The petrographic, mineralogical and geochemical features suggest that the apatite in DaG 978 should have formed during metamorphism in the presence of a fluid. Therefore, the apatite age represents an absolute age for fluid activity in an asteroidal setting. An impact event could have provided the heat to activate this young fluid activity in ESS.Chondritic meteorites, consisting of the materials that have formed in the early solar system (ESS), have been affected by late thermal events and fluid activity to various degrees. Determining the timing of fluid activity in ESS is of fundamental importance for understanding the nature, formation, evolution and significance of fluid activity in ESS. Previous investigations have determined the relative ages of fluid activity with short-lived isotope systematics. Here we report an absolute  Chondritic meteorites formed in the early solar system and may tell us about primary processes at that time. Here, Zhang et al. report an absolute 207Pb/206Pb isochron age  of apatite from a carbonaceous chondrite constraining timing of fluid activity in meteorites. Previous investigations have dated a few secondary minerals  that have formed by low-temperature and metamorphic processes in the presence of fluids, based on short-lived isotopic systematics  are composed of the solid minerals that have formed in the early solar system (ESS). They are the most precious samples to constrain primary processes that have been prevailed in the solar nebula1345(PO4)3 in chondrites. Apatite and merrillite [Ca9NaMg(PO4)7] in chondrites are thought to be secondary minerals, due to the large difference in condensation temperatures for Ca and P  carbonaceous chondrite with a low abundance of volatile elements2O3 contents (<0.2 wt%). A maximum metamorphic temperature of ∼580–680 °C was deduced, based on the compositions of coexisting spinel and olivineThe detailed petrography and mineralogy of DaG 978 have been reported in ref. 2O  has three major textural occurrences in DaG 978 . First, 2O . Compare2O . The oxygen isotopic compositions are similar to those of anorthite and mesostasis in a sapphirine-bearing Al-rich chondrule from DaG 978   and Th  . The thr 2 s.d.; . The excThe mineralogical features in DaG 978 indicate that both metamorphic process and metasomatic process have affected this chondrite1962O5 up to 3.5 wt%  could have formed though fluid-assisted metamorphism, their REE geochemistry and petrographic textures may reflect various contributions from metamorphic processes and metasomatic processes. Most of apatite grains in type 4–6 OCs exhibit negative Eu anomalies119911207Pb/206Pb isochron age  of the apatite obtained in this study has two important implications. First, in the literature, all ages  about fluid activity in ESS are relative ages or model ages727The 26Al and 60Fe . HoIn addition, recent studies suggested that some large asteroids  have water evaporation from localized regions, indicating subsurface fluid activityIn summary, the apatite in DaG 978 has an origin by fluid-assisted metamorphism and was formed at 4,450 Myr, ∼117 Myr later than CAI formation. Its age is the first absolute age about fluid activity in ESS. The late formation of apatite in DaG 978 represents a young metamorphic event in the presence of fluid in the parent body setting. An impact event at ∼4,450 Myr may have provided the heat to activate this fluid activity in late ESS. Alternatively, this meteorite might be ejected from a large (possibly Ceres like) asteroid, where subsurface fluid activities could last up to ∼117 Myr after CAI formation.Petrographic textures of apatite in DaG 978 were mainly observed with JEOL 7000F field-emission scanning electron microscope (SEM) at Hokkaido University and JEOL 6490 SEM at Nanjing University. Both SEM instruments are operated at an accelerating voltage of 15 kV. Mineral chemistry of apatite was determined using a JEOL 8100 electron probe micro-analyzer (EPMA) at Nanjing University. A defocused beam (10 μm in diameter) at a beam current of 10 nA and an accelerating voltage of 15 kV was used. A few natural and synthetic materials were used standards. All EPMA data are reduced with the ZAF (atomic number–absorption–fluorescence) correction procedure. Before EPMA analysis, all Ca–phosphate minerals were qualitatively measured by energy dispersive spectrometers (EDS) installed on the SEM instruments. Since SEM–EDS results show high chlorine and a very low intensity of fluorine, Cl and F are the first and second elements to be measured, respectively.+ ions of 20 keV and the beam spot size was 8–10 μm in diameter. A primary beam current of 100 pA was used to obtain a count rate of negative 16O ions of ∼2 × 107 c.p.s. A normal-incident electron gun was used for charge compensation of the sputtered area. A mass resolving power of ∼5,500 was used to separate 17O from 16OH. Negative 16O ions were detected with an axial Faraday cup, while negative 17O and 18O ions were detected with an axial electron multiplier detector, in magnetic peak-jumping mode. The instrumental mass fractionation effect was corrected using San Carlos olivine. The reported uncertainties of the individual analyses are expressed in 2 s.d., which were estimated by considering both the internal error of each measurement and the reproducibility of the standard measurements. After the measurements, all spots were evaluated using filed-emission SEM.Oxygen isotope compositions of apatite and associated silicate minerals were determined with the Cameca IMS-1270 instrument at Hokkaido University. Before measurements, the polished sections of DaG 978 were carbon-coated. The primary ion beam was mass filtered positive Cs16O− of –14.5 keV and irradiated on the sample surface to a diameter of ∼25 μm. The primary beam current is 10–15 nA. Kinetic energy filtering was used to reduce interferences from molecular ions by offsetting the sample acceleration voltage (–100 eV). The energy bandwidth was 20 eV. The exit slit was set to a mass resolving power of ∼500. Positive secondary ion intensities were counted for 10 s with an electron multiplier detector in magnetic peak-jumping mode. Each analysis included 15 cycles. Relative sensitivity factors between secondary ion intensity and concentration for each REE (relative to Ca) were determined using the Takashima augite, for which REE contents have been well determined by instrumental neutron activation analysisThe REE compositions of apatite and merrillite were determined with the Cameca IMS-6f instrument at Hokkaido University. Before measurements, the polished sections of DaG 978 were carbon-coated. The procedure was similar to that described in refs 2− primary beam was accelerated at −13 kV, with an intensity of ∼10 nA. The ellipsoidal spot is ∼20 × 30 μm in size. The secondary ions were extracted at an initial energy of 10 keV. The entrance slit and field aperture were set to a mass resolution power of ∼8,000. The energy slit was closed to a bandwidth of 60 eV to reduce the energy dispersion. Detection of secondary ions took the advantage of both mono-collector mode and multi-collector mode. For each analysis in this study, secondary ion beam intensities of 40Ca231P16O4+, 238U+ and 238U16O2+ were detected in L1-EM by peak-jumping sequences. In the second sequence, 204Pb, 206Pb and 207Pb were detected with multi-collector electron multipliers L2, L1 and C, respectively. In the third sequence, 238U, 232Th16O and 238U16O were measured with L1, H1 and H2, respectively. The Pb/U ratios were calibrated with an empirical correlation between Pb+/U+ and UO2+/U+ ratios, normalized to the 1,160 Myr apatite standard from the Prairie Lake alkaline–carbonatite complex in Ontario, Canada13207Pb/206Pb isochron and total Pb/U isochron ages were calculated at the 95% confidence level using the Isoplot/Ex 3.0 softwareThe U–Pb isotopic system of apatite was determined using the Cameca IMS-1280HR instrument at the Institute of Geology and Geophysics, Chinese Academy of Sciences, China, following the analytical procedure of refs The data shown and discussed in this paper are presented in the How to cite this article: Zhang, A-C. et al. Young asteroidal fluid activity revealed by absolute age from apatite in carbonaceous chondrite. Nat. Commun. 7:12844 doi: 10.1038/ncomms12844 (2016).Supplementary figure 1-2, Supplementary table 1-6


Metastatic clear cell renal cell cancer (mccRCC) portends a poor prognosis and urgently requires better clinical tools for prognostication as well as for prediction of response to treatment. Considerable investment in molecular risk stratification has sought to overcome the performance ceiling encountered by methods restricted to traditional clinical parameters. However, replication of results has proven challenging, and intratumoural heterogeneity (ITH) may confound attempts at tissue-based stratification.n = 183) of geographically separated mccRCC cohorts from the SuMR trial  and the SCOTRRCC study . Tumour protein levels quantified by reverse phase protein array (RPPA) were investigated alongside clinical variables. Regularised wrapper selection identified features for Cox multivariate analysis with overall survival as the primary endpoint.We investigated the influence of confounding ITH on the performance of a novel molecular prognostic model, enabled by pathologist-guided multiregion sampling  37.9, 95% confidence interval 4.1–353.8) and 2-year survival rates . Comparisons with established clinico-pathological scores suggest favourable performance for NEAT . Limitations include the relatively small cohorts and associated wide confidence intervals on predictive performance. Our multiregion sampling approach enabled investigation of NEAT validation when limiting the number of samples analysed per tumour, which significantly degraded performance. Indeed, sample selection could change risk group assignment for 64% of patients, and prognostication with one sample per patient performed only slightly better than random expectation (median logHR = 0.109). Low grade tissue was associated with 3.5-fold greater variation in predicted risk than high grade (p = 0.044).The optimal subset of variables in the final stratification model consisted of N-cadherin, EPCAM, Age, mTOR (NEAT). Risk groups from NEAT had a markedly different prognosis in the validation cohort  to inform the development of reproducible molecular risk stratification methods.The online version of this article (doi:10.1186/s12916-017-0874-9) contains supplementary material, which is available to authorized users. There is a great unmet need for better treatment and diagnosis of kidney cancer, which remains the most lethal of all genitourinary malignancies. Five-year survival in renal cell cancer (RCC) is approximately 40% overall, 10% in metastatic disease . Clear cSunitinib is a first-line treatment for metastatic ccRCC (mccRCC), doubling median progression-free survival compared with older immunotherapies such as IL-2 and interferon-α , 10. SunAdvances in prediction of treatment response and prognostication may be severely hindered by intratumoural heterogeneity (ITH) –16. Inden = 22, London [n = 22, Scotland [n = 8), similar targeted agents (n = 3) or no drug (n = 11). These cohorts were enriched for patients with a poor or intermediate prognosis, in line with the SuMR trial selection criteria [n = 45) which included an additional patient [p values were two-tailed and corrected for multiple hypothesis testing [This study examined two geographically separated cohorts of mccRCC patients with multiregion tumour sampling Table . Excludi, London ). The vaScotland , 19). Thcriteria . Median  patient . Compari testing . Net rec testing .Table 1C3 pieces; cryostat sections of each piece were examined to confirm ccRCC status and for morphological classification. Up to four samples per morphologically distinct region in each tumour were selected for protein extraction; each of these samples reflected circa 50–75 mm3 of tissue.Details of multiregion tissue mapping and sample preparation are given in . Briefly3 spatially mapped region [p < 0.05) were described previously [F test only when relevant assumptions held, assessed by the Lillefors and Fligner-Kileen tests [F-test assumptions and so were not assessed in our previous work using the ANOVA framework. Therefore, six proteins , EPCAM, mTOR (MTOR), MLH1, BCL2) were candidate molecular variables input into feature selection (described in the following section). The antibodies used for these candidate variables are listed in (supplementary) Table S1 of Additional file Fifty-five protein targets were investigated by RPPA, selected according to prior knowledge and validated antibody availability . Each tud region . Proteineviously , 25. Brien tests . RankingVariables were selected for Cox proportional hazards regression to overall survival on the development cohort using wrapper feature selection with backward elimination regularised by Bayesian information criterion (BIC) , 27. BacIMDC and MSKCC scores were calculated according to the relevant clinical parameters , 4. Suffp value for stratification into 'high' and 'low' risk groups defined by NEAT logHR = 0 were calculated. This analysis was performed on 106 datasets per MNTS examined, where each dataset represented a unique combination of samples across all patients in the validation cohort. Therefore, every patient was represented in each of the 106 datasets; thus, 106 NEAT HR and log-rank p values were generated for each MNTS, representing predictive performance distributions across the different tumour sample combinations.In order to evaluate tumour sampling effects on NEAT performance, a subsampling procedure produced datasets taking a maximum number of tumour samples (MNTS) of 1, 2 or 3 per tumour (and thus per patient). This approach employed Sobol sampling ; see supn = 44, Fig. n = 44) has a bimodal OS distribution that aligns with that of subgroups identified in larger mccRCC cohorts [The two mccRCC cohorts were similar across many characteristics Table , althoug cohorts , 4, 30.Fp = 1.18 × 10−4, and all selected variables were individually significant in the multivariate model . The proportional hazards assumption was not invalidated C index) [NEAT performed well on the geographically separated validation and development cohorts Fig. . This woC index)  values fC index)  values fp value were generated to reflect sampling 1, 2 or 3 regions per tumour in the validation cohort; these distributions capture NEAT performance for different sample combinations taken across tumours and patients. For example, consider three patients, each with RPPA data from four different tumour samples. If a single sample is taken from each patient for NEAT analysis, there would be 43 (i.e. 64) unique combinations of tumour samples across the three patients. Validation power rose significantly at each increase in the number of tumour samples taken per patient, and the full dataset with a median of four spatially separated samples per tumour appeared adequate, conferring good predictive power. NEAT overall performance on the validation cohort was poor when limited to a single tumour sample per patient, and was significantly impaired with two samples per patient ; strong statistical significance is due to the large datasets studied. Taking two samples per tumour gave improved stratification performance over a single sample , and substantial further improvement was found when taking three samples . Application of NEAT to different subsets of tumour samples per individual patient changed risk group assignment for 64% of the validation cohort . In order to further investigate the independent prognostic power of individual tumour regions, we compared prediction using expression values averaged across all available samples for each individual against the best possible results obtained using only one sample per tumour. Validation using all of the available samples per tumour outperformed even the most predictive single sample taken (p < 10−6).The overall approach to investigate the effects of tumour sampling on predictive performance is summarised in Fig. ent Fig. . In the ort Fig. . Interescirca 50–75 mm3) relative to standard approaches based on tissue sections from formalin-fixed paraffin embedded material such as tissue microarray analysis (<0.2 mm3 per region). Therefore, the RPPA data analysed cover a higher proportion of the overall tumour volume relative to standard approaches. The sampling approaches may be an important enabling factor in NEAT reproducibility and hence good validation performance, despite the relatively small cohorts studied. The RPPA technique offers potential as a quantitative alternative to IHC and has already been applied in a clinical setting through the Clinical Laboratory Improvement Amendments (CLIA) facility certification process [This study examines the effect of sampling on the performance of a novel molecular prognostic approach, NEAT, using protein measurements from 183 regions across 44 mccRCC tumours. The unique development cohort from the SuMR trial allowed for selection of proteins that had increased intratumoural expression variance with treatment; we hypothesised that these proteins may be markers of aggressiveness and therefore useful in prognostication. Although the cohorts are relatively small, NEAT gave statistically robust stratification of the independent validation cohort by overall survival Fig. . The tre process , 37. Then = 44 total), which is associated with increased risk of type II error and wide confidence intervals on predictive performance. Cytoreductive nephrectomy is standard clinical practice, and the use of upfront tyrosine kinase inhibitor (TKI) treatment is variable, limiting recruitment of a uniform cohort  for NEAT development. A further limiting factor on the size of cohorts in our study was the availability of appropriately consented fresh-frozen material with multiregion sampling and pathology assessment for RPPA analysis. Our approach to discover resistance biomarkers required multiregion sampling of tumour tissue from patients treated with upfront sunitinib in order to enable comparison of candidate marker variance in sunitinib-exposed and sunitinib-naïve material. Therefore, the cohorts received different treatment regimens and also had significant differences in some clinical characteristics. NEAT performed well on both cohorts despite these differences, and so might be broadly useful for prognostication of mccRCC. Further study of NEAT performance on an independent upfront sunitinib cohort would be of interest to further explore potential clinical utility, such as to inform decision making about performing a cytoreductive nephrectomy [The NEAT development and validation cohorts were relatively small . NEAT compares well with established clinico-pathological scores on a geographically separate independent validation cohort who received current standard therapy. Results show that evaluation or attempted use of any molecular prognostic and predictive methods with few tumour samples will lead to variable performance and low reproducibility. We demonstrate parameters  that may be used to inform sampling in order to enhance biomarker reproducibility, and results underline the critical importance of addressing heterogeneity to realise the promise of molecular stratification approaches. Through studies such as TRACERx , we antiRecommendations arising from our research include the following: (1) biomarker validation studies should implement tumour sampling protocols that match as closely as possible to the discovery work; (2) clinical biomarker research and ultimately front-line diagnostic approaches may benefit from greater tumour sampling rates; (3) clinical parameters  can guide sample selection, and investigation of additional parameters to inform sampling may be useful; (4) optimisation of tumour sampling rate and sample selection protocols are important research areas to enable advances in stratified cancer medicine.Additional file 1:Supplementary methods, tables and summary of the Additional file Additional file 2:NEAT model, custom computer code and relevant datasets for investigation of limiting sample number on NEAT performance (ZIP archive). (ZIP 13 kb)Additional file 3:CSV). (CSV 141 kb)Reverse phase protein array data for multiregion tumour sampling . (CSV 962 bytes)Clinical data; columns are: anonymised patient identifier, age, grade, event, overall survival, Heng class, MSKCC class. Anonymised identifiers correspond with those given in Additional file


The aim of this study was to evaluate two RGD radiotracers radiolabelled with fluorine-18 or gallium-68, in detecting angiogenesis in grafted human tumours and monitoring their treatment with the anti-angiogenic agent bevacizumab.18F-RGD-K5 and 68Ga-RGD were acquired before treatment (baseline), after three bevacizumab injections (t1) and after seven bevacizumab injections (t2).Sixteen mice bearing an U87MG tumour in one flank and a contralateral A549 tumour were treated with intravenous injections of bevacizumab twice a week for 3 weeks. PET images with 3. The decrease in tumour uptake of both RGD tracers was non-significant. Therefore it was not possible to predict this efficacy on tumour growth based on RGD PET results, whereas ex vivo measurements showed a significantly lower tumour uptake of both tracers in mice sacrificed at t2 vs. at baseline.In A549 tumours, the treatment stopped the tumour growth, with a tumour volume measured by calliper remaining between 0.28 and 0.40 cm18F-RGD-K5, this decrease in tumour SUVmax became significant at t1, whereas it was also observed with the 68Ga-RGD tracer, but only at t2. 18F-RGD-K5 appeared more efficient than 68Ga-RGD in the visualisation and follow-up of U87MG tumours.In U87MG tumours, the uptake measured on PET decreased during treatment, reflecting the partial therapeutic effect observed on tumour volume, consisting in a decrease in the slope of tumour growth. Using The comparison of those results with those of immunohistochemistry at baseline and at t2 favoured the hypothesis that tumour RGD uptake reflects other cancer properties than just its angiogenic capacity. Two models were tested, either grafting the U87MG cell line of human GB, which is highly vascular and ανβ3 positive, or grafting A549 cell line of human lung carcinoma, which is moderately vascular and ανβ3 positive, at a lower level than U87MG . PET analyses were made using Syntegra–Philips software . Time activity curves (TACs) were obtained by drawing regions of interest (ROI) on dynamic images; counts in the ROI were reported to the total counts in the mouse derived from the dynamic PET data sets. Tumour uptake measured on the static PET data sets was reported as maximum standardised uptake value (SUVmax), which was calculated as the maximal value of the SUVs in the ROI.PET acquisitions were performed using the Mosaic animal PET machine . For each PET tracer, 1–6 MBq . Uptake of tumours and tissues was expressed as mean ± SD percentage of injected activity per gram of tissue (%ID/g), corrected for 18F or 68Ga decay.One hour and twenty minutes after injection of All tumours were embedded in paraffin according to standard procedures and different stains were performed: haematoxylin-eosin (HE) for evaluating tissue appearance, tumour-proliferating antigen Ki67 for the tumour aggressiveness and CD31 for expressing the relative microvessel density (MVD) (%). The tumour slices were observed with an optical microscope in transmitted light . All quantitative analyses of the immunostaining were expressed as the percentage of positive area over the area of the entire section; they were measured by using Image J software.


NF2 gene. In this study, we found age-dependent differences in the clinical parameters of sporadic VSs. Young patients were characterized by progressive tumour behaviours, including earlier onset of initial symptoms, shorter symptom duration and larger tumour size. An increased rate of “two-hits” of both NF2 alleles, usually by mutation and allelic loss, was observed in young cases compared to older, and this correlated with the loss of protein and mRNA expression. In contrast, the tumours with a single mutation (referred to as ‘one-hit’) exhibited obvious expression levels. Moreover, a mixture of merlin-expressing tumour cells and non-expressing tumour cells was observed in ‘one-hit’ schwannomas, suggesting that a subset of ‘one-hit’ tumour cells was present in these tumours. To mimic the growth promoting effects by the second hit, we performed lentivirus-mediated NF2 knockdown in the ‘one-hit’ schwannoma cultures. Following the loss of NF2 expression, schwannoma cultures demonstrated increased proliferation rates. Above all, we have identified a correlation between the NF2 status and the growth patterns of sporadic VSs. The treatment decision-making, microsurgery or “wait and scan” strategy, should be carried out according to the tumour’s genetic background.The great majority of sporadic vestibular schwannomas (VSs) are due to the inactivation of the  In rare cases, they occur as bilateral tumours within the context of the inherited familial cancer syndrome Neurofibromatosis Type 2 (NF2). NF2 is also associated with schwannomas of other cranial, spinal, and cutaneous nerves; meningiomas and, less frequently, ependymomas.Vestibular schwannomas (VSs) are benign tumours that originate from myelin-forming schwann cells that surround the vestibular branches of the 8th (auditory) cranial nerve. The vast majority (95%) of VSs are unilateral and sporadic schwannomas, which occur predominantly in the 4th to 6th decades of lifeNF2 tumour-suppressor gene was first isolated in 1993, when mutations were demonstrated in NF2 patients3. The NF2 gene is located on chromosome 22q 12.2 and encodes a 595-amino acid protein, designated merlin or schwannomin. Alterations in the gene that lead to the development of NF2-related VSs are considered to follow Knudson’s two-hit model4, either by mutation in both alleles or by mutation in one allele and the loss of the other allele in conjunction with a loss of heterozygosity (LOH) on chromosome 227. Currently, the genetic profiles of sporadic VSs have not been fully characterized. The “two-hits” inactivation of the NF2 gene that results in total merlin loss has also been demonstrated in sporadic VSs with detection rates ranging from 27% to 61%11, which is much less than expected. Additionally, conflicting results from merlin expression analyses have been reported in studies on sporadic VSs15. They are classified by varying protein levels into none (or faint), medium or strong. A possible factor that may impair the sensitivity of these analyses is stromal contamination in the tumour tissues11.The 17. However, the biological background of these phenotypical heterogeneities is largely unknown. Some studies have reported that fast-growing tumours tend to occur in younger individuals18, suggesting there may be a correlation between age at diagnosis and tumour growth rate in sporadic VSs. To understand the genetic events responsible for the clinical behaviours of sporadic VSs, we divide the tumours into three age groups at equally spaced intervals, including 38 young cases (<30 years old), 191 middle-age cases (31–60 years old) and 53 elderly cases (>60 years old), and perform comparisons of the clinical and genetic characteristics between age groups. We are also interested in the implications of protein/mRNA expression and tumour-cell proliferation in response to different NF2 gene statuses. Therefore, we intend to assess the prognostic impact of the NF2 gene alterations on tumour growth.The therapeutic management of sporadic VSs mainly includes two strategies: microsurgery or the “wait and scan” strategy. An important aspect in the determination of the most suitable therapy is the tumour growth rate. Some tumours remain stable for years, whereas others grow relatively fastAll experimental protocols were approved by the Research Ethics Review Committee of Shanghai Jiao Tong University. The methods and experimental protocols in human samples were carried out in accordance with relevant guidelines, and all study participants signed the informed consent form. This study conformed to the provisions of the Declaration of Helsinki.2. Five cases of NF2-related VSs were also included as positive controls of genetic analyses. All tumour specimens were resected by the same surgeon, and were then subjected to histological examinations to confirm the diagnosis of schwannomas. Tumour size was measured in terms of the maximal dimension of the lesion on magnetic resonance (MR) imaging. As controls, 10 cases of normal vestibular nerves from vestibular neurectomy for Meniere’s disease were also included. Peripheral blood samples were collected from all patients prior to operation with written informed consent.A total of 282 patients with sporadic VSs were included in a multi-center study from March 2008 to September 2015. The patients were divided into three age groups at equally spaced intervals: 38 young cases (less than 30 years old), 191 middle-age cases (between 30 and 60 years old) and 53 elderly cases (more than 60 years old). The two ages (30 and 60 years) were chosen to define age categories because sporadic VSs often occur after 30 years and the incidence rates tend to decrease after 60 yearsNF2 gene. DNA extraction from the tumour tissues and blood lymphocytes was performed using the TIANamp Genomic DNA Kit , as previously described19. The whole coding sequence and the exon–intron boundaries of the gene were amplified by polymerase chain reaction (PCR) using standard methods and underwent bidirectional sequencing as previously described. The sequence data were analysed using Sequencer 4.9 software  and compared with the NF2 sequence (NM_016418) in GenBank. Mutations were described according to the standard nomenclature for DNA sequence changes reported by the Human Genome Variation Society (HVGS).Bidirectional sequencing was conducted to detect microlesions in the NF2 gene, we used a commercial MLPA kit for the analysis . The NF2 probemix contains probes for each of the exons of the NF2 gene. The procedure was based on the manufacturer’s protocol with an initial DNA amount of 75 ng on the 3500 Genetic Analyser . Relative peak heights of all amplicons of each test sample were compared to a normalized average of three vestibular nerves. The results were analysed with MRC-Coffalyser software (MRC-Holland) and the Dosage Quotient (DQ) was used to describe the copy number status. A range of 0.8 < DQ < 1.2 was considered normal, and 0.4 < DQ < 0.7 was considered to show a heterozygous deletion. In addition, three probes located in other regions  on 22q 12.2 were included as positive controls to identify the occurrence of a loss of heterozygosity (LOH) on chromosome 22. Eleven reference probes were included to detect different autosomal chromosomal regions.To identify large/exonic deletions that were not detected by direct sequencing, and to measure the copy number of the 3 in size and digested with 0.25% trypsin. The cells were collected and re-suspended in culture medium: DMEM , 2.5% Nu-Serum (BD), 0.5 µM forskolin (Sigma) and 10 nM β1-heregulin . To establish merlin-knockdown cultures, short hairpin RNAs (shRNA) were synthesized, annealed, and inserted into a lentivirus vector, containing an independent open reading frame for green fluorescence protein (GFP). Lentiviral shRNA (Lv-shRNA) vectors were constructed using three different shRNA sequences against NF2/merlin expression including 5′-ACTTCAAAG ATACTGAC AT-3′ (sh1), 5′-TCTGGATA TTCTGCACAAT-3′ (sh2), and 5′-TTCGTGTTAATAAGCTGAT3′ (sh3). A nonsense shRNA was also constructed using the target sequence 5′-TTCTCCGAACGTGTCACGT-3′ . The lentivirus-mediated shRNAs were used to transfect schwannoma cells at a multiplicity of infection (MOI) of 20. To screen the target for the most effective viral transfection, the percentage of GFP-positive cells in the total cell numbers was evaluated under the fluorescence microscope at day 3 following transduction.Human Schwann cells (HSCs) were purchased from ScienCell Research Laboratories , and cultured in Schwann Cell Medium . For primary schwannoma cell culture, tumour tissues were cut to 2 mm−ΔCt (NF2/GAPDH)). The PCR primers were listed as follows: Forward, 5′-GCAGATCAGCTGA AGCAGGA-3′ and reverse, 5′-ACCAATG AGGT TGAAGCTTGGTA-3′ for NF2; Forward, 5′AAGGTG A AGGTC GGAG T CAACG 3′ and reverse, 5′ -CAGCCTTCTCCATGGTGGGAA-3′ for GAPDH.RNA extraction was done using TRIzol reagent according to standard methodology. High-quality RNA was reverse-transcribed into cDNA by a reverse transcription kit . PCR amplification was run with SYBR® Premix Ex Taq™ (Takara). Amplification and qPCR measurements were performed on the CFX96 Real-Time PCR Detection System (Bio-Rad). The results were normalized to GAPDH as an endogenous reference  containing a protease inhibitor (phenylmethanesulfonyl fluoride). Western blotting analysis was performed with antibodies specific for merlin  and cyclinD1 . The β-actin antibody  was used to ensure equal loading of total protein. The protein bands were detected using an chemiluminescence HRP substrate (Millipore). The band densities were quantified using the Imager Lab Software (Bio-Rad). For immunohistochemistry, after deparaffinization and serial ethanol treatment, tumour slices were processed for microwave antigen retrieval pretreatment, digested with 0.3% trypsin, quenched with 3% H2O2, blocked with pre-inoculated goat serum and incubated with anti-merlin antibody  at 4 °C overnight. Biotin-conjugated goat antirabbit IgG secondary antibody and HRP-conjugated avidin were applied at a 1:200 dilution. As a chromogen, 3,3-diaminobenzidine tetrahydrochloride was used, and the sections were counterstained with Haematoxylin. For immunofluorescence, normal cranial nerve samples were processed as in the immunohistochemistry staining (without H2O2 quench). HSCs and schwannoma cells were plated on glass slides, fixed with 4% paraformaldehyde for 20 minutes, and then permeabilized in 0.1% Triton X-100. The tissue slides and the glass slides were blocked with pre-inoculated goat serum before incubation with the anti-S100 , and anti-merlin . Alexa-488– and Alexa-594–conjugated goat anti-mouse and goat anti-rabbit antibodies were used as secondary antibodies. The Alexa Fluor® 488 Phalloidin  was used for the staining of the F-actin cytoskeleton. The sections were also nuclear counterstained with 4,6-Diamidino-2-phenylindole .3 cells/well) and grown for 0, 24, 48, or 72 h. At each specified time point, 10 μL/well CCK-8 solution was added and the cells were incubated for an additional 4 h at 37 °C. The absorbance of the wells was measured using a SpectraMax190 microplate reader , and the absorbance values  obtained from three aliquots were averaged to produce a single value to represent cell viability. The absorbance was then expressed in numerical values that were finally subjected to statistical analysis.The 5-ethynyl-20-deoxyuridine  labelling was used to assess the DNA synthesis activity. Briefly, after treatment, the cells were exposed to 50 µL EdU for an additional 24 h at 37 °C, washed with phosphate buffered saline, fixed with 4% formaldehyde for 20 min and incubated with 0.3% Triton X-100 for 20 min. Subsequently, the cultures were reacted with 80 μL of 1 × Apollo® reaction cocktail for 20 min. The DNA contents of the cells in each well were then counterstained with DAPI for 15 min. The effect of merlin knockdown on cell proliferation was determined by a Cell Counting Kit-8 (CCK-8) from Dojindo . Briefly, cells were seeded into 96-well plates . The statistical analysis of the clinical parameters of the three age groups was performed using the Chi-square test, the one-way ANOVA test, or the Kruskal-Wallis one-way ANOVA. The comparisons of merlin expression between tumours and the cell proliferation index in different treatments were performed using the Student’s t-test or the one-way ANOVA. All data generated or analysed during this study are included in this published article (and its Supplementary Information files).p = 0.026) and a larger tumour size  compared to the eldery cases. These findings suggested that fast-growing schwannomas tend to occur in young individuals.During the study period, 282 patients with sporadic VSs ranging in age at diagnosis from 8 to 85 years (average age = 45.4 years) were identified. For comparison, these individuals were classified in to three age groups: the young group , the middle-age group  and the elderly group . As described in Table NF2 mutations relative to tumour behaviours, the detection for the mutation (including exonic deletions) and the copy number of the NF2 gene was performed using a combination of Direct sequencing and MLPA analysis among the two groups with different clinical characteristics, including 38 cases of young patients and 53 cases of elderly patients with sporadic VSs . Five cases of NF2-associated VSs were also included as positive controls for the genetic analyses. The deletion of exon 1 was by far the most common deletion mutation identified in the lymphocytes of NF2 patients20. Consistent with the previous finding, four out of the five NF2-related tumours in this study were found to have an exon 1 deletion by Dosage Analysis . These mutations were demonstrated in their matched blood samples, supporting them as germline mutations. Additionally, positive dosage results were demonstrated in tumours in another three regions on chromosome 22, suggesting that the allelic loss of the NF2 gene occurred in conjunction with a loss of heterozygosity (LOH) event. However, in sporadic VSs, germ-line mutations were not observed in any blood samples, but somatic mutations were detected in 54 (59.3%) of the 91 cases, including two cases with a deletion of a single exon . As previously reported21, most mutations identified were truncating mutations  throughout the first 15 exons of the gene. As shown in Table NF2 protein (exon 1 to 8), but no particular mutational hot spots were found. Evans et al.22 reported that the ratio of frameshift to nonsense mutations increased significantly with increasing age at diagnosis in sporadic VSs. However, in our study, the distribution of mutation type did not differ significantly between the two age groups. A second inactivation event was identified in 19 of 26 mutated tumours in the young groups and in 11 of 28 mutated tumours in the elderly groups, including 24 cases with mutation in one allele and a loss of the other, and only 6 cases with two different mutations on each allele. Interestingly, in total, three out (2 young and 1 elderly) of 20 ‘non-mutated’ tumours exhibited allelic loss. Considering these particular cases, the frequency of “two-hits” tumours in the young cases was estimated to be 67.9% (19/28), which was much higher than in the elderly cases . In contrast, the tumours with a single mutation (here referred to as ‘one-hit’) appeared to occur more frequently than the ‘two-hits’ tumours in elderly patients, with a ratio of 17:11. Therefore, the association of tumour behaviours with the differential NF2 gene status, i.e., the ‘two-hits’ or the ‘one-hit’, seemed to be established through our comparisons of the clinical and genetic characteristics between the young and elderly patients.In an attempt to evaluate the VSs Fig. . Five csis Fig. . These xon Fig. . As preNF2 gene status, an antibody recognizing an epitope close to the carboxy-terminus of merlin was used to assess the expression levels of the full-length merlin in schwannomas. The density values of merlin 70-kDa bands were corrected for the difference in the density of actin bands. Protein samples from eight ‘two-hits’ tumours  and eight ‘one-hit’ tumours  were prepared. One normal vestibular nerves and six NF2-related schwannomas were also included as the positive and negative controls, respectively. The Immunoblot analysis by anti-actin antibody demonstrated that equal amounts of protein samples were used  will influence the transcription, splicing or translation of the NF2 gene. Because the Tumour #93 showed some expression of full-length merlin, this mutation may serve as a hypomorphic mutation, which might be expected to retain expression of a hypomorphic protein. In contrast, most, if not all, of the batch II tumours were found to exhibit distinct intensity of the 70-kDa bands compared to the bands of batch I, suggesting that the full-length merlin was expressed in batch II tumours, which was consistent with the results from the mutational analyses.To investigate the association of merlin expression with the detected 12. Merlin has been argued to mediate tumour suppression by directly modulating mitogenic signal transduction at or near the plasma membrane, where merlin and the F-actin cytoskeleton were co-localized25. The merlin protein was found to be strongly expressed at or near the cell membrane in normal vestibular nerves and HSCs, as expected  and non-expressing (hypothesized as the ‘two-hits’) tumour cells. As seen in HSCs, the colocalization of merlin and F-actin at the membrane cytoskeleton was shown in schwannoma cultures  tumours and 10 group II tumours  were included. For the convenience of comparisons, the averaged value obtained for each tumour batch was divided by that in 10 normal vestibular nerves to obtain an indicator ratio. As described in Fig. NF2 gene statuses . Merlin mRNA levels remained high in normal vestibular nerves relative to both ‘one-hit’ group I and ‘two-hits’ group II tumours. The statistical analysis revealed that in group II tumours the ratios (0.12 ± 0.17) were much lower than in group I tumours , indicating that the second inactivation hit, either mutation or allelic loss, may induce further repressed expression levels of NF2 transcripts, resulting in the universal loss of merlin expression in the ‘two-hits’ tumours.The mutant 29. Merlin knockdowns have been demonstrated to cause an increased proliferation rate of human schwann cells (HSCs) in vitro30. In contrast, over-expression or reintroduction of the wild-type NF2 could inhibit the proliferation of HSCs and primary schwannoma cultures by arresting cell cycle at the G0/G1 phase31. In the expression analyses, a subset of ‘one-hit’ tumour-cells was suggested in the primary schwannoma cultures from a ‘one-hit’ schwannoma (#168). The presence of merlin expression in ‘one-hit’ schwannomas should be attributed to the existing ‘one-hit’ tumour cells, in which the merlin mRNA was actively expressed by qRT-PCR analysis. To knockdown the merlin expression in these tumour cells, transfection of lentivirus-mediated shRNAs into the schwannoma cell populations was performed. Every experiment was carried out between the second and the fourth passage, when proliferation rates were the best and the number of fibroblasts was negligible . On day 3 after transfection, each of the knockdown groups and the control groups were harvested and divided into two parts: one part was sent for Western-Blotting/qRT-PCR analyses to verify the presence of efficient merlin silencing and if successful, the remaining part was subjected to an analysis for cell proliferation. As shown in Fig. NF2 shRNAs  exhibited an excellent extent of reduction in both mRNA and protein expression levels, as compared to the controls. EdU staining was conducted to detect the newly synthesized DNA to determine the proliferation activities. As shown in Fig. NF2 shRNAs than in those with nonsense controls. The above results were further confirmed by CCK-8 assays  of schwannomas.Consistent with its role as a tumour suppressor, merlin plays an important role in controlling the proliferation of a variety of cell typesays Fig. , indicatres Fig. . These fNF2 gene, and compared these alterations with the clinical characteristics. Sporadic VSs were most commonly seen in the middle-aged individuals with a proportion of 67.8% (191/282), and the tumours in young and elderly individuals were uncommon with proportions of 13.5% (38/282) and 20.6% (53/282), respectively. Here, we found age-dependent differences between the clinical parameters of the three age groups, including the age at the onset of initial symptoms, the symptom duration and the tumour size. The differences were much more significant in the young group than in the elderly group. The progressive clinical behaviours observed in young sporadic VSs led us to explore the underlying genetic mechanisms.This study investigated the genetic alterations in sporadic VSs, including mutations and allelic loss of the 34. Schwannomas are prone to contamination by non-tumourous cells such as macrophages, lymphocytes and cells on the blood vessels11. Stromal contamination can reduce the detection concentration of tumour cells, and therefore mask the presence of mutation in some samples. This notion may explain the failure to identify a mutation on the NF2 gene in all of our cases.Tumour analysis is more complex than standard lymphocyte DNA analysis. The tumour tissues are not solely composed of tumour cells; they are a heterogeneous collection of both tumour cells and surrounding non-tumourous  cellsNF2 gene in NF2-related VSs, the familial forms of vestibular schwannomas. The NF2 gene mutation occurs as the first hit during the development of vestibular schwannomas7. In the current study, we found somatic mutations in 54 (59.3%) of the 91 Chinese patients. The results were consistent with the 53–82% detection rates reported in previous studies11. The allelic loss of the NF2 gene in conjunction with LOH on chromosome 22 represents the most common form of the second hit in schwannomas7. The step-by-step inactivation hits were confirmed by comparisons between mutation patterns of blood and tumour DNA in NF2-related patients. In the genetic study, the mutated sporadic schwannomas however exhibited different NF2 gene statuses, i.e., the ‘one hit’ and the ‘two-hits’. The presence of only one mutational event in sporadic VSs has also been reported by many studies11. In young individuals, the ‘two-hits’ schwannomas were present more often than the ‘one-hit’ tumours and the frequency ratio was estimated to be 19:7. In contrast, the ‘one-hit’ appeared to occur more frequently than the ‘two-hits’ in elderly patients, with a ratio of 17:11. The comparison between the genetic profiles of the two age groups with different clinical characteristics may establish a relationship between the detected NF2 gene status and tumour behaviours. In schwannomas considered as ‘one-hit’ tumours, the methylation of the promoter-associated CpG island may represent the event secondary to the inactivation of the NF2 gene. Kino et al.35 reported that the site-specific methylation of the promotor elements was important for NF2 silencing in sporadic VSs. However, another three studies that included 93 sporadic VSs detected no aberrant methylation of the NF2 gene in any tumours37, suggesting that promoter methylation is an uncommon mechanism of NF2 inactivation in sporadic VSs.The ‘two-hit’ model, which implies a germ-line mutation in one tumour suppressor gene (TSG) allele followed by somatic loss of the remaining wild-type allele, has held true for most TSGs in the explanation of cancer predisposition. The hypothesis has been strongly demonstrated in the NF2 gene status in schwannomas by our detection tools may not reflect this fact. The development of schwannomas has been reported to be only seen in conditional homozygous NF2 knockout mice, and not in NF2 hemizygous mice6. Regardless of the stromal cells in schwannoma tissues, it seems unlikely that the so-called ‘one-hit’ tumours contain pure ‘one-hit’ tumour cells. A mutation that causes structural abnormalities in the region of the NF2 gene may make the locus more susceptible to damage, resulting in the second inactivation hit38. It is possible that a subset of ‘one-hit’ tumour cells at risk of being transmitted to ‘two-hits’ may have been present in these schwannomas26. The notion was further supported by the merlin staining in the ‘one-hit’ tumour tissues and isolated primary cultures. In contrast to the staining homogeneity in HSCs, the schwannoma cell populations contained a mixture of merlin-stained (hypothesized as ‘one-hit’) and non-stained tumour cells (hypothesized as the ‘two-hits’). The MLPA analysis for the detection of allelic loss may not be sensitive enough to obtain positive results when a high content of ‘one-hit’ tumour cells with a remaining normal allele were present in tumour tissues. The fast-growing schwannomas may have a much larger percentage of ‘two-hits’ tumour cells relative to ‘one-hit’ tumour cells, resulting in positive results in the dosage analyses of tumour DNAs. In other words, the lower percentage of ‘two-hits’ tumour cells in slow-growing tumours may lead to negative results.In expression studies, most of the ‘one-hit’ schwannomas exhibited distinct merlin-bands, but weak or non-bands were observed in all of the ‘two-hits’ tumours, suggesting that the total loss of merlin expression should require the inactivation of both alleles. The ‘one-hit’ NF2-shRNAs into the schwannoma cultures to knockdown merlin expression. Following the loss of merlin expression, schwannoma cultures demonstrated increased proliferation rates, as expected. The finding suggested that ‘two-hits’ tumour cells exhibited a stronger proliferation activity than the ‘one-hit’ tumour cells. Therefore, it was not surprising that higher detection rates of the ‘two-hits’ were observed in young schwannomas characterized by progressive behaviours. The finding also suggested that the ‘two-hits’ tumour cells may play a leading role in the clinical progression of sporadic schwannomas.To investigate the implications of cell proliferation in response to merlin loss by the occurrence of the second hit, we performed transfection of lentivirus-mediated NF2 gene status was correlated with the clinical behaviors of sporadic VSs. The ‘one-hit’ schwannomas, probably containing a high content of ‘one-hit’ tumour cells, may exhibit slower growth rates compared to their ‘two-hits’ counterparts. Therefore, the treatment decision-making, the microsurgery or the “wait and scan” strategy, should be chosen according to genetic backgrounds. The cerebral spinal fluid (CSF) circulates through the CNS and has a large interface with the vestibular schwannoma tissues. Recent studies have demonstrated the great potential of tumour-derived cell-free DNA (cfDNA) from CSF for cancer prognosis41. Future improvements of the non-invasive detection techniques concerning CSF cfDNA may provide detailed genetic information on patients with sporadic VSs, especially the proportions of ‘one-hit’/‘two-hits’ tumour DNA, generating a prognostic impact on the clinical treatment strategies.According to current studies, we postulated that the detected Supplementary informationSupplementary Table S1Supplementary Table S2Supplementary Table S3


Osteocytes, the major type of bone cells embedded in the bone matrix and surrounded by the lacunar and canalicular system, can serve as biomechanosensors and biomechanotranducers of the bone. Theoretical analytical methods have been employed to investigate the biomechanical responses of osteocytes in vivo; the poroelastic properties have not been taken into consideration in the three-dimensional (3D) finite element model. In this study, a 3D poroelastic idealized finite element model was developed and was used to predict biomechanical behaviours  of the osteocyte-lacunar-canalicular system under 150-, 1000-, 3000-, and 5000-microstrain compressive loads, respectively, representing disuse, physiological, overuse, and pathological overload loading stimuli. The highest local strain, pore pressure, and fluid velocity were found to be highest at the proximal region of cell processes. These data suggest that the strain, pore pressure, and fluid velocity of the osteocyte-lacunar-canalicular system increase with the global loading and that the poroelastic material property affects the biomechanical responses to the compressive stimulus. This new model can be used to predict the mechanobiological behaviours of osteocytes under the four different compressive loadings and may provide an insight into the mechanisms of mechanosensation and mechanotransduction of the bone. Bone is a dynamic biological structure that can adapt to its mechanical environment by changing its structure , 2. The Osteocytes are known to regulate bone remodelling and are located within their lacunae surrounded by the perilacunar matrix (PCM) embedded in the bone matrix (extracellular matrix or ECM). Osteocytes are connected to each other with slender cell processes located within small tubes called the canaliculi within the bone matrix. It is believed that, through this osteocyte cell body-cell process-lacunar-canalicular system, the osteocytes are the mechanosensors/mechanotranducers in bones, which can sense the mechanical loadings and transduce them into biochemical signals regulating bone remodelling –11.To investigate bone tissue mechanosensing/mechanotransduction and biomechanical behaviours of osteocytes, a number of researchers have studied the osteocyte-lacunar-canalicular system by theoretical analytical models. The mathematical model has been a good tool to explain the phenomenon of mechanotransduction in the lacunar-canalicular system , which iSince nutrients and exchanges of metabolic products or biochemical signals of osteocytes depend on the movement of interstitial fluid , basicalPoroelasticity is a well-developed theory for investigating the interaction of fluid and solid phases in a fluid-saturated porous medium –41, whicμε, the physiological load was between 200 and 2500 με, and the overuse loading range was 2500–5000 με ]E), Poiseveloped –66. The essed as  67]. Th. ThE), Ptermined .In order to investigate the biomechanical behaviours of the osteocyte-lacunar-canalicular system, the compressive loadings were applied to the 3D FE model. In this study, the displacements were used as loadings rather than the forces. In the current study, the uniaxial ramped static loading was applied on the model.Uniaxial compressive loads were applied in the long bone axis , with glTo prevent rigid body motion, the symmetrical boundary conditions were applied to the nodes on the opposing faces of the applied displacement loading, and only 1/8 of the symmetry model was used in the FE analyses. Furthermore, the initial fluid pore pressure was assumed to be zero and imposed on the external surfaces of the model due to the lack of osmotic pressure in the osteocyte-lacunar-canalicular system .Distributions of the maximum principal strains of the osteocyte-lacunar-canalicular system FE model were predicted under global compressive loads of 150 microstrains (disuse), 1000 microstrains , 3000 microstrains (overuse), and 5000 microstrains , respectively. The maximum principal strains of the FE model were found to be ~633, 4272, 12820, and 21528 microstrains, respectively. The strain distribution of the model under the 1000-microstrain compressive loading is shown in T), and the maximum pore pressure is located at the junction areas of canaliculi, PCM, and processes.The maximum pore pressures on the cell and processes were 8.98, 74.24, 175.9, and 285.7 kPa when the model was subjected to global compressive loads of 150, 1000, 3000, and 5000 microstrains, respectively. Previously, the maximum pore pressure of the cortical bone was found to be up to 250 kPa under 1 MPa uniaxial stress . Thus, tμm/s when the model was subjected to global compressive loads of 150, 1000, 3000, and 5000 microstrains, respectively. t = 0.2T. In a previous study, the peak fluid flow velocity in canaliculi of the bone lacunar-canalicular system was ~60 μm/s under a surface strain of 400-microstrain loading [μm/s under 298- and 510-microstrain loading [The maximum fluid velocities of the FE model were found to be 2.69, 18.19, 56.65, and 97.98  loading . Recentl loading . CompareIn order to investigate the biomechanical responses of the cell body and processes, five points of the model in one straight line were selected for analyses . BecauseDetailed analyses of the maximum principal strains with loading time at these five different locations are shown in Figures It is now widely believed that fluid that flows through the osteocyte-lacunar-canalicular system can affect and control the bone adaptation since when the osteocytes are stimulated by fluid stress, they can produce biological signals that guide the recruitment/activity of osteoclasts and/or osteoblasts on the surface of the bone . Thus, tThe simulation results of our poroelastic FE model under the compressive loads were found to be in good agreement with the previous results , 69, 76.−18, 10−19, and 10−20 m2, respectively [Pore fluid pressure occurred when the osteocyte-lacunar-canalicular system is subjected to loads . The ostectively . In addiectively , and theectively . Our preμm/s under 150, 1000, 3000, and 5000 microstrains, respectively, suggesting that the fluid velocity increases with the increasing global loading. Fluid velocity is another important characteristic of the response of an osteocyte to the mechanical loading stimuli, since fluid movement within the lacunar-canalicular system caused by mechanical loading can cause small deformations of bone, deliver nutrients to, and remove wastes from the osteocytes [Furthermore, using the present model, fluid velocity was also predicted and simulation was conducted for fluid flow within the osteocyte-lacunar-canalicular system in situ. In our model, the maximum fluid velocities were 2.69, 18.19, 56.65, and 97.98 teocytes , and theteocytes .T, followed by declining and finally reaching the steady states. Overall, these trends of changes are coincident with the mode of loading with the load increasing starting from the initial point and reaching the highest load at 0.1T. Furthermore, among the selected locations analysed, the proximal side (near to the cell) of a process was found to bear the highest local strain, pore pressure, and fluid velocity. In addition, among the various degrees of loads applied, it was found that the degrees of biomechanical responses increase with mechanical loads. In other words, the local strain, pore pressure, and fluid velocity of the cell and a process increase with the global loading. Because the loading time and the ramp time were kept the same, the high compressive loading occurred with sharper loading ramps and caused much higher velocity, pressure, and principal strains peaks.Moreover, the distributions of the strain, pore pressure, and fluid velocity of five selected points in the cell and a process were investigated over the whole loading time period and under different degrees of compressive loading. It was found that the values of these parameters increased firstly and reached the peak values at about the 0.1In the current study, the poroelastic material property was applied in all simulations. The osteocyte-lacunar-canalicular system is considered to be fully saturated, having only a solid matrix phase and a fluid phase with no air voids, and it consists of the fluid spaces surrounding the osteocytes and their processes. In most previous studies, the porosity of osteocyte-lacunar-canalicular system was assumed to be 0.05 , 72, 78.−25 to 7.172 × 10−17 (m2) [-20 m2 or smaller. In our study, the permeability values are 0.6 × 10−19, 4 × 10−20, and 5.0 × 10-15 m2 in the cell body and process, in PCM and canaliculi, and in the ECM, respectively. The permeability is not a constant value and thus the strain-dependent permeability was implemented in the present FE model. Our result is consistent with Darcy's law that the fluid velocity and pore pressure vary with the hydraulic permeability. Our study indicates that the poroelastic material property can affect the biomechanical responses to the mechanical stimulus and the permeability is important in controlling the fluid flow behaviours in the poroelastic model.Moreover, the permeability (the ease with which the fluid can move within a porous system) which is related to porosity, is also a very important measure for the poroelastic model. In the current study, viscosity was assumed 0.001 Pa s . Permeability depends on the number, orientation, and size of the canaliculi, as well as on the amount of filling by osteocytes and their processes. While it is too hard to determine it directly, there have been estimations of permeability of the osteocyte-lacunar-canalicular systems ranging over 8 orders of magnitude , with th−17 (m2) , 83, 84.−17 (m2) , 52, 83,−17 (m2) , 78, and−17 (m2) . Such va−17 (m2) . Accordi−17 (m2) , to be aμm/s when the model was under 150 microstrains (disuse), 1000 microstrains , 3000 microstrains (overuse), and 5000 microstrains , respectively. The values of the strain, pore pressure, and fluid velocity, which were found to be the highest at the proximal region of cell processes, increase with the global loading. This new model can potentially be used to predict the mechanobiological behaviours of osteocytes under physiological or pathological loadings, which may provide an insight into understanding the mechanisms of mechanosensation and mechanotransduction of the bone.Although there have been some previous studies using FE analysis models mostly under compressive loads to examine biomechanical behaviours of the osteocyte-lacunar-canalicular system, these studies had not taken the bone poroelastic properties into account. The current study has developed a 3D poroelastic idealized FE model to investigate the biomechanical responses of the osteocyte-lacunar-canalicular system under different degrees of compressive loading stimuli. It was found that predicted maximum principal strains of osteocytes were ~633, 4272, 12820, and 21528; the maximum pore pressures were ~8.98, 74.24, 175.9, and 285.7 kPa; and the maximum fluid velocity values were ~2.69, 18.19, 56.65, and 97.98


Toronto, CanadaInformations: Nancy BushPhone: 4169782719info-SUR1726@cpdtoronto.caE-mail: www.cpd.utoronto.ca/lungtransplant/Site: Toronto, CanadaInformations: Nancy BushPhone: 4169782719info-SUR1726@cpdtoronto.caE-mail: www.torontothoracicrefresher.ca/Site: Windsor, United KingdomInformations: EACTSinfo@eacts.co.ukE-mail: www.eacts.org/academy/2017-programme/Site: Windsor, United KingdomInformations: EACTSinfo@eacts.co.ukE-mail: www.eacts.org/academy/2017-programme/Site: Marseille, FranceInformations: Judith MoonenPhone: + 31 6 29 229 655office@fecect.orgE-mail: www.fecect.orgSite: Berlin, GermanyInformations: EACTSinfo@eacts.co.ukE-mail: www.eacts.org/academy/2017-programme/Site: Paris, FranceInformations: Raul Wilsonannualmeeting.conferenceseries.com/cardiologists/Site: Catanzaro, ItalyInformations: Prof.PasqualePhone: +39 09613695851umgaorta@unicz.itE-mail: maori.unicz.it/Site: Colorado Springs, United StatesInformations: Heather Nuttermeetings@westernthoracic.orgE-mail: meetings.westernthoracic.org/Site: Chicago, United StatesInformations: ASAIO Headquarterswww.asaio.comSite: Las Vegas, United StatesInformations: Joseph BashaPhone: 318-623-0890www.TheNewOrleansConference.comSite: Windsor, United Arab EmiratesInformations: EACTSinfo@eacts.co.ukE-mail: www.eacts.org/academy/2017-programme/Site: Chicago, United StatesInformations: Suzanne Williamsvascular.cmesociety.com/Site: Toronto, CanadaInformations: Karlin ZoePhone: 7025085200Fax: 7025085200braininjury2017@protonmail.comE-mail: http://braininjury.alliedacademies.com/Site: New York, United StatesInformations: EJ WeldonPhone: 978-927-8330info@InternationalCoronaryCongress.comE-mail: http://InternationalCoronaryCongress.comSite: Informations:Phone: 662-547-0930Fax: 662-589-9321cdihearts@gmail.comE-mail: http://www.heartcareheart.orgSite: Astana, KazakhstanInformations: Stella Papandreopoulouinfowscts@gmail.comE-mail: www.wscts.netSite: Houston, United StatesInformations: Alan P. Stolz, M.Ed.Phone: 832-355-9930Fax: 832-355-9931astolz@bcm.eduE-mail: www.cme.texasheart.orgSite: Amsterdam, NetherlandsInformations: Jessie AlisonPhone: 7025089022Fax: 7025089022Singapore, SingaporeInformations: Evahttps://www.ATSOP.sgSite: Birmingham, United KingdomInformations: L.R. Associates - Ms. L. RichardsonPhone: 01296 733 823Fax: 01296 733 823lorrainerichardson1@btinternet.comE-mail: New York, United StatesInformations: Office of Continuing Medical EducationPhone: 646-227-2025cme@mskcc.orgE-mail: www.mskcc.org/IPcourseSite: Informations: Kathie JohnsonPhone: 8282143944heartdiseases@alliedconferences.orgE-mail: http://heartdiseases.alliedacademies.com/Site: Iaşi, RomaniaInformations:https://www.eab2017romania.org/Site: Surgery Conference Cartagena, ColombiaInformations: Damon MarquisPhone: 312-202-5800Fax: 312-202-5801info@CardiovascularSurgeryConference.orgE-mail: http://www.cardiovascularsurgeryconference.orgSite: Orlando, United StatesInformations: Lindsay OppedisanoPhone: 800-253-7657; 203-263-0006 ext 120Fax: 203-263-4839loppedisano@cine-med.netE-mail: innovation.surgery.duke.ed/coursesSite: San Antonio, United StatesInformations: Daniella MosbyPhone: 4084292646heartsurgery@cmesocietyconferences.comE-mail: http://heartsurgery.cmesociety.com/Site: Williamsburg, United StatesInformations: Carol Ann RosenbergPhone: 434-924-5310uvacme@virginia.eduE-mail: www.cmevillage.comSite: Hamburg, GermanyInformations: Sue Hesfordsue@ests.org.ukE-mail: www.ests.orgSite: Rome, ItalyInformations: Akif Ündar, PhDPhone: 717-379-5020Fax: 717-531-0355congressi@opbg.netE-mail: https://www.ispmcs.org/annual-conference/Site: Lecce, ItalyInformations: Gaetano Di Rienzo, MD, FETCSPhone: +39/349/6398344Fax: +39/832/661608gaetanodirienzo1@gmail.comE-mail: http://www.endoscopiatoracica.itSite:


Gastrointestinal stromal tumour (GIST) is the most common primary mesenchymal tumour of the gastrointestinal tract. Spindle cell monophasic synovial sarcoma (SS) can be morphologically similar. Angiogenesis is a major factor for tumour growth and metastasis. Our aim was to compare the angiogenic expression profiles of high-risk GIST and spindle cell monophasic SS by histological, immunohistochemical and molecular characterisation of the neovascularisation established between xenotransplanted tumours and the host during the initial phases of growth in nude mice.The angiogenic profile of two xenotransplanted human soft-tissue tumours were evaluated in 15 passages in nude mice using tissue microarrays (TMA). Tumour pieces were also implanted subcutaneously on the backs of 14 athymic Balb-c nude mice. The animals were sacrificed at 24, 48, and 96 h; and 7, 14, 21, and 28 days after implantation to perform histological, immunohistochemical, and molecular studies (neovascularisation experiments).Morphological similarities were apparent in the early stages of neoplastic growth of these two soft-tissue tumours throughout the passages in nude mice and in the two neovascularisation experiments. Immunohistochemistry demonstrated overexpression of pro-angiogenic factors between 24 h and 96 h after xenotransplantation in both tumours. Additionally, neoplastic cells coexpressed chemokines  and their receptors in both tumours. Molecular studies showed two expression profiles, revealing an early and a late phase in the angiogenic process.This model could provide information on the early stages of the angiogenic process in monophasic spindle cell SS and high-risk GIST and offers an excellent way to study possible tumour response to antiangiogenic drugs. High-grade sarcomas can be implanted into immunodeficient mice, where they grow as xenografts supported by the murine stroma blood supply . In geneIn recent years, much research has focused on the role of angiogenesis in tumour development, growth, invasion, and metastasis , 7. It hKIT- or PDGFRA-activating mutations 11].RNA was extracted from 50 to 200 mg of tumour samples obtained from the NU335 and Nu407 series.The whole methodology and studied genes  are alsoComparing speed of growth in murine passages, we observed a similar average growth speed in SS (0.142 mm/day) and in GIST (0.103 mm/day).No morphological changes were observed between passages in GIST; however, a high number of mitoses were clearly observed in the passages in both tumours  and 2F. The IHC study of SS showed positivity for EMA , cytokerDouble-immunofluorescence staining demonstrated that chemokine ligand expression in general was slightly higher in the xenograft passages than in the original tumour . There wIn our neovascularisation experiments, during the first hours after xenografting, peritumoral haemorrhagic areas with inflammatory infiltration compounded by lymphocytes, plasma cells, neutrophils, karyorrhectic, and apoptotic figures were observed in SS and GIST. Small capillaries surrounded the xenograft associated with mesenchymal angioblastic and non-angioblastic cells included in a loose matrix.Patchy hypoxic necrosis in SS appeared within the first 96 h after implantation, reaching a peak extension in the third week. The SS presented characteristic adipose tissue infiltrate and peritumoral skeletal muscle mouse fibres, but without the pseudocapsule observed in GIST. In GIST, the massive necrosis appeared during the first week, earlier than in the SS. After the fourth week, the histological picture of the GIST was re-established, with features similar to those of the human control, re-establishing also the amount of mitoses and the remission of necrosis, which became patchy and scant. During the third week after xenografting, the inflammatory component decreased. At this time, newly formed capillary vessels were remodelled and penetrated or sprouted into the tumour.Areas of massive necrosis were associated with a lower proliferative index in both tumours. Ki67 was lower in the early stages after tumour xenografting in both tumours. In the last weeks, the increase in Ki67 expression was also inversely correlated with HIF1α in both neoplasms.Angiogenic factors represented by the VEGF family and their receptors presented a different expression profile in the two tumours. In SS, maximum VEGF positivity presented 24 h after implantation and was also expressed in the extracellular matrix, while VEGF positivity was lower in GIST and appeared 96 h after xenografting . VEGFR2 Double-immunofluorescence staining showed chemokine expression  in the tumour cell cytoplasm/nucleus and deposited in the extracellular matrix. This was also the case for their receptors (CXCR3 and CXCR2)  and 4H. Gene expression profiles  in GIST RNA samples corresponding to the third week of SS and to the fourth week of GIST were not viable for analysis.Angiogenesis is critical for the growth and metastasis of tumours. Early in tumorigenesis, an angiogenic switch is activated by hypoxia, promoting the expression of pro-angiogenic growth factors, such as the VEGF family, its receptors, and HIF1α among others . Recent HIF1α is a principal regulator of cellular and systemic homeostatic response to hypoxia as it activates many genes, including those involved in angiogenesis . In our Anthracyclines and ifosfamide, either alone or in combination, are the gold standard treatments for advanced SS . HoweverCluster analysis performed on our qRT-PCR expression studies revealed two additional groups of genes clearly separated into two stages, corresponding to early angiogenic induction where VEGF and PDGF family genes among others play an important role, and the later remodelling phases where other angiogenic genes are overexpressed. Apparently the high-risk GIST behaved biologically as high-grade sarcoma in the passages and neovascularisation experiments similar to our previous molecular results . HoweverIn addition to angiogenic factors, chemokines also play an important role during angiogenic induction. The coexpression of ligands and chemokine receptors in neoplastic cells and extracellular matrix suggests that autocrine and paracrine stimulation by the tumour cells results in production of angiogenic factors in response to hypoxia during the first stages of tumour growth, as reported in other neoplastic and non-neoplastic conditions , 30, 41.This model provides information on the early stages of the angiogenic process in monophasic spindle-cell SS and high-risk GIST. We suggest that different angiogenic molecular profiles could predict different biological and clinical behaviour and determine the response to antiangiogenic treatment. We also demonstrate the importance of chemokine expression as a therapeutic target of tumour growth.The fact that angiogenesis is a dynamic, changing and multistep process over time should be taken into consideration when developing future therapeutic strategies in soft-tissue tumours.The authors declare that they have no conflict of interest.


The use of clinical MRI scanners to conduct pre-clinical research facilitates comparisons with clinical studies. Here the utility and sensitivity of anatomical and functional MRI data/biomarkers acquired from transgenic mouse models of neuroblastoma using a dedicated radiofrequency (RF) coil on a clinical 3T scanner was evaluated.T2-weighted imaging, quantitation of the native longitudinal relaxation time (T1) and the transverse relaxation rate (R2*), and dynamic contrast-enhanced (DCE)-MRI, was used to assess tumour volume, phenotype and response to cyclophosphamide or cabozantinib.Multiparametric MRI of transgenic mice bearing abdominal neuroblastomas was performed at 3T, and data cross-referenced to that acquired from the same mice on a pre-clinical 7T MRI system. T2-weighted image contrast enabled clear tumour delineation at 3T. Significant correlations of tumour volume  and R2*  measured at 3 and 7T were established. Mice with neuroblastomas harbouring the anaplastic lymphoma kinase mutation exhibited a significantly slower R2* (P<0.001), consistent with impaired tumour perfusion. DCE-MRI was performed simultaneously on three transgenic mice, yielding estimates of Ktrans for each tumour . Cyclophosphamide elicited a significant reduction in both tumour burden (P<0.002) and native T1 (P<0.01), whereas cabozantinib induced significant (P<0.01) tumour growth delay.Excellent Simultaneous multiparametric MRI of multiple tumour-bearing animals using this coil arrangement at 3T can provide high efficiency/throughput for both phenotypic characterisation and evaluation of novel therapeutics, and facilitate the introduction of functional MRI biomarkers into aligned imaging-embedded clinical trials. High field (>4.7T) small animal dedicated MRI systems that provide high resolution and signal to noise ratios (SNR) have and continue to be the preferred platform for pre-clinical imaging investigations. The physical contrast characteristics and imaging performance of such pre-clinical scanners differs substantially from clinical MRI systems. The use of clinical scanners to conduct pre-clinical research is attractive, as it facilitates a more direct comparison with clinical studies, the application and development of clinically applicable imaging protocols, matching of field strength-related mechanisms such as relaxation, and provides evidence to support the clinical relevance of functional MRI data/biomarkers . This apMost clinical scanners operate at between 1 and 3T, having lower SNR levels than pre-clinical high field systems, and hence reduced image quality if standard clinical imaging coils are used. This is exacerbated particularly when imaging the small fields-of-view necessary while using rodents, but with a high enough resolution to be able to accurately resolve lesions and acquire meaningful functional data. One approach of increasing SNR is to use small dedicated radiofrequency (RF) receiver coils. Such coils can be designed to fit closely to the object of interest, giving a better coupling between the object and coil (high filling factor), a stronger signal and thus improved image quality.in vivo, transgenic mouse models, in which tumours are driven by expression of the target gene of interest and arise spontaneously within the native tissue of origin, are being increasingly exploited  Act 1986, the United Kingdom National Cancer Research Institute guidelines for the welfare of animals in cancer research and the ARRIVE guidelines  were imaged in both scanners at ambient temperature. These solutions were transferred into 5 mm NMR tubes, which in turn were placed in 50 ml falcon tubes and restrained using dental paste to limit motion artefacts. The phantoms were scanned four times on each scanner, and the average value used to assess the T1 field dependence.To assess the linearity of MYCN is a potent oncogenic stimulus in cancer. MYCN expression correlates with an aggressive tumour phenotype, enhanced tumour angiogenesis and poor clinical prognosis in neuroblastoma  gene in neuroblastoma, and its close association with MYCN amplification, stimulated the development of a novel double transgenic mouse model for ALK and MYCN  underwent imaging on the 3T platform. Of these, 10 mice  were scanned at both 3 and 7T within 24 h of each other in order to compare tumour volume determination and quantification of tumour R2* at 3 and 7T. Dynamic contrast-enhanced (DCE)-MRI was performed simultaneously on three Th-MYCN mice at 3T. To evaluate the 3T platform for imaging-embedded intervention trials, anatomical and functional (native T1 and R2*) MRI measurements were acquired from Th-MYCN tumours prior to and 24 h post-treatment with either saline  or a single 25 mg kg−1 i.p. dose of the DNA alkylating agent cyclophosphamide , or prior to and 48 h post treatment with a daily oral dose of either water  or 30 mg kg−1 of the multi-kinase inhibitor cabozantinib   using a dedicated high-resolution RF coil  . The coi−1), fentanyl (0.05 mg kg−1), and medetomidine (0.5 mg kg−1) in sterile water. For 3T data acquisition, up to three anaesthetised mice were positioned supine with their abdomens in the centre of each individual element of the coil. Mouse core body temperature was maintained by a built-in heating system controlled within the ‘Mouse Hotel’ coil.Anaesthesia was induced by intraperitoneal injection of midazolam . T2-weighted images were used to assess the extent of the disease and aid axial sequence planning, which was performed across the central part of the tumour. Proton density and T1-weighted images were then acquired using a 3D spoiled gradient echo sequence with variable flip angle . Multigradient echo (MGE) images were acquired using a 2D gradient echo sequence with multiple echo times . No fat suppression was employed.Initially, whole body anatomical −1 gadopentate dimeglumine (Gd-DTPA) was simultaneously administered to all three mice at a rate of 2 ml min−1, 30 s after the start of the dynamic acquisition. The total 3T protocol scanning time was less than 15 min.For the acquisition of DCE-MRI data, a lateral tail vein of each mouse was cannulated with a 27G butterfly catheter connected to a 10 m long line of polyethylene tubing , and attached through the available wave-guides to syringe pumps located in the scanner control room, thereby enabling the simultaneous remote intravenous administration of contrast agent to each mouse. DCE-MRI data were acquired using the 3D spoiled gradient echo sequence with 200 time points, a temporal resolution of 2.5 s and a total acquisition time of 8 mins 33 s. A bolus injection of 0.1 mmol kgT2-weighted images for each tumour-containing slice, where clear delineation of tumour could be determined, using OsiriX Lite (Pixmeo). The T1-weighted images were analysed using dedicated software  3D spoiled gradient echo used to provide estimates of the native tumour spin-lattice relaxation time T1 (ms) (R2* (s−1) maps calculated by fitting a single exponential to the signal intensity echo time curve on a voxel-by-voxel basis using a Bayesian maximum a posteriori approach (Ktrans (min−1), the volume transfer constant between blood plasma and extracellular extravascular space, Ve , the initial area under the gadolinium concentration curve from 0 to 60 s after injection of Gd-DTPA.Tumour volumes were extrapolated from manually drawn regions of interest (ROIs) on T1 and R2* of tumours arising within Th-MYCN and Th-ALKF1174L/Th-MYCN transgenic mice was performed on a 7T horizontal bore microimaging system  using a 3-cm birdcage volume coil, as previously described  was found between the T1 values obtained from the 3T clinical scanner and the 7T pre-clinical scanner, with the median T1 values being higher at 7T.Measurements from the T2-weighted MR images of tumour-bearing mice acquired at 3 and 7T are displayed in in vivo. No significant difference was found between volumetric measurements acquired at the different field strengths , and which were significantly correlated , impossibility of achieving the exact same positioning and acquiring data from the same central slice on the two imaging platforms, and the absence of any B1 correction for the data acquired at 3T.Parametric T1 and R2* obtained at 3T from representative Th-MYCN and Th-ALKF1174L/Th-MYCN mice are shown in T1 and R2* values was apparent in tumours arising in the Th-ALKF1174L/Th-MYCN mice. The quantitative data are summarised in T1 between the Th-MYCN and the Th-ALKF1174L/Th-MYCN mice , R2* was significantly faster in tumours in the Th-MYCN mice when compared to the Th-ALKF1174L/Th-MYCN cohort .Parametric maps of native in vivo. Pharmacokinetic modelling was employed to derive and spatially map DCE-MRI parameters. Parametric Ktrans maps obtained from each tumour-bearing mouse are shown in Ktrans, Ve and IAUGC60 acquired from the three simultaneously imaged animals were 0.161 min−1 (0.124–0.202 min−1), 0.396 (0.374–0.415) and 3.28 (2.38–4.27) mM.s, respectively.DCE-MRI was successfully performed on three transgenic mice simultaneously using the 3T platform, yielding data with sufficient temporal and spatial resolution to evaluate vascular biomarkers of each imaged tumour MYCN mice prior to and 24 h after treatment with saline or CPM are shown alongside individual tumour volume, T1 and R2* values in P=0.0015) reduction in tumour burden, and was associated with a significant decrease in native T1 (P=0.0085). Collectively this translated to a significant difference in both relative tumour volume (P=0.0004) and native T1 (P=0.0035) between the control and CPM groups (P=0.0076). There was however no associated significant change in relative native T1 or R2* with CBZ, and no significant treatment-induced change in any parameter over 48 h in the control group  on clinical scanners, used to inform on either treatment stratification or treatment response  and spectroscopy are considered. This can contribute to a decrease of In conclusion, we have demonstrated the utility and sensitivity of a high resolution 3-channel/3-animal MR RF coil for performing informative anatomical and functional MRI studies in transgenic mouse models of neuroblastoma on a clinical 3T system. In this way, simultaneous data acquisition in multiple tumour-bearing animals can provide high efficiency/throughput for both phenotypic characterisation and trials of novel therapeutics, with additional mechanistic insight provided by multiparametric MRI, improving the accuracy of pre-clinical data and facilitating the introduction of functional MRI biomarkers into aligned imaging-embedded clinical trials.


The incidence of delayed graft function in patients undergoing kidney transplantation remains significant. Optimal fluid therapy has been shown to decrease delayed graft function after renal transplantation. Traditionally, the perioperative volume infusion regimen in this patient population has been guided by central venous pressure as an estimation of the patient’s volume status and mean arterial pressure, but this is based on sparse evidence from mostly retrospective observational studies. Excessive volume infusion to the point of no further fluid responsiveness can damage the endothelial glycocalyx and is no longer considered to be the best approach. However, achievement of adequate flow to maintain sufficient tissue perfusion without maximization of cardiac filling remains a challenge. Novel minimally invasive technologies seem to reliably assess volume responsiveness, heart function and perfusion adequacy. Prospective comparative clinical studies are required to better understand the use of dynamic analyses of flow parameters for adequate fluid management in kidney transplant recipients. We review perioperative fluid assessment techniques and discuss conventional and novel monitoring strategies in the kidney transplant recipient. Despite improvements in outcomes for patients undergoing renal transplantation, delayed graft function (DGF) remains a significant complication and is a predictor of the subsequent clinical course . DelayedThe kidneys receive approximately 25% of the body’s total cardiac output (CO) and are essential for maintaining tonicity of body fluids and for adjusting extracellular volume. Patients with renal failure often have electrolyte imbalances and tend to oscillate between hypovolemia and hypervolemia . This reCentral venous pressure (CVP)-guided volume infusion is the traditional approach in renal transplantation , 8 and iPrevious studies on the assessment of intravascular volume and optimization of CO and renal blood flow have indicated that conventional monitoring provides insufficient data for adequate fluid management –16. HoweIn this article, we review perioperative fluid assessment techniques and discuss conventional and novel monitoring strategies in this challenging patient population.The definition of DGF is not consistent in the literature. At least 18 different heterogeneous criteria were identified in a systematic review . DelayedThe reported incidence of DGF varies from 2 to 70% in deceased kidney transplants , 23, notThe underlying pathophysiology of DGF can be donor, recipient or surgeon related and can be due to hemodynamic (ischemic–reperfusion) or immunological (particularly T-lymphocyte) processes. A key factor is occult imbalance between oxygen delivery and consumption in the graft .Acute tubular necrosis (ATN) is the most common cause of DGF. Acute tubular necrosis can be already present at the time of organ procurement if the deceased donor’s kidney is injured. Prolonged warm and cold ischemia time, as well as the manner of preservation, also can lead to ATN . Acute tSeveral epidemiological studies in different patient populations have confirmed an association between stages of AKI and short-term and long-term outcomes –33 as weIn summary, DGF is predominantly caused by ATN. Adequate perfusion of the transplanted kidney is required, but to date no specific therapeutic intervention has reduced the incidence of ATN and recovery . The rolEuvolemia: a state in which the vascular volume is adequate for filling of the heart and maintenance of an appropriate CO to generate the appropriate oxygen supply for tissue needs.Fluid responsiveness: a state in which an increase in vascular volume increases the stroke volume (SV) and CO. The notion of fluid responsiveness does not necessarily mean that the patient will benefit from or need fluid therapy. Parameters of perfusion should guide the decision to assess the fluid response rather than responsiveness in a compromised patient.Fluid overload: a state in which excessive accumulation of fluid in the vascular system is caused by excessive parenteral infusion or deficiencies in cardiovascular or renal fluid volume regulation.Hypovolemia: a state in which effective circulatory volume is decreased and there is insufficient oxygen delivery to tissues, possibly resulting in organ dysfunction.The assessment of a patient’s intravascular volume for achievement of appropriate fluid management is a major challenge for the clinician and has been shown to improve postoperative outcomes . UnderstDespite a clear definition, there is no gold standard for the diagnosis and treatment of hypovolemia. Both acute and chronic conditions interact with fluid homeostasis, and medical history must always be considered. Progressive loss of renal function results in various adaptive and compensatory changes to maintain homeostasis with glomerular filtration rates below 10 ml/min, and these abnormalities typically have clinical consequences. A patient with end-stage renal disease has a disturbed acid–base and electrolyte balance and compromised hemodynamic autoregulation. Inflammatory mediators can alter renal hemodynamics and cause glomerular, tubular and interstitial damage, if uncontrolled .In addition to the uncertainty associated with the patient’s volume state, appropriate targets remain unclear. To determine which monitoring method provides the most accurate information in the context of kidney transplantation, and which target to rely on, we will review previously implemented methods, their advantages and disadvantages, and their relative utility in predicting fluid responsiveness and tissue perfusion. Methods range from conventional techniques such as clinical history and physical examination, measurement of blood pressure (BP), heart rate (HR), urine output (UOP), CVP and pulmonary artery pressure (PAP), to novel technologies such as echocardiography, cardiorespiratory interactions and dynamic analyses of flow parameters.Clinical experience and use of traditional parameters such as BP, HR, UOP, CVP  and PAP Hypovolemia in the perioperative setting often can be misdiagnosed clinically  due to aIn the case of hypovolemia and hypoperfusion, physiological compensatory mechanisms can maintain effective vascular volume and perfusion by increasing the vascular retention of an infused volume and decreasing vascular capacitance. Plasma levels of renin and aldosterone increase and atrial natriuretic factor decreases, followed by arterioconstriction which decreases precapillary hydrostatic pressure and filtration , 42. A 3Blood pressure is the most commonly used vital parameter for organ perfusion, and functions as an indirect measurement of renal perfusion in clinical practice. Although classic physiological studies suggest that autoregulation of renal blood flow is maintained at lower levels of mean arterial pressure (MAP 50–60 mmHg) , 45, retGiven the expected large oscillations in hemodynamic parameters during kidney transplantation, invasive arterial BP monitoring is commonly used. In a recent study of the biochemical outcomes of renal transplant recipients, no difference was observed in the creatinine levels of patients with a MAP between 95 and 131 mmHg, but MAP was continuously maintained at > 95 mmHg via the judicious use of fluids and dopamine . ConsistEarlier studies have indicated that the magnitude of UO is one of the most clinically used predictors for graft function immediately after reperfusion  and propA decrease in CVP in the post-transplant period is observed in a majority of kidney recipients, independent of their intravascular fluid status , but thiUse of pulmonary artery catheters has fallen out of favor due to concerns about complications such as pulmonary embolism among high-risk patients , pulmonaStatic cardiac filling pressures such as CVP and PAP correlate poorly with the intravascular volume and are notoriously unreliable in predicting accurate fluid responsiveness compared to dynamic parameters , 60. In Thus, the CVP should not be used alone to guide fluid responsiveness  and shouThe primary goal in the perioperative setting is to avoid tissue hypoxia, the major cause of organ dysfunction. Traditional indicators can be normal in the presence of tissue hypoxia and cannot be used to predict an imbalance between oxygen demand and consumption, particularly if they are not interpreted in the context of perfusion markers such as CO, lactates and central venous saturation –15, 61.Sufficient oxygenation and tissue perfusion are crucial for all metabolic needs of cells and are based on hemoglobin concentration, arterial oxygen content and CO. Therefore, the percent change in any one of these three variables equally alters oxygen delivery, with CO usually having the largest changes . As CO iAccording to Frank–Starling’s law of the heart, administering fluids can increase the preload and thereby increase SV and CO. Under physiological conditions, both ventricles work on the ascending portion of the Frank–Starling curve, and afterload, contractility and HR are assumed to remain constant during the volume infusion . This meAmong the many devices and monitors currently available for measuring cardiac function and output, we will review the intraoperative use of transesophageal echocardiography (TEE) and emphasize the utility of noninvasive devices that calculate reliable trends in CO and dynamic indices based on simple physiology.Transesophageal echocardiography can be a useful diagnostic tool for evaluating right and left heart function, potential outflow tract obstruction and the presence of a pericardial effusion. It also can be used to obtain Doppler measurements of CO. However, TEE is operator dependent, and errors in the measurement of the diameter of the left ventricular outflow tract skew results. Presence of an irregular HR also affects the final calculation of CO.Use of intraoperative TEE on renal allograft outcomes has only been evaluated in one study, in which living donor renal transplants were examined. Guiding fluid therapy in 110 patients by corrected flow time obtained with continuous transesophageal Doppler (TED) monitoring did not improve immediate graft function compared to fluid administration guided by CVP. However, significantly less fluid was used in the TED-monitored group, and the incidence of postoperative complications related to fluid overload was reduced .Transesophageal eechocardiography does not permit continuous monitoring for instantaneous evaluation of fluid responsiveness. Transesophageal echocardiography is also time consuming and expensive, and requires intensive operator training. Careful attention must be given to the limitations of the assessment of CO by TEE, which may be beyond the level of expertise of a physician with only moderate experience. New studies with more robust data are required to support the use of TEE for fluid management in this patient population.Cardiac output alone can be a poor predictor of fluid responsiveness, although its response to a preload-modifying maneuver such as mechanical ventilation can provide useful information about fluid responsiveness without the administration of fluid .2 rebreathing [Currently there are several noninvasive CO monitoring technologies including pulse contour analysis (PCA), pulse wave transit time, thoracic electrical bioimpedance/bioreactance and COreathing . We willreathing .Dynamic variation of arterial waveform-derived parameters , pulse pressure variation (PPV) and stroke volume variation (SVV)) in mechanically ventilated patients provides a precise indication of fluid responsiveness, particularly when compared to static indices . HoweverThe fundamentals underlying these parameters are based on simple physiology and are well represented in the Frank–Starling curve Fig. . The cycDynamic analyses of flow parameters need to be restricted to patients under controlled mechanical ventilation with tidal volumes between 8 and 10 ml/kg ideal body weight because heart–lung interactions are more complicated with spontaneous breathing efforts with or without a ventilator . When riThe use of PPV may be preferable to SPV and SVV because it is measured directly from the arterial pressure tracing and uses more advanced digital software . DespiteIn a recent study from Japan, SVV was compared with CVP and pulmonary artery diastolic pressure as estimates of the right and left ventricular preload in patients undergoing renal transplantation. As expected, SVV better predicted volume responsiveness . A retroAn overview of studies monitoring and targeting fluid therapy in kidney transplantation and their main outcomes is presented in Table Accumulating evidence supports the concept that fluid therapy should be individualized and based on dynamic indices of the intravascular volume. Conventional monitoring does not provide sufficient information for this approach, but novel technologies may allow titration of therapeutic interventions to ensure that oxygen delivery matches tissue need. One approach using dynamic indices has been goal-directed therapy (GDT). Goal-directed therapy decreased postoperative complications, hospital length of stay, mortality and hospital costs in high-risk surgical patients –19. HoweIn contrast to GDT, which is guided by “goals”, flow-directed therapy dictates fluid administration based on “triggers” . The triBy optimizing perioperative hemodynamic management in kidney transplantation, we hopefully can improve patient and kidney outcomes. The most recent studies of fluid therapy in the perioperative setting provide more questions than answers and challenge faith in traditional concepts. Effective perioperative fluid administration in kidney transplantation remains a black box, reinforced by the fact that fluid requirements are highly variable among patients and surgical procedures and the assessment of an individual patient’s intravascular volume remains challenging. It is unlikely that there will be a standardized all-inclusive algorithm, and a more highly individualized evidence-based approach adapted to each patient’s physiological needs and responses to volume and pharmacological therapy might be more helpful.Prospective comparative clinical studies are warranted to better understand the use of dynamic analyses of flow parameters for optimal fluid management in specific patient populations such as kidney transplant patients, who often have impaired cardiovascular physiology and reduced hemodynamic autoregulation due to end-stage kidney disease.