Non-invasive methods to monitor tumour growth are an important goal in cancer drug development. Thermographic imaging systems offer potential in this area, since a change in temperature is known to be induced due to changes within the tumour microenvironment. This study demonstrates that this imaging modality can be applied to a broad range of tumour xenografts and also, for the first time, the methodology’s suitability to assess anti-cancer agent efficacy. Mice bearing subcutaneously implanted H460 lung cancer xenografts were treated with a novel vascular disrupting agent, ICT-2552, and the cytotoxin doxorubicin. The effects on tumour temperature were assessed using thermographic imaging over the first 6 hours post-administration and subsequently a further 7 days. For ICT-2552 a significant initial temperature drop was observed, whilst for both agents a significant temperature drop was seen compared to controls over the longer time period. Thus thermographic imaging can detect functional differences (manifesting as temperature reductions) in the tumour response to these anti-cancer agents compared to controls. Importantly, these effects can be detected in the first few hours following treatment and therefore the tumour is observable non-invasively. As discussed, this technique will have considerable 3Rs benefits in terms of reduction and refinement of animal use. As a result, the effect is not tracked in the same mouse over time and a large number of mice are required. There is therefore a need for techniques which enable evaluation of drug efficacy, particularly in the first few hours after administration, to circumvent the issues raised.Human subcutaneous (s.c.) tumour xenografts play a key role in the pre-clinical development of novel anti-cancer agents. Human tumour cells or tumour fragments are subcutaneously transplanted into the flanks of immunodeficient mice, and once tumour is detectable visually or by palpation, drug treatment commences, with tumour growth measured using callipers to determine tumour cross sectional area as a function of tumour volume. Tumour growth delay following treatment is compared with untreated controls to determine any drug effects. These models often suffer from low accuracy and poor reproducibilityOne powerful solution to this problem is to employ non-invasive imaging techniques to monitor tumour growth and vasculature changes. To this end, a wide variety of methods have been explored. Techniques such as MRI and ultrasound are known to be effective in estimating tumour size and drug-tumour interactionsThermographic imaging can monitor both tumour growth and vasculature changes in s.c. implanted xenografts. Infrared thermography measures infrared radiation in the 7,000–15,000 nm range, which is then related to the temperature of the object being imagedThermographic imaging has already been applied to the detection of tumours, as a means to aid diagnosis of clinical breast cancer, where due to inflammation local to the tumour, it is warmer than the surrounding tissue78In this study we directly assess the potential for thermographic imaging to be applied to the evaluation of novel therapeutics in the s.c. tumour xenograft model. Firstly, the detection of xenografts of different tissue origins is studied to confirm that the technique can be applied to tumour types beyond breast cancer. Secondly the suitability of the methodology to assess the efficacy of anti-tumour agents with distinctly different mechanisms of action is evaluated.To demonstrate the extent to which tumours can be discriminated based on the temperature differential with the surrounding tissue, corresponding thermal and digital images were taken of the same H460 tumour xenograft. It can be seen that the area of significant temperature drop was contained within the borders of the tumour area as marked out on digital images taken of the same tumour xenograft . In addiBoth agents demonstrated efficacy  in the H460 tumour model using standard calliper measurement . AssessiBetween 24 hours and 7 days an overall drop in temperature for all three groups was seen . Linear 3 (p < 0.001), doxorubicin showed a reduction of 0.0050 °C/mm3 (p < 0.001) and ICT-2552 showed a reduction of 0.0054 °C/mm3 (p < 0.001). Interestingly, treatment groups showed a larger drop in temperature with increase in volume compared to the control group, suggesting that therapeutic intervention has remodelled the tumour with an associated reduction in vascularisation with a consequence of reduced temperature/tumour volume.When the relationship between tumour temperature and absolute tumour volume was investigated, a linear relationship between the 2 parameters was observed . To quanSignificantly more bodyweight loss was observed over the course of the experiment for mice treated with doxorubicin, compared to those treated with ICT-2552 (p < 0.01) or untreated controls (p < 0.001), with no difference between ICT-2552 and controls (p = 0.61) . When boIn this study the application of the thermographic imaging technique to detection of xenografts of different tumour types has been demonstrated, expanding on previously published studies which focused solely on growth of breast cancer xenografts7The main finding of the work presented here is that thermographic imaging can be used to non-invasively monitor the therapeutic and toxic effects of administering different classes of anti-cancer agents to mice bearing s.c. tumour xenografts. Crucially we have shown that this monitoring is possible in the first 6 hours after treatment, a period in which the tumour is not sufficiently grown to measure using conventional methods, e.g. callipers. This will bring significant 3Rs benefits, since monitoring can be carried out in the same animal over the entire length of the experiment rather than having to use cohorts of animals for each time pointSpecifically, we have successfully demonstrated that thermographic imaging can be readily utilised to monitor the early onset effects of a vascular disrupting agent (VDA), the experimental compound ICT-2552. In previous studies it has been demonstrated that vascular shutdown occurs within the first 6 hours of compound administration for this12With doxorubicin, which has a different mechanism of action to ICT-2552Although this study was not specifically focused on the application of thermographic imaging to monitor off-target drug toxicities, the relationship between body weight loss and temperature drop would suggest that thermographic imaging could also be utilised to monitor overt treatment toxicity.Whilst other non-invasive technologies are available to monitor therapeutic effects in cancer, such as the use of ultrasound, positron emission tomography or magnetic resonance imagingIn conclusion, we have demonstrated that thermographic imaging can be used to assess tumour growth and therapeutic intervention non-invasively in a broad range of tumour types in preclinical cancer models. Whilst further research is required to fully understand the biology underpinning the concept behind therapeutic agent-associated temperature reduction, and on translating the technology for clinical use, thermographic imaging is shown to be a rapid, reliable and cost-effective technology for use in preclinical studies of anticancer agents.An FLIR ThermoVision™ A-Series Infrared Camera A320  was used. This uses a focal plane array sensor with uncooled microbolometer and has a spectral range of 7.5–13 μm. The captured image has a size of 320 × 240 pixels, at 16 bits per pixel, and a temporal frequency of 9 Hz. The imaging system has a fixed aperture of 1.3 F stops, a focal length of 18 mm and a minimum focus distance of 40 cm. All measurements were taken in a temperature-controlled room. The camera was calibrated by imaging a 37 °C-preheated squash ball at the beginning and end of every session.The experimental vascular disrupting agent (VDA), azademethylcolchicine (ICT-2552) was synthesized in-house as described previouslyBalb/c immunodeficient nude mice , between the ages of 6 and 8 weeks were used. Throughout the study, all mice were housed in air-conditioned rooms in facilities approved by the United Kingdom Home Office to meet all current regulations and standards. All procedures were carried out under a Project Licence (PPL 40/3670) issued by the UK Home Office according to government legislation, following approval of the work by the local Animal Welfare Ethics Review Board at the University of Bradford, and in accordance with the UK National Cancer Research Institute Guidelines for the Welfare of Animals3  mice were randomised into groups (n = 3) and received either ICT-2552 at 20 mgkg−1, or doxorubicin at 10 mgkg−1, as a single intraperitoneal dose on day 0. For comparison, a control group was left untreated since extensive previous studies in-house using the excipients in this study have not demonstrated any effect on tumour growth, and therefore it was not deemed ethical to set up excipient only control groups in addition to the untreated control. Tumour volume was determined using callipers as follows: 2-dimensional calliper measurements of the tumours were taken, with volumes calculated using the formula (a2 × b)/2, where a is the smaller and b the larger diameter of the tumour. Tumour volume was then normalised to the respective volume on day 0, and semi-log plots of relative tumour volume (RTV) versus time were made. Animal body weights were recorded throughout the experiment to monitor for any deleterious effects. Mann-Whitney U tests were conducted to determine the statistical significance of any differences in growth rate (based on tumour volume doubling time) between control and treated groups. In addition thermographic images were captured as described below.The H460 lung adenocarcinoma model was selected for these studies. Once tumour volumes reached approximately 32 mmTo capture thermographic images, non-anaesthetised mice were held at a fixed distance of 50 cm from the camera and a sequence of images captured over a 3 second period, generating approximately 30 images. Individual images were chosen from the sequence using visual assessment, with the images demonstrating the sharpest edges selected to reduce the effect of motion blur caused by mouse movement. Immediately following thermographic imaging, digital images were captured using a Panasonic Lumix SZ1 digital camera used in macro mode with flash disabled. The thermal images were analysed with ThermaCam Researcher Pro 2.10 software . The mean, minimum and maximum temperatures across each tumour were calculated by drawing a straight line along the maximum tumour diameter and capturing temperature data across the line.Two-tailed t-tests were carried out at 95% confidence interval, including Bonferroni correction for multiple comparisons, and, to compare the relationship between tumour temperature and both body weight and tumour volume, a linear regression to fit a linear model to the data was used, with an F-test used to assess accuracy.How to cite this article: Hussain, N. et al. The use of thermographic imaging to evaluate therapeutic response in human tumour xenograft models. Sci. Rep.6, 31136; doi: 10.1038/srep31136 (2016).


Maintenance fluids in critically ill brain-injured patients are part of routine critical care. Both the amounts of fluid volumes infused and the type and tonicity of maintenance fluids are relevant in understanding the impact of fluids on the pathophysiology of secondary brain injuries in these patients. In this narrative review, current evidence on routine fluid management of critically ill brain-injured patients and use of haemodynamic monitoring is summarized. Pertinent guidelines and consensus statements on fluid management for brain-injured patients are highlighted. In general, existing guidelines indicate that fluid management in these neurocritical care patients should be targeted at euvolemia using isotonic fluids. A critical appraisal is made of the available literature regarding the appropriate amount of fluids, haemodynamic monitoring and which types of fluids should be administered or avoided and a practical approach to fluid management is elaborated. Although hypovolemia is bound to contribute to secondary brain injury, some more recent data have emerged indicating the potential risks of fluid overload. However, it is acknowledged that many factors govern the relationship between fluid management and cerebral blood flow and oxygenation and more research seems warranted to optimise fluid management and improve outcomes.The online version of this article (doi:10.1186/s13054-016-1309-2) contains supplementary material, which is available to authorized users. Fluid management in critically ill brain-injured patients is aimed at maintaining adequate cerebral blood flow (CBF) and oxygenation. However, fluid management in brain-injured patients has several distinctive features compared with non-brain-injured critically ill patients: 1) fluid tonicity is a more pertinent issue; (2) tissue oedema not only results in oxygen diffusion impairments but may also impair CBF due to the unfavourable volume–pressure characteristics of the intracranial content; 3) fluid management is commonly regarded as ‘basic care’ in brain injury, whereas fluid management in other critically ill patients is commonly guided by haemodynamic monitoring, rendering it ‘intensive care’; and 4) optimising CBF with adequate fluid management seems intrinsically more challenging than systemic circulation, because sophisticated monitoring tools for CBF and cerebral oxygenation are generally less well implemented in clinical practice. These distinctive features of fluid management in brain-injured patients deserve scrutiny, because recent data  suggest that the ‘basic care’ of fluid administration in brain-injured patients may have an impact on outcome [ fluid to fluid ma optimisiThe aim of this narrative review is: to summarize existing guidelines and contemporary literature on routine (maintenance) fluid management in critically ill brain-injured patients (traumatic brain injury (TBI), subarachnoid haemorrhage (SAH), intracerebral haemorrhage (ICH), ischaemic stroke), with a focus on the amounts and types of fluids and volume and circulatory status monitoring; and to discuss practical issues of fluid management.Some basic concepts are relevant to understand effective fluid management in brain injury. The influence of fluid administration or volume status on CBF and cerebral oxygenation is complex because many factors determine the influence of the first on the latter Fig. . In addiOsmolality of plasma and brain interstitial fluid and CSF are equal under normal circumstances . HypotonCerebral oedema is stratified depending on location (intracellular or extracellular) and BBB disruption. Cytotoxic oedema is the cellular oedema of neurons or astrocytes and is the result of mainly sodium and water shifts into the cells after an insult with ATP depletion and mitochondrial dysfunction , 12. VasAutoregulation concerns the capacity of the blood vessels in the brain to sustain CBF by vasodilation or vasoconstriction over a wide range of systemic blood pressures, and in a more general sense may be regarded as the capacity of brain vessels to regulate blood flow in response to changes in metabolic needs. The connection between volume status and intact autoregulation relates to increased CBF to preserve oxygen delivery in response to fluid loading and decreased haematocrit or to maintaining constant CBF through vasodilation when blood pressure drops due to hypovolemia.Perfusion pressure determinants are both upstream and downstream pressures, with upstream pressures being arterial and downstream pressures being venous. Both lower arterial pressures and higher venous pressures will theoretically result in lower perfusion pressures, albeit with different consequences (i.e. low flow versus tissue oedema) . IncreasContemporary recommendations for routine fluid and intravascular volume management are available from several guidelines and consensus conferences , 19–21. The current guidelines on fluid management in brain injury recommend using fluid balances to guide volume status Table . A non-sThe mean fluid intake was around 3–4 L/day in SAH patients who were treated with normovolemia or received fluid management based on volumetric haemodynamic monitoring versus 4–5 L/day in patients managed with hypervolemic treatment which often included CVP or pulmonary artery occlusion pressure (PAOP)-directed management. Fluid balances generally did not differ between both treatment groups and varied around neutral balance −0.5 to +1 L) even in a study where mean daily fluid intake was >8 L . Only on L even in = 5400) reported a temporal association between increased fluid intake and mortality when administered in the pre-DCI period in SAH patients (days 1–3 after the bleed), although it seemed to be beneficial in the DCI risk period (days 4–14) [In the trial on prophylactic hypervolemia after aneurysm clipping after SAH by Lennihan et al.  the hypeys 4–14) . The datys 4–14) . In lineys 4–14) . The ICPys 4–14) . Studiesys 4–14) , 45, 48.A recent review summarized current knowledge on risks and benefits of different types of fluids that are used in traumatic brain injuries , and then = 16) highlighted several important findings [A comprehensive literature search by delegates from a 2010 SAH consensus conference that selected studies on clinical monitoring and volume status  showed findings .In SAH patients, TPT monitoring seems a feasible method of assessing volume status and may help to improve outcome , 25, 34.2)), which was indeed nicely shown in a recent study in SAH patients: fluid responsiveness was associated with improvements in PBrO2 and cerebral perfusion pressure [2. Intravascular pressures, especially CVP, have not been shown to be particularly useful as clinical parameters to assess fluid responsiveness [Fluid responsiveness  in patients with cardiac output monitoring may help to improve cerebral oxygenation , which makes its assessment and definition much more difficult. For comparison, clinical examples outside neurocritical care are oliguria in fluid-overloaded septic and decompensated heart failure patients representing venous congestion . ObviousA practical approach to fluid management in brain-injured patients may include: maintenance fluid volumes routinely administered, the type(s) of fluids allowed and their tonicity; and triggers for more advanced haemodynamic monitoring. Monitoring may include invasive methods (e.g. TPT-guided) or less invasive methods  . FurtherAn algorithm has been used with success by the author in critically ill SAH patients to significantly reduce fluid intake whilst maintaining sufficient cardiac output and indices of cardiac preload Fig. . This alThe scarce available evidence indicates that fluid management in brain-injured patients should generally be targeted at euvolemia using isotonic fluids. Consequently, it seems that not only ‘too dry’ but also ‘too wet’ is detrimental , 69. AvoIt is intriguing that fluid balances seem less clearly associated with secondary brain injuries than fluid intake . This may indicate that ‘fluid throughput’ may be harmful , but it 2 [Endpoints of fluid management should be clearly defined in future investigations; these endpoints may concern derived parameters of cerebral perfusion and oxygenation when direct effects of (systemic) fluid management on the brain are examined, such as PBrO2 , or clin2  or prosp2 . The inc2 , 46, 47.Although monitoring and treatment aimed directly at the injured brain is an important area of intense research, the data presented seem to indicate that appropriate fluid management is clinically relevant. This notion is in line with previous research indicating that systemic complications and management may have a major impact on mortality in critically ill brain-injured patients .Routine fluid management may influence clinical outcomes in brain-injured patients. However, the impact of fluid management on brain pathophysiology is complicated due to many intermediate factors governing their relationship. More recent literature has indicated that hypervolemia may be detrimental similar to non-brain-injured critically ill patients. However, research on consequences of fluid overload is seriously hampered by a lack of uniform definitions and the fact that cerebral oedema is difficult to routinely assess. Although the general aim of fluid management in critically ill brain-injured patients is euvolemia using isotonic fluids, ascertainment of euvolemia is problematic in routine clinical practice without haemodynamic monitoring. Therefore, awareness of potential harm from both hypovolemia and hypervolemia may currently be insufficient.


Results of rheology evaluations show that both the apparent viscosity and the thermal stability of the fracturing fluid decreased with the regeneration number of times increasing. In addition, the apparent viscosity of the fluid which was without regeneration was more sensitive to the shear action compared with that of the fluid with regeneration once or twice. When the fracturing fluid was without regeneration, the elasticity was dominating due to the three-dimensional network structure of the formed gel; the viscosity gradually occupied the advantage when the fracturing fluid was regenerated once or twice. The settling velocity of proppant was accelerated by both the regeneration process and the increasing temperature, but it was decelerated when the proppant ratio increased. Results of core damage tests indicate that less permeability damage was caused by the PVOH/OBT fracturing fluid compared with that caused by the guar gum fracturing fluid after gel breaking.A regenerable polyvinyl alcohol/organic boron fracturing fluid system with 1.6 wt% polyvinyl alcohol (PVOH) and 1.2 wt% organic boron (OBT) was studied, and its main regeneration mechanism is the reversible cross-linking reaction between B(OH) Hydraulic fracturing is a favorable technique applied to the productivity improvement of oilwells located in reservoirs with the characteristics of low porosity and poor permeability. A hydraulic fracturing job consists of a series of stages, including successively injecting a preflush fluid, a proppant carrier fluid, and a displacement fluid . The fraGuar gum is a kind of natural and synthetic polymer mainly constituted by the polymannose –5. DespiThe three kinds of fracturing fluids  are most commonly-used at present. However, in the process of gel breaking and fracture clean-up, the guar gum and polymer with the high molecular weight will be irreversibly degraded and cannot be reused. Polyvinyl alcohol (PVOH) can cross-link with cross-linkers such as boron and chromium to form the fracturing fluid gel . BecausePolyvinyl alcohol  with a molecular weight of 70 kDa, an effective purity of 97% and a degree of alcoholysis of 88%±2% was used as the polymer. Hydroxypropyl guar gum  with a molecular weight of 300 kDa and an effective purity of 96% was also used to conduct the contrast test.The organic borate cross-linker (OBT) was self-made in laboratory using the sodium borate, glycerin, diethanolamine, sodium hydroxide and deionized water. The brief synthetic process of OBT was: (1) putting 15 wt% glycerin, 1.8 wt% sodium hydroxide and 48.2 wt% deionized water into a 3-mouth flask at the same time to prepare an even solution; (2) adding 20 wt% sodium borate and stirring the solution at 60°C for 30 min to ensure the uniform dissolution; (3) further adding 15 wt% diethanolamine into the above solution, elevating the reaction temperature to 80°C and sealing the flask for 5 h. Then the organic borate cross-linker (OBT) with a color of faint yellow and a pH value of about 9.5 was obtained.4)2S2O8) was used as the gel breaker. Sphere ceramic particles within a diameter range of 0.40 to 0.60 mm and an average apparent density of 2.78 g/cm3 were used as the proppant. Synthetic formation brine was used in experiments and its compositions are listed in Sodium hydroxide (NaOH) was applied to change the pH of fracturing fluids and the ammonium persulfate . In the process of shear stability tests, the apparent viscosity of each fracturing fluid sample was tested at 60°C with a shear rate of 170 1/s and a shear time from 0 to 3000 s. In the process of thermal stability tests, the apparent viscosity of each fracturing fluid sample was tested from 60°C with a shear rate of 170 1/s and a heating rate of 5°C/min. When measuring the viscoelasticity, the storage modulus G' and loss modulus G'' of each fracturing fluid system were achieved by changing the frequency scanning from 0.01 Hz to 100 Hz.The scanning electron microscope  was applied to analyze the microscopic structure of gelled fracturing fluids. The brief preparation process of the scanning sample was shown as follows: (1) uniformly smearing a small volume of gel on a frosted glass plate; (2) quickly putting the frosted glass plate into a blast dryer to remove the moisture from the gel sample; (3) putting the frosted glass plate in a vacuum coating apparatus to coat a metal film which can be conductive on the dried gel sample; (4) immigrating the frosted glass plate with the coated gel sample into the sample chamber of the scanning electron microscope. Then microscopic images can be obtained using the imaging software.4)2S2O8 was prepared as the gel breaker. The gelled fracturing fluid was broken within 1 h at 60°C after the (NH4)2S2O8 solution was added, and then the apparent viscosity of the residual fracturing fluid decreased to only 8 mPa·s. During the regeneration process, the NaOH solution (20 wt%) was used to regulate the pH from 5 to 9–10, and then the fracturing fluid gel was generally reformed. Repeated the above process and the fracturing fluid could regenerate again. In following experiments, the fracturing fluid system without regeneration was named system 1, and systems regenerated once and twice were named system 2 and system 3, respectively. After each regeneration reaction, the regenerated gel was separated with the uncross-linked fluid using a filter screen with the screen size of 75μm. The recovery ratio of the regenerated fracturing fluid system was defined as the ratio of the recovered volume of the regenerated gel to the initial volume of the system before regeneration. The recovery ratios of system 2 and system 3 were 0.77 and 0.61, respectively.In the gel breaking process, a solution with 2.0 wt% (NHStatic proppant settling tests including the single particle settling test and settling tests using different proppant ratios (the volume ratio of proppant to fluid) were performed to explore the proppant carrying capacity of fracturing fluids. To eliminate the effect of the air bubble on the proppant particles, ceramic particles were soaked in the gelled fracturing fluid at least 12 h prior to the settling tests. 1,000 mL gelled fracturing fluid was put into a transparent cylinder container (diameter 6 cm × height 60 cm) with scale in millimeter, and then soaked ceramic particles were putted into the container; meanwhile, a high-speed video device  was used to track particles via the middle 20 cm height of the container, and then the static settling velocity of particles was computed using the mobile image analysis software. The proppant ratios were 3, 7 and 10%, respectively.ϕ 25 mm× L 80 mm) with similar initial water permeability of about 60 mD were used to conduct core damage tests, and the brief experimental procedure was: (1) vacuumizing dried cores using a vacuum pump and saturating them with the synthetic formation brine; (2) injecting 5.0 PV (pore volume) the broken fracturing fluid into cores; (3) placing cores into an oven at 60°C for 2 h; (4) flooding the fracturing fluid out by inversely injecting the formation brine until the injection pressure leveled off, and measuring the final water permeabilities of cores.Four Berea cores  with a measurement range of 0.02 to 2,000 μm was applied to measure the particle size distribution of the diluted fracturing fluid after gel breaking. The laser wavelength was 450 nm and the experimental temperature was25°C. The median size was calculated to characterize the distribution range of broken gel particles.4- are slowly generated :η=:η=16]:η and the natural logarithm of γ:Eq  can be mη and ln γ was show in η and ln γ, and it is consistent with the expression shown in the Eq (The effect of shear rate (in the range of 1 to 170 1/s) on the apparent viscosity of each gelled system was studied and the relation between ln n the Eq . Furthern the Eq  can be sn the Eq –(7) to dk and the fluid behavior index n of each system were calculated using the slope and the intercept in Eqs  and  and k anThermal tolerance is an important parameter for the evaluation of a fracturing fluid. There is no doubt that a fracturing fluid with a good thermal stability has relatively wide application ,18. Fig For system 1 which was without regeneration, the initial viscosity at 60°C was 488.2 mPa·s; with the increase of temperature to 65°C, the viscosity changed slightly (474.5 mPa·s). When the temperature was over 65°C, the viscosity greatly decreased and it was down to about 50 mPa·s at 102°C. In the present study, the temperature at which the apparent viscosity of the fracturing fluid decreased to 50 mPa·s was defined as the critical temperature. For system 2 and 3, the temperatures corresponding to the cross-points were same with the critical temperatures and they were 88°C(system 2) and 75°C(system 3), respectively. Data indicate that the thermal stability of the fracturing fluid was weakened with the increase of the number of regeneration. The high degree of the cross-linking reaction between PVOH and OBT of system 1 was responsible for its better thermal stability.The viscoelasticity of the gelled fracturing fluid can be described using the storage modulus G' and the loss modulus G'' . G' reprδ indicates that the loss modulus holds the dominant position in the deformation process [δ is the loss tangent, G' is the storage modulus in Pa, and G'' is the loss modulus in Pa.The loss tangent tanδ was employed to further explore the change of the relationship between the elasticity and the viscosity of three fracturing fluids, as shown in the Eq , and the process ,24.tanδδ values of three systems were in the range of 0.2–0.4 and slightly changed; with a further increase of the scan frequency, tanδ sharply increased because of the great increment of G'' than that of G'. Moreover, the differences among three tanδ curves were gradually enlarged after the scan frequency exceeded 10 Hz. Final tanδ values of systems 1, 2 and 3 were 0.59, 0.91 and 1.37, respectively.δ indicate that the viscosity property of the regenerated fracturing fluid gradually took the advantage compared with the elasticity property, and it reflects a fact that the network structure of the gelled fracturing fluid was destroyed after regeneration.The analyses of G', G'' and tanThe influence of the proppant ratio on the proppant settling velocity of three fracturing fluid systems was also investigated at 60°C and the results are shown in The degree of the core permeability damage of three fracturing fluid systems after gel breaking was measured according to the procedure described in the section 2.5; meanwhile, the common guar gum (0.35%) fracturing fluid was also used as a contrast. For system 1, the median size of more than 80% of the gel particles was in the range of 10–45 μm, and they were in the range of 5–40 μm for system 2 and 4–40 μm for system 3, respectively. Data in A regenerable polyvinyl alcohol/organic boron fracturing fluid system with 1.6 wt% PVOH and 1.2 wt% OBT is optimized, and the suitable pH range for the formation of the PVOH/OBT fracturing fluid when its apparent viscosity exceeds 400 mPa·s is 8.5–10.The apparent viscosity of the fracturing fluid decreases with the regeneration number of times increasing. The viscosity of system 1 which is without regeneration and owns a three-dimension network structure is more sensitive to the shear rate compared with that of system 2 or system 3.The thermal stability of the fracturing fluid became weaker with the increase of the regeneration number of times. When the fracturing fluid is without regeneration, its elasticity property takes the advantage, but the viscosity property gradually occupies the advantage when the fracturing fluid is regenerated once or twice.Both the regeneration process and the experimental temperature can accelerate the proppant settling velocity, but the proppant settling velocity is decelerated with the increase of the proppant ratio in the fracturing fluid.Less permeability damage is caused by the PVOH/OBT fracturing fluid compared with that caused by the commom guar gum fracturing fluid after gel breaking.


Preclinical researches involving this drug mostly concern Triple Negative Breast Cancer (TNBC) murine models. Here, we explored the therapeutic efficacy of sunitinib on a PyMT-derived transplanted model classified as luminal B (HER2-positive) and monitored the response to treatment using both n = 7) or 14 (n = 8) days with sunitinib at 40 mg/kg or with vehicle only. Response to therapy was assessed in vivo by monitoring glucose tumour metabolism and hypoxia using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and [18F]fluoromisonidazole ([18F]FMISO) Positron Emission Tomography (PET). After primary tumour excision, ex vivo digital microscopy was performed on treated and control samples to estimate vascular density (CD31), apoptosis (Tunel), proliferation (Ki-67), Tumour-Associated Macrophage (TAM) infiltration (F4/80), metabolism (GLUT1) and cellular response to hypoxia . The drug impact on the metastasis rate was evaluated by monitoring the PyMT gene expression in the lungs of the treated and control groups.Tumour-induced animals were treated for 9  vs control (n = 3) tumours (p < 0.05). With this small sample, [18F]FMISO PET showed a non-significant decrease of hypoxia in treated vs control tumours. The drug triggered a 4.9 fold vascular volume regression (p < 0.05), as well as a 17.7 fold induction of tumour cell apoptosis (p < 0.001). The hypoxia induced factor 1 alpha  expression was twice lower in the treated group than in the control group (p < 0.05). Moreover, the occurrence of lung metastases was not reduced by the drug.Concomitant with sunitinib-induced tumour size regression, [18F]FDG and [18F]FMISO PET were relevant approaches to study the response to sunitinib in this luminal B (HER2-positive) model. The sunitinib-induced vascular network shrinkage did not significantly increase tumour hypoxia, suggesting that tumour regression was mainly due to the pro-apoptotic properties of the drug. Sunitinib did not inhibit the metastatic process in this PyMT transplanted model.[The online version of this article (doi:10.1186/s12885-015-1540-2) contains supplementary material, which is available to authorized users. Based on encouraging preclinical data, the sunitinib drug, a multi-kinase inhibitor, has been investigated in several clinical studies in association with various cytotoxic drugs but led to disappointing results in breast cancer patients. Classically, breast cancers are classified according to the expression levels of the estrogen (ER) and progesterone (PR) receptors, and of the human epidermal growth factor receptor 2 (HER2) oncogene. The large majority of studies with sunitinib involved advanced and heavily treated breast cancer focusing on the HER2-negative (or trastuzumab (TZM) insensitive) subtypes  mouse models, and all found that the drug delayed the tumour growth at doses ranging between 20 to 60 mg/kg/day. Interestingly, sunitinib treatment induced tumour regression in a MCF7 xenograft model , which iin vivo Positron Emission Tomography (PET) with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and [18F]fluoromisonidazole ([18F]FMISO) radiotracers, which allow to monitor tumour glucose metabolism and hypoxia respectively. Furthermore, in vitro analyses were used to quantify the chemotherapy impact on several cancer-associated parameters, namely vascularization (CD31), apoptosis (TUNEL), proliferation (Ki-67), hypoxia , TAM infiltration (F4/80), metabolic activity (GLUT1) and metastasis.For this purpose, we used the MMTV-PyMT murine model whose oncogenesis is induced by expression of the polyoma virus middle T oncoprotein under control of the Mouse Mammary Tumour Virus (MMTV) promoter (PMID: 1312220). Following the recommendations of Varticovski et al.  about thAnimal studies were approved by the animal ethics committee “Comité d'EThique en Expérimentation Animale” (CETEA DSV n°44) under reference 12–036 and conducted in accordance with the Directives of the European Union.FVB/N-Tg (MMTV-PyMT)634Mul/J (PyMT) 12-weeks-old mice were used as tumour donor. Aseptically collected mammary tumours from PyMT mice were minced and immersed in cold Dulbecco's Modified Eagle's Medium . Mechanical cell dissociation was performed using Medicon disposable chambers . The cell suspension was then progressively filtered using Filcon filters with pore sizes of 500 μm, 200 μm and 70 μm (BD bioscience). Finally, cells were aliquoted in freezing medium  and stored in liquid nitrogen.in vitro culture step, in the mammary fat pad of the posterior nipple in FVB mice. The tumour volumes were calculated using calliper measurements and the approximated formula for a prolate ellipsoid, given by:After freezing medium removal and enumeration, the tumour cells were directly inoculated, without any To evaluate drug toxicity, body animal weights were also monitored.n = 4) and control (n = 3) groups. The treated one received per os a daily dose of sunitinib at 40 mg/kg in 20 mM dimethyl sulfoxyde . The control group received only the DMSO solution. Drug administration was performed during 9 consecutive days.Two sets of mice were used in this study. For the main set A, 7 animals were implanted with 3 million viable cells. PyMT tumours were allowed to grow for 21 days. The mice were randomized into treated (n = 4) and control (n = 4) mice was obtained implanting 400 000 viable cells. Treatment began at day 25 post implantation. It continued for 14 days until resection at day 39. For both sets A and B, the primary tumours were surgically removed after treatment and the mice were kept alive for 60 supplementary days before euthanasia to analyse the lungs for metastasis content.To further explore the neoadjuvant therapy effects on the metastatic incidence, we extended primary tumour growth and treatment times before mammary tumour surgical resection. Thus, a secondary set B of treated  algorithm after Fourier rebinning, with a voxel size of 0.5 × 0.5 × 0.8 mm3 . Radioactivity uptake in regions of interest (ROIs) was measured using BrainVISA 4.0 and Anatomist 4.0.2  and expressed in Standardized Uptake Value (SUV) calculated using:FDG uptake remained stable during sunitinib administration, from 1.1 ± 0.2 to 1.1 ± 0.2 (n = 4) in SUV (NS). As a result, after 5 days of treatment, the [18F]FDG uptake was significantly lower, by a factor of 1.5 (p < 0.05), in the treated versus the control tumours  and the control group , although lower in the control group. After 6 days of sunitinib or vehicle only administration, the tracer uptake remained stable in the control group  and decreased in the treated one , thus reducing the initial differences between the two groups  and 45 ± 20 mm3 (n = 4) respectively (p < 0.01). The nine day sunitinib treatment induced a 4.9 fold regression of the vascular volume reported to the hematoxylin volume, with values of 3.6 ± 1.8 % (n = 3) and 0.7 ± 0.05 % (n = 4) in the control and treated groups respectively (p < 0.05) , an increase of the small size vessels  and no evolution for medium size vessels  in control tumours to 5.7 ± 3.5 % in treated ones . TUNEL labelling revealed a high induction of apoptosis by a factor of 17.7. Indeed, in nutrient supplied expanding tumours, very low programmed cell death was observed, representing 4.1 ± 0.3 % in volume (n = 3), while this value was 72.6 ± 10.7 % in treated tumours   were over-expressing Ki-67 in non-treated tumours and the therapeutic agent reduced this population to 730 ± 334 proliferating cells per mm3 . This represents a 1.8 fold reduction of the tumour proliferation process . In the sunitinib treated mice, this value was at 546 ± 169 macrophages per mm3 (n = 4) . Finally, GLUT1 whole tumour expression was enhanced by a factor of 2.57 in the sunitinib treated group when compared to control , and reached 40.0 ± 12.3 % after the 9 days-long treatment .Digital microscopy analysis was exclusively performed on set A of mice. In the control and treated groups, the mean hematoxylin volumes estimated using digital microscopy were 126 ± 6 (05) Fig. . The sunels Fig.  and 5a-b1) Figs.  and 5c-dss Figs.  and 5e-f4) Figs.  and 5g-hol Figs.  and 5i-jn = 4) and control (n = 3) groups. In set B with a 39 days tumour growth duration, including 14 days of sunitinib or DMSO treatment, the incidence of lung metastasis was of 50 % in the treated (n = 4) and control (n = 4) groups  breast cancers –25. The Src family , ii) the phospholinositide 3-kinase (PI3K) activating the Akt/mTOR cell-survival pathway, iii) the mitogen-activated protein kinases (MAPK/ERK) pathway through recruitment of the Shc adapter protein and iv) the phospholipase Cγ1 (PLC-γ) pathway inducing the protein kinase C (PKC) activation and a cytosolic Ca2+ concentration increase FDG or [18F]FMISO PET. The closest related work describes a [18F]FDG PET monitoring of the sunitinib response on lung metastases in a 4T1 intravenously induced metastatic model [18F]FDG signal in the lungs of the sunitinib-treated mice compared to the control mice, which correlated with an enhanced seeding of lung metastases associated with sunitinib administration. In our [18F]FDG PET data, the mean SUV increased during the 5 day-tumour growth in the control group, whereas it remained stable in the treated tumours. We checked that the stable [18F]FDG uptake in treated tumours that were concomitantly decreasing in size was not due to partial volume effect (PVE) [18F]FDG has been previously studied emphasizing the role of the GLUT protein family [18F]FDG uptake in sunitinib-treated tumours might be at least partly explained by the lower levels in vascularisation, TAM infiltration and cell viability in sunitinib treated by comparison to control tumours. Yet, the overall conclusion is therefore that [18F]FDG PET evidenced the response to sunitinib treatment in this tumour model.In addition to the decrease in tumour size, the vascular network evaluation demonstrated that sunitinib impacts vascular density and maturity. Moreover, the treated tumours were characterised by a higher level of apoptosis by comparison to controls. Previously, this multi-kinase inhibitor has already been shown to present operties , 30. Regoperties . To our ic model  and showct (PVE)  and founn family . In our 18F]FMISO PET scans, randomization did not yield two perfectly equivalent groups regarding the hypoxia levels as expressed in SUV. Nevertheless, the untreated tumours remained stable in hypoxia over the treatment course, whereas the sunitinib administration tended to reduce hypoxia, although the difference was not significant in our small sample. Therefore, despite the reduction in blood supply, the treated tumours did not become more hypoxic than before the sunitinib administration. This might seem paradoxical as the sunitinib-induced anti-angiogenic effects are often associated with an increase in hypoxia due to the tumour starvation in nutrients. This enhanced hypoxia phenomenon has for instance been described by Welti et al. [18F]FMISO PET therefore appeared useful to characterise the hypoxia level inside the tumours, and also to unveil the preferential way of action of the drug on this model.In our FDG PET and [18F]FMISO PET imaging results suggest that in addition to its anti-angiogenic effects, the sunitinib efficacy on this model is mostly due to its direct pro-apoptotic properties.We showed that the luminal B (HER2-positive) type PyMT model was particularly sensitive to sunitinib compared to other preclinical breast cancer models, such as TNBC models that have been extensively used to study the effects of this drug. Our histology, [


Fluid therapy after initial resuscitation in critically ill, septic patients may lead to harmful overloading and should therefore be guided by indicators of an increase in stroke volume (SV), i.e. fluid responsiveness. Our objective was to investigate whether tissue perfusion and oxygenation are able to monitor fluid responsiveness, even after initial resuscitation. Thirty-five critically ill, septic patients underwent infusion of 250 mL of colloids, after initial fluid resuscitation. Prior to and after fluid infusion, SV, cardiac output sublingual microcirculatory perfusion (SDF: sidestream dark field imaging) and skin perfusion and oxygenation (laser Doppler flowmetry and reflectance spectroscopy) were measured. Fluid responsiveness was defined by a ≥5 or 10 % increase in SV upon fluids. In responders to fluids, SDF-derived microcirculatory and skin perfusion and oxygenation increased, but only the increase in cardiac output, mean arterial and pulse pressure, microvascular flow index and relative Hb concentration and oxygen saturation were able to monitor a SV increase. Our proof of principle study demonstrates that non-invasively assessed tissue perfusion and oxygenation is not inferior to invasive hemodynamic measurements in monitoring fluid responsiveness. However skin reflectance spectroscopy may be more helpful than sublingual SDF. In critically ill, septic patients, fluid administration to improve tissue perfusion and oxygenation is generally guided by systemic haemodynamic parameters; fluid responsiveness is defined by a cardiac preload challenge by fluid infusion resulting in augmented stroke volume (SV) and cardiac output (CO) , 2. CurrTherefore, we studied whether tissue perfusion and oxygenation is able to predict and monitor fluid responsiveness, in critically ill, septic patients considered hypovolaemic on clinical grounds after initial resuscitation.2 saturation (ScvO2) <65 %, increasing vasopressor requirements, decreasing urine output and mottled skin. Patients were included >8 h up to 10 days after ICU admission to allow for initial resuscitation by fluid administration and vasopressor infusion and to obtain informed consent. Exclusion criteria were admission for intracranial catastrophes, known intra-abdominal hypertension, known extensive peripheral vascular disease and known congestive heart failure. Patients were treated by intensive care staff, with, among others, appropriate broad-spectrum antibiotics, source control and, if needed, intubation and mechanical ventilation according to guidelines for standard practice in our institution. None of the patients received drotrecogin alpha activated or hydrocortisone. One patient received intravenous nitroglycerin. Settings of the ventilator and of vasoactive agent infusions were unaltered during the study.This single center study was performed in the general intensive care unit (ICU) of the Erasmus MC University Medical Centre. Ethical approval for this study was provided by the ethical committee of the Erasmus MC University Medical Centre (MEC-2009-112). Written informed consent was obtained from each patient or his or her legal representative. Consecutive patients admitted in our general ICU with sepsis and on haemodynamic monitoring with a central venous catheter and a femoral artery catheter connected to a PiCCOplus™ device  were eligible. These monitoring tools are standard in our institution when sepsis is accompanied by hypotension and extensive fluid administration and vasopressor support is considered or performed. Sepsis was defined as the presence of two or more systemic inflammatory response criteria with a suspected or confirmed infection . The sec®, Fresenius Kabi, Bad Homburg, Germany) were infused in 15 min, after which measurements were repeated.Patients were studied in the supine position; 250 mL of colloid solution  were also continuously measured. Calibration of the pulse contour-derived CO was performed by transpulmonary thermodilution involving three separate central venous injections of 20 mL of ice cold NaCl 0.9 %, at baseline. To calculate cardiac index CO was divided by body surface area. Sublingual microvascular blood flow was evaluated using SDF . Image acquisition and subsequent analyses were performed according to published consensus criteria /mm and for proportion of perfused vessels 100 [100–100] % (unpublished data). To determine the intrarater reproducibility of the sublingual microvascular parameters, the complete image analysis on 75 randomly selected SDF sequences was repeated at a later time point, in the absence of knowledge of interventions and the intraclass correlation coefficient on consistency was calculated, considered good when ≥0.6. The intraclass correlation was 0.79 for microvascular flow index, 0.76 for functional capillary density, 0.73 for vessel density and 0.74 for proportion of perfused vessels. LDF and RS were performed using an O2C device  applied to a finger. The tissue was illuminated with a pulsed 830 nm class 1 laser diode and the backscattered light was spectrally analysed to assess the velocity-dependent frequency shifts caused by flowing red blood cells. The microvascular haemoglobin oxygen saturation and relative haemoglobin concentration were measured by illuminating tissue with visible white light (500–630 nm), which is backscattered and changed in colour according to its O2 saturation. The mean laser Doppler flow and microvascular haemoglobin oxygen saturation was recorded and averaged over a stable period of 1 min. In our healthy volunteers average values (median and interquartile ranges) for laser Doppler flow is 352 [185–488]AU, for microvascular haemoglobin oxygen saturation 72 [68–79] % and for relative haemoglobin concentration 47 [41–52] AU (unpublished data).At baseline, patients were placed in supine position and after calibration and zeroing to atmospheric pressure at mid-chest level, mean arterial pressure (MAP) was taken from the femoral artery catheter and heart rate (HR) from the recorded electrocardiogram. They were measured continuously throughout the experiment. The pulse contour-derived SV and CO . Groups were compared using Mann–Whitney or Fisher exact tests, where appropriate. Intragroup comparisons were done with help of the Wilcoxon matched pairs test. Receiver operating characteristics (ROC) were calculated and compared to assess predicting and monitoring values of parameters for fluid responsiveness. Baseline values were used for prediction and the changes in parameters following the fluid challenge were used for monitoring. The areas under curves (AUC) ± standard error and sensitivity, specificity, positive and negative predictive values  are given. An AUC >0.70 was considered clinically useful. The cut-offs were determined as the cut-off values with the highest sensitivity and specificity combined. The Spearman correlation coefficient was used to express relations. The intraclass correlation coefficient was used to evaluate reproducibility of microcirculation measurements. Data are expressed as median and interquartile ranges. A two-sided P<0.05 was considered statistically significant. Exact P values are given unless <0.001.In line with other studies and 50 % fluid responses, we estimated that 35 patients would be sufficient to reach the proof of principle study goals. Patients in whom the 250 mL fluid infusion induced an increase in SV by ≥5 % were defined as fluid responders, since the 10 % cutoff usually applies to 500 mL infusion. We nevertheless also evaluated fluid responses of ≥10 %. Non-parametric test were used because of relatively small numbers, even though most variables were distributed normally  and µHbSO2 .The increase in SV related to the increase in rHb  by fluid infusion in responders. However, the effect of fluid infusion and the subsequent increase in SV on the parameters of tissue perfusion was relatively small in our study, and changes in SDF variables in responders did not differ from that in non-responders. We included patients beyond the initial phase of fluid resuscitation and SDF measurements suggest that the sublingual microcirculation was sometimes hyperdynamic at this stage. De Backer et al.  noted amTissue perfusion parameters seemed, at least in part, dependent on systemic haemodynamics. This is in agreement with some studies  but in cThe limitations of our study include the relatively small number of patients. Future research on guiding fluid resuscitation by tissue perfusion parameters is also needed to study benefits of this approach of increasing tissue oxgenation by fluid administration in critically ill, septic patients, since our study suggests reasonable intrarater and intrapatient reproducibility of some of these non-invasive measurements. Additionally, we cannot exclude that prediction and monitoring values of microcirculatory parameters are different from ours in patients with less prior resuscitation. Finally, real time analysis would be needed to utilise SDF images for guiding fluid administration, the necessary off-line analysis makes it currently unsuitable for clinical use.In conclusion, the value of non-invasively assessed skin perfusion and oxygenation for monitoring fluid responsiveness in critically ill, septic patients after initial resuscitation is not inferior to that of invasive hemodynamic measurements. This may help fluid management in septic patients, even though an outcome benefit has yet to be demonstrated.


Ondřej Novák and Lukáš Spíchal are not affiliated with #3 but with #2 Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, CZ-78371, Czech Republic.The affiliation for the 12In the Funding section, the grant number from the funder Fondo Europeo de Desarrollo Regional (Spain) is listed incorrectly. The correct grant number is: BIO2013-49125-C2-1-P.


Goal-directed therapy used in the perioperative period of patients undergoing cardiac surgery shortens the length of ICU stay . We aimeWe have been using the restrictive fluid strategy since March 2013 in our institute. Patients who were on the liberal fluid regime were analyzed retrospectively. From March 2013 until today, patients who were on restrictive fluid strategy were analyzed prospectively. A total of 125 patients were included in the study. Age, duration of anesthesia, type of fluids given intraoperatively, fluid index (ml/kg/hour), fluid intake/output balance, creatinine and lactate levels were compared with pulmonary and renal morbidity, and length of stay in ICU, using multivariate analysis.P < 0.05) was established between the amount of crystalloid given intraoperatively, fluid index and fluid balance with pulmonary morbidity (n = 52). The fluid index and inotropes usage were correlated with the postoperative creatinine levels (P < 0.05). There was no correlation between perioperative lactate levels with fluid balance and fluid index. Intraoperative blood loss, the amount of given crystalloid, colloid, blood and FFP, fluid balance, duration of anesthesia and postoperative blood transfusion were found to be related (P < 0.05) with the length of ICU stay. Four percent of the patients required renal replacement therapy and the overall mortality was 0.8%.A significant correlation , without causing any vital organ dysfunction.


The telomeric protein TRF2, involving in telomeric and extratelomeric DNA damage response, has been previously reported to facilitate multidrug resistance (MDR) in gastric cancer cells by interfering ATM-dependent DNA damage response induced by anticancer drugs. Rap1 is the TRF2-interacting protein in the shelterin complex. Complex formation between Rap1 and TRF2 is essential for their function in telomere and end protection. Here we focus on the effects of Rap1 on TRF2 function in DNA damage response induced by anticancer drugs. Both Rap1 and TRF2 expression were upregulated in SGC7901 and its MDR variant SGC7901/VCR after etoposide treatment, which was more marked in SGC7901/VCR than in SGC7901. Rap1 silencing by siRNA in SGC7901/VCR partially reversed the etoposide resistance. And Rap1 silencing partially reversed the TRF2-mediated resistance to etoposide in SGC7901. Rap1 silencing did not affect the TRF2 upregulation induced by etoposide, but eliminated the inhibition effect of TRF2 on ATM expression and ATM phosphorylation at serine 1981 (ATM pS1981). Furthermore, phosphorylation of ATM targets, including γH2AX and serine 15 (S15) on p53, were increased in Rap1 silencing cells in response to etoposide. Thus, we confirm that Rap1, interacting with TRF2 in the shelterin complex, also has an important role in TRF2-mediated DNA damage response in gastric cancer cells treated by etoposide. The TRF2-Rap1 complex possesses higher ability to remodel telomeric DNA than either component alone.10 Rap1 could be potentially involved in the function of TRF2 in DNA damage response,11 whereas it remains unknown whether TRF2 requires Rap1 for its function in DNA damage response induced by anticancer drugs.Rap1 is the TRF2-interacting protein in the shelterin complex. Complex formation between Rap1 and TRF2 is essential for their function in telomere and end protection. Rap1 relies on TRF2 for stable expression.In this paper, we aimed to investigate changes of Rap1 and TRF2 expression in human gastric cells (SGC7901 and SGC7901/VCR) treated by etoposide, summarize effects of Rap1 silencing on the TRF2 expression and DNA damage response and identify the phosphorylation of ATM targets. Subsequently, the association between Rap1 and TRF2 was discussed to further the relevant study on the possible mechanism of multidrug resistance and develop effective methods against the multidrug resistance.11 we detected the presence of co-upregulated TRF2 and Rap1 expression in gastric cancer cells after etoposide treatment. Treated with 20 μg/ml etoposide for 6 h,7 the mRNA and protein levels of TRF2 and Rap1 in SGC7901 and SGC7901/VCR cells were determined by western blot and real-time PCR. As shown in In previous work, we reported that etoposide or adriamycin (ADR) treatment upregulated TRF2 expression in gastric cancer cells, which occurred at early stage of DNA damage response and showed dose-dependency. On the basis of the significance of the Rap1-TRF2 complex,Two Rap1 siRNA eukaryotic expression vectors were constructed and transiently transfected into SGC7901/VCR, respectively, to identify whether Rap1 participates in TRF2-mediated resistance to etoposide in gastric cancer cells. As shown in 7 On the basis of this, cotransfection of Rap1 siRNA vector and TRF2 eukaryotic expression vector was performed into SGC7901 cells transiently, resulting in TRF2 overexpression, low Rap1 expression and increased sensitivity to etoposide as compared with SGC7901–TRF2 cells . Correspondingly, the phosphorylation of ATM targets, including γH2AX and serine 15 (S15) on p53, were also reduced  supplemented with 10% (v/v) heat-inactivated fetal calf serum and antibiotics at 37 °C with 5% CO2 in a humidified incubator .Human gastric cancer cell line SGC7901 and its multidrug-resistant cell variant SGC7901/VCR were obtained from State Key Laboratory of Cancer Biology .m Tris (pH 8.0), 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 100 μg/ml PMSF, 1 μg/ml Aprotinin, 1%NP-40, 0.5% sodium orthovanadate). Proteins were resolved by SDS–PAGE and then electrotransferred to nitrocellulose membranes . Nonspecific binding was blocked with 5% fat-free milk for 1 h at room temperature. The membrane was then separately incubated with anti-TRF2 antibody , anti-Rap1 antibody , anti-ATM antibody , anti-phospho-ATM (S1981) antibody , anti-γH2AX antibody , anti-phospho-p53(S15) antibody  or β-actin antibody  overnight at 4 °C. After binding of the horseradish peroxidase-coupled secondary antibody  at room temperature for 2 h, antigens were visualized by enhanced chemiluminescence . All results were representative of three independent experiments.Cells were collected, and total cellular proteins were prepared with lysis buffer , and cDNA was synthesized using the PrimeScript RT reagent kit , according to the manufacturer's recommendations. A LightCycler FastStart DNA Master SYBR Green I System  was used for the real-time PCR. The 25-μl reaction contained 12.5 μl of SYBR Green qPCR master mix , 10 nmol of each primer, 2.0 μl of the DNA template and 8.5 μl of dHβ-actin, 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′ (forward)and 5′- CAC CTTCTACAATGAGCTGCGTGTG-3′ (reverse);and Rap1, 5′-GCCACCCGGGAGTTTGA-3′ (forward)and 5′-GGGTGGATCATCATCACACATAGT-3′ (reverse);and TRF2, 5′-CTGAGCTCACACCACTGGAA-3′(forward)and 5′-GCATCTTCTGCTGGAAGGTC-3′ (reverse).Two pairs of hairpin siRNA template oligonucleotides for Rap1 based on pSilencer3.1-H1 vector  were designed by siSearch. Oligo-1:7 and pSilencer3.1-Rap1 siRNA plasmids were cotransfected into SGC7901. pcDNA3.1 and pSilencer3.1-H1 were cotransfected as negative control.5′-GATCCGTGTAGCTCGGAGGATTGAATTCAAGAGATTCAATCCTCCGAGCTACA TTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAATGTAGCTCGGAGGATTGAATCTCTTGAATTCAATCCTCCGAGCTACACG-3′. Oligo-2: 5′-GATCCGTTGGATGTATTTACAGCTGTTCAAGAGACAGCTGTAAATACATCCAA TTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAATTGGATGTATTTACAGCTGTCTCTTGAACAGCTGTAAATACATCCAA CG-3′. These oligonucleotides were annealed and ligated into vector, respectively. According to the manufacturers' instructions, pSilencer3.1-Rap1 siRNA plasmids 1 or 2 were transiently transfected into SGC7901/VCR using LipofectamineTM 2000 (Invitrogen). pSilencer3.1-H1 alone was transfected as negative control. Further studies were taken at 72 h post transfection. Similarly, previously constructed pcDNA-TRF24 cells per well) and incubated for 24 h. Etoposide was added and incubated for 72 h. Then treated cells were incubated with 20 μl of MTT solution (5 mg/ml) in PBS for 4 h. After removing the medium, 150 μl of dimethylsulfoxide (Sigma) was added to each well to dissolve crystals. Absorbance at 490 nm was measured with a microplate reader BP800 (Biogit). Dose-effect curves of etoposide were drawn on semilogarithm coordinate paper and IC50 values were determined.Sensitivity of gastric cancer cells to etoposide was evaluated using MTT assay. Cells were seeded onto 96-well plates . A value of


Page 1: In the Funding Information section, grant number “09-546-2-206” should read “6-059-2-023.”Volume 4, no. 2,


Previous research has shown that adding a small amount of a second immiscible fluid to particulate suspension can result in a significant influence on viscosity. In this study, the effects of the second fluid addition over a small dosage range on the rheological properties of particle suspension were investigated. As the dosage of the second fluid was increased, the viscosity and yield stress initially decreased then increased and finally decreased again. The microstructure of the suspension was observed using a confocal laser scanning microscope (CLSM) and showed three different states with the increasing dosage of the second fluid: a dispersive sate, cluster state and cell state in sequence. The presence of these states interpreted the non-monotonic trend of viscosity and yield stress in the suspensions. The rheology of suspension is a complex function of the interactions between the particles, including van der Waals forces, electrostatic forces, steric interactions, hydrodynamic interactions, and Brownian forces1234678111013et al.et al. found that a second fluid could greatly decrease the viscosity and yield stress of suspensionset al.et al.Different from results reported by Koos −5. The solubility of kerosene in the aqueous solution of MF (0.754 wt%) is less than10−4, and that of kerosene in water is less than 10−5. So the solubilization effect of MF for kerosene in water is insignificant.In the all particle suspensions, water is denoted as the primary fluid, while a small amount of kerosene is denoted as the second fluid, immiscible with the primary fluid. The all suspensions were composed of particles, water, a little amount of dispersant and kerosene (or without kerosene). Dispersant possess both hydrophobic and hydrophilic groups, i.e. it is a surfactant. Methyl naphthalene sulfonate formaldehyde condensation (MF) was chosen as the dispersants in this paper, which has a polymer chains and negative charge. Two preparation methods were used in this study  displayed a different trend compared to those prepared by method (I). For HGB suspensions, the apparent viscosity did not vary significantly as the dosage of the secondary fluid was increased from 756 mPa s (the second fluid 0 vol%) to 709 mPa s (the second fluid 0.60 vol%) . SimilarIn order to evaluate changes in the hydrophobicity of the particle surface modified by the second fluid through method (I), the contact angles of the composite particles at both turning points in air were measured. As shown in The microstructures of suspensions prepared through different methods were observed using a CLSM . HydrophThe behavior of the secondary fluid in HGB suspension prepared by method (II) is shown in As the dosage of the second fluid was increased in method (I), the viscosity and yield stress of different particle suspensions displayed similar trends, namely an initial decrease followed by an increase and then a rapid decrease . The sol1819The kerosene content varied between the first and the second turning points, which induced an increase in suspension viscosity , the second fluid failed to form hydrophobic films and existed in the form of micro-droplets. This state is termed “isolated state” . The exiet al.et al.In this study, multiple effects of the second fluid addition on the rheology of particle suspensions containing dispersant were investigated. In method (I), the second fluid pre-coated particles and dramatically changed the rheological properties. When the dosage of the second fluid was increased, the viscosity of the suspension initially decreased and then increased, and lastly decreased again. The yield stress followed a similar trend. Three states  were observed by the characterizations, and found to follow the non-monotonic trends. The second fluid was found to reduce viscosity through the formation of hydrophobic films. A further increase in dosage resulted in an increase in viscosity, as composite particles adhered together to form liquid bridges and network structures. Higher dosages of the second fluid were found to rapidly reduce the viscosity due to the formation of cells, which exhibited lower hardness and wider size distributions. The trend obtained unifies the different discoveries from Koos Findings from this study suggest that method (I) may be a promising technology for industrial applications, due to its convenience and propensity to reduce the viscosity of suspensions. The viscosity can be selectively tuned over a narrow range of second fluid dosages. One such application for method (I) may be in the preparation of highly concentrated coal water slurries used in clean coal technology. This pre-coating method of the second fluid exploits a new way for industrial production.Hydrophilic hollow glass beads (HGB) were purchased from Suzhou Zeer Chemical Products Company (Jiangsu). Polyethylene (PE) was obtained from Yilufa Plastic Material Sales Department . Bituminous coal (BC) originated from Inner Mongolia. Methyl naphthalene sulfonate formaldehyde condensation (MF) was chosen as the dispersant, and the molecular structure is shown in HGB and PE particles were dried in an oven at 70 °C for 24 h. BC samples were dried at 105 °C for 24 h, and then comminuted using a ball mill. The resulting BC particles were then sieved through 40 and 200 mesh sieves to obtain two particle size distributions. Coarse and fine BC particles were mixed in a mass ratio of 6:4 for use in experiments.The blend of 1.0 wt% dispersant (dry solid particle basis) and weighted deionized water was added into HGB, PE, and BC, respectively. Suspensions were mixed for 20 min by vortex at 1000 rpm.As the second fluid, kerosene was added to particle mixtures using two methods: (I) First, kerosene was added into the dry solid particles in a flask and stirred for 30 min to ensure homogeneity. Particles coated by the second fluid were called composite particles. Then the composite particles were mixed with dispersant and deionized water for the final suspensions; (II) First, dry solid particles, dispersant, and deionized water were mixed and kerosene was added in proportion followed by sufficient agitation.Particle size was determined by automatic laser granularity analyzer  by suspending particles in ethanol and subjecting the suspension to ultrasonic diffusion.−1 to 100 s−1 over a period of 100 s. The value at the shear rate of 100 s−1 was used as the apparent viscosity of the suspension. The yield stress is defined as the stress at which the sample begins to deform plastically. Yield stress measurements were performed as follows: a shear stress sweep from high to low stress with 50 sample points over 500 s. Measurements were repeated three times to ensure that the results were reproducible.Rheological properties of suspensions were measured using a Bohlin CVO rheometer  at 25 ± 0.1 °C. The rheometer consisted of a cup centered on a turntable with a rotor concentrically suspended within it. The sample was placed in the annular space between the inner rotor and outer cylinder for measurement. The viscosity of suspensions was measured by logarithmically increasing the shear rate from 0.01 sThe microstructures of dried particles were observed using a scanning electron microscope (HITACH SU1510), as shown in Solid particles and a certain amount of kerosene were mixed to obtain composite particles, namely particles coated by kerosene. The composite particles were pressed into non-porous discs with a diameter of 13 mm and thickness of 1 mm at a pressure of 12.5 MPa. These discs were stored separately in valve bags to prevent volatilization prior to analysis. Three discs were made for each sample. Three measurements were conducted at different locations on each disc, thus obtaining the mean values of nine measurement results for each particle type.Contact angles were water contact angles in air, which were measured using a static drop method with an optical tensionmeter (Theta Lite). Photographs were taken at the interface of the disc and water and contact angles were obtained using analytical software.How to cite this article: Zhang, J. et al. Multiple Effects of the Second Fluid on Suspension Viscosity. Sci. Rep.5, 16058; doi: 10.1038/srep16058 (2015).


Exact expressions of velocity, temperature and mass concentration have been calculated for free convective flow of fractional MHD viscous fluid over an oscillating plate. Expressions of velocity have been obtained both for sine and cosine oscillations of plate. Corresponding fractional differential equations have been solved by using Laplace transform and inverse Laplace transform. The expression of temperature and mass concentration have been presented in the form of Fox-H function and in the form of general Wright function, respectively and velocity is presented in the form of integral solutions using Generalized function. Some limiting cases of fluid and fractional parameters have been discussed to retrieve some solutions present in literature. The influence of thermal radiation, mass diffusion and fractional parameters on fluid flow has been analyzed through graphical illustrations. A great deal of efforts have been put to study the phenomena of mass transfer and radiative heat flux in free convective flows because of its applications in many industrial and chemical processes. In principal, many physical and biological configurations and working owe their appearance and existence to mass and heat transfer. In manufacturing and chemical industry, many processes include radiative heat flows such as construction of satellites, oil and other chemicals’ filtration, construction of accessorizes using solar power, nuclear power processes, fuel combustion and drying of porous mediums’, etc. The process of mass transfer is very similar to heat flow in fluids and this similarity has prompted researchers to study both processes simultaneously. In particular, for the cases of low mass transfer and for low concentration in the fluid, the process of mass transfer and heat flow behave in almost the same way. The study of combined effects of mass transfer and radiation of heat in fluid flow has become a central topic of investigation in recent times. Furthermore, the study of motion of magnetohydrodynamic (MHD) fluid bears its own importance in the context of its vast use in many industrial and nuclear processes.Exact solutions for various Newtonian and non Newtonian fluids over oscillating bodies for various physical settings have been calculated. Penton  seems toSimilarly many authors have worked on determining exact solutions for free convection flows. Soundalgekar  was firsBut the exact solutions of constitutive models of fluid motion with fractional derivatives are rare in present literature. The study of effects of radiation and mass transfer on free convective flows using fractional calculus tools is even more rare and is a motivation for present investigation. Fractional constitutive relationship model has an advantage over customary constitutive relationship model as it readily assesses the properties of viscous and molecular mediums that are sometimes overlooked by ordinary derivative models. Fractional calculus approach is useful when it comes to generalization of complex dynamics of fluid motion. Many authors ,T, C,g, D are velocity of the fluid, its temperature, species concentration in the fluid, kinematic viscosity, gravitational acceleration, coefficient of thermal expansion, coefficient of expansion with concentration, thermal conductivity of the fluid, radiative heat flux, specific heat at constant pressure, density of fluid and mass diffusion coefficient, respectively.Above assumptions and Boussinesq’s approximation lead to the following set of governing equations of unsteady flowAlso in Eq. , \documeFollowing Cogly-Vincentine-Gilles equilibrium model based on assumption of optically thin medium with relative low density, we haveIntroducing Eq.  in Eq. , we havementclass2pt{minim of Eqs.  and 8)  8\docume of Eqs.  and 6),,8\documeM and F are Prandtl number, Schmidth number, thermal Grashof number, mass Grashof number, Hartmann number and dimensionless thermal radiation parameter, respectively.The following dimensionless quantities have been introducedUsing dimension less quantities  in goverTo obtain analytical formulas for velocity, temperature and concentration, we use fractional derivative approach. In particular, we consider Caputo fractional differential operator. Equations , 11) an an11) anPodlubny \documentAnalytical solutions will be obtained by means of Laplace transform and inverse Laplace transform.C.Applying Laplace transform to Eq.  and usinC, we write Eq. 21\documeu, we apply discrete Laplace transform to Eq. . Also, We can also write the above expression in terms of Fox-H function,m to Eq.  and obtaving Eq. , 22) an an25\docm to Eq. , 48), 30\documey, t) solutions of governing equations in ordinary differential operator. Some significant limiting cases have been discussed below.For  in Eqs. , 22) an an\docum in Eqs. , we can The absence of magnetic field i.e. 1 in Eq.  lead to m to Eq.  and 51)\document-s)dsEq.  and 53)\documenterfc(.) represents complementary error function.Assuming radiative heat flux to be constant along y-direction of plate, F = 0 \documentM, F, Many interesting physical aspects of radiative heat flow have been brought into light through graphs. These graphs also represent the influence of physical parameters t ad for fixed values of M, F, f and t. Also, it can be seen in both graphs that velocity is vanishing for higher values of y as was expected because the impact of oscillations on fluid gets weaker as fluid gets farther away from the plate.Figures M and other fluid and fractional parameters are taken to be fixed. Besides the different shape of velocity profiles for sine oscillations and cosine oscillations, it is observed that velocity increases with increase in M. The influence of parameters M, F and M and thermal radiation parameter, F even if the pattern of velocity profiles is different for sine and cosine oscillations. It is also noted that velocity responds to the changes of M faster than the changes in F.Figures F i.e. increasing values of F decrease the temperature of fluid.Figures It can be seen from Figs. mentclass2pt{minimmentclasspt{minimap is a positive integer,r] denotes the integer part of the real number r. According to Table Finally, in order to have a clearer idea about the accuracy of analytical solutions that have been established, a comparison between the numerical and analytical results was prepared for concentration. The corresponding results have been included in Table Stehfest  for calcStehfest . This almentclass2pt{minimExact solutions have been calculated for fractional MHD free convective viscous fluid over a vertical oscillating plate and influence of thermal radiation and mass diffusion on fluid motion have been analyzed. Expressions of velocity field, temperature and mass concentration of fluid have been obtained by applying Laplace transform to fractional differential equations governing present fluid flow problem. In particular, Caputo fractional differential operator is favored and motivation to employ fractional calculus tool is generalization of dynamics of such fluid flow problems.Expressions of velocity field have been obtained for both sine and cosine oscillations of plate and are presented in series form and in the form of integral solutions. The part of velocity corresponding to oscillations of plate is nicely presented in the form of Fox- H function and the part of velocity corresponding to thermal radiation, mass diffusion and magnetic field effects has been presented in integral solutions form, employing the concept of Generalized function. The expression of mass concentration of fractional MHD fluid has been presented in the form of general Wright function and the exact expression of temperature is written in the form of Fox- H function form.All solutions satisfy initial and boundary conditions.Some significant limiting cases of fractional and fluid parameters have also been taken into account and expressions of mass concentration and temperature, present in literature, have been retrieved for t, eventually.The velocity of fluid for both sine and cosine oscillations increase with increasing M, thermal radiation parameter, F and Schmidth number, The velocity has inverse relation with fluid parameters Hartmann number, F.Temperature of fluid increases for decreasing values of Prandtl number, Mass concentration of fluid is negatively influenced by Schmidth number, A contrasting behavior of velocity profiles for different values of oscillating frequency, The influence of fractional parameters on fluid motion is also studied through graphs. These graphs show that for decreasing values of C, the accuracy of obtained analytical solutions has been checked by making a comparison between the numerical and analytical results. Numerical data is in good agreement with analytical results. Same can be done for temperature and velocity field.For concentration of fluid To analyze the behavior and influence of fluid and fractional parameters on free convective flow, graphs of velocity, temperature and mass concentration have been drawn and following observations are made:


The following information is missing from the Acknowledgements section: The research leading to these results has partly received funding from the EU-FP7-SOCIOEC project under grant agreement No. 289192.


We newly developed an in situ hybridization (ISH) method to provide early and direct evidence of bacterial infection in ascites in patients with SBP. This paper will review the diagnosis of SBP, including our novel approach with ISH method to detect bacterial DNA in SBP ascitic fluid.Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication in cirrhotic patients with ascites. Although identifying the pathogen(s) plays a major role in the management of infectious diseases, ascitic fluid cultures often show negative results in patients with clinical signs and symptoms of SBP, and ascitic fluid cell analyses are the gold standard method for diagnosing SBP. SBP is generally diagnosed based on an increased number of polymorphonuclear neutrophils in the ascitic fluid (>250/mm Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae, are able to efficiently translocate from the lumen of the gut to the mesenteric lymph nodes [Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication in patients with cirrhosis and ascites. SBP is a bacterial infection that occurs in the absence of an evident intra-abdominal and surgically treatable source of infection, such as the perforation or inflammation of intra-abdominal organs –4. Althoph nodes , 6. Sinc3) without an evident intra-abdominal surgically treatable source of infection [3) PMN count in the ascitic fluid in cases in which there is no obvious source of bacteria spreading to the ascites, regardless of whether ascitic fluid cultures are positive [ in situ hybridization (ISH) method for detecting bacterial DNA in the ascites of patients with SBP.The prevalence of SBP in cirrhotic hospitalized patients with ascites ranges from 10% to 30% , 7. Althnfection . Althougnfection , 10. Thepositive , 11–13. Paracentesis is extremely important, as the PMN count in the ascitic fluid plays an essential role in obtaining a diagnosis of SBP. Diagnostic paracentesis should be performed in all patients who present with 1) compatible signs or symptoms ; (2) impairment of the hepatic or renal function; (3) unexplained hepatic encephalopathy; (4) gastrointestinal bleeding , 11–13.  compatib3 in the absence of an intra-abdominal and surgically treatable source of infection. The cutoff value of 250 PMN cells/mm3 has the greatest sensitivity, whereas 500 PMN cells/mm3 exhibits the greatest specificity [3  in order to adjust for the presence of blood in the ascites [Despite the use of a sensitive method , ascites cultures often show negative results, even in patients with an increased ascitic PMN count and clinical symptoms suggestive of SBP , 10, 18.cificity –21. Howe ascites , 11–13.The PMN count in the ascitic fluid may be determined according to a hematological method using either a light microscope and manual counting chamber or an automated cell counter –24. The 3). Therefore, it is recommended to inoculate the ascitic fluid into blood culture bottles at the patient's bedside in order to increase the sensitivity of the bacterial culture [3) and negative cultures exhibit a clinical presentation similar to that of bacteriologically confirmed SBP [Conventional bacterial culture methods, such as laboratory analyses of fluid collected in syringes or tubes, effectively detect bacteria in less than 50% of ascites samples with an elevated PMN count  and (2) at least two of the following: a total protein level of >1 g/dL, a serum lactate dehydrogenase level above the upper limit of normal, and a glucose level of <50 mg/dL. In addition, both an alkaline phosphatase level of >240 U/l and carcinoembryonic antigen level of >5 ng/mL in the ascitic fluid have been reported to exhibit good diagnostic performance for detecting gut perforation into the ascitic fluid, with a sensitivity of 92% and specificity of 88% [Various parameters available at the time of paracentesis have been proposed to assist in rapidly detecting secondary peritonitis. Parameters in the ascitic fluid in patients with secondary peritonitis, as proposed by Runyon and Hoefs , are as y of 88% . Howevery of 88% , 34, 35.It takes several hours to obtain the results of an ascitic fluid cell count. Therefore, the use of leukocyte reagent strips (LERS) has been proposed as a fast and inexpensive method for diagnosing SBP. These reagent strips, which were originally developed to diagnose urinary tract infections, detect leukocytes based on their esterase activity according to a colorimetric method . However3; however, the diagnostic performance of a reagent strip test calibrated for ascitic fluid with a cutoff of 250 PMN cells/mm3 has recently been reported [Most of the above strips were developed for use in urine with a threshold of >50 PMN cells/mmreported . That stThe levels of proteins, such as granulocyte elastase  and lactBacterial cultures require several days to obtain results. Hence, bacterial DNA detection and sequencing is increasingly being used to diagnose various infectious diseases –45. SomeA new strategy using an ISH method for detecting the genomic DNA of bacteria phagocytized in neutrophils and macrophages was recently developed to identify causal bacteria in cases of sepsis –56. The The concept of the ISH method for detecting bacterial DNA in SBP patients with ascites is shown in Several cDNA fragments corresponding to the 23S rRNA genes of various bacteria were obtained using PCR. Since we found no single cDNA probe able to detect all types of bacteria universally, we mixed plural cDNA fragments to create a new probe cocktail. This cocktail was able to detect the genomic DNA of all 59 bacterial strains examined, including the leading species accounting for SBP . The newThe ISH tests showed positive results in 10 of 11 SBP ascites samples, whereas negative results were obtained in all remaining 40 non-SBP ascites samples. These findings suggest that the ISH test yields high sensitivity (91%) and specificity (100%) for detecting phagocytized bacterial DNA in leukocytes the ascites of SBP patients. Importantly, the ISH tests showed positive findings in seven cases with negative culture results, thus suggesting that the ISH method can be used to identify bacterial infections that are not detected using the bacterial culture method . FurtherRapid diagnosis and treatment play a key role in the management of SBP. In general, potentially fatal cirrhosis-related infectious disease is diagnosed based only on an increased PMN count in the ascitic fluid, and the identification of the causal pathogen is sometimes not taken into consideration. Although no ideal method for detecting causal bacteria has been established, our novel ISH test may be used to provide early and direct evidence of bacterial infection in SBP patients with ascites. The current findings therefore shed new light on the management of SBP.


Both cetuximab and bevacizumab inhibited tumour growth in vivo, and this effect was associated with selectively perturbed glucose metabolism and reduced hypoxic volumes based on PET/MRI imaging. Global proteomic profiling of xenograft tumours  revealed alterations in proteins involved in glucose, lipid and fatty acid metabolism , as well as hypoxic regulators and vasculogenesis . These findings correlated with western immunoblotting (xenograft lysates) and histological examination by immunohistochemistry. These results define important mechanistic insight into the dynamic changes in metabolic and hypoxic response pathways in colorectal tumours following treatment with cetuximab and bevacizumab, and highlight the ability of these therapies to selectively impact on tumour cells and extracellular microenvironment.Angiogenesis and epidermal growth factor receptor (EGFR) inhibition has been shown to have anti-tumour efficacy, and enhance the therapeutic effects of cytotoxic chemotherapy in metastatic colorectal cancer. The interplay of signalling alterations and changes in metabolism and hypoxia in tumours following anti-VEGF and anti-EGFR treatment is not well understood. We aimed to explore the pharmacodynamics of cetuximab and bevacizumab treatment in human colon carcinoma tumour cells  Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death in men and women worldwide, with nearly 1.4 million new cases diagnosed in 2012, representing 9.7% of cancers worldwide . Early sK-Ras tumours. These targeted agents, now validated in mCRC and other tumour types, target and perturb critical cell-signalling pathways that regulate (stimulate) tumour angiogenesis and growth.Significant advances have been made in the treatment of metastatic colorectal cancer (mCRC) . RefinemBevacizumab disrupts angiogenesis by binding to vascular endothelial growth factor (VEGF)-A, reducing availability of this ligand to its receptors, and preventing their activation. Based on pre-clinical models, bevacizumab has been shown to have both anti-vascular and anti-angiogenic effects, resulting in regression of existing tumour vasculature -13, and K-Ras are associated with resistance to cetuximab therapy  for 10 h in TTBS  at 4°C, followed by incubation with either IRDye 800 goat anti-mouse IgG or IRDye 700 goat anti-rabbit IgG . Fluorescent signals were detected using the Odyssey Infrared Imaging System, .® (C18) 150 × 0.15-mm i.d. reversed phase UPLC column (Waters), using an Agilent 1200 HPLC, coupled online to an LTQ-Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) [Proteomic experiments were performed in duplicate. Tumour xenograft lysates (20 μg) were separated using SDS-PAGE and proteins visualized by Imperial™ Protein Stain (Thermo Fisher Scientific). Each gel lane was cut into 17 individual gel bands (1-2 mm width) and individual gel slices destained (50 mM ammonium bicarbonate/acetonitrile), reduced  for 30 min), alkylated (50 mM iodoacetic acid (Fluka) for 30 min) and trypsinized (0.2 μg trypsin (Promega Sequencing Grade) for 16 h at 37°C), as described , 53, 54.entific) . The colentific) . Up to fwww.proteinatlas.org) was used as an annotated resource to assess the tissue expression of proteins identified in this study [Raw data were processed using Proteome Discoverer  and searched with Mascot , Sequest , and X! Tandem (v2010.12.01.1) against a database of 125,803 ORFs . Data was searched with a parent tolerance of 10 ppm, fragment tolerance of 0.6 Da, minimum peptide length 6, maximum peptide length 144, and max delta CN 0.05. Peptide lists were generated from a tryptic digestion with up to two missed cleavages, carbamidomethylation of cysteines as fixed modifications, and oxidation of methionines and protein N-terminal acetylation as variable modifications. Peptide spectral matches (PSM) were validated using Percolator based on q-values at a 1% false discovery rate (FDR) , 57. Witis study .Significant spectral count normalised (Nsc) and fold change ratios (Rsc) were determined as previously described , 52, 54.t-tests (GraphPad v5.0) were calculated, with *p < 0.05 and **p < 0.01 considered statistically significant.Student's