Blood vessels are important tissue structures that deliver oxygen and nutrition. In tumour tissue, abnormal blood vessels, which are hyperpermeable and immature, are often formed; these tissues also have irregular vascularisation and intravasation. This situation leads to hypoperfusion in tumour tissue along with low oxygen and nutrition depletion; this is also called the tumour microenvironment and is characterised by hypoxia, depleted nutrition, low pH and high interstitial pressure. This environment induces resistance to anticancer drugs, which causes an increase in anticancer drug doses, leading to increased side effects. We hypothesised that normalised tumour blood vessels would improve tumour tissue perfusion, resupply nutrition and re-oxygenate the tumour tissue. Chemotherapy would then be more effective and cause a decrease in anticancer drug doses. Here we report a neovascularisation-inducing drug that improved tumour vascular abnormalities, such as low blood flow, blood leakage and abnormal vessel structure. These results could lead to not only an increased chemo-sensitivity and tissue-drug distribution but also an up-regulated efficiency for cancer chemotherapy. This suggests that tumour blood vessel normalisation therapy accompanied by angiogenesis may be a novel strategy for cancer therapy. Several studies have shown that tumour vessels are different from normal microvessels in both structure and functionHypoxia-inducible factors (HIFs) are key transcriptional factors that are cellular adaptive responses to hypoxia. They play a pivotal pro-survival role in ischaemic tissues. Therefore, there have been numerous reports of attempts to overcome ischaemic conditions by stabilising HIFs1011The aim of this study was to assess whether PHD inhibitor administration induced normalisation of tumour vessel features, such as the vessel lumen, tight junction formations and pericyte coverage. We examined not only the functional features associated with the tumour microenvironment, such as tissue perfusion, vessel permeability and hypoxia, but also the features resulting in chemotherapy efficiency. Here we show that transient PHD inhibition led to the normalisation of tumour blood vessels, which could enhance anticancer drug sensitisation in a tumour mouse model.To study the effects of PHD inhibitor on tumour blood vessels, we administered DMOG to Lewis lung carcinoma (LLC) model mice. Microvessels consist of a single layer of endothelial cells and pericytes covered with an endothelial layer on the outside. However, tumour blood vessels usually lack structure. Therefore, we focused on the tumour blood vessels, particularly the endothelial cells (ECs), which were stained for CD31. The DMOG treatment drastically changed aspects of the tumour blood vessels; this slightly increased the CD31-positive area, markedly increased the average vessel diameter and decreased the blood vessel density . MoreoveWe then evaluated whether or not those reconstituted tumour blood vessels had normal structures. In general, normal microvessels consisted of an EC layer covered with pericytes, and the ECs formed tight junctions between each other; however, tumour blood vessels were poorly covered with pericytes. Representative fluorescent images for NG-2, a marker for pericytes, are shown in To confirm whether the reconstituted tumour vessel was functionally normal, we evaluated the tissue blood perfusion, vessel leakage and drug distribution. We used high molecular weight fluorescence-labelled Dextran (200 K), which did not leak from normal vessels. Tumour tissue perfusion was recovered after DMOG treatment . The leaHypoxic regions, which were found in tumour tissues, were feasible environments for tumour progression, and tumour cells with chemo- and radio-resistance had a poor prognosis. To evaluate if vessel normalisation improved the hypoxic environment in the tumour, a hypoxia probe was administered to tumour-bearing mice to detect regions of tissue hypoxia. Hypoxic regions in the tumour tissue are shown with immunofluorescence staining in To assess the anti-cancer drug activity, we evaluated the results of vessel normalisation and functional improvement after DMOG treatment. Typically, overexpression of HIFs is considered a bad prognostic factor; therefore, we assessed if DMOG treatment itself affects tumour growth, metastases, cell proliferation and survival term; consequently, DMOG treatment was not found to affect any of them . The micOur results demonstrated that tumour blood vessel normalisation accompanied by angiogenesis after treatment with PHD inhibitor led to the repair of sensitivity to chemotherapy based on the structural alterations in the tumour vessels and promoted tight junction formation and pericyte coverage.Blood vessel ECs are equipped with oxygen sensors. The signalling pathways for HIFs, such as PHD2 and HIF2α, allow the vessels to readjust their shape to optimise blood flow13Lacks of pericytes and tight junction formation in the blood vessels are popular features of tumour vessels, unlike normal vessels. These were regulated by several factors, such as VEGF, which was controlled by PHD2 in the ECsThese structural changes have often been accompanied by the normalisation of vessel function1193. In our study, CDDP administration was started when the mean tumour volume reached >1,000 mm3. In general, larger tumours exhibit more resistance to anticancer therapies than smaller tumours23Tumour microenvironments enhance resistance to anti-cancer therapies and radiation therapy; many chemotherapeutics rely on oxygen radical formation to kill cancer cells, and tumour hypoxia reduces their efficacyOriginally, we expected that PHD inhibitor treatment would promote tumour growth, but surprisingly, the growth was not stimulated after PHD treatment. The PHD inhibitory effect appeared to be transient; however, normalised vessels induced the oxygenation of the ischaemic tumour tissue, which enhanced the activation of innate and adaptive immune responses and cell death programsIn this study, we found that tumour vessel normalisation after the administration of a PHD inhibitor enhanced the efficacy of chemotherapy without accelerating the primary tumour growth. It may be an effective new strategy for anticancer treatments. In addition, it may be more beneficial for the treatment of large advanced cancers without increasing chemotherapy-associated adverse events.All animal experiments were approved by the Institutional Animal Care and Use Committee of Tottori University (approved number: 14-Y-12) that was approved by the Japanese Association for Accreditation for Laboratory Animal Care, and the animal research and handling were performed in strict conformance with federal Institutional Animal Care and Use Committee guidelines.ad libitum in a 12-h light–dark cycle at 22 ± 1 °C.The C57BL/6 female mice were obtained from CLEA Japan, Inc. and housed in cages with food and water available 2 and 95% room air at 37 °C.The LLC cells were maintained in Dulbecco’s Modified Eagle Medium containing 10% foetal bovine serum and penicillin/streptomycin in 5% CO7 cells/mL) in phosphate-buffered saline (PBS). Some of the cells (1 × 106 cells) were subcutaneously transplanted into the right flank of the mice which were aged at 8–12 weeks. The mice were treated with 400 mg/kg DMOG  or 50 mg/kg FG4592  intraperitoneally 10 days after the tumour transplant. Once every two days, the tumours were measured in two dimensions using a calliper. The tumour tissue volume was calculated using the formula: V = π (length × width2)/6. The mice were sacrificed at a defined time point or when the tumour volume reached 4500 mm3.These cells were harvested and re-suspended  , anti-NG2 (1:400) , anti-ZO-1 (1:400) , anti-CC3 (1:400)  and anti-The tumour tissues were sliced into frozen sections of 8-μm thickness at −20 °C and were dried in air. The sectioned tissue samples were rehydrated with PBS for 10 min and fixed with 4% (w/v) cold paraformaldehyde for 10 min. The sections were washed with PBS and permeabilised with 0.5% (v/v) TritonThe quantification of the immunofluorescent images which were taken using a microscope  was performed using its application software. At least 15 fields of images at 100, 200 or 400 magnification were analysed for each sample. Each experiment used at least three animals from each group.Dextran–FITC  was intravenously administered to a tail vein 10 min prior to sacrifice. The tumour tissues were excised and immediately frozen with liquid nitrogen. The tumour samples were stored at −80 °C until the sectioning. The tumour tissues were partitioned into sections of 8- and 20-μm thickness and were observed using a fluorescent microscope. Furthermore, the 8-μm sections were prepared for immunofluorescence staining with anti-CD31 antibody and were evaluated for vessel permeabilisation.Drug distribution in the tumour tissues was evaluated using Hoechst 33342 . The tumour-bearing mice, which had been treated with or without DMOG 6 days prior, were injected with Hoechst 33342 50 mg/kg in PBS into the tail vein 10 min prior to sacrifice. The tumours were excised and immediately frozen with liquid nitrogen. The tumour samples were stored at −80 °C until sectioning. The tumour tissues were then partitioned into frozen sections of 20-μm thickness. The sectioned samples were observed under a fluorescent microscope, images were obtained and we quantified the percentage of the Hoechst-positive area.The hypoxic regions of the tumour tissues were detected using the Hypoxyprobe™ kit , which included pimonidazole, as per the manufacturer’s protocol. In brief, pimonidazole was injected at 60 mg/kg into the tumour-bearing mice. Ninety minutes later, the mice were sacrificed; we collected the tumours and cryosectioned the tissue into 4-μm thick slices. The sections were fixed with ice-cold acetone for 10 min, washed with PBS and then incubated with rabbit anti-pimonidazole anti-sera (1:20) overnight at 4 °C. The sections were then incubated with AlexaFluor 488 conjugated goat anti-rabbit antibody (1:1000) for 1 h. The images were obtained with the KEYENCE BZ-9000 microscope, the ratio of the pimonidazole-positive area was quantified using the equipped software.The sensitivity of cancer chemotherapy was assesses with CDDP administration. Tumour-bearing mice had been treated with or without DMOG; 6 days later, these mice were treated with or without 2.5 mg/kg cisplatin  intraperitoneally for 3 days in a row. The tumours were measured once every two days after the DMOG administration until a defined time point. The tumour volume was calculated using the formula described above. The apoptosis and DNA damage analysis in the tumour tissue was evaluated with CC3 and γH2AX at 48 h after the final administration of CDDP. The nuclei of the tumour cells that had been treated with CC3, and the γH2AX foci were quantified as a percentage of the total nuclei within 15 fields of view in a magnification of 400.p value of <0.05, but values of p < 0.01, p < 0.001 and p < 0.0001 are also shown to indicate the level of confidence.Statistical analyses were performed using the unpaired t-test followed by a Bonferroni’s post-hoc test. All statistical analyses were performed using the GraphPad Prism (version 6.02) software. Statistical significance was defined as a How to cite this article: Koyama, S. et al. Tumour blood vessel normalisation by prolyl hydroxylase inhibitor repaired sensitivity to chemotherapy in a tumour mouse model. Sci. Rep.7, 45621; doi: 10.1038/srep45621 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Fluid overdose can be harmful in critically ill patients. Since central venous pressure (CVP) is currently considered to be an inappropriate indicator of preload, much attention is being given to predicting fluid responsiveness, i.e., the response of stroke volume (SV) or cardiac output (CO) to fluid challenge. However, when fluid responsiveness was evaluated in critically ill patients, including sepsis, only 40–50% of the patients responded. Moreover, most fluid responders do not show significant hemodynamic improvement after fluid administration. In this review, we discuss why fluid responsiveness based on the Starling mechanism did not work well in the clinical setting.According to the Starling mechanism, a patient whose SV/CO significantly increases after a fluid challenge is considered to be a fluid responder and judged to need fluid therapy. However, the currently recommended fluid challenge dose of crystalloid 250–500 mL has little effect on increasing blood volume and is not sufficient to increase the preload of the Starling curve. Especially in septic patients, due to their vascular hyperpermeability, increase in blood volume is even smaller. Furthermore, Infusion induced hemodilution is known to reduce blood viscosity and hematocrit, as a result, decreasing afterload. This indicates that the increased SV/CO after fluid challenge is caused not only by increased preload but also by decreased afterload. For these reasons, fluid responsiveness with small crystalloid challenge is questionable as a clinical indicator of fluid therapy. Fluid therapy has been used to prevent or to treat circulatory failure. However, excessive fluid in critically ill patients has been recognized to cause cardiac complications, including pulmonary edema and heart failure , 2. It iThe venous blood is theoretically separated into stressed and unstressed volume . The unsHemodynamic monitoring has been used to guide fluid therapy. Central venous pressure (CVP) has been used as an indicator for fluid therapy for a long time. Since the compliance of the vein is 30 times higher than that of the artery , CVP varInstead of CVP, attention is being paid to the assessment of fluid responsiveness . Fluid rIn the 1920s, E.H. Starling, an English physiologist, conducted a study on the heart and lungs of dogs and showed that the CO increased as the right atrial filling pressure increased by elevating the height of the venous blood reservoir . He alsoIf rapid fluid challenge improves SV/CO, the fluid responsiveness is considered positive. The patient is considered hypovolemic, and both CO and tissue perfusion are expected to increase by fluid therapy Fig. .If SV is not improved by fluid challenge, the fluid responsiveness is considered negative. Aggressive fluid therapy in this situation increases the risks for both pulmonary edema and heart failure Fig. .If left ventricular function is decreased, the SV/CO response is minimal even if the preload is increased, consequently, the above interpretation cannot be applied, Fig. .The fluid responsiveness can be explained using the Starling curve as follows .If rapidThe SV/CO changes due to fluid challenge have been evaluated using both pulmonary artery catheterization and Doppler transesophageal echocardiography. Recently, devices to measure stroke volume variation (SVV) and pulse pressure variation (PVV) have been developed to evaluate fluid responsiveness . SVV andDose the right atrial filling pressure increase in parallel with the infused volume?Fluid challenge with 6 mL/kg (250–500 mL) of crystalloid for 15 min is currently recommended, and patients with an SV increase of 10–15% are determined to be fluid responders . HoweverTo explain the fluid responsiveness using the Starling curve, preload or right filling pressure is used as the horizontal axis and stroke volume as the longitudinal axis. However, except for patients after cardiac surgery, the right filling pressure cannot be measured. Fluid responsiveness was evaluated under the assumption that the preload was increased by the fluid challenge. But, it is not clear whether the administered fluid increases preload in a volume-dependent manner.A clinical study showed that when 1.5 L of lactated Ringer’s solution was infused for 30 min prior to cesarean delivery, only 25% of the infused solution remained in the blood and the fluid increased the blood volume only by 7% . Since bFurthermore, individual differences in the effect of infusion on blood volume have also been found. Svensén et al. administered 25 mL/kg of lactated Ringer’s solution to patients who underwent abdominal surgery for 45 min during surgery and found that 40% (4/10) of the patients were fluid responders in whom the CO increased, and the others were non-responders . Ueyama Sepsis is characterized by diffuse endothelial injury and shedding of the endothelial glycocalyx layer, which induces capillary hyperpermeability. Consequently, crystalloid and colloid solutions cannot be expected to remain in the intravascular space of septic patients. Studies have shown that only 5% or less of the crystalloid infusion remained in the intravascular volume after 1 h in septic patients , 16.Decreased Afterload by Fluid-Induced HemodilutionIn summary, the blood volume expansion effect of 250–500 mL of crystalloid is not only insufficient but also variable among individuals, so this menu is inappropriate for fluid challenge. Furthermore, the mathematical effect of fluid challenge does not translate to septic patients because the blood volume expansion effect of fluid was less in septic patients. Therefore, problems remain with the use of the SV/CO response to small amounts of infusion.CO is affected not only by preload but also by afterload. In Starling’s experiment, the blood was used as preload. However, unlike the blood, fluid reduces afterload, i.e., systemic vascular resistance (SVR) by hemodilution.Hemodilution decreases blood viscosity and hematocrit.When the blood is diluted, the thickness of the plasma layer in the arteriole increases, leading to decreased vascular resistance with the arteriolar wall , 18.Infusion is considered to decrease SVR due to the following effects on blood flow characteristics.When colloid was administered to patients who underwent peripheral vascular surgery, the CO actually increased but the SVR and mean arterial pressure (MAP) decreased .In septic patients, hemodilution due to volume preload also induced a decrease in SVR. Monge-Garcia et al. administered 500 mL of either crystalloid or colloid for 30 min and evaluated the SV/CO using Doppler transesophageal echocardiography and reported a 10% decrease in the SVR by infusion in fluid responders (67%) .Approximately 50% of septic patients develop myocardial damage caused by inflammatory cytokines and by vascular hyperpermeability earlier at onset, and cardiac function decreases at the early stage of sepsis . In the Does infusion increase SV/CO similar to the Starling curve?Hence, decreased afterload due to hemodilution has a large impact on the interpretation of the fluid responsiveness in fluid challenge.If fluid administration behaved like the Starling curve, the increase in blood volume by infusion should linearly increase CO. However, this does not agree with the past findings that evaluated the relationship between percent change in blood volume and in CO after crystalloid or colloid administration in parturients . This reOne of the objectives of fluid therapy for critically ill patients is to increase both blood pressure and tissue perfusion pressure by increasing the CO. MAP is defined as follows.−5) × CO (L/min) ÷ 80MAP (mmHg) = SVR  in 2001 by Rivers et al. , the morIt is not clear whether fluid responsiveness guided fluid therapy improve the prognosis of critically ill patients or not. However, as fluid responders are recommended to repeat fluid therapy until responsiveness disappears, it may induce overhydration and worsen the prognosis. Furthermore, fluid challenge itself is considered to be harmful in some situations. For instance, when a patient falls into hypotension five times a day, a total of 2500 ml of fluid is required for five times 500 ml of fluid challenge . For thiInstead of fluid challenge, some authors report the effectiveness of the passive leg raising (PLR) . This maFrom these results, indication of fluid therapy needs to be judged from various examinations and physical findings.Starling’s law explains that shifting blood from unstressed volume to stressed volume restores decreased SV/CO. Since Starling’s law does not take into consideration the decrease of afterload due to fluid-induced hemodilution, it is not an appropriate model to explain fluid responsiveness.Like watering a plant, infusion replenishes the whole body and circulates through the whole body and does not remain in a particular compartment. Liquid taken orally is absorbed in the intestine and retained in the interstitium and then spreads from the blood vessels to the cells. As this is the case, the storage reservoir of fluid is the interstitium, not the blood vessels. It is only natural that infused solution moves rapidly from the blood vessels to the interstitial space. Thus, the effect of fluid on SV/CO is short-lived and limited.The effect of fluid challenge on SV/CO is more complicated than we thought, and interpretation is difficult. In my opinion, fluid responsiveness based on the Starling curve would not work in the past, present, and future.


Tumour progression is dependent on the interaction between tumour cells and cells of the surrounding microenvironment. The tumour is a dynamic milieu consisting of various cell types such as endothelial cells, fibroblasts, cells of the immune system and mesenchymal stem cells (MSCs). MSCs are multipotent stromal cells that are known to reside in various areas such as the bone marrow, fat and dental pulp. MSCs have been found to migrate towards inflammatory sites and studies have shown that they also migrate towards and incorporate into the tumour. The key question is how they interact there. MSCs may interact with tumour cells through paracrine signalling. On the other hand, MSCs have the capacity to differentiate to various cell types such as osteocytes, chondrocytes and adipocytes and it is possible that MSCs differentiate at the site of the tumour. More recently it has been shown that cross-talk between tumour cells and MSCs has been shown to increase metastatic potential and promote epithelial-to-mesenchymal transition. This review will focus on the role of MSCs in tumour development at various stages of progression from growth of the primary tumour to the establishment of distant metastasis. It is now understood that tumour cells do not act alone. Cancer cells interact with their surrounding stroma and these interactions lead to an ‘activated state’ resulting in increased release of pro-inflammatory cytokines and growth factors . The tumThe tumour stroma varies between each cancer type and the heterogeneous nature of the tumour makes it complicated to study. It is important to develop an understanding of what drives non-cancerous cells toward an activated state, what that activated state is and what it subsequently means for tumour cell progression.MSCs are multipotent stem cells originally found to have the capacity to differentiate into the tri-lineages - osteoblasts, chondrocytes and adipocytes . They arMSCs reside in the bone marrow stroma alongside haematopoietic stem cells (HSCs), osteoblasts, osteoclasts, adipocytes, endothelial cells (ECs) and monocytes , 21. MSCKnowledge of these characteristics as well as their differentiation capacity has caused excitement in the field of regenerative medicine and use of MSCs has potential for therapeutics in a range of fields such as cardiology, immunology and neurology. However, in the field of cancer research many studies suggest that MSC activity may contribute to poorer outcomes –27.In recent studies, it has been shown that MSCs can also home to tumour sites and contribute to tumour growth and progression –29. AnalMSCs have been implicated in the promotion of tumour growth in numerous cancer types such as follicular lymphoma , head anKarnoub and colleagues showed that co-injection of human bone marrow MSCs with only one of four breast cancer cell lines (MCF7) into mice led to accelerated tumour growth, however, co-injection with all cell lines  led to increased metastasis . SimilarAn increase in tumour growth was also found in mice following co-injection of human adipose tissue derived MSCs with the prostate cancer cell line MDA-PCa-118b . In anotSome research groups have investigated the tumour promoting function of MSCs isolated from the tumour, arguably a more realistic approach to understanding the role of MSCs within the tumour microenvironment. Co-injection of MSCs isolated from human head and neck carcinoma , gastricFrom the studies described thus far, we can ascertain that MSCs are important players in the promotion of tumour growth and progression. Key thoughts to consider at this point would be whether naïve MSCs can induce such an effect upon arrival at the tumour through paracrine signalling and cell-cell contact, or do tumour microenvironment exposed MSCs transition to a determined ‘activated’ or reprogrammed state. The studies discussed above describing MSCs directly isolated from the tumour give evidence to the latter whereby tumour derived MSCs differed in activity to naïve bone marrow derived MSCs. Taking this into account, future studies should consider further investigation into the functional and molecular differences that occur in MSCs isolated from various tumour types. Are they functionally, morphologically and molecularly the same or does it depend on the tumour source?In contrast to the research described above there is evidence to suggest that MSCs can also have an inhibitory effect on tumour growth. Suppression of tumour growth has been noted in breast cancer , Kaposi’MSCs are a heterogeneous population of cells containing subpopulations with differing differentiation capacities . MoreoveOtsu et al. showed that murine bone marrow MSCs had a cytotoxic effect on the tumour in a melanoma mouse model through the release of reactive oxygen species when in contact with ECs present at the capillaries. This induced apoptosis of the ECs and reduced tumour growth. However, the cytotoxic effect of the MSC was only observed when implanted at high concentrations. MSCs seeded onto EC derived capillaries in matrigel evoked a cytotoxic effect at a EC:MSC ratio of 1:1 or 1:3. Cytotoxicity decreased when the MSC number was reduced by an order of magnitude  and giveAnother explanation for the contrasting results is that like macrophages there is a polarisation of MSCs in response to secreted factors from the tumour that either drives the cells toward a tumour promoting or suppressive function. Tumour infiltrating macrophages can become induced by the stromal microenvironment and are referred to as tumour associated macrophages (TAMs) , 53. DepMSCs were previously found to express toll-like receptor (TLR)- 1, 2, 3, 4, 5 and 6 and TLR-agonist interaction stimulated MSC migration and immunomodulatory factor secretion . In partCancer associated fibroblasts (CAFs) are a heterogeneous population of fibroblast-like cells with a tumour promoting function. The heterogeneity may be due to varying cell origins and the molecular constitution of tumour stroma from which the cell fate is determined. CAFs have been found to originate from bone marrow MSCs, fibroblasts and by transdifferentiation of epithelial and endothelial cells –63. The 1 chain of type I (pro)collagen [Evidence to suggest CAFs can be derived from MSCs was found in in vivo studies whereby genetically tagged bone marrow derived cells, injected into mice, were found at the tumour site with myofibroblast morphology and expressing α smooth muscle actin (α-SMA) and the αcollagen –69. A sucollagen .Further evidence to support the hypothesis that CAFs can originate from MSCs comes from in vitro studies where MSCs are cultured long-term in tumour cell conditioned medium. In a study by Mishra et al. human MSCs were cultured for up to 30 days in the breast cancer cell line MDA-MB-231) conditioned medium . The res conditioOn the other hand, it must be noted that MSCs and CAFs share many similarities. A study has shown that CAFs share many of the same surface markers as MSCs such as CD29, CD44, CD73, CD90, CD106 and CD117, and have the capacity to differentiate to osteocytes, chondrocytes and adipocytes, and express vimentin . An inteMSCs interact with cancer cells at multiple stages of cancer progression. At the primary tumour MSCs have been shown to drive tumour cells toward an invasive, pro-metastatic state. Human MSCs injected alone into mice with mammary carcinoma xenografts resulted in a 42% occurrence of metastatic lesions, compared with 17% in the control treated mice . SimilarTracking of MSCs using magnetic resonance imaging in a mouse xenograft model has shown that MSCs were more likely to home to the lung metastatic site than to the primary tumour . A studyThe presence of MSCs in the tumour stroma may stimulate EMT of the cancer cells. Research has shown that direct co-culture of breast or gastric cancer cells with human bone marrow derived MSCs resulted in the upregulation of EMT markers N-cadherin, vimentin, Twist and Snail and the downregulation of E-cadherin , 81. CorA study by Karnoub and colleagues investigated the effect of MSCs on breast cancer cell motility and migration to the site of metastasis . Human bvβ1, αvβ3, αvβ5, αvβ6, α8β1 and α5β1, on many cell types [OPN is a chemoattractant with adhesive properties and can facilitate invasion through the binding of integrins, mainly αll types –88. Incrll types –92. OPN ll types . OPN facll types –98. Chanll types , 100.MSCs are bone marrow resident cells and given the poor prognosis in patients diagnosed with metastatic bone cancer, it is a key area in which to explore their role . MSCs plEntry of cancer cells into the bone marrow may be facilitated by MSCs through adherence of the metastatic cell to bone marrow ECs . SeveralCells of the bone marrow including HSCs, megakaryocytes, macrophages and myeloid-derived suppressor cells have been implicated in developing a hospitable metastatic niche . HoweverAn interesting study by Joseph et al. investigated the interaction between HSCs derived from the bone marrow of mice implanted with prostate cancer cell lines that formed either osteoblastic or osteolytic metastatic lesions. They found that HSCs derived from the mice with osteoblastic lesions stimulated osteoblastic differentiation of MSCs through BMP2 signalling, while HSCs derived from mice with osteolytic lesions enhanced the differentiation of mixed marrow mononuclear to osteoclasts through IL-6 signalling . It is tIL-6 is a pro-inflammatory cytokine that is known to mediate cell proliferation, cell survival and lymphocyte differentiation . IL-6 maIt is now understood that MSCs interact with and influence tumour cells at various stages of progression. It is not clear however, whether the effect is predominantly tumour promoting or suppressive. Explanations that could account for the conflicting results include differences in experimental design, the heterogeneity within the MSC population or varying responses dependent on the stimuli . NonethCancer therapies classically target tumour cells yet, what remains is an activated stroma that provides an encouraging microenvironment for any surviving tumour cells. Evidence to support this comes from studies in breast cancer in which stromal-related gene expression or gene signatures was predictive of clinical outcome , 116. Mo


This article provides the useful data information on multiscale modeling of liquid storage laminated composite cylindrical tank under seismic load. The investigation process is divided into three levels. Within the numerical homogenization, the hexagonal microstructure one-eighth and full RVE model is assumed on micro-scale level. Homogenization of unidirectional lamina is done in four steps. For effective material properties of the whole laminate, the laminated representative volume element is assumed on meso-scale level. The numerical homogenization is done by using the Finite Element Method in the program ANSYS. Effective modulus of elasticity is taken into the calculation of internal forces of the laminated composite cylindrical tank on macro-scale level. The base shears bottom of tank wall and tank base, the bending moment above the base plate, and overturning moment bellow the base plate as function of the tank fluid filling are presented. Data inform about seismic response of liquid laminated composite tank during seismic even in Slovak Republic, respecting of Eurocode 8 recommendations. Specifications tableValue of the data•Approach of numerical homogenization of unidirectional lamina for the hexagonal microstructure.•Interaction of fluid-laminated composite tank due to earthquake.•Comparison of base shears of tank wall bottom and tank bottom as function of the tank fluid filling.•Comparison of the bending moments above the base plate and overturning moments bellow the base plate depending on the tank fluid filling.11.1On the micro-scale level, the material properties of one laminated composite layer are obtained for an unidirectional fiber reinforced composite layer consists of isotropic fibers and isotropic matrix. The fiber volume fraction and fiber diameter were found from electron microscope digital shot. Each layer of the laminate has the same thickness. The material properties of each layer were used from obtained average stresses 1.2NS, where N=20 for the wall and for the base slab. The effective material properties of the whole laminate are observed from meso-scale level The circular cylindrical tank is made of the laminate [0/0/90/90]1.3ag=1.5 m s−2, B category of the subsoil. The seismic response data of fluid filled laminated composite cylindrical tank was obtained.Then, these material properties are used for calculation of internal forces of laminated composite ground supported cylindrical tank 2For heterogeneous materials, a large number of material properties are needed Within the numerical homogenization, the hexagonal microstructure was assumed. Four steps of numerical homogenization of unidirectional lamina were applied:Step 1Ci1 with i=1, 2, 3, , the strain x1. The coefficients Ci1 are found by using In order to determine the components Step 2Ci2 with i=1, 2, 3, , the strain x2. The coefficients Ci2 are found by using In order to determine the components Step 3Ci3 with i=1, 2, 3, , the strain x3. The coefficients Ci3 are found by using In order to determine the components Step 466, the strain C55 and C66 are found by using In order to determine the component CWe considered different fluid filling heights of tank: 0.5 m, 1 m, 1.5 m and 2 m.–comparison of the base shears components in fluid impulsive mode and fluid convective mode, and the base shear components of the tank wall and tank bottom depending on the tank fluid filling,–comparison of components of the bending and overturning moments in impulsive and convective mode of fluid, and moments components of tank wall and tank bottom as function of the tank fluid filling.V consists of the sum Vi, Vc and Vw and on the other side the total base shear at below the tank bottom V* comprises of the sum Vi, Vc, Vw and Vb. The total bending moment at the bottom of the wall M consists of the sum Mi, Mc and Mw, and the total overturning moment immediately bellow of the tank base plate M* is given as the sum of Mi, Mc, Mw and Mb. Index “i” means fluid impulsive mode component, “c” fluid convective mode component, “w” tank wall component, and “b” base bottom component.The total base shear at the bottom of the wall –V of tank wall bottom and tank bottom V* as function of the tank fluid filling,comparison of the total base shears –M and total overturning moments immediately bellow the base plate M* depending on the tank fluid filling.comparison of the total bending moment immediately above the base plate


Maternal COS inclusion had enhanced (P < 0.05) the foetal survival rate and size at 35 days. COS supplementation had both increased (P < 0.05) SOD, CAT and T-AOC activities and elevated (P < 0.05) IL-10, IgG and IgM concentrations in the amniotic fluid. Moreover, COS had affected (P < 0.05) the amniotic fluid’s lysine, citrate, glucose and hypoxanthine levels. Overall, COS inclusion induced amniotic fluid antioxidant status and metabolic profiles modifications characterising improvements in foetal survival and growth in a pig model.Foetal loss and intrauterine growth restriction are major problems in mammals, but there are few effective ways in preventing it. Intriguingly, chitosan oligosaccharide (COS), a biomaterial derived from chitosan, can promote foetal survival and growth. Therefore, we have investigated how COS affects foetal survival and growth in a pig model. Fifty-two sows were divided into two treatment groups (n = 26) and fed either solely a control diet or a control diet that includes 100 mg/kg COS. Amniotic fluid and foetus samples from six sows that were of average body weight in each group were collected on gestation day 35. We applied a  Chitosan oligosaccharide (COS), which is a depolymerised product of chitosan, holds great potential applications in the food, pharmaceutical, agricultural and environmental industries1246Pregnancy is associated with the onset of many adaptation processes that will likely change over the gestation course9111H NMR)-based metabolomics is an established technique for studying complex biological samples 131417Currently, proton nuclear magnetic resonance  on gestation day 35 compared to the CON group. Notably, the sow foetal size (crown-to-rump length) was elevated (P < 0.05) at 35 days of COS inclusion.As shown in –related gene  mRNA expression levels in foetuses are presented in P < 0.05) in BMP2, BMP4 and PPARγ mRNA expression levels was observed in sows fed with COS-supplemented diets, compared to those in the CON group. However, there was no difference (P > 0.05) in the expression level of OB-R among the two treatment groups.Foetal developmentP < 0.05) in amniotic fluid SOD, CAT and T-AOC activities respectively by 14.62%, 44.60% and 23.48%. Meanwhile, noticeably increased (P < 0.05) ASA and AHR activities were found in the amniotic fluid after COS ingestion, and no changes (P > 0.05) of GSH and MDA content in the amniotic fluid were noted among the two groups.The antioxidant parameters changes in the amniotic fluid observed after COS supplementation are listed in P > 0.05) between the two treatment groups. Meanwhile, the anti-inflammatory cytokines IL-10 concentration was significantly increased (P < 0.05) by COS supplementation. The amniotic fluid concentrations of IgG and IgM were respectively 64.06% and 29.74% higher (P < 0.05) in the COS-supplemented sows compared to the control sows. However, no significant difference (P > 0.05) was found in the amniotic fluid IgA concentration between the two treatments.1H NMR spectra of sow amniotic fluid from the CON group and the COS-treated group. In total, 16 metabolites were assigned in the amniotic fluid (ic fluid . MoreoveP < 0.05) the amniotic fluid lysine, citrate and glucose levels, but decreased (P < 0.05) the hypoxanthine level.PCA was initially performed on the amniotic fluid spectral data. Two principal components were calculated for the treatment groups, in which 70.7% and 12.7% of the variables were explained by PC1 and PC2, respectively. The PCA results  demonstrAmniotic fluid is the clear, watery liquid that surrounds growing foetuses within the amniotic cavity, and serves several important functions during foetal developmentet al. showed that anti- inflammatory cytokines such as IL-10 may have crucial roles in preventing feto-placental damage that could ensue from local inflammationIt is now increasingly clear that the feto-placental unit survival and growth are influenced by a complex interactive network of cytokines, some of which are produced by local immune components and others by reproductive tissues1H NMR-based metabolomics approach has been applied to investigate COS-induced foetal survival and growth alterations in a pig model. In the current study, glucose, which is a major energy substrate and plays a role in animal growth and developmentMaternal amniotic fluid metabolic profiles are valuable sources of information about foetal development, and can be potentially useful in the diagnosis of pregnancy disordersAmino acids are a crucial part of a proper energy balance and in the maintenance of the tricarboxylic acid cycle span by providing carbon backbone exchange via various anaplerotic and cataplerotic pathwaysde novo synthesis, which might partly account for COS’s growth-promotion property. In addition, proteomics studies must be performed to indicate changes in the foetal proteome after maternal COS inclusion.Maternal nutrition during gestation is closely associated with foetal growth and development in humans and animals, because it alters the foetal genome expression39In summary, COS supplementation could alter amniotic fluid antioxidant and immune status, in addition to the metabolic profiles, and thus create an optimal internal environment for foetal growth. Metabolic variations could be attributed to the functional variations in amino acid metabolism, glucose metabolism, the tricarboxylic acid cycle and oxidative protection, which have important practical implications in enhancing foetal survival and development. These novel findings might transfer to some extent into the clinical arena in the future.All experimental procedures in the present study were approved by the Animal Management Rules of the Ministry of Health of the People’s Republic of China and the Animal Care and Use Committee of Sichuan Agricultural University. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.Fifty-two multiparous sows , whose parities were in the range 3–4 were selected from a commercial pig farm  and transported to Sichuan Agricultural University . The sows were individually housed in gestation crates (1.5 × 2.0 m) in a pregnancy room. The ambient temperature in the pregnancy room was maintained at 15–18 °C.All sows were determined to be in the oestrous stage and were then inseminated twice with unfrozen semen via artificial insemination 3–5 days after weaning. The sows were randomly allotted to one of two treatments (26 sows/treatment) from day 1 of mating to ensure that each group had the same number of sows of similar parity. The treatment groups were as follows: (1) control diet without supplementation (CON); (2) control diet with COS added at a concentration of 100 mg/kg (COS).ad libitum access to water.The diets were formulated to meet or exceed the nutrient requirements recommended by the National Research Council (NRC) (2012)At day 35 of gestation, 12 hours after their last meal, six sows of average body weight for each group were chosen. Thereafter, the selected sows were prepared for anaesthesia (15 min) and then hysterectomised to obtain conceptuses . Approximately 4 mL of amniotic fluid from each foetus was immediately collected for metabolomics and biochemical assays. Next, the foetal survival rate and size (crown-to-rump length) were recorded as previously describedTM RT Reagent kit  according to the manufacturer’s instructions, and reverse transcription was performed at 37 °C for 15 min and 85 °C for 5 s in a Thermal Cycler PTC0200 .Total RNA was extracted from frozen foetal tissue (approximately 100 mg) with a TRIzol Reagent  according to a previous study® Green PCR I PCR reagents  using a CFX96 Real-Time PCR Detection System . All foetal samples were detected in triplicate. The reaction mixture (10 μL) contained 5 μL of freshly SYBR®Premix Ex TaqTM II , 1 μL forward primers (4 μM) and 1 μL reverse primers (4 μM), 1 μL reverse transcription products and 2 μL nuclease-free water. The PCR conditions were pre-run at 95 °C for 10 s, and 40 cycles of denaturation steps at 95 °C for 5 s, followed by an annealing temperature of 55.7 °C for 30 s, and a 72 °C extension step for 10 s. After amplification, melting curve analysis was performed to confirm each product’s specificity. Melting curve conditions were 1 cycle of denaturation at 95 °C for 10 s and then 65 °C changed to 95 °C at a temperature change velocity of 0.5 °C/s. The standard curve of each gene was run in triplicate to obtain reliable amplification efficiency values. The correlation coefficients of all standard curves were >0.99, and the amplification efficiency values were 90–110%. GAPDH expression was used as a reference gene to normalise the mRNA expression of the target genes, and the relative quantification of gene expression among the treatment groups was analysed using the 2−ΔΔCt methodAll primers were synthesised commercially by Invitrogen  and shown in Amniotic fluid antioxidant status evaluation was performed using a microplate reader  that contained the malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), and the total antioxidant capacity (T-AOC). All antioxidant-related kits were furnished by the Nanjing Jiancheng Bioengineering Institute .et al.MDA content was examined according to a procedure used by Livingstone SOD activity was measured spectrophotometrically at 550 nm using a method described in a previous studyet al.2O2 at 37 °C for 1 s per millilitre of amniotic fluid.CAT activity was examined using the colorimetric method described by Özmen GSH content was spectrophotometrically determined at 412 nm because the reaction between reduced GSH and 5,5′- dithiobis-p-nitrobenzoic acid can form yellow-colour 5-thio-2-nitrobenzoic acid, which is readily detectableet al.2− was generated by the reaction of xanthine and xanthine oxidase. A colouration reaction is developed using the Griess reagent after the addition of the electron acceptor; the colouration degree is directly proportional to the amount of superoxide anion in the reaction.The amniotic fluid’s ASA capacity is expressed in U per litre of amniotic fluid, where one U is the quantity of superoxide anion free radicals scavenged within 40min per llitre of amniotic fluid, which is equal to each microgram of vitamin C–scavenging under the same conditions.ASA was measured in accordance with the method of Jiang et al.− was generated on the basis of the Fenton reaction (Fe2+ + H2O2/Fe3+ + OH− + •OH). A coloration reaction is developed using the Griess reagent after the addition of the electron acceptor. The coloration degree is directly proportional to the quantity of hydroxyl radicals in the reaction. Amniotic fluid AHR capacity is expressed in U per millilitre of amniotic fluid, where one unit is defined as the amount that decreases 1 mmol/L H2O2 within 1 min per millilitre of amniotic fluid.AHR was measured in accordance with the method of Jiang 3+ to Fe2+. Fe2+ can then be combined with phenanthroline to form stable and coloured chelates. T-AOC was estimated at 550 nm and expressed as U per millilitre of amniotic fluid. Here, 1 U represents the 0.01 increase in the absorbance value in 1 minute per millilitre of amniotic fluid.T-AOC determination would enable the evaluation of the total activities of several parameters, including polyphenol complexes, protein thiol groups, glutathione and vitamins C and E, all of which can convert FeAll immune indices were analysed using commercially available porcine Enzyme-Linked Immunosorbent Assay kits purchased from R&D system . Amniotic fluid cytokines  and immunoglobulin  concentrations were detected following manufacturer’s instructions. Cytokine and immunoglobulin concentrations were presented as pg/mL and μg/mL of amniotic fluid, respectively.2PO4/K2HPO4, pH 7.4, 100% D2O). The mixture was then centrifuged at 16,000× g at 4 °C for 10 minutes, and 550 μL of the supernatant was transferred into 5 mm NMR tubes .Aliquots of amniotic fluid were thawed at room temperature for 1 hour prior to the analysis. The amniotic fluid analysis was proceeded with 200 μL of amniotic fluid reconstituted into 400 μL of phosphate buffer  with a 2 s recycle delay and a 100 ms tm. The 90° pulse length was adjusted to about 10 μs for each sample and 64 transients were collected into 32,000 data points over a 20 ppm spectral width. Metabolite assignments were usually obtained by considering chemical shifts, coupling constants and relative intensities.NMR spectra were recorded using an Agilent DD2 600 MHz NMR spectrometer  operating at 599.93 MHz 1H NMR spectra were corrected for phase and baseline distortions using Topspin 3.0 (Bruker Biospin). The chemical shifts in amniotic fluid spectra were referenced to the anomeric proton signal of α-glucose at δ 5.23.All free induction decays were multiplied by an exponential weighting function corresponding to a 1 Hz line-broadening before Fourier transformation. All 1H NMR spectrum (δ 8.6–0.5) was automatically data reduced to 1700 integral segments of equal length (0.002 ppm) using MestReNova (Mestrelab Research). The water (5.17–4.28 ppm) and urea (6.50–5.50 ppm) regions were removed before data analysis to eliminate the effects of variation in the suppression of the water and urea signals. Integrated data were normalised to the total sum of the spectrum prior to principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA) to give the same total integration value for each spectrum.Each amniotic fluid sample P < 0.05, which was determined according to the significance test of the Pearson’s product–moment correlation coefficient.PCA was performed using SIMCA-P 11.5 . PCA is an unsupervised pattern recognition method, and can be used for viewing “clusters” within multivariate data. Multivariate data can be displayed in a few principal components as a set of “scores” that highlight general trends and outliers. PLS-DA and OPLS-DA were performed using a unit variance-scaled approach. PLS-DA is a supervised pattern recognition method that explains the maximum separation between defined class samples in multivariate data. PLS-DA is performed by PLS regression against a specific cluster of samples as a dummy variable. Once a PLSDA model is calculated and validated, it can be used to predict class membership for unknown samples. OPLS-DA was performed with the NMR data to facilitate interpretation of loading. The model coefficients were back-calculated from the coefficients incorporating the weight of the variables and plotted with color-coded coefficients to enhance the model’s interpretability. Thus, the metabolites responsible for the differences between samples detected in the scores plot could be extracted from the corresponding loadings with the weight of the variable contributing to the discrimination. A correlation coefficient of r > 0.755 was the significance cut-off value based on the discrimination significance at t-test of SAS 9.0 . The foetal survival rate data were analysed using a chi-square test within SAS. Each sow was considered as a statistical unit. Data are presented in the format mean ± standard deviation. P < 0.05 was considered significant when used to compare the differences between the two groups.All amniotic fluid antioxidant and immune parameters, as well as foetal data , were statistically analysed using the Student’s How to cite this article: Wan, J. et al. Amniotic fluid metabolomics and biochemistry analysis provides novel insights into the diet-regulated foetal growth in a pig model. Sci. Rep.7, 44782; doi: 10.1038/srep44782 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Jag1, Dll1, Dll4, or Dll1 plus Dll4 in Lgr5+ve cells was induced by a Cre-mediated gene recombination, and differentiation or proliferation of the intestinal epithelial cells was examined by immunohistochemistry. These data are the extension of the data presented and discussed in the paper entitled “Indispensable role of non-canonical Notch signaling in the proliferation of Apc-deficient intestinal tumors“  The data on the immunohistochemical analysis of conditional Notch ligand knockout mice is presented. Targeted deletion of  Specifications Table[please fill in right-hand column of the table below]Value of the data•Dll1, Dll4, and Jag1 depletion in Lgr5+ve cells of the mouse intestine.Data presented displays the outcome of •+ve cell-dependent intestinal epithelial cell homeostasis.These data serve as a benchmark for future research work regarding the role of Notch ligands in Lgr5•The data is valuable for future research works focused on the functional relevance of Notch signaling in intestinal epithelial cell differentiation and proliferation.1Jag1, Dll1, Dll4, or Dll1 plus Dll4 genes in LGR5+ve cells  and ROSA26-tdTomato mice (Stock No. 007909) were purchased from The Jackson Laboratory . Jag1-floxed (Jag1fl/fl) mice Dll1-floxed (Dll1fl/fl) mice Dll4-floxed (Dll4fl/fl) mice LGR5-EGFP-ires-CreERT2; ROSA26-tdTomato; Jag1fl/fl(Jag1fl/fl) mice, LGR5-EGFP-ires-CreERT2 ROSA26-tdTomato; Dll1fl/fl(Dll1fl/fl) mice, LGR5-EGFP-ires-CreERT2; ROSA26-tdTomato; Dll4fl/fl (Dll4fl/fl) mice, and LGR5-EGFP-ires-CreERT2; ROSA26-tdTomato; Dll1fl/fl; Dll4fl/f  mice. LGR5-EGFP-ires-CreERT2; ROSA26-tdTomato mice served as the control (Control). Cre-mediated gene recombination was induced by intraperitoneal injection of tamoxifen  for 5 consecutive days, as previously described All the animal experiments were approved by the Animal Welfare Committee of Tokyo Medical and Dental University . All animal procedures were carried out in compliance with the institutional standards for use of laboratory animals. 2.2The primary antibodies used are as follows: anti-Dll1 , anti-Dll4 , anti-RFP , anti-tdTomato , anti-Hes1  2.3Immunohistochemistry of mouse intestinal tissues was performed as previously described MEXT/JSPS KAKENHI grant number 25293170 , grant number 23102003 (to RO and TN), grant number 15K15286 (to RO), grant number 16H05284 (to RO) and grant number 226221307 ; the Research Center Network Program for Realization of Regenerative Medicine fromJapan Agency for Medical Research and Development, AMED, grant number 16bm0304001h0004 ; the Practical Research Project for Rare / Intractable Diseases from Japan Agency for Medical Research and Development, AMED, grant number 16ek0109188h0001 (to RO).This study was supported by


Hypoxia is a condition of insufficient oxygen to support metabolism which occurs when the vascular supply is interrupted, or when a tumour outgrows its vascular supply. It is a negative prognostic factor due to its association with an aggressive tumour phenotype and therapeutic resistance. This review provides an overview of hypoxia imaging with Positron emission tomography (PET), with an emphasis on the biological relevance, mechanism of action, highlighting advantages, and limitations of the currently available hypoxia radiotracers.A comprehensive PubMed literature search was performed, identifying articles relating to biological significance and measurement of hypoxia, MRI methods, and PET imaging of hypoxia in preclinical and clinical settings, up to December 2016.A variety of approaches have been explored over the years for detecting and monitoring changes in tumour hypoxia, including regional measurements with oxygen electrodes placed under CT guidance, MRI methods that measure either oxygenation or lactate production consequent to hypoxia, different nuclear medicine approaches that utilise imaging agents the accumulation of which is inversely related to oxygen tension, and optical methods. The advantages and disadvantages of these approaches are reviewed, along with individual strategies for validating different imaging methods. PET is the preferred method for imaging tumour hypoxia due to its high specificity and sensitivity to probe physiological processes in vivo, as well as the ability to provide information about intracellular oxygenation levels.Even though hypoxia could have significant prognostic and predictive value in the clinic, the best method for hypoxia assessment has in our opinion not been realised. Hypoxia generally refers to sub-physiologic tissue oxygen levels <5–10 mmHg). Tumour hypoxia, a hallmark of malignancy, is a common and important feature of the tumour microenvironment. It is the consequence of an oxygen delivery versus consumption mismatch that occurs when cell proliferation outstrips neoangiogenesis during tumour growth. This results in very low oxygen levels (<5 mmHg) in tumours versus 40–60 mmHg in healthy tissues 0 mmHg. T. The latThe origin of chronic hypoxia in human tumours was postulated by Thomlinson and Gray in 1955 . ChronicBrown and colleagues  were theHypoxia represents a unique tumour vulnerability to be exploited in the context of newly emerging personalised medicine strategies. Undoubtedly, both chronic and acute tumour hypoxia directly affect clinical responses to therapy by influencing tumour growth, ability to metastasize, and resistance to cell death.A comprehensive PubMed literature search was performed, identifying articles relating to types of hypoxia, biological significance of hypoxia, measurement of hypoxia, MRI methods, and PET imaging of hypoxia in preclinical and clinical settings, up to December 2016. Search terms that were used to identify such articles were “hypoxia imaging,” “MRI,” “FMISO”, “FAZA”, “FETNIM”, “EF5”, “HX4”, “RP-170”, “Cu-ATSM”, and “PET” or “positron emission tomography.” Original publications in English were selected for inclusion in this review.Tumour hypoxia is frequently seen in solid tumours, and tumour cells survive by activating different signalling pathways leading to a plethora of temporally or spatially heterogeneous changes in tumours Table , elicite2 levels less than 10–15 mmHg, cells become radioresistant and gene expression of hypoxia-regulated genes under control of hypoxia-inducible factor (HIF1) increases. Decreased adenosine triphosphate (ATP) synthesis is seen at pO2 levels less than 10 mmHg and together with decreased protein synthesis leads to lower oxygen consumption by cells. Finally, pO2 levels less than 1 mmHg reduce oxidative phosphorylation and conversely enhance glycolysis to maintain adequate ATP levels misonidazole autoradiography [18F]labelled nitroimidazoles and Cu-labelled diacetyl-bis(N4-methylthiosemicarbazone) analogues [18F]labelled drug to be clinically tested was fluoromisonidazole (FMISO) MISO intracellular trapping is shown in Fig. 2-Nitroimidazole compounds were originally developed as hypoxic cell radiosensitisers and were introduced as hypoxia markers in the 1970s  after administration to obtain good contrast  with a h in vivo , metabol in vivo , and fai h after  in vivo –163.Fig.18F]Fluoroazomycin-arabinofuranoside (FAZA) is more hydrophilic than FMISO. Consequently, it has faster clearance kinetics, resulting in improved tumour-to-reference tissue ratios, and thus hypoxia-to-normoxia contrast. Head-to-head comparisons between FAZA, [124I]IAZA, and FMISO in preclinical animal studies imaged at 3 h after injection demonstrated faster vascular clearance of FAZA, resulting in an increased tumour-to-blood ratio (5.19) relative to that of [18F]fluoromisonidazole (3.98). More recently, clinical studies have successfully evaluated the feasibility of FAZA for imaging hypoxia in gliomas 2-(2-nitro-1H-imidazol-1-yl)-N--acetamide (EF5), first investigated as a hypoxia PET tracer in 2001 3-fluoro-2-(4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propan-1-ol (HX4), a next-generation 2-nitroimidazole tracer contains a 1,2,3-anti-triazole moiety (as a synthetic convenience) rendering it more hydrophilic than FMISO, specifically designed to maximize pharmacokinetic and clearance properties. Initial studies in humans demonstrate rapid renal clearance and urinary excretion of HX4, with a favourable dosimetry profile similar to that of FMISO Fluoroerythronitromidazole (FETNIM) accounts for its rapid renal clearance and low liver uptake, compared with FMISO. This also could explain the positive correlation between tumour blood flow and initial tumour FETNIM uptake RP-170). The hypoxic selectivity of RP-170 was demonstrated in glioma patients on the basis of significant correlations between uptake, oxygen tension measurements, and HIF1alpha immunostaining Cu-ATSM and [64Cu]Cu-acetate had almost identical uptake in vivo over 2–16 h, post injection. However, up to 1 h post injection, [64Cu]Cu-acetate had a superior tumour-to-muscle ratio [64Cu]Cu-ATSM may still be clinically relevant as a tracer for hypoxia, perhaps HIF1 status, as suggested by some investigators FAZA PET image intensities and tumour oxygenation was demonstrated by Tran et al. [18F]FAZA accurately showed improved uptake when rats with subcutaneous rhabdomyosarcomas were treated with air, in contrast to carbogen. This correlated well with an invasive OxyLite probe, although the probe demonstrated a relatively high heterogeneity in the oxygen value measured depending on the specific point within the tumour. Functional MRI  in 24 patients with glioblastoma . HypoxicThese studies suggest that both MRI and PET could complement each other and provide a future direction in selecting the best modality to image hypoxia.There is evidence from numerous clinical studies across a range of tumour types to support the existence and importance of the “hypoxia driver” phenotype both in pre-clinical –219 Tab and clinThe clinical significance of hypoxia PET imaging is to identify individuals with poor prognosis and those likely to benefit from hypoxia-targeted therapy. Several studies have shown that hypoxia PET imaging predicts outcome. High FMISO retention has been associated with higher risk of loco-regional failure and shorter progression-free survival in head-and-neck , 252–254These findings have been discussed in a recent meta-analysis of PET hypoxia studies which have demonstrated a common tendency towards predicting outcome in tumours showing higher tracer accumulation . DecreasIt is well known that tumours demonstrate temporal changes and/or heterogeneity in the spatial distribution of hypoxia. Identification of these areas with PET hypoxia scans enables image guidance and hence, radiation dose escalation to radioresistant sub-volumes , 259. BoHypoxia research has a long history; however, accurate and reproducible measurement of clinically relevant hypoxia with high sensitivity continues to evade the scientific community. Although radionuclide measurements of hypoxia started in the early 1980s we are yet to have a widely accepted method. In addition to studies with oxygen electrodes, imaging utilising exognous probes including FMISO-PET, FAZA-PET, HX4, and immunohistochemistry with pimonidazole have been the mainstay of hypoxia assessment in clinical studies; the EU-funded METOXIA consortium for example utilises HX4 for assessment of hypoxia . In the Notably, despite design of newer nitroimidazoles with substantially different physicochemical properties—high hydrophilicity—concomitant improvements in signal-to-noise ratio and, thus, reduction in imaging times, have not been achieved, indicating that the ideal chemical design has not yet been realised. There is also paucity of studies examining heterogeneity of hypoxia using parametric imaging to detect the influence of hypoxia sub-volumes and whether this additional detail will have prognostic or predictive value. With the advent of PET-MRI scanners, it will be feasible to multiplex imaging modalities to provide addition information such as perfusion to increase accuracy of hypoxia measurements or provide complementary information with higher predictive value. Whatever selected method will require assessment of precision of measurement which is non-trivial with such a spatio-temporally evanescent phenomenon as hypoxia.Thus, it is accepted that hypoxia could have significant prognostic and predictive value in the clinic; however, the best method for hypoxia assessment has in our opinion not been realised.


The present address should have appeared as shown above.Volume 7, no. 2, doi:


Educators are increasingly interested in applying neuroscience findings to improve educational practice. However, their understanding of the brain often lags behind their enthusiasm for the brain. We propose that educational psychology can serve as a bridge between basic research in neuroscience and psychology on one hand and educational practice on the other. We evaluated whether taking an educational psychology course is associated with increased neuroscience literacy and reduced belief in neuromyths in a sample of South Korean pre-service teachers. The results showed that taking an educational psychology course was associated with the increased neuroscience literacy, but there was no impact on belief in neuromyths. We consider the implications of these and other findings of the study for redesigning educational psychology courses and textbooks for improving neuroscience literacy. There is a growing interest in applying neuroscience findings to further educational theory, practice, and policy ,2. The bThis paper addresses two questions. The first is how best to bridge between neuroscience research and educational practice. The second is how best to improve neuroscience literacy and reduce belief in neuromyths among educators, so that they can better understand student learning and make informed evaluations of “brain-based” instruction. We propose that both questions can be answered by considering the mediating role played by educational psychology.With respect to how best to bridge between neuroscience and education, Bruer  argued tWe propose addressing this problem by recursing: by bridging between cognitive psychology and education via the intermediate discipline of educational psychology; see Educational psychology is also well positioned to improve the neuroscience literacy of teachers and reduce their belief in neuromyths. Most teacher training curricula include an educational psychology course. This course covers concepts from cognitive and developmental psychology that are relevant for neuroscience. Critically, it also introduces the scientific method, which may be new to many pre-service teachers, and which is essential for understanding how to reason validly from theoretical hypotheses to experimental results, and ultimately to practical applications. Prior research has shown that taking psychology courses improves reasoning about research methods and making statistical inferences from noisy data better than taking chemistry courses ,16. For Neuroscience concepts include the basic theoretical terminology of brain science, and the structures and functions of the brain. Neuroscience methods are the techniques by which neuroscientists study the brain and produce neuroscience data. Neuroscience applications are when neuroscientists use neuroscience findings to improve brain function and address real-world problems. Our definition of neuroscience literacy overlaps with prior definitions [We follow the OECD  definitiinitions ,18.Prior studies have found that educators have low neuroscience literacy in the sense that they have difficulty interpreting neuroscience findings ,19. ThisThe lack of neuroscience literacy is particularly problematic because people defer to neuroscience explanations and brain images when evaluating scientific evidence –23. EducAttempts to bridge between neuroscience and education have been plagued by the recalcitrant presence of neuromyths. These are widely believed–but incorrect–statements about brain structure, function, and development. Common examples include overemphasizing the importance of hemisphere dominance and the existence of critical periods during early development . Some neRegardless of their origins, neuromyths are widespread and have proven difficult to dispel. For example, many educators (and members of the public) believe that the left hemisphere is logical, associated with language and analytic thinking, whereas the right hemisphere is intuitive, associated with spatial reasoning and creativity. This neuromyth likely has its origins in misunderstandings of seminal neuroscience studies of the cognitive deficits of split-brain patients ,28. In fThere are many other prevalent neuromyths. These include an overemphasis on early interventions using “enriched environments”; that people only use 10% of their brains; that there exist multiple intelligences or learning styles, each with a separate neural correlate; and that physical exercises can improve communication between the left and right hemispheres ,9. TheseThe current study investigates whether taking an educational psychology course is associated with reduced belief in neuromyths. This might be because educational psychology includes concepts from cognitive and developmental psychology that are critical for understanding the results of research in cognitive and developmental neuroscience. This might also be because educational psychology courses typically cover the scientific method, which may be new to many pre-service teachers. An understanding of the scientific method might transfer to better understanding and evaluation of neuroscience studies. As noted above, there is evidence that taking psychology courses improves reasoning about research methods and inferences from data ,16.There have been several prior attempts to measure neuroscience literacy and belief in neuromyths. Herculano-Houzel  construcHoward-Jones et al.  adapted increased belief in neuromyths, a result that runs counter to Howard-Jones et al. [Dekker et al.  compareds et al. .These findings have been replicated and extended in more recent studies using larger samples and more diverse participants, including members of the general public and educators in Latin America, Europe, and the United States –37. Of pThe current study investigated the potential of educational psychology to bridge between education and neuroscience. Specifically, it evaluated whether taking an educational psychology course is associated with increased neuroscience literacy and reduced belief in neuromyths in pre-service teachers. Although conventional educational psychology courses do not provide much coverage of neuroscience, they do present concepts from cognitive and developmental psychology for interpreting the findings of cognitive and developmental neuroscience studies. They also emphasize the scientific method, research methods, and reasoning from theory and data to applications–skills that might transfer to the evaluation of neuroscience research ,16.There were three research questions: (1) Is taking an educational psychology course associated with increased neuroscience literacy? (2) Is taking an educational psychology course associated with decreased belief in neuromyths? (3) Do factors such as (i) the information sources consulted to learn about neuroscience findings and (ii) prior coursework in the biological sciences modulate neuroscience literacy and belief in neuromyths in pre-service teachers?To address these research questions, we focused on a sample of South Korean pre-service teachers majoring in elementary education. We chose this population for two reasons. First, neuroscience literacy and belief in neuromyths have been comparatively understudied in Asian countries , with onM age = 20.86 years, SD = 2.33) and the control group consisted of 49 participants . They received course credit for their participation.Participants were recruited from a public university in Seoul, South Korea. All were sophomore pre-service teachers in the elementary teacher training program. To earn an elementary teaching certification in South Korea, pre-service teachers must complete the required teacher training curriculum at one of 13 government-designated universities for elementary education. The experimental group consisted of pre-service teachers taking the required educational psychology course, and were tested during the Spring 2014 semester. The control group consisted of pre-service teachers who had completed the same coursework as the experimental group but who had not taken and were not enrolled in the required educational psychology course; they were tested during the Spring 2017 semester. Participants in both groups completed the survey twice, once at the beginning of the semester and once at the end. The experimental group consisted of 50 participants  about the neural correlates of intelligence, memory, dreaming, music, and bilingualism. Section II. Brain function contained 8 items (3 correct and 5 incorrect) about the localization and lateralization of brain function. Section III. Brain development contained 10 items (7 correct and 3 incorrect) about brain development in young children and adolescents. Section IV. Brain structure contained 12 items (7 correct and 5 incorrect) about basic brain anatomy and physiology. Section V. Neuroimaging contained 6 items (3 correct and 3 incorrect) about what neuroimaging measures reflect and their valid interpretation. Finally, section VI. Applying neuroscience results contained 10 items (5 correct and 5 incorrect) about applying neuroscience findings to reason about drugs, diet, and diseases of the brain. Within each section, correct and incorrect statements were randomly ordered.Items were organized into six sections, each representing a different domain of interest. Section Participants judged each statement as correct (“Yes”), incorrect (“No”), or responded “I don’t know.” The number of correct answers across all 59 items constituted the neuroscience literacy score . We defined a subset of the items as neuromyths items according to two criteria, as described below in the Results section. The number of incorrect answers across the neuromyth items constituted the belief in neuromyths score.The survey also collected background information about participants. This included their age, gender , number of biological sciences courses taken , and information sources consulted to learn about neuroscience research . Age had an open response format and all other items had a multiple-choice format. For the information sources item, multiple options could be selected.The survey was created in English and translated into Korean. The accuracy of translation was confirmed by double-blind peer review.Educational Psychology: Windows on classrooms (9thedition). This textbook covers cognitive, social, and emotional development; learning, memory, and complex cognition; behaviorism; motivation; instruction; individual differences and intelligence; exceptional learners; classroom management; assessment; and standardized testing.The goal of the educational psychology course is to present theories and evidence from psychology relevant for education to pre-service teachers. The instructor was a professor trained in education and cognitive psychology. The course materials included textbook readings, lectures, assignments, and exams. The textbook was a Korean translation of Eggen and Kauchak’s  EducatioThe neuroscience-relevant content of the course included a section of the textbook and the instructor’s lecture notes. The textbook chapter on cognitive and language development describes the structure of neurons, the function of the cerebral cortex, the nature of myelination, and the controversy surrounding “brain-based learning.” In addition, it debunks two neuromyths that were measured by the neuroscience knowledge survey, about hemispheric differences and critical periods. The instructor lecture notes introduced the structure of brain stem, the limbic system, and the cerebral cortex, and also specify how information is transmitted in the brain. Overall, the educational psychology course was conventional in structure.The study was approved by the Institutional Review Board at the University of Minnesota (#1306P37021). The pre-test was administered at the beginning of the semester. The consent form explained that the purpose of the study was to investigate educators’ understanding of neuroscience research related to learning and instruction. In particular, the terms “neuroscience literacy” and “neuromyths” were not used. Participants provided informed consent and then completed both parts of the neuroscience knowledge survey.Participants were not informed that there would be post-test at the end of semester to ensure that they did not engage in extra-curricular study of neuroscience. The instructor was not shown the neuroscience knowledge survey during the semester to ensure he did not tailor his instruction to its contents.The post-test was administered three months later, at the end of the semester. The items were same at post-test as at pre-test, but their order was changed within each subsection. For the participants in the experimental group, the post-test was introduced as an assessment tool for evaluating the impact of taking an educational psychology course on their understanding of neuroscience relevant to education. For participants in the control group, it was introduced as an assessment tool for evaluating the impact of their overall program during the semester. Again, the terms “neuroscience literacy” and “neuromyths” were not used. Participants provided informed consent and completed just the first part of the survey. (The second part was not administered because it was assumed that their background information had not changed.)F = 6.680, p = .011, ηp2 = .064) and time  = 8.152, p = .005, ηp2 = .078). Critically, these were qualified by a significant interaction  = 11.898, p = .001, ηp2 = .109), shown in M = 28.62, SD = 8.2) and the control group  (p = .440). However, at post-test, the experimental group  had significantly higher scores than the control group  (t(76.393) = 3.954, p < 001, d = 0.80). This provides evidence that taking an educational psychology course improves pre-service teachers’ neuroscience literacy.The first research question is whether taking an educational psychology course is associated with increased neuroscience literacy. To answer this question, we analyzed neuroscience literacy scores in a two-way repeated measures ANOVA with between-subjects factor group  and within-subjects factor time . There were main effects of group (ts(49) > 2.25, ps < .047, ds > 0.33), with the largest effect for section III. Brain development (d = 0.81). The only section that showed no improvement was section I. General knowledge, although the trend was in the predicted direction. By contrast the control group showed no pre-post gains on any of the six sections (ps > .152).To better understand this finding, we analyzed the pre-post improvement of each group on each of the six sections see . The expThe second research question is whether taking an educational psychology course is associated with decreased belief in neuromyths. To address this question, we defined neuromyths in two ways. The first definition was operational, as incorrect items that were answered incorrectly  by at least 50% of the experimental or control participants at pre-test. Note that larger scores indicate greater belief in neuromyths. This definition was inspired by prior research on psychological misconceptions ,55. By tF = 19.319, p < .001, ηp2 = .166), no main effect of time (p = .905), and no interaction of group and time (p = .087); see M = 5.84, SD = 1.7) believed more neuromyths than participants in the control group , and the absence of an interaction indicates that taking an educational psychology course does not close this gap.Given this operational definition, we analyzed whether taking an educational psychology course reduced belief in neuromyths. A two-way repeated measures ANOVA with between-subjects factor group  and within-subjects factor time  revealed a main effect of group  = 21.083, p < .001, ηp2 = .179), with participants in the experimental group  believing more neuromyths than their counterparts in the control group , and again neither the main effect of time (p = .712) nor the interaction of group and time (p = .793) reached significance; see A potential limitation of defining neuromyths to be the most recalcitrant incorrect statements is that this may have stacked the deck against finding an effect of taking an educational psychology course. We therefore considered a broader definition of a neuromyth as any incorrect statement in the survey, and computed the number of the 31 incorrect items that were answered incorrectly . This broader definition makes sense because most of the incorrect items were taken from prior studies of belief in neuromyths, specifically the Herculano-Houzel  study ofz-tests found that no difference in the proportion of each group that consulted each information source (ps > .322), with the exception of commercial products, which a greater proportion of the experimental group reported consulting (p = .043). Across all participants, the most frequently consulted information sources were public media , books , and “no sources” .The third research question concerned factors that modulate neuroscience literacy and belief in neuromyths. We first investigated the information sources that participants reported consulting to learn about neuroscience findings see . We did ps > .05); see We analyzed whether particular information sources were associated with higher neuroscience literacy or lower belief in neuromyths. This analysis was complicated by the fact that participants could select multiple information sources and by the fact that some information sources were selected by very few participants. We therefore constructed a between-subjects factor, information source, with four levels: only public media, only books, “no sources”, and multiple sources. Eighty-eight participants could be assigned to one of these four levels. We conducted one-way ANOVAs with information source as the factor on each of three dependent measures: neuroscience literacy, belief in neuromyths defined operationally, and belief in neuromyths defined broadly. These were the measures collected at pre-test, before the experimental group experienced the educational psychology course. There was no effect of information source on any of the dependent measures  studies conducted in the laboratory to improve student learning in the classroom. These might serve as models for valid inferences about applying neuroscience findings to educational practice.Neuroscience applications use neuroscience findings to improve brain function and address real-world problems. Educational psychology textbooks sequester their coverage of neuroscience from their coverage of psychological and educational topics. As a result, they provide little support for applying neuroscience findings to understand whether instructional interventions will work, and if so, why. Nevertheless, taking an educational psychology course was associated with increased knowledge of section not reduce belief in neuromyths. This was surprising. One reason may be because educational psychology is just one of several bridging disciplines between education and neuroscience [Contemporary educational psychology textbooks discuss and debunk neuromyths. For example, the textbook used in the current study explicitly addressed neuromyths about hemispheric differences and critical periods . Neverthoscience ,60. ImprStable neuromyths are incorrect beliefs that continue to be widespread today, even in the face of disconfirming evidence. Examples include the neuromyth about exaggerated differences between the left and right hemispheres and the neuromyth about the effectiveness of physical exercises on the integration of hemispheric function. These neuromyths have been documented by prior studies [Declining neuromyths are incorrect beliefs that were widespread in the past but that are becoming less prevalent. For example, a neuromyth that was once widely believed is that exposing infants to classical music increases their educational achievement later in life . However, this neuromyth was believed by less than 50% of pre-service teachers in our sample.A broader consideration of the literature suggests that the neuromyths that people believe may be changing over time. In particular, we propose two classes of neuromyths.  studies ,38, and may also explain the surprising finding that participants in the experimental group believed more neuromyths than participants in the control group. This is because the control group data were collected three years after the experimental group data, and the difference may reflect a general decline in belief in some neuromyths Click here for additional data file.S1 Dataset(TXT)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.


A 49‐year‐old Pakistani male presented with “heaviness” in his chest. Chest radiograph and computed tomography (CT) confirmed a massive left‐sided pleural‐based opacity. Three years ago, he was investigated for a left‐sided lymphocytic, exudative pleural effusion following an episode of dengue fever. Tube thoracostomy removed 1.3 L of fluid. Pleural biopsy and bronchial washings were non‐contributory. He received empirical anti‐tuberculosis treatment and remained asymptomatic until this presentation. To investigate the new pleural mass, he underwent a video‐assisted thoracoscopic surgery, which revealed a 2.2 kg mass in the pleural cavity involving the anterior mediastinum and chest wall and adhered to the visceral pleura. Following conversion to an open thoracotomy, the mass was completely excised, which involved non‐anatomical lung resection. Histopathology and immunohistochemistry of the resected tumour were consistent for a desmoid tumour. He was followed up for 9 months with no evidence of tumour recurrence. Predominantly pleural‐based desmoid tumour is rare but should be included in the differential diagnosis of spindle cell tumours. Pleural tumours and associated effusions are common clinical presentations. Most pleural tumours are malignant although benign ones (e.g. solitary fibrous tumour of the pleura (SFTP)) occasionally occur. This case describes the presentation of desmoid tumour as a rare cause of pleural tumour.A 45‐year‐old businessman was hospitalized for 5 days for dengue fever in 2011 during the peak of a dengue epidemic in Pakistan. He was managed with supportive treatment. Other than controlled hypertension, his past medical and family histories were unremarkable. A week post‐discharge, he re‐presented with left‐sided chest pain, exertional dyspnoea, cough and a medium‐sized, multi‐loculated left‐sided pleural effusion. Baseline haematological, coagulation and biochemical profiles were normal. A diagnostic pleural aspiration revealed a straw‐coloured lymphocyte‐predominant exudate. Bronchoscopy and bronchial washings were non‐informative. A closed Abrams’ biopsy yielded only non‐specific pleural inflammation. A chest tube was inserted and 1.3 L of fluid was removed. In accordance with local practice for undiagnosed lymphocytic effusions, he was given an empirical trial of quadruple anti‐tuberculosis (TB) therapy for TB pleuritis which he took for 4 months before being lost to follow‐up.He re‐presented in June 2015 with “heaviness” in his chest. A chest X‐ray (CXR) and computed tomography  or from metastatic spread of extrapleural cancer. Benign pleural tumours, for example, SFTP, are significantly less common. A desmoid tumour predominantly involving the pleural cavity has rarely been reported. Desmoid tumours, or desmoid‐type aggressive fibromatoses, are clonal fibroblastic proliferations characterized by locally aggressive tumours which do not metastasize but often recur despite resection Desmoid tumours can affect any part of the body. In patients with familial adenomatous polyposis (FAP), intra‐abdominal desmoids (involving the bowel and mesentery) predominate. In a series of 234 desmoid tumours, the largest extra‐abdominal sites were in the foot, shoulder, thigh and calf Most desmoid tumours arise sporadically. Genetic mutations (e.g. that of CTNNB1 gene) which activate β‐catenin are common He had an unusual presentation of a preceding lymphocytic effusion nearly 4 years before presenting with this desmoid tumour. It is unclear if the two events are causally related. However, antecedent trauma .No conflict of interest declared.Appropriate written informed consent was obtained for publication of this case report and accompanying images.Y. C. G. Lee is a National Health and Medical Research Council (NHMRC) Career Development Fellow and receives research project grant funding from the NHMRC, New South Wales Dust Disease Board, Sir Charles Gairdner Research Advisory Committee, Westcare and the Cancer Council of Western Australia.