospective study would be able to clarify the relationships between the variables. However, the present study has been performed as retrospective design. Although all the medical records were reviewed carefully, the limitations inherent in a retrospective study were inevitable. In addition, TFCd0% detects the changes in intra- and extravascular volume status based on the electrical conductance in the patient's chest, but it does not distinguish intravascular from extravascular volume change. However, when a large amount of fluid is administered to maintain a stable hemodynamic state, large fluid shift to the third space and tissue edema occurs more often than otherwise. In this case, since TFCd0% reflects the intraoperative fluid balance well, the measurement of TFCd0% can be useful to detect and prevent the side effects of large fluid administration. Thus, in addition to hemodynamic parameters and TFCd0%, the simultaneous use of the dynamic parameters derived from heart–lung interactions, indicative of fluid responsiveness, would improve intraoperative fluid management –17.In the clinical field, fluid balance is difficult to estimate accurately, due to conditions such as continuous surgical bleeding in the operating field, absorption of surgical bleeding by gauzes, and the insensible loss of body fluid. Furthermore, estimates based on the intake and output of intraoperative fluid are time-consuming. In pediatric cardiac surgery, patients require continuous monitoring for intraoperative fluid balance. The TFCd0%, which correlated well with body weight gain, body weight gain%, and intraoperative fluid balance, may be more useful indicator than the conventional methods for fluid management.In conclusion, TFCd0%, derived using a bioreactance technique, correlated well with body weight gain, body weight gain%, and intraoperative fluid balance. It is therefore a useful indicator in the intraoperative fluid management of pediatric patients undergoing a Fontan procedure.


Unfortunately, after publication of this article , it was AcknowledgementsWe are grateful for the technical support by Aruna Visavadiya, Ying Li, and Rhesa Dykes. Dr. Britta Engelhardt (Theodor Kocher institute) is thanked for providing the bEnd5 cells.FundingThis work was supported by NIH grant NS45734 and in part by NIH grant C06RR0306551 and the ETSU College of Medicine.


Introduction. Correct diagnosis of peritoneal infectious disease can be extremely difficult due to non-specific clinical features. Thus, careful assessment with thorough histopathological work-up is essential. Here, we report the first case of human peritoneal cysticercosis mimicking peritoneal carcinosis.Case presentation. The patient presented with recurring ascites and a tumour in the Douglas cavity accompanied by elevated tumour markers. There were no signs of systemic infection. On laparoscopy, the tumour was resected completely. Histology revealed a granulomatous reaction and a diagnosis suspicious of tuberculosis was made. Only after additional sections, avital cestode-fragments were visible and Taenia martis DNA was detected. Further staging by computerized tomography scan of the lung and brain turned out negative and the patient recovered quickly.Conclusion. Laparoscopy and histopathological examination can be extremely helpful for correct diagnosis and management in uncertain recurrent ascites. This case clearly demonstrates that orphan infectious diseases should also be considered. Only complete histopathological examination with serial sections and additional molecular testing can lead to the appropriate diagnosis. CRP, C-reactive protein; MRI, magnetic resonance imaging.Peritoneal infectious disease is difficult to diagnose. Elevated C-reactive protein (CRP), eosinophilia or signs of systemic infectious disease can lead to the correct diagnosis. However, often those signs are missing, thus delaying appropriate treatment.Taenia martis cystercercosis worldwide.In our case, not only were specific symptoms of an infectious disease, such as fever, night sweats or weight loss, missing, but also some observations such as recurrent ascites, a tumour in the Douglas cavity and elevated laboratory tumour markers prompted the diagnosis of peritoneal carcinosis. Only laparoscopy and complete histological work-up led to the diagnosis of peritoneal cysticercosis. Therefore, we want to draw attention to the importance of complete histopathological work-up with specific stains for a correct diagnosis. It is also important to broaden the spectrum of differential diagnosis of peritoneal (infectious) disease, as this is the first case of human peritoneal cysticercosis among the three reported cases of human A 36-year-old woman presented to her gynecologist with recurring, asymptomatic ascites, which progressed slightly over a period of 8 weeks. There were no signs of systemic infectious disease with normal CRP, normal white blood cell count and no evidence of eosinophilia. However, laboratory tumour marker Cancer Antigen 125 (CA125) was elevated at initial presentation with normal carcinoembryonic antigen (CEA), Cancer Antigen 19.9 (CA19.9) and Cancer Antigen 72.4 (CA72.4). Medical ultrasound of the abdomen did not reveal any further findings, but with magnetic resonance imaging (MRI) a tumour (1.6×1.6 cm) could be detected in the Douglas cavity.The patient wanted to become pregnant and was alarmed by the initially elevated laboratory tumour markers. Therefore, the decision for laparoscopy was made. Abdominal laparoscopy was performed, and a sharply demarcated tumour with a smooth surface was detected within the Douglas cavity at the posterior cervical wall. The tumour could be resected completely  and was On gross examination, a 1.6×1.6×1.6 cm, circumferential tumour with a white to yellow cut surface was visible.Mycobacterium turned out negative, and the case was sent to the national reference centre of haematopathology in Wuerzburg (Germany) to rule out a lymphoma with a granulomatous reaction. Serial sections were performed with almost complete sectioning of the tumour. Histopathology assessment revealed a tumour mass with central necrosis and aggregates of epitheloid cells with intermingled multinucleated giant cells of Langhans type in the periphery . Interestingly, ELISA for Echinococcus granulosus was positive – very likely due to antigen cross-reactivity within the taeniid cestode family.After detection of the degenerated larva, diagnosis of a helminthic parasite infestation was confirmed by microbiological analysis. Nucleic acid testing with pan-taeniid cestode-specific primers directed against 12S mitochondrial rDNA . There was no increase in eosinophilic granulocytes; however, to rule out a helminth infection, serial sections of the tumour were performed. Only after almost complete sectioning of the tumour an avital degenerated larva indicative of a helminthic parasite infestation could be detected in the central necrosis. Therefore, this case clearly demonstrates that only after step by step pathologic work-up the correct diagnosis could be rendered.A sharply demarcated tumour was resected and given for histopathological analysis. Granulomatous reaction with loose aggregates of epitheloid histiocytes with intermingled mature lymphocytes was found; however, no well-formed granulomas were detected. The differential diagnosis of granulomatous reaction is long, including tuberculosis, foreign body reaction, sarcoidosis, helmithic infection or lymphoma . No mycobacterial DNA could be identified, and the material was sent to the reference centre of haematopathology to rule out lymphoma. Lymphocytes were mature and composed of a mixed population of B- and T-lymphocytes with a predominance of T-lymphocytes, and no Hodgkin or Reed Sternberg cells were visible, so there was no indication of lymphoma. Foreign body material could not be detected, and no fungi-like T. martis (ENA accession number LT837855).Despite being instructive for proceeding with clinical and histopathological analysis and differential consideration of recurrent peritoneal fluid, this case turned out to be even more exciting. Microbiological nucleic acid testing with ubiquitous taeniid cestode primers revealed 99 % identity to T. martis is a tapeworm, which dwells as the adult stage in the small intestine of carnivores. Rodents serve as an intermediate host, in which T. martis cysticercus larva stages mainly develop in the peritoneal and pleural cavity. In Europe, T. martis has been observed in martens and rodents in Italy, Germany, The Netherlands, Belgium, Spain, Poland, Belarus and Switzerland [T. martis cysticercosis have been reported worldwide [T. martis cysticercosis has only been observed once in a non-human primate, in a Tonkean macaque in the Alsace region [T. martis larvae, thus limiting the risk of generalization and recurrence. Transmission to intermediate hosts occurs via the oral route by way of food or water contaminated by faecal matter. The former two cases involved patients who were recreational gardeners, which could be a potential risk factor. No history of gardening was reported by our patient, rather extensive recreational hiking in the Alps; however, without contact with wild animals. This indicates the possibility of contamination of the environment with eggs of this tapeworm, and it is highly probable that a natural T. martis life cycle existed in the environment of the patient's living place with small rodents and carnivores in close vicinity.tzerland . So far,orldwide . Cysticee region . All casT. martis should be added to the differential diagnosis of unusual peritoneal tumors.Even though the number of diagnosed cases is low, we cannot rule out the possibility that cases may have been missed in the past due to the absence of appropriate analysis. We therefore want to stress the importance of a thorough histopathological analysis with additional molecular analysis and strongly recommend that cysticercosis of


Currently, if recurrence occurs, then extraction of the teeth adjacent to the lesion is commonly performed, which may influence the aesthetics or masticatory function. Thus, a more effective and less aggressive treatment method is urgently demanded, particularly for the recurring lesion. This study investigated the effects of the intralesional pingyangmycin (PYM) injections for the recurrent granulomatous epulis and assessed the complications.Relapse of granulomatous epulis is common after surgery because of local irritations, hormonal level A total of 16 patients with recurrent granulomatous epulis underwent intralesional PYM injections, between July 2010 and June 2014. The effects and complications of the treatment were retrospectively reviewed.The total number of injections performed was 48 . The median number of injections per patient was three . All cases completely recovered with no recurrence and resorption of the alveolar bone after a follow-up of more than 12 months. The complications included slight bleeding, local swelling and pain following injection. All these symptoms resolved 7 to 10 days after the injection.In summary, intralesional PYM injections may be a preferred option for recurring granulomatous epulis.Key words:Granulomatous epulis, recurrence, pingyangmycin, sclerotherapy. Epulis is a clinically diagnostic term referring to a reactive focal connective tissue proliferation in the gingiva, and its exact histological nature is unknown. Epulis occurs at any gender and age, but is more common in female and young people . AlthougGranulomatous epulis, a smooth or lobulated exophytic lesion with a deep red or purplish colour , is alsoTherefore, more effective and less aggressive treatment methods should be discovered for the recurrent granulomatous epulis. In this study, the intralesional pingyangmycin  injection was used to treat the 16 patients with recurrent granulomatous epulis. All cases completely recovered without recurrence, which indicated that PYM sclerotherapy may be an additional treatment method for the recurrent granulomatous epulis.Between July 2010 and June 2014, 16 patients with recurrent granulomatous epulis were prospectively enrolled for the intralesional PYM solution injection treatment at the Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Wuhan University .2. Pregnant patients.3. Lesions that were definitely caused by foreign material.* Approach and techniqueAfter recurrence, all patients were recommended to receive intralesional PYM injection. All patients with follow-up of more than 12 months after the final treatment were included in this study.PYM has been described previously . After tAmong the 16 cases, 12 cases experienced one surgery, 4 cases underwent multiple surgeries, and 7 cases had tooth extraction. All of the lesions relapsed no more than one month after the surgery. The total number of injections performed was 48. The median number of injections per patient was three . The median duration of follow-up was 18 months . Overall, in this series, all of the patients completely recovered with no recurrence  and peripheral giant cell granuloma, based on the histological feature. However, other literature classified epulis into acanthomatous epulis, giant-cell epulis, ossifying epulis and periodontal fibromatous epulis. In 2008, the Dental Dictionary categorised epulis into congenital of newborn, epulis fissuratum, epulis-giant cell, and epulis granulomatosa. Furthermore, some categorizsation was also based on the age group. These different classifications result in the confusing diagnoses and treatments.Therefore, Chinese histopathologists and doctors  summarizGranulomatous epulis, which is also referred to as gingival pyogenic granuloma, is the most common disease among oral pyogenic granuloma . AlthougWith regard to the treatment, a comprehensive treatment is the strategy to cure granulomatous epulis, generally including aetiological treatment  and surgical procedures (excision and biopsy of the lesion). However, the argument about treatment focuses on removing the teeth involved by the lesion, some clinicians considering the high rate of recurrence suggest that removing the lesion and the involved teeth together , others A more effective and less aggressive therapy is exigently acquired to treat granulomatous epulis, particularly for recurring lesions. In the present cases, the intralesional PYM injection was used for the relapsed lesions. The exciting result indicated that PYM sclerotherapy may be an additional method for the granulomatous epulis.Streptomyces Pingyangensisn with lower pulmonary toxicity than BLM (PYM is BLM A5 made in China extracted from than BLM ,7, whichthan BLM . The cytthan BLM . Furtherthan BLM  and lympthan BLM  malformathan BLM  indicateGranulomatous epulis share similar histopathological and clinical features with infantile haemangiomas . This evBeside PYM, there are also some sclerosing agents which are used for granulomatous epulis or oral pyogenic granuloma, including absolute ethanol , sodium Taken together, intralesional PYM injection was safe and highly effective in the treatment of granulomatous epulis. In the present cases, the lesions were completely involuted by PYM with no recurrence and no tissue destruction, whilereas the teeth adjacent to the lesions were reserved. This exciting result indicates that PYM sclerotherapy may become a preferred treatment modality for relapsing granulomatous epulis.


To investigate the changes in mRNA expression levels of telomerase-related significant proteins in several types of cancer.Human telomerase reverse transcriptase, pontin, reptin and dyskerin expressions were measured in normal and tumour tissues obtained from 26 patients with colorectal, breast and gastric cancers, using the real-time reverse transcriptase-polymerase chain reaction method.For all patients, no significant difference was found in mRNA expressions of human telomerase reverse transcriptase and dyskerin (p>0.05), although their levels in tumour tissues were found to be higher than in normal tissues. However, pontin and reptin mRNA expressions were significantly higher in tumour tissues than in normal tissues (p<0.01). While human telomerase reverse transcriptase showed a high correlation with only pontin (p<0.001) in normal tissues, high positive correlations were observed between human telomerase reverse transcriptase with pontin (p<0.005), reptin (p<0.01) and dyskerin (p<0.01) in tumour tissues.The increased mRNA expressions of all four genes in tumour tissues may suggest a role in cancer development. Correlations of pontin, reptin and dyskerin with human telomerase reverse transcriptase support the hypotheses describing their roles in telomerase complexes. Telomerase enzyme activity is required for immortality of tumour cells and the carcinogenesis process. In human cancers, telomerase is upregulated where it serves tumour proliferation by helping in the protection of functional telomeres . The telPontin and reptin are the helicase ATPases associated with diverse cellular activities (AAA+). Pontin and reptin are known as RuvB-like protein 1 (Ruvbl1) and Ruvbl2, respectively . Pontin The aim of this study was to compare the mRNA expressions of hTERT, pontin, reptin and dyskerin in normal and tumour tissues, and to examine the relationships between pontin, reptin and dyskerin with hTERT.Thirteen patients with colon cancer, 7 patients with breast cancer and 6 patients with gastric cancer were included in this study between October 2009 and February 2011. Seventeen patients were female (aged 31-74 years) and 9 were male (aged 46-68 years). All the patients had recently been diagnosed using the biopsy method and had not previously received any medical treatment. The histopathological diagnosis was adenocarcinoma in all patients whether they had colorectal, gastric or breast tumours. Tumour and non-tumour tissue samples were obtained from patients during surgical resection of the tumour. Macroscopically, tumour-free margins were obtained during surgery and non-tumour tissue samples were collected from macroscopically normal areas in the stomach, colon, rectum and breast. Patients were classified according to the TNM system, developed by the American Joint Committee on Cancer . InformeTM RNA Stabilization Reagent  during surgical removal, frozen and stored at -80 °C until further use.Sufficient amounts of distant non-tumour tissues of the colon, breast and stomach were obtained from the patients, as control tissues. All tumour tissue samples and distant normal tissue samples were immediately placed in microtubes filled with RNA SaferFrozen tissue samples (approx. 100 mg) were defrosted and homogenized in a Tripure Isolation Reagent in a Roche MagNA Lyser instrument, and RNA phase was separated. The method for RNA isolation was applied according to the manufacturer's instructions. The RNA samples obtained were kept frozen at -80 °C, to be used later. cDNA was synthesized by real-time (RT)-PCR using a Roche Transcriptor High Fidelity cDNA Synthesis Kit.cDNA samples were amplified in RT-PCR with a Roche Light Cycler 480 Probes Master Kit using Taq DNA polymerase . This method was applied according to the manufacturer’s instructions and specific forward and reverse primers and probes were used for each gene amplification designed by the kit manufacturer. The hTERT, pontin, reptin and dyskerin expression profiles were determined human β-actin was amplified as an reference gene. mRNA expression measurements in tumour and normal tissues were repeated 3 times. Relative quantification (RQ) values were calculated using the formula shown below:T - CT] / 2[CT  - CT]RQ = 2[CStatistical analyses were performed using SPSS 22.0 software . The data were presented as means (standard deviation), and statistical analyses were performed using the paired-sample t-test and Wilcoxon signed-rank test. Pearson correlation coefficients were calculated to determine the relationship between the parameters. For all analyses, a p-value <0.05 was regarded as statistically significant.mRNA expressions of hTERT  and dyskIn all the patients, 53.8% (14/26) of the samples for hTERT, 73.1% (19/26) of those for pontin, 57.7% (15/26) of those for reptin and 53.8% (14/26) of those for dyskerin showed higher mRNA expression ratios (RQ>1) in tumour tissues than in normal tissues.Three genes, but not hTERT, were expressed in both normal and tumour tissues of all patients. Only ten of the patients showed both tumour and normal tissue expressions of hTERT. In the other 16 patients, 7 showed expression of hTERT in their tumour tissues, but not in normal tissues.For all patients, there were significantly positive correlations between hTERT, pontin and reptin, in both normal and tumour tissues . DyskeriIt has been shown that telomerase activity is determined frequently in tumour tissues but rarely in normal tissues . Most maAdditional factors are needed for full activity of the telomerase complex besides TERT and TERC. Dyskerin, which is one of them, directly binds to TERC and stabilizes it in the complex. Loss of the function of dyskerin decreases the steady-state levels of TERC, reduces telomerase activity and causes premature telomere shortening . MontanaPontin and reptin are eukaryotic AAA+ proteins and they participate in different pathways . The expIt is claimed that TERT complexes are dynamic and thus there are at least two different forms of TERT complexes. One of these forms is the pontin/reptin/TERT complex (pretelomerase complex), which has low telomerase catalytic activity. The other one is the TERT/TERC/dyskerin complex (mature telomerase complex), which has high enzymatic activity. In a step-by-step assembly model, a pretelomerase complex is formed, then reshaped to form a mature telomerase complex . So, altIn conclusion, this is the first study to compare hTERT, pontin, reptin and dyskerin expressions in normal and tumour tissues of the same patients. Mostly, increased mRNA expressions, in tumour tissues of all genes, show their role in cancer development. In addition, correlations of pontin, reptin and dyskerin with hTERT support the hypotheses describing their roles in the formation of the telomerase complex in cancer. Furthermore, due to the lack of expression of hTERT in some normal tissues, while being present in tumour tissues, it may be a potentially valuable diagnostic marker of cancer, especially of breast cancer.