Since its inception in 2001, in utero electroporation (IUE) has been widely used by the neuroscience community. IUE is a technique developed to introduce plasmid DNA into embryonic mouse brains without permanently removing the embryos from the uterus. Given that IUE labels cells that line the ventricles, including radial fibers and migrating neuroblasts, this technique is an excellent tool for studying factors that govern neural cell fate determination and migration in the developing mouse brain. Whether IUE has an effect on microglia, the immune cells of the central nervous system (CNS), has yet to be investigated.pCIG2, pCIC-Ascl1, or pRFP-C-RS expression vectors to label radial glia lining the ventricles of the embryonic cortex and/or hypothalamus. Specifically, we conducted IUE at E14.5 and harvested the brains at E15.5 or E17.5. Immunohistochemistry, along with cytokine and chemokine analyses, were performed on embryonic brains with or without IUE exposure.We used IUE and the pCIG2, pCIC-Ascl1, or pRFP-C-RS vectors alone altered microglia morphology, where the majority of microglia near the ventricles were amoeboid and displayed altered expression signatures, including the upregulation of Cd45 and downregulation of P2ry12. Moreover, IUE led to increases in P2ry12− cells that were Iba1+/IgG+ double-positive in the brain parenchyma and resembled macrophages infiltrating the brain proper from the periphery. Furthermore, IUE resulted in a significant increase in cell death in the developing hypothalamus, with concomitant increases in cytokines and chemokines known to be released during pro-inflammatory states . Interestingly, the cortex was protected from elevated cell death following IUE, implying that microglia that reside in the hypothalamus might be particularly sensitive during embryonic development.IUE using the Our results suggest that IUE might have unintended consequences of activating microglia in the embryonic brain, which could have long-term effects, particularly within the hypothalamus.The online version of this article (10.1186/s12974-018-1213-6) contains supplementary material, which is available to authorized users. Microglia are the resident immune cells of the central nervous system (CNS). Murine microglia are generated from yolk sac erythro-myeloid progenitors (EMPs), which are a distinct lineage from hematopoietic stem cells –3. DurinIncreasingly, microglia are being shown to play a role in neural development. For example, in the embryonic brain, microglia influence neural progenitor maintenance , as wellIn utero electroporation (IUE) is a technique developed to introduce plasmid DNA into embryonic mouse brains, while the animals are still alive in the uterus –22. ManypCIG2, pCIC-Ascl1, or pRFP-C-RS IUE, embryonic brains demonstrated high numbers of amoeboid microglia that displayed altered expression signatures within 24 h following electroporation, including the upregulation of Cd45 and downregulation of P2ry12. IUE also resulted in a significant increase in cell death in the developing hypothalamus, including changes in cytokines and chemokines known to be released during pro-inflammatory states. Taken together, our results demonstrate that embryonic microglia become activated following IUE, and suggest that the hypothalamus is particularly sensitive to inflammation.Given our interest in radial glia and microglia interactions, and since we routinely employ IUE to label radial glial cells, we wanted to explore any potential effects of the IUE technique on microglia. Following CD1 mice (Charles River) were used for all experiments. Animal protocols were approved by the University of Calgary Animal Care Committee and followed the Guidelines for the Canadian Council of Animal Care.pCIG2 expression vector, which contains a β-actin promoter/CMV enhancer and an IRES–EGFP cassette, was used for IUE shown in the primary figures. In addition, the pCIC-Ascl1 expression vector, which contains a β-actin promoter/CMV enhancer upstream of the Ascl1 sequence and an IRES–mCherry cassette, and the pRFP-C-RS expression vector , which contains a CMV promoter and a tRFP cassette, were used in Additional file pCIG2 DNA was injected at a concentration of 0.5–0.7 μg/μL into the lateral ventricle of E14.5 brains. Following DNA injection, 7 mm BTX platinum plated electrodes (Harvard Apparatus) and a BTX ECM 830 Electro Square Porator (Harvard Apparatus) were used to pulse  embryonic brains five times, separated by intervals of 950 ms. Once the embryos were placed back inside the pregnant dam, the cavity was filled with warm saline and the peritoneum was sutured shut, which was followed by suturing closed the abdominal wall. Following the stop of anesthesia, 2 mL of Ringer’s solution was injected into the back of the pregnant female, which was placed on a heating pad to aid in recovery.The IUE procedure has been described elsewhere . In brieTwenty-four or 72 h following IUE, E15.5, or E17.5 brains were collected in ice-cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. The brains were then washed in PBS and equilibrated in 20% sucrose/PBS overnight at 4 °C. Brains were embedded in Clear Frozen Section Compound  and cryosectioned (10–20-μm sections). For immunohistochemistry (IHC), cryosections were rehydrated in PBS, washed with PBT (PBS with 0.1% Triton-X), blocked using 5% normal donkey or goat serum  for 1 h at room temperature (RT), and exposed to rabbit anti-Fezf1 , chicken anti-GFP , rabbit anti-Iba1 , goat anti-Iba1 , rat anti-Cd45 FITC , rabbit anti-P2ry12 , rabbit anti-cleaved active caspase 3 , goat anti-Sox9 , mouse anti-NeuN , and/or goat anti-Vegfr2  at 4 °C overnight. Slides were then washed with PBT and exposed to secondary antibody  for 2 h at RT. Sections were mounted using Aqua Poly/Mount (Polysciences Inc.). Fluorescent IHC images were captured on a Zeiss Axioplan 2 fluorescent microscope. Brightness and/or contrast of the entire image was adjusted using Adobe Photoshop CS5.1 if deemed appropriate.v/v) 56.4% DMEM , 28.2% F-12 , 5% fetal bovine serum, 5% horse serum, 2% B-27 supplement , 1% N2 supplement , 1% GlutaMAX , 1% penicillin/streptomycin , and 0.4% fungizone . Brains were placed in a 37 °C incubator with 5% CO2. For the cytokine/chemokine assay, culture media was collected 6 h following the start of culturing. The collected culture media was spun down at 3000×g for 10 min to remove debris, and the middle 200 μl of the culture media was removed and flash frozen with liquid nitrogen. The resulting samples were sent to Eve Technologies  and analyzed using the Mouse Cytokine Array/Chemokine Array 31-PLEX (MD31). Each sample was analyzed for eotaxin, G-CSF, IL-1β, IL-6, IL-10, IP-10, MCP-1, MIP-1α, MIP-1β, MIP-2, RANTES, and TNFα .Embryonic wild-type and IUE (E14.5) brains were collected at ~ E14.75 (6 h following IUE) or at E17.5 (3 days following IUE) and placed in a 48-well tissue culture plate  with 300 μl of culture media containing . ImageJ was used to determine the relative downregulation of P2ry12 staining between E15.5 wild-type and IUE brains. Quantitative results for all cell counts and cytokine/chemokine protein levels are represented by mean scores ±SEM and were analyzed by two-tailed unpaired t tests using Prism 6 (GraphPad Software).The first four 20-μm sections prior to the start of Fezf1 staining  were used for all cortex cell counts, and the first four 20-μm Fezf1+ sections were used for all hypothalamic cell counts  14.5 brains that were still intact in the uterine horns but temporarily removed from the body cavity of pregnant dams. Any plasmid injected into the lateral ventricle diffuses into the third ventricle given that the ventricles are still connected at this time. Twenty-four hours following the IUE procedure, we stained E15.5 brains for GFP to detect the patch of cells successfully electroporated. GFP-labeled cell bodies and projections were observed lining the lateral ventricles in the cortex  brains as compared to wild-type . Similarly, quantification of P2ry12 staining in the hypothalamus . Although altered morphology and changes in expression were observed elsewhere in the brain . Given that macrophages have Fc receptors that bind IgG, and that IgG antibodies are present at a high quantity in the peripheral circulation, cells that have recently entered the brain parenchyma from the periphery often have some IgG bound by their Fc receptors [+/IgG+ cells that were P2ry12− might represent a population that had recently entered the brain from the periphery.Given the accumulation of Iba1eceptors –33. Ther+/IgG+ double-positive cells within the parenchyma of IUE brains  brains as compared to wild-type , consistent with the notion that IUE causes peripheral myeloid cells to invade the developing brain.Immunohistochemistry (IHC) with Iba1 and IgG antibodies demonstrated an increase in Iba1pCIG2 IUE (E14.5) brains, (2) E15.5 brains electroporated with electrode paddles only (at E14.5), (3) E15.5 brains injected with elution buffer (EB) only (at E14.5), or (4) E15.5 brains punctured with the glass needle (at E14.5). We detected a significant increase in the number of Iba1+ cells that upregulated Cd45 in the hypothalamus of E15.5 brains electroporated with paddles alone  and injected with EB alone ; however, brains that received the entire IUE procedure showed a striking increase in Cd45 expression in the hypothalamus . At the same time, a decrease in the microglia-specific marker P2ry12 was observed in Iba1+ cells, as compared to wild-type , was the E15.5 brain electroporated with electrode paddles at E14.5 . Furthermore, P2ry12 was similarly downregulated in Iba1+ cells in the cortex  E15.5 pCIG2 DNA at a concentration of 0.5 μg/μL into the lateral ventricle of E14.5 brains, which were pulsed using 30 V (50 ms) four times, separated by intervals of 1000 ms (versus 45 V (50 ms) five times separated by intervals of 950 ms, as used previously). Similar to what we observed using the original IUE parameters, IUE with the gentler parameters still resulted in the upregulation of Cd45 , consistent with previous results [p = 0.0281). While the majority of the CC3+ cells within the hypothalamus appeared to be apoptotic bodies , interleukin 1 beta , and interleukin 6 , as well macrophage inflammatory protein 2 , regulated upon activation normal T cell expressed and secreted , and interferon gamma-induced protein 10  in IUE brains as compared to wild-type brains. IUE brains also showed elevated levels of monocyte chemotactic protein 1 , macrophage inflammatory protein 1 alpha , macrophage inflammatory protein 1 beta , ganulocyte colony-stimulating factor , and eotaxin  as compared to wild-type brains. Together, the results suggest that IUE induces a pro-inflammatory response, which is supported by the changes in microglia morphology and expression signatures, in addition to the infiltration of peripheral myeloid cells.Given that IUE alters microglia morphology and expression signatures in a way that resembles activation of microglia during a pro-inflammatory immune response, we next measured and compared cytokine and chemokine levels in wild-type and IUE brains. To do this, we cultured whole ~ E14.75 wild-type and IUE (E14.5) brains for 6 h and collected the conditioned media to analyze cytokine and chemokine protein levels. Specifically, we found an upregulation of the pro-inflammatory cytokines and chemokines: tumor necrosis factor alpha , which can be produced by activated macrophages in order to suppress macrophage activation and the further production of pro-inflammatory cytokines/chemokines [+/Cd45high cells within the parenchyma of the hypothalamus  and cortex  showed that E17.5 IUE brains and wild-type brains were comparable by E17.5. Although microglia morphology and expression signatures appeared to recover in E17.5 IUE (E14.5) brains , IL-1β , and IL-6 , as well as MIP-2 , RANTES , and IP-10 , were all trending towards higher levels in IUE brains. Similarly, some E17.5 IUE (E14.5) brains also showed elevated levels of MCP-1 , MIP-1α , MIP-1β , G-CSF , and IL-10  but again, not at the level of significance. In fact, eotaxin  was the only marker that had normalized fully to levels observed in wild-type brains. Together, the results suggest that IUE induces an immediate pro-inflammatory response, which appears to recover at different rates in different embryos.Given the upregulation of the anti-inflammatory cytokine interleukin 10 in IUE brains  following IUE, and if so, whether embryonic microglia respond to the transfected cells using toll-like receptors (TLRs), TAM receptors , or other mechanisms shown to be used to recognize viral DNA or foreign cytoplasmic DNA [Given that microglia originate from yolk sac-derived Cd45ammation . Howevern proper –33, 37. n proper –40. For n proper –45. Resin proper , 47. It smic DNA –48.Considering that IUE involves both the uptake of foreign DNA and the generation of a foreign protein , future experimentation should also determine whether it is the presence of a foreign DNA plasmid or the generation of a foreign protein that is causing the cells in the CNS to mount an immune response following IUE. Given that fluorescent proteins are expressed in a number of transgenic mouse lines, in which we have not observed an immune response (data not shown), we propose it is the uptake and presence of foreign DNA itself in the cell’s cytoplasm that is triggering the immune response we observe following IUE. Moreover, considering that most IUE experimental procedures use constructs coding for a fluorescent protein to label the transfected cells, the current study has demonstrated that careful controls are needed when using this approach. Nonetheless, further investigation of whether the foreign DNA is in fact stimulating the neuroimmune response following IUE is needed, as well as a deeper appreciation of the consequences of the resulting immune response.During microglia activation and the subsequent infiltration of peripheral myeloid cells, inflammatory cytokine and chemokine levels are often elevated , 49, 50.pCIG2) in addition to a wild-type control, to enable comparison for any long-term consequences of the IUE procedure itself.Along these lines, we did observe an increase in apoptosis specifically in the hypothalamus, suggesting that at least in this brain region, IUE might permanently affect development. During a pro-inflammatory state, cell death programs are often initiated in order to instigate phagocytic cells such as macrophages and microglia to clear foreign materials. Thus, observing an increase in apoptotic cells within the hypothalamus following IUE suggests that this brain region is particularly sensitive to IUE and inflammation during development. Furthermore, considering inflammation during development can have long-term consequences on learning and memory as well as other behavioral alterations –19, we ppCIG2, pCIC-Ascl1, or pRFP-C-RS IUE alone activates microglia and alters microglial expression signatures. Importantly, IUE was shown to induce cell death in the developing hypothalamus but not the cortex. Moreover, we observed universal upregulation of pro-inflammatory cytokines and chemokines throughout the embryonic brain, consistent with the notion that IUE causes inflammation. These findings demonstrate that IUE might have the unintended consequence of activating microglia in the embryonic brain, which could have long-term consequences in a region-specific manner, particularly within the hypothalamus.By exploring the effect of IUE on microglia, we demonstrated that Additional file 1:Supplementary Figures 1–9. (PDF 31554 kb)


Second, to evaluate interobserver agreement and reader delineation confidence.To compare maximum tumour diameter (MTD) and gross tumour volume (GTV) measurements between T2-w and high-b-value DWI images and compared between sequences; T staging was derived from MTD. Interobserver agreement was assessed and delineation confidence scored (1 to 5) by each observer.The staging MRI scans of 45 SCCA patients  were assessed retrospectively by two independent radiologists . MTD and GTV were delineated on both T2-w sequences by 14.80%/9.98% (MTD) and 29.70%/12.25% (GTV) for each reader, respectively, causing T staging discordances in approximately a quarter of cases. Bland-Altman limits of agreement were narrower and intraclass correlation coefficients higher for DWI. Delineation confidence was greater on DWI: 40/42 cases were scored confidently (4 or 5) by each reader, respectively, versus 31/36 cases based on T2-w images.GTV and MTD were significantly and systematically lower on DWI versus TSequence selection affects SCCA measurements and T stage. DWI yields higher interobserver agreement and greater tumour delineation confidence.MTD and GTV measurements are significantly lower on DWI than on T2-w MRI.• Such differences cause T staging discordances in up to a quarter of cases.• DWI results in higher agreement between inexperienced and experienced observers.• DWI offers greater tumour delineation confidence to inexperienced readers.•  The incidence of squamous-cell carcinoma of the anus (SCCA), commonly referred to as anal cancer, has increased steadily over the past 4 decades in the Western world , 2. The 2-weighted (T2-w) sequences, obtained in the appropriate planes, provide detailed anatomical depiction of the anorectal region thanks to optimal soft-tissue contrast [Magnetic resonance imaging (MRI) is recommended in Europe as the imaging modality of choice for loco-regional staging of SCCA  and has contrast –8 and arb-value sequences, facilitating detection and delineation. Anal cancers typically appear restricted on DWI [Diffusion-weighted imaging (DWI) is now routinely included in body MRI protocols in most European oncological imaging centres: it has been shown to aid the diagnosis and response assessment of a variety of malignancies –13 and td on DWI .th ed.) criteria [Maximum tumour diameter (MTD) is an important measurement in anal cancer, as it determines the T stage according to current TNM ; the primary tumour was deemed undetectable on either sequence by secondary consensus reading (n = 2); the presence of MRI image artefact precluded accurate tumour measurements (n = 0).A review board waiver was granted for this retrospective analysis of anonymised imaging data acquired as part of normal clinical care. Fifty patients with biopsy-proven SCCA undergoing pelvic MRI for locoregional staging prior to definitive chemoradiation were identified from the picture archiving and communication system (PACS) of two tertiary-referral cancer centres, between July 2007 and June 2015. Cases were excluded if the tumour was incompletely imaged on either T2-w sagittal turbo spin echo (TSE) sequence covering the pelvis , a T1-w axial TSE sequence for pelvic nodal detection , a T2-w axial TSE sequence of the pelvis  and high-resolution small-field-of-view T2-w TSE sequences perpendicular and parallel to the anal canal . DWI consisted of a single shot spin echo-echo planar imaging (SE-EPI) axial diffusion-weighted sequence  encompassing the pelvis with three b-values in all cases . Vendor-generated apparent diffusion coefficient (ADC) maps were automatically created at the time of acquisition. Patients did not undergo any additional preparation prior to the examination.Patients were scanned supine on one of three 1.5-T MRI scanners  using a pelvic phased array coil. The examination protocol included a T2-w and axial high-b-value (b = 800) DWI sequences, with a 1-week interval between the two reading sessions; DWI was read in conjunction with apparent diffusion coefficient (ADC) maps. Free-hand perilesional regions of interest (ROIs) were drawn on each slice with visible tumour and GTVs obtained by computing the ROI volumes. MTDs were obtained from sagittal T2-w sequences and sagittal reformats of axial high-b-value DWI, choosing the plane yielding the longest measurement on a case-by-case basis and using straight-line measurements  with 1 year prior MRI experience but no previous experience in staging SCCA and a subspecialty gastrointestinal radiology fellow (DP) with 5 years’ experience of staging gastrointestinal cancers evaluated the scans independently using all available sequences. Anonymised scans were downloaded from the local PACS onto a standalone workstation  and presented in randomised order in OsiriX v.7.5.1 ; readers were blinded to all clinical information. GTV delineation was performed separately on high-resolution axial-oblique T2-w and DWI sequences using a 5-point scale ; 3, tumour boundaries identified with confidence on approximately half of the images; 4, tumour boundaries identified confidently on most images (> 75%); 5, tumour boundaries identified confidently on all images).Each observer rated their confidence at contouring each tumour GTV on both T2-w vs. DWI comparisons; measurements were compared using the independent samples t-test and correlated using Pearson’s r. Interobserver agreement between the readers’ MTD and GTV measurements was assessed using the 95% Bland-Altman limits of agreement [P value < 0.05 was taken to represent statistical significance for all analyses.Statistical analysis was performed using IBM SPSS Statistics, version 23. Mean values between the two readers’ MTD and GTV measurements were used in Tgreement . IntraclThe final cohort consisted of 45 patients, 25 females and 20 males, with a mean age of 62 years  and corresponding to 45 MRI data sets analysed by each observer.2-w sequences and DWI are summarised in Table 2-w and DWI for both observers  (Table Reader-specific tumour diameters and volumes measured on T1) Table . As expe2-w and -0.14 (-2.38 to +2.10) cm on DWI. Agreement was considerably superior on DWI for GTV. Mean GTV difference was -3.96 (-17.91 to +9.97) cm3 on T2-w and 0.87 (-6.75 to +8.50) cm3 on DWI  between the two readers was -0.46 (-2.89 to +1.97) cm on T2-w sequences by both readers. This gap in confidence was more substantial for the inexperienced reader: they assigned a low confidence score (1 to 3) to 14 cases on T2-w versus 5 cases for the experienced reader on DWI and a high confidence score to 31 cases on T2-w versus 40 cases on DWI. Full confidence score results are reported in Figs. Tumours were outlined with greater confidence on DWI than on T2-w sequences.We found that tumour volumes and maximum diameters measured on functional DWI were significantly lower than those measured on anatomical T3 on DWI versus 21.8 cm3 on T2-w, a relative difference of 17.43%; similarly, Regini et al. measured smaller GTVs on DWI, with relative differences of 3.04% -7.62% not reaching statistical significance. Squamous cell carcinomas are typically associated with markedly impeded diffusion and display high signal intensities on DWI; adenocarcinomas, conversely, only appear moderately restricted because of their glandular structure and presence of mucin [2-w sequences.While no previous paper has investigated anal cancer, similar differences have been found in primary rectal cancer –21: Curvof mucin . This pa2-w and DWI sequences by both the inexperienced and experienced observer. With the wider implementation of personalised radiotherapy protocols, MTD and, consequently, T stage may also affect the GTV to clinical target volume (CTV) margin, the dose to the primary tumour and the use of simultaneous boost; the PLATO  protocol, for example, mandates an isocentric GTV-CTV margin of 10 mm for tumours up to 4 cm in MTD versus 15 mm for larger tumours [Tumour greatest dimension is the only measurement determining T stage in SCCA according to AJCC TNM criteria : based o tumours . To our Accurate tumour delineation is critical to radiotherapy planning. With the implementation of intensity modulated radiotherapy (IMRT) in the treatment of SCCA, it has become possible to escalate the dose to the target volume whilst maintaining the same or reducing dose to the surrounding normal tissues, resulting in steep dose gradients. To ensure appropriate dose delivery, both tumour and normal tissues must be delineated in 3D with high precision in reference to advanced diagnostic imaging techniques, including functional imaging . The imp2-w sequences represent the bedrock of pelvic MRI for detailed anatomical interpretation but require an advanced level of knowledge of the relevant cross-sectional anatomy. Signal intensities of tumour, muscle, fat and bowel contents are often very similar and can be challenging to tell apart with confidence. We believe our results partly reflect the challenges of distinguishing tumour from normal tissue in the anorectum on anatomical T2-w sequences alone. Fourteen and nine cases were assigned a low confidence score (1 to 3) by the inexperienced and experienced observer, respectively; these corresponded to either small (T1/T2) tumours with irregular margins and an infiltrative behaviour through the anal sphincter complex or anorectal junctional tumours surrounded by mucosal oedema and/or luminal fluid , emphasising the importance of taking the learning curve into account when approaching DWI.A drawback of the most commonly used single-shot echo-planar-imaging (EPI)-based DWI sequence is that it is prone to artefacts and susceptibility-related geometrical distortions, potentially detrimental in the setting of radiotherapy planning. These issues are being addressed through the development of distortion-correction strategies  and the Regarding the potential implications of underestimating vs. overestimating tumour length/volume, it is worth stressing that the current research focus in patients with early disease is radiotherapy dose de-escalation, given the low rates of locoregional failure and significant toxicity at current dose regimens , 23. Conb-values were nevertheless consistent. We did not evaluate spatial concordance and volume overlap between T2-w and DWI, as performed by Burbach et al. for rectal cancer [This study has a number of limitations: its retrospective nature meant that minor variations in the imaging acquisition across different 1.5-T scanners could not be avoided; the sequences used for measurements and DWI l cancer , though 2-w sequences to the inexperienced observer and even to the experienced in the case of small tumours infiltrating the anal sphincter complex or at the anorectal junction.In summary, this study has shown that anal cancer MTD and GTV measurements are consistently and significantly lower on DWI than on T2-w sequences, with consequent intersequence T staging discordances and potential implications for radiotherapy target volume delineation. This highlights the need for more specific guidelines on the subjects. Based on these findings and our clinical experience we would recommend the inclusion of DWI in anal cancer staging/radiotherapy planning MRI protocols and its use alongside anatomical sequences. DWI measurements resulted in higher agreement between observers with differing levels of experience. DWI offered greater tumour delineation confidence over T


The advent of array-based genome-wide DNA methylation methods has enabled quantitative measurement of single CpG methylation status at relatively low cost and sample input. Whereas the use of Infinium Human Methylation BeadChips has shown great utility in clinical studies, no equivalent tool is available for rodent animal samples. We examined the feasibility of using the new Infinium MethylationEPIC BeadChip for studying DNA methylation in mouse.In silico, we identified 19,420 EPIC probes (referred as mEPIC probes), which align with a unique best alignment score to the bisulfite converted reference mouse genome mm10. Further annotation revealed that 85% of mEPIC probes overlapped with mm10.refSeq genes at different genomic features including promoters (TSS1500 and TSS200), 1st exons, 5′UTRs, 3′UTRs, CpG islands, shores, shelves, open seas and FANTOM5 enhancers. Hybridization of mouse samples to Infinium Human MethylationEPIC BeadChips showed successful measurement of mEPIC probes and reproducibility between inter-array biological replicates. Finally, we demonstrated the utility of mEPIC probes for data exploration such as hierarchical clustering.Given the absence of cost and labor convenient genome-wide technologies in the murine system, our findings show that the Infinium MethylationEPIC BeadChip platform is suitable for investigation of the mouse methylome. Furthermore, we provide the “mEPICmanifest” with genomic features, available to users of Infinium Human MethylationEPIC arrays for mouse samples.The online version of this article (10.1186/s12859-017-1870-y) contains supplementary material, which is available to authorized users. Epigenetic mechanisms refer to dynamic processes that integrate internal and external signals and regulate gene expression in a spatiotemporal manner. DNA methylation is the most studied epigenetic mechanism and involves the covalent addition of a methyl group to cytosine primarily in the context of CpG dinucleotides. DNA methylation is established by the de novo methyltransferases, DNMT3A and DNMT3B, and is maintained during cellular division by DNMT1, thereby assuring propagation of the methylation patterns . FinallyThe recent development and optimization of methods for quantification of DNA methylation genome-wide have mainly focused on human genomic DNA, with special interest in reducing the sample input while improving both accuracy and coverage. Genome-wide DNA methylation arrays allow absolute measurement of single CpG methylation status located at various regulatory regions throughout the human genome at relatively low cost, thereby giving great utility in clinical studies. The use of Infinium HumanMethylation450 BeadChips (HM450) in epigenome-wide association studies in large cohorts has proven a very promising approach in deciphering putative pathogenic mechanisms influenced by both genetic and environmental factors , 4. HowePrior investigation of the feasibility of using the HM450 platform on non-human primate and mouse genomes has revealed common probes mapping to bisulfite converted reference genomes . The newhttp://www.novocraft.com) could also be considered. However, comparison of these three alignment tools for former versions of DNA methylation arrays (HM27 and HM450) has demonstrated Bismark to have the largest percentage of uniquely mapped probes overlapping between the three alignment tools [To identify EPIC probes with the potential of detecting DNA methylation in mouse, we first pursued an in silico approach whereby probe sequences were mapped to the mouse genome. The most recent genome of the widely used C57BL/6 mouse strain, referred to as mm10 or GRCm38), was downloaded from Ensembl and used as a reference genome Fig. . EPIC pr, was dowBismark relies on Bowtie for mapping, which by default uses a 5′-end seed of 28 nucleotides to initiate the alignment process . ImportaIn order to verify our mapping strategy EPIC probes were aligned to the human genome (hg19/GRCh37) using Bismark with default setting (−l 28 and -n 1), which returned 866,663 uniquely mapped hits corresponding to a mapping efficiency of 100% Fig. .mEPIC probes in the rest of the manuscript.We thus identified 19,420 EPIC probes  located in chromosome 11 and a minimum number of probes (513) in chromosome 16 Fig. . To annoOverlap analysis revealed that approximately 84%  of mEPIC probes overlapped with annotated mm10.refSeq genes, but that each gene was targeted by only a few probes Fig. , therefoComparison of mouse and human gene names for individual mEPIC probes revealed that a large fraction (> 80%) were common between the two species, thus suggesting that genomic regions covered by mEPIC probes are highly conserved between humans and mice. Considering that high sequence homology generally occurs within exonic regions we hypothesized that mEPIC probes primarily map to exons. To test this, we examined mEPIC probes in the light of RefSeq transcript information (exon start and end positions) extracted from the UCSC genome table browser. Overlap analysis between mEPIC probes and mm10.RefSeq exons revealed that 72% of gene-associated mEPIC probes  were indeed located in exons, corresponding to an overall 60% of the total 19,420 mEPIC probes.We subsequently examined gene- and CpG-related features of the mEPIC probes Fig.  as conveAnnotations of mEPIC probes are summarized in Fig. P-value cut-off.Finally, we aimed to validate mEPIC probes experimentally by performing DNA methylation analysis of mouse samples using Infinium MethylationEPIC BeadChips. To reduce sample heterogeneity as a possible bias for DNA methylation analysis we used genomic DNA extracted from sorted myeloid cells with >95% purity of C57BL/6 mice. Six samples arising from 3 cell types in biological duplicates were hybridized on two slides. Idat files were processed through scripts adapted from the ChAMP Bioconductor Package  which fiP-value cut-off of 0.01 (default settings), thus contrasting with the 19,420 mEPIC probes identified in silico. This discrepancy might result from ambiguous hybridization or, on the contrary, might suggest that additional signals could be used for DNA methylation studies in mouse samples. We first addressed this issue in silico by loosening the mapping criteria of EPIC probes and applying a higher mismatch allowance within the Bismark alignment. However, the number of uniquely mapped hits peaked at only 20,337 when 2 mismatches were permitted, as additional mismatch allowance resulted in ambiguous mapping, i.e. mapping of probes to multiple target regions. We then addressed whether a shorter part of the EPIC probe could be responsible for hybridization and subsequent generation of DNA methylation signals. Reduction of the length of the probe sequence to 20 nucleotides in silico and subsequent mapping to the mouse genome resulted in marked decrease in uniquely mapped hits, concomitant with an increase in ambiguous hits. We next examined the DNA methylation density distribution and observed that the 263,029 probes passing the detection P-value cut-off peaked at 0.3  were considered, the DNA methylation values displayed an expected bimodal distribution peaking at low (~0.1) and high (~0.85) beta values  did contain a CpG dinucleotide. The remaining probes predominantly targeted non-CpG (or CH) sites (13%), i.e. CpA (9%), CpT (2%) or CpC (2%) and TpG dinucleotides (9%) of which the latter could result from C > T transitions, a common DNA methylation-mediated mutation caused by deamination of 5-methylcytosine [Due to pooled samples with mixed gender, 633 mEPIC probes mapping to chromosome X (none of the mEPIC probes mapped to chromosome Y), as well as 31 mEPIC probes known to target non-CpG sites in humans, had been filtered prior to the analysis. Of the 18,756 remaining mEPIC probes, 18,559 (~99%) passed the detection cytosine . Moreovecytosine  we predo probes 7% did con2 > 0.999   or BMIQ 2 value of 0.996 , as the human counterpart. A summary of mEPIC probe characteristics is listed in the “mEPICmanifest” available in Additional file In conclusion, our study demonstrates that human Infinium MethylationEPIC BeadChip array is a valid and affordable platform for studying DNA methylation in mouse samples.−ckit+sca1−CD34+CD16/32int or Lin−ckit+CD115+Ly6C+CD11b− populations using a BD influx sorter for cell type 2 and 3, respectively.All C57BL/6 mice were bred and maintained under specific pathogen-free conditions in the animal facility at Karolinska University Hospital . Extraction of genomic DNA was performed using QIAamp DNA Micro Kit (Qiagen). Three cell populations were isolated from mouse bone marrow using: Monocyte isolation kit (Miltenyi Biotec) for cell type 1 and sorting of Linhttp://support.illumina.com/array/array_kits/infinium-methylationepic-beadchip-kit/downloads.html), converted to fasta format and processed to their reverse/complements. Processed EPIC probes are available in Additional file http://www.ensembl.org/info/data/ftp/index.html/), whereas different mouse strain genomes were downloaded from the Mouse Genomes Project (ftp://ftp-mouse.sanger.ac.uk/REL-1504-Assembly/). The “bismark_genome_preparation” function of Bismark (version 0.14.5) [EPIC probes were downloaded from the Illumina website ( 0.14.5)  with def 0.14.5)  was usedMismatch information was extracted from Bismark-derived BAM files using the calmd –e function of Samtools (version 1.5) . Since Bhttps://genome.ucsc.edu/cgi-bin/hgTables). 1500 nucleotides were subsequently added to the “txStart” of each transcript to ensure inclusion of proximal promoters in the annotation. Overlap analyses between mEPIC probes and RefSeq transcripts/exons were conducted with the “intersect” function of BEDTools (version 2.25.0) [http://fantom.gsc.riken.jp/5/datafiles/latest/extra/Enhancers/ and overlap with mEPIC probes was determined with the intersect function of BEDTools (version 2.25.0) [http://support.illumina.com/array/array_kits/infinium-methylationepic-beadchip-kit/downloads.html). Target site sequence context was retrieved via http://togows.org/api/ucsc/mm10/TargetSite_GenomicCoordinates.fasta.RefSeq genes were downloaded from UCSC genome table knownGenes (mm10.refGene) ( 2.25.0) . Count o 2.25.0) . Noticea 2.25.0) . FANTOM5 2.25.0) . InformaThe Infinium Human MethylationEPIC BeadChip (Illumina) platform was used for DNA Methylation profiling. Samples were randomized on 2 slides and processed by the core facility for Bioinformatics and Expression Analysis (BEA), Karolinska Institutet, Huddinge campus. Idat files were loaded into R using scripts adapted from the ChAMP package (version 2.6.0)  referredAdditional file 1:EPICprobes. (TXT 54191 kb)Additional file 2:mEPICmanifest. (XLS 5446 kb)Additional file 3: Figure S1.Human_EPIC. (PDF 692 kb)Additional file 4:Rat_EPIC. (XLS 1680 kb)Additional file 5:GuineaPig_EPIC. (XLS 1914 kb)Additional file 6:Rabbit_EPIC. (XLS 2083 kb)Additional file 7:Sheep_EPIC. (XLS 2552 kb)Additional file 8:Pig_EPIC. (XLS 2701 kb)Additional file 9:Cow_EPIC. (XLS 2971 kb)Additional file 10:Dog_EPIC. (XLS 3095 kb)Additional file 11:Cat_EPIC. (XLS 3076 kb)Additional file 12:Macaque_EPIC. (XLS 6450 kb)Additional file 13:Chimpanzee_EPIC. (XLS 6473 kb)Additional file 14:129S1_SvImJ_EPIC. (XLS 1809 kb)Additional file 15:A_J_EPIC. (XLS 1819 kb)Additional file 16:AKR_J_EPIC. (XLS 1818 kb)Additional file 17:BALB_cJ_EPIC. (XLS 1810 kb)Additional file 18:C3H_HeJ_EPIC. (XLS 1812 kb)Additional file 19:CAROLI_EiJ_EPIC. (XLS 1813 kb)Additional file 20:CAST_EiJ_EPIC. (XLS 1816 kb)Additional file 21:CBA_J_EPIC. (XLS 1808 kb)Additional file 22:DBA_2J_EPIC. (XLS 1811 kb)Additional file 23:FVB_NJ_EPIC. (XLS 1799 kb)Additional file 24:LP_J_EPIC. (XLS 1810 kb)Additional file 25:NOD_ShiLtJ_EPIC. (XLS 1801 kb)Additional file 26:NZO_HlLtJ_EPIC. (XLS 1812 kb)Additional file 27:Pahari_EiJ_EPIC. (XLS 1807 kb)Additional file 28:PWK_PhJ_EPIC. (XLS 1813 kb)Additional file 29:SPRET_EiJ_EPIC. (XLS 1806 kb)Additional file 30:WSB_EiJ_EPIC. (XLS 1812 kb)Additional file 31:mEPIC.loading.Script.R. (R 2 kb)Additional file 32:Champ.load.mEPIC.Script.R. (R 6 kb)Additional file 33:Champ.norm.mEPIC.Script.R. (R 10 kb)


Crystalloid fluid and vasoactive drugs are used in the early treatment of sepsis. The purpose of the present study was to examine how these drugs alter plasma volume expansion, peripheral edema, and urinary excretion.Twenty-five anesthetized sheep were made septic by cecal puncture and a short infusion of lipopolysaccharide. After 50 min, a slow infusion of isotonic saline was initiated: the saline either contained no drug, norepinephrine (1 μg/kg/min), phenylephrine (3 μg/kg/min), dopamine (50 μg/kg/min), or esmolol (50 μg/kg/min). Ten min later, 20 mL/kg Ringer´s lactate solution was given over 30 min. Central hemodynamics, acid-base balance, and the urinary excretion were monitored. Frequent measurements of the blood hemoglobin concentration were used as input in a kinetic analysis, using a mixed effects modeling software.1-receptors accelerated, while beta1-receptors retarded, the distribution and elimination of fluid. A pharmacodynamic Emax model showed that Ringer´s lactate increased stroke volume by 13 ml/beat. Alpha1-receptors, but not beta1-receptors, further increased stroke volume, while both raised the mean arterial pressure. Modulation of the beta1-receptors limited the acidosis.The fluid kinetic analysis showed slow distribution and elimination of Ringer´s lactate, although phenylephrine and dopamine accelerated the distribution. Once distributed, the fluid remained in the peripheral tissues and did not equilibrate adequately with the plasma. Overall, stimulation of adrenergic alpha1-receptors with vasoactive drugs accelerated, while beta1-receptors retarded, the distribution and elimination of fluid. The tendency for peripheral accumulation of fluid was pronounced, in particular when phenylephrine was given.Stimulation of adrenergic alpha Crystalloid fluid loading and the administration of vasoactive drugs are essential steps in the early treatment of septic patients with suspected hypovolemia and tissue hypoperfusion , 2. The In healthy sheep, vasoactive drugs markedly change the distribution and elimination of crystalloid fluid, thereby altering the plasma volume expansion, urinary excretion, and the risk of peripheral edema . HoweverThe purpose of the present study was to use volume kinetics to describe the distribution and elimination of crystalloid fluid in an experimental sepsis model, based on serial measurements of the hemodilution during and after an infusion –8, and hTwenty-five healthy male sheep weighing 14–26 kg  were studied. The protocol and the experimental procedures were approved by the Animal Care and Use Committee of the Shaoxing People’s Hospital , and the study was conducted in adherence with the Guide for Care and Use of Laboratory Animals.After an overnight fast, anesthesia was induced with propofol (5 mg/kg). The animals were intubated via tracheotomy and ventilated at a tidal volume of 10 ml/kg and respiratory rate of 15 breaths per minute. The anesthesia was maintained with 1.5–2.5% sevoflurane and intermittent doses of sufentanil (0.6 μg/kg) and cisatricurium (0.2 mg/kg), as needed.In a sterile operating environment, a 3-lumen vascular catheter was placed in the right internal jugular for drug administration and fluid infusion, as well as for registration of the central venous pressure (CVP). One femoral artery was cannulated for measurement of blood pressure and for sampling of blood. A middle laparotomy was performed and a cystostomy catheter was placed in the bladder for collection of urine.1-receptor blocker and is not used to support hemodynamics in septic patients. This drug was included to increase the width of our manipulation of the beta1-receptors and for its ability to increase urinary excretion, and the dose corresponds to the one used in a study of volume kinetics during laparoscopic surgery  and of pH . In contrast, dopamine receptors offered marginal prevention [–1.2 (3.0) versus –1.7 (2.8) mmol/L, P = 0.08).Modulation of the betak12 30–50 10−3 min-1) was achieved with phenylephrine and dopamine.The animal model used here created a moderately severe sepsis with hyperkinetic circulation and was used to investigate the effect of vasoactive drugs on the kinetics of Ringer´s lactate. The infused fluid expanded a central fluid space (the plasma) of between 1.3 and 2.7 L, which is large when considering the small size of these animals (20 kg). The distribution of fluid from the central to the peripheral fluid space in septic sheep without vasoactive drug was quite slow, when compared to previous work in conscious healthy sheep . A more Vt) is less pronounced [k12 and k21, where normal fluid exchange implies that the former rate constant should be about twice as high as the latter [Vc and Vt was clearly not normal. The rate constant k21 even attained negative values, which has previously only been described for the "transurethral resection syndrome" [A slow distribution could be regarded as a benefit, as it increases the plasma volume expansion resulting from the infusion. Cardiac preload is then better maintained due to the larger fraction of the infused fluid that remains in the circulating blood. Moreover, oxygenation of peripheral tissues will be better preserved because the diffusion distance becomes smaller when the edema in peripheral tissues  can be understood on the basis of the use of general anesthesia [The negative value denotes that fluid accumulated in peripheral tissues without equilibrating adequately with the plasma, indicative of a shock-like situation. The combination of a normal ovolemia . Edema westhesia , and theBetter insight into the fluid kinetics was sought by performing the full population kinetic analysis where the relative effects of the drugs on adrenergic receptors were used as covariates . Populat1-adrenergic receptors accelerated, while beta1-adrenergic receptor stimulation retarded, the distribution of crystalloid fluid. The magnitude of these influences could be quantified  at the expense of extravascular fluid accumulation (Vt) is beneficial to oxygenation of the tissues [The results show that alphaantified  and illuantified . The pro tissues .1-receptor stimulation increased while beta1-adrenergic receptor stimulation decreased the urinary excretion. Hence, these effects were opposite to those exerted by the adrenergic receptors on the rate of fluid distribution. They are supported by a previous study in conscious sheep [k10 caused by a combination of sepsis, general anesthesia, and hypovolemia. Our previous work with non-septic sheep [The present findings also indicate that alphaus sheep , and alsus sheep . In the ic sheep , and humic sheep , suggest1- and/or beta1-adrenergic receptors increased MAP, but only the alpha1-adrenergic receptor stimulation increased SV. Interestingly, modulation of the beta1-adrenergic receptors limited the acidosis, which is an effect that should be evaluated further.The pharmacodynamic analyses suggest that the effects of volume expansion (plasma dilution) on the changes in SV and MAP were quite small. The adrenergic receptors had a relatively stronger influence on the hemodynamics. Stimulation of alphak12, k21 and k10), while Vc can be regarded as a conversion factor between plasma dilution and plasma volume. The size of Vc represents the plasma volume but, more precisely, the body fluid volume that equilibrates very quickly with the plasma water in arterial blood. Plots created using these three rate constants illustrate how the body handles the fluid, with a barrier  that retards distribution between Vc and a peripheral space, Vt [Vt is not given directly from the model, but it can be calculated in retrospect as k12Vc / k21. Given this equation, we can understand the degree of difficulty in interpreting a negative rate constant. The size of Vt then attains a negative value, which shows that fluid is virtually suctioned into Vt, without having clearly defined borders.Simulations of fluid distribution, as depicted in pace, Vt . The siz1-adrenergic receptors help to limit this aberrant fluid distribution, and also reduce the systemic acidosis that is typical of sepsis. The beneficial influence of beta1-adrenergic receptor stimulation on the distribution of infused fluid corroborates earlier findings in conscious sheep [1- and beta1-adrenergic receptor stimulation both acted to sligthly raise MAP, but a strong and preferential beta1-adrenergic effect could be considered to be a more beneficial characteristic of any vasoactive drug used during sepsis.From a translational perspective, the results of the present study suggest that crystalloid fluid given in an early phase of sepsis has a marked tendency to accumulate in extravascular, peripheral tissues. Drugs that exert a strong stimulating effect on betaus sheep . Alpha1-Our study has several limitations. The current international consensus guidelines for sepsis state that vasopressors therapy should be initiated as an adjunct therapy when hypovolemia and hypotension is not resolved through volume resuscitation. Here, the infusion of vasopressor was initiated 10 min before volume loading, as the purpose was to examine how the vasopressors affect fluid kinetics. The grading of the strength of the drugs on adrenergic receptors was based on literature that relates to non-septic, conscious subjects . This grOnly one dose of each drug was tested. The results still provided information about how fluid kinetics varies with the degree of the alpha- and beta-receptor stimulation, as these characteristics differed greatly between the drugs used. The administered amounts of drug are not as high as they might seem, because extrapolation of doses between species should be made by considering the "effective body size," which is the 2/3 power of the body weight . However1-receptors accelerated, while beta1-receptors the retarded, the distribution and elimination of Ringer´s lactate. Peripheral edema was pronounced due to the virtual absence of redistribution of fluid from peripheral tissues. The effectiveness of plasma volume expansion for improving hemodynamics was limited. Modulation of beta1-receptors reduced acidosis.In conclusion, infusion of vasoactive drugs in septic sheep with moderate hypovolemia and hypotension showed that stimulation of alphaS1 Dataset(XLS)Click here for additional data file.


The determination of fluid density and viscosity using most cantilever-based sensors is based on changes in resonant frequency and peak width. Here, we present a wave propagation analysis using piezoelectrically excited micro-cantilevers under distributed fluid loading. The standing wave shapes of microscale-thickness cantilevers partially immersed in liquids , and nanoscale-thickness microfabricated cantilevers fully immersed in gases  were investigated to identify the effects of fluid-structure interactions to thus determine the fluid properties. This measurement method was validated by comparing with the known fluid properties, which agreed well with the measurements. The relative differences for the liquids were less than 4.8% for the densities and 3.1% for the viscosities, and those for the gases were less than 6.7% for the densities and 7.3% for the viscosities, showing better agreements in liquids than in gases. Viscosity and density are the fundamental properties of a fluid, and their measurements are important in many applications, from the quality control of combustible gases  to bloodIn our previous study , we presThe measured transfer function and wavenumber of the cantilever presented more reliable information over a wide range of frequencies, whereas commonly used resonance-based methods require a frequency range close to the resonant frequencies and are subject to a significant amount of uncertainties in case of liquids due to their low quality factors . In thisρ is the density of the fluid, ω is the radial frequency, b is the width, and When a cantilever is excited in a fluid, the fluid-structure interaction leads to the resistance and dissipation of the wave propagation on the cantilever . The ints number , Re=ρωb2essed as :(2)ΓrecFor the cantilever immersed in a fluid, the quality factor depends on the cantilever bending stiffness and the Reynolds number, and both microscale- and nanoscale-thickness cantilevers were used in this study. When a microscale-thickness cantilever was completely immersed in a liquid, the decay rate of the wave propagation on the cantilever was too large to induce standing waves. Therefore, the microscale-thickness cantilevers were partially immersed in liquids to improve the sensitivity of dynamic property measurements, whereas the nanoscale-thickness cantilevers were fully immersed in gases.D is the bending stiffness, w is the deflection, x is the lengthwise coordinate with the origin at the interface between vacuum (or air) and fluid, and bM is the mass per unit length of the cantilever. Assuming harmonic vibration, the response of the cantilever partially immersed in a fluid can be given as:H is the Heaviside step function, and If the cantilever has a small thickness and width compared to the lengthwise dimension, the effects of the shear deformation and the rotary inertia are negligibly small compared to those of the bending deformation. The equation of motion for a section of a cantilever immersed in a fluid can be expressed as :(3)D∂4w(After substituting Equations (1) and (4) into (3), the wavenumbers of the cantilever beam in a fluid is derived from Equation (3) as:To solve for the unknown coefficients, Λ and Then the transfer function between the vibration response at lated as :(7)Λeiϕ=The estimation of the fluid properties from the fluid structure interaction above requires a numerical process. The numerical method was applied to estimate the effects of fluid loading on the wave propagations on the cantilever. The transfer function in Equation (7) is a function of the complex wavenumber, Consequently, the fluid density and viscosity were simultaneously estimated from the measured complex wavenumber.3, 0.70 ± 0.20 ng) fully immersed in a gas and the microscale-thickness cantilever  partially immersed in a liquid, respectively. Considering the size of the cantilever chips, the nanoscale-thickness cantilever chip (5 × 5 × 0.7 mm3) was attached to a 10 × 10 × 0.5 mm3 PZT chip, while the microscale-thickness cantilever chip (1.5 × 3.5 × 0.3 mm 3) was fixed to a 4 × 6 × 0.5 mm3 PZT chip. Two wires were connected to the PZT chips to apply the excitation voltage. The nanoscale-thickness cantilever chip with the PZT chip was placed inside a gas chamber .v/v glycerol were measured using the microscale-thickness cantilever. The cantilever chip with the PZT chip was fixed on a slide glass, and a syringe needle  which contains a liquid inside, was set to slide parallel to the cantilever length direction to adjust the immersion length ), and then the transfer function was calculated by Equation (7). The transfer functions of the cantilevers in vacuum and water  are shown in The effects of the fluid-structure interaction on the wave propagation of the cantilever were analyzed by the change in the wavenumber. To measure the dynamic stiffness of the cantilever, air at 760 mmHg was used for a reference fluid, as it has known values of density and viscosity. For the calculation of the density and viscosity, the wavenumbers of the cantilever in the fluids were applied to the proposed process. The densities and viscosities b were mev/v) glycerol in water, or acetone (L is the length of the cantilever). The fluid-structure interaction reduced the wave speed significantly since the densities of liquids are three orders of magnitude higher than those of gases. The vibration amplitude in liquids was lower than that in vacuum due to the larger dissipation of the wave energy. In fact, the resonant vibration responses were very small when completely immersed in liquids. As the cantilever length in liquid decreased, the natural frequency and overall vibration amplitude increased. Therefore, the cantilevers were controlled to be partially immersed so that the vibration resonance was sufficient for measuring cantilever oscillation and its interaction with the surrounding fluids. The influence of fluid-structure interaction was significant at low frequencies, and decreased with increasing frequency as predicted by Equation (5). The wavenumber variation also decreased for the cantilever in vacuum with increasing frequency.We have presented an alternative method to measure the density and viscosity of liquids and gases using a wave propagation analysis on microscale- and nanoscale-thickness cantilevers. The microscale-thickness cantilevers were partially immersed in liquids to achieve sufficient vibration resonance. With this configuration, the wave propagations were measured to determine the interaction between the cantilever and the fluid, providing the exact vibration solution of the cantilever, and hence the density and viscosity of the fluids. In the proposed method, the distributed fluid loading was analyzed in a wide frequency range of measurements, whereas previously an oscillating sphere of an effective radius for a specific mode was assumed to analyze the influence of the fluid loading. The exact solution of the cantilever vibration without prior information on the vibration mode was advantageous for securing the flexibility of the experimental setup related to the immersed length in the testing liquid. This method also allowed for the precise and robust determination of the surrounding fluid properties with minimal influence from external noise because the transfer function was obtained from the relative displacement between two locations on the cantilever. The wave approach for the vibration analysis allows for the verification of the experimental setup since defective parts or improper boundary conditions induce completely different vibration behaviors . The nat


Aberrant genetic and epigenetic variations drive malignant transformation and are hallmarks of cancer. Using PCR-free sample preparation we achieved the first in-depth whole genome (hydroxyl)-methylcytosine, single-base-resolution maps from a glioblastoma tumour/margin sample of a patient. Our data provide new insights into how genetic and epigenetic variations are interrelated. In the tumour, global hypermethylation with a depletion of 5-hydroxymethylcytosine was observed. The majority of single nucleotide variations were identified as cytosine-to-thymine deamination products within CpG context, where cytosine was preferentially methylated in the margin. Notably, we observe that cells neighbouring tumour cells display epigenetic alterations characteristic of the tumour itself although genetically they appear “normal”. This shows the potential transfer of epigenetic information between cells that contributes to the intratumour heterogeneity of glioblastoma. Together, our reference (epi)-genome provides a human model system for future studies that aim to explore the link between genetic and epigenetic variations in cancer progression. In cancers, 5hmC patterns undergo considerable changes4 that have been linked to genome instability6 and remodelling of the DNA methylation pattern.7 Previous studies revealed that 5hmC is consistently found at significantly reduced levels in various solid tumours.10 Indeed, epigenetic regulators such as DNA methyltransferases (DNMT), ten-eleven-translocation (TET) proteins or isocitrate dehydrogenases (IDH), are crucial for normal and malignant cellular developement.11 Very few studies however, have effectively mapped the distribution of 5hmC in normal or cancer tissues. Herein, we present the first single base resolution maps of whole genomes, methylomes, and hydroxymethylomes for matched human glioblastoma and tumour margin samples.Genetic and epigenetic alterations to the genome shape the development of human malignancies. The patterns of the DNA methylation mark 5-methylcytosine (5mC) become aberrant in human malignancies and affect cellular functions.12 Total RNA sequencing of all three samples was also performed. We employed oxidative bisulfite sequencing (oxBS-seq)13 and bisulfite sequencing (BS-seq) to generate high-depth (80×) sequence coverage and built single-base resolution maps that distinguished 5mC and 5hmC modifications -methylation changes in the promoter in connection to transcript levels of key epigenetic regulators Fig. . Apparenour Fig. . TET2 pr22 Interestingly, even when 5mC is high and 5hmC levels shift from low to high  and MYC signalling pathways (SMARCB1 and LZTR1), and have been recently described in a genomic characterisation of IDH(+) glioma patients.24 Additionally, complex patterns of somatic structural variants were characteristic of the tumour  and DNMT1/3B (chromosomes 19 and 20) were haploid and polyploid in tumour respectively, which suggests a connection between the observed hypermethylation (DNMT polyploidy) and 5hmC loss (diminished TET function) in the tumour.We analysed the mutational landscape by identifying single nucleotide variants (SNVs) in tumour and margin using the blood sample as a reference. Although we identified 8169 SNVs in the tumour Fig. , the marour Fig. . We obsep-value < 2.2e–16, two-sided). Conversely, we found that SNV sites were significantly less hydroxymethylated in the margin compared to non-SNV sites  with no apparent differences for all types of cytosine mutations   Fig. . Regions A) Fig.  did not  A) Fig. .Fig. 4LiTumour purity was estimated to be 71% by genetic analysis and 60% by immunohistochemistry (see Methods). Based on tumour purity, we calculated the expected global level of 5hmC to have a lower limit of 7% .5mCpG sequences have previously been reported to be mutational hotspots in human genetic disease and cancer-relevant genes, probably through the deamination of 5mC to T.33 The concept of field defects34 has been used to describe the early events in the stepwise transformation of the cancer that can potentially lead to further oncogenic changes. To the best of our knowledge, this is the first observation of acquired, early changes in 5hmC in proximal cells.Our observation that 5hmC levels in the tumour are significantly lower than the estimated lower limit suggests a loss of 5hmC in the genetically “normal” cells within the tumour mass. Earlier studies reported that genome-wide methylation changes in tumours can be acquired in adjacent normal cells linking 5mC to field defects.Accurate, high resolution whole genome maps have allowed us to discern a role for 5hmC in protecting the genome against somatic mutations, observe global hypermethylation in a tumour and provide new evidence that associates 5hmC with epigenetic transformation in genetically normal cells adjacent to the tumour. Epigenetic analysis of 5hmC, in addition to 5mC, may foster diagnostic approaches in the future.35 After rapid recovery she underwent six weeks of radiotherapy (60Gy in 30 fractions) with concurrent temozolomide (TMZ) treatment. She then completed two cycles of adjuvant TMZ treatment during 12 weeks. This was stopped because of poor tolerance. While her 9-months post-operative scan was clear of tumour, her 11-months check demonstrated tumour recurrence and she died 3 months later from the combination of tumour progression and thromboembolic complications.A 71-year old woman underwent supra-total resection of a right frontal intrinsic tumour with maximum diameter of the enhancing component of 19 mm, surrounded by only minimal oedema. Histological examination confirmed the diagnosis of glioblastoma multiforme (WHO 9440/3). Tissue collection protocols were compliant with the UK Human Tissue Act 2004 (HTA License ref. 12315) and approved by the Local Regional Ethics Committee (LREC ref. 04/Q0108/60). Informed consent was obtained from the patient before surgery. Surgical samples were taken from the enhancing tumour mass and corresponding non-enhancing margin using advanced surgical techniques described previously.Brain tumour biopsies were fixed in 10% formalin and embedded in paraffin wax from which 4-µm-thick sections were cut and stained with haematoxylin and eosin (H&E). The diagnosis of glioblastoma multiforme (WHO 9440/3) was made by light microscopical examination of H&E-stained sections. Glioblastoma was defined by WHO-2016 criteria of an infiltrative astrocytoma with proliferative activity, necrosis and/or microvascular proliferation.IDH1 status by immunohistochemistry, the 4-µm-thick sections were dried at 60 °C for 2 h and further processed on a Bond Max (Leica). After a 60-min pre-treatment with cell conditioner 2 (pH 6) the slides were incubated with 1:80 diluted H09 anti-IDH1 R132H antibody  at room temperature for 30 min. A standard 3,3’-diaminobenzidine (DAB) detection kit was used for chromogenic detection. The common IDH1 mutation (R132H) was not detected.To determine the MGMT promoter methylation, H&E-stained slides were reviewed and neoplastic cell-rich tissue was dissected from consecutive unstained sections. DNA was extracted from the dissected tissue using the QIAamp DNA FFPE Tissue Kit (Qiagen) and was bisulphite-converted using the EpiTect Bisulphite Kit (Qiagen). The MGMT promoter methylation was determined by pyrosequencing of four CpG sites (CpGs 76-79) in exon 1 of the MGMT gene using the CE-Marked therascreen MGMT Pyro Kit on a Pyromark Q24 System (Qiagen). Significant levels of MGMT promoter methylation were not detected.To determine the To determine the tumour cell purity of the samples, H&E-stained slides were reviewed and cell counting was performed on multiple high-powered fields. The tumour cell purity of both the tumour and its margin was assessed by a pathologist prior to molecular profiling. No tumour cells were detected in the margins and the tumour was estimated to consist of 60% tumour cells.Each tumour and margin sample was frozen to −80 °C immediately on collection. The frozen samples were then defrosted and a 50 mg portion taken for DNA/RNA extraction. The samples were homogenised and then divided for DNA and RNA extraction. DNA extraction of both tissues and blood was performed using the QIAGEN DNeasy Blood and Tissue Kit, RNA extraction was performed using the QIAGEN RNeasy Mini Kit. Quantification was performed using the Qubit quantification assay. The tumour/normal pair used in this study was the sample judged most pure by comparison with blood DNA using whole genome sequencing.12 For the generation of PCR-free libraries for oxBS, libraries were oxidised (TrueMethyl Kit from CEGX) prior to bisulfite treatment. In brief, 350 and 175 ng of sonicated DNA from tumour and margin respectively were end repaired and dA-tailed before ligation of customized adapter pair. After (oxidation)-bisulfite treatment, degraded fragments were recovered through primer extension before dA-tailing and second adapter ligation. Libraries were subsequently immobilized on streptavidin coated magnetic beads, washed with binding buffer, eluted with 50 mM NaOH at 60 °C for 15 min. BS and oxBS-seq libraries were sequenced on an Illumina HiSeq 2500 platform, V4 chemistry, 2x101+7 index cycles.PCR-free ReBuilT libraries for bisulfite sequencing were generated following our previously published method.Libraries of the tumour and margin samples were prepared using TruSeq RNA Access kit (Illumina), using an input of 40 and 20 ng respectively. The libraries were sequenced for 2 × 75 cycles on HiSeq 2500 in Rapid mode achieving an average coding coverage of 108x (F2), 136X (F3), 277x (M1), 272x (M3).https://github.com/sblab-bioinformatics/epigenetics-of-glioblastoma).Details of the analysis are available in the manuscript's GitHub repository  was performed by Strelka.37 Large copy number variants and structural variants were respectively called with Canvas38 and Manta.39 The full workflow can be found in the Tumour Normal application of the Illumina BaseSpace platform .Libraries were sequenced on an Illumina HiSeq 2500 platform. Alignment to the human reference genome GRCh37 and quality control was performed using Isaac.40 and aligned to the human reference genome hg19 using bwameth,41 a wrapper around bwa-mem.42Raw reads were trimmed to remove adapter sequences ligated to the 3'-end using cutadapt43 and quantified using kallisto.44As above, reads were trimmed to remove adapter sequences using cutadapt and aligned to reference transcripts obtained from Ensembl38 provided us with an estimate of 71% purity of the tumour sample.45 This software calculates coverage and tumour SNV allele frequencies at heterozygous germline positions along the genome. It then assigns copy numbers per genomic regions and infers genome-wide ploidy and purity by fitting the data to expected models for each copy number state given purity and diploid coverage level combinations. Purity is then derived from the best fitting model.The copy number variant analysis using Canvaswww.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5171Data are available in the ArrayExpress database (Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Table 1Supplementary Table 2Supplementary information


The motion along the basilar membrane in the cochlea is due to the interaction between the micromechanical behaviour of the organ of Corti and the fluid movement in the scalae. By dividing the length of the cochlea into a finite number of elements and assuming a given radial distribution of the basilar membrane motion for each element, a set of equations can be separately derived for the micromechanics and for the fluid coupling. These equations can then be combined, using matrix methods, to give the fully coupled response. This elemental approach reduces to the classical transmission line model if the micromechanics are assumed to be locally-reacting and the fluid coupling is assumed to be entirely one-dimensional, but is also valid without these assumptions. The elemental model is most easily formulated in the frequency domain, assuming quasi-linear behaviour, but a time domain formulation, using state space method, can readily incorporate local nonlinearities in the micromechanics. Examples of programs are included for the elemental model of a human cochlea that can be readily modified for other species. •General formulation of an elemental model for cochlear mechanics.•Reduce to the transmission line model for locally-reacting micromechanical and 1D fluid coupling.•Incorporation of non-uniform areas, 3D fluid coupling and non locally-reacting micromechanics.•MATLAB programs for the elemental model in the frequency domain and time domain. These models can have various levels of abstraction, depending on what features of the cochlear mechanics they are attempting to describe. Wave models, for example, are good at describing the global behaviour of the motion along the cochlea without getting too involved in the detailed physical processes that give rise to the wave motion. The most widely used wave model uses the WKB method , in whicAn alternative approach to modelling the mechanics of the cochlea is to divide the length of the cochlea into a finite number of elements and describe the individual physical processes involved in cochlear mechanics. These equations can be coupled together in a numerical model that makes no explicit assumptions about the type of wave propagation. The earliest example of this approach is the “transmission line” model , in whicAn elemental approach to the formulation of cochlear mechanics can be viewed as a generalisation of the transmission line model, in that the fluid motion and the organ of Corti dynamics are analysed separately and then coupled together. In the elemental model, however, the organ of Corti dynamics are not restricted to being locally-reacting and the fluid flow is not restricted to being proportional to the pressure difference between adjacent elements. The elemental model reduces to a transmission line model if these restrictions are imposed, but can also account for the more general case.This paper first introduces the elemental model, for the case of a locally-reacting basilar membrane and 1D fluid coupling in a uniform box model. If the BM response is linear, the coupled solution can be solved efficiently in the frequency domain and this formulation is considered first. A time domain formulation is considered at the end of this paper which leads to a state space implementation of the elemental model that can be used to simulate the non-linear response of the cochlea but is also important in assessing the stability of linear models. It is shown how the fluid coupling part of the formulation can be adapted to model non-uniform distributions of fluid chamber area and the near field component of the pressure that is present when the fluid coupling is analysed in 3D. More general forms of the BM response are also then considered, where both longitudinal mechanical coupling along the BM and non-symmetrical feedforward behaviour along the organ of Corti can be accounted for.The theoretical formulation behind the fluid coupling within the cochlea and its passive micromechanics are reasonably well understood. The intention of the present review paper is to demonstrate how these individual responses may be integrated into a numerical formulation that can be readily used to predict the coupled response of the cochlea under different conditions. Although there have been a number of micromechanical models proposed for the active cochlea, there is still considerable debate about the form of such a model and how it should couple into the fluid. Since this is still unclear, and also for reasons of brevity, the elemental model will be developed and illustrated here only using passive micromechanical models, on the understanding that active models can be incorporated with a more general form of BM admittance.2eiωt. The essence of the elemental model is that the length of the cochlea is divided into a finite number of sections and that the interaction between the fluid in the chambers and the mechanical response of the BM is described in terms of the transverse modes of the element in each section. The general form of the assumed model is illustrated in x, a radial variable, y, and a transverse variable, z. The aim of the elemental model is to reduce the complicated interaction that occurs between the fluid and the motion of the BM in all three directions to a single set of discrete equations, as a function of only the longitudinal variable, x.In this section, we assume a linear micromechanical response, and an excitation of the cochlea at a single frequency, so that the amplitude and phase of all the dynamic variables can be considered complex and proportional to 2.1ψ(y) and v is the complex BM velocity distribution in the longitudinal and radial directions at a frequency ω, the dependence of which is suppressed for notational convenience, then the single variable can be used to express the complex BM velocity at the longitudinal position x, as defined asW is the width of the cochlear partition and the normalisation of the mode shape is assumed to be such thatIn general, there may be multiple transverse modes across the basilar membrane, but in practice the fluid coupling is relatively insensitive to the exact form of the radial BM velocity distribution  and so op1 and p2 are the three-dimensional pressure distributions in the upper and lower fluid chambers.In a similar way, a single variable describing the difference in pressures in the two main fluid chambers that act on the element is defined to bev(x) and p(x), are now spatially sampled to give a finite set of BM velocities and pressures at a number of discrete points, vn and pn, where the integer variable, n, runs from 2 to N. The first element in the set of velocities and pressures, n = 1, in these arrays is reserved to describe the excitation at the oval window from the middle ear, and the final element, n = N, is used to define the conditions at the helicotrema. Although it is not essential to the formulation, we assume here that the spatial sampling is uniform, so that if the length of the cochlear is L then the length of the elements in the longitudinal direction is always equal to L/N, which is defined as Δ. The number of elements and hence their length is determined by the shortest wavelength that needs to be reproduced in the longitudinal direction. A rule of thumb commonly used in finite element analysis is that they should be at least six elements within the shortest wavelength, so in practice it is common to use about 500 elements to describe the wave motion, which may have a wavelength as small as 0.5 mm , to that of the complex BM velocity distribution, v(x), both proportional to eiωt, ash is the effective height of the fluid chambers, given by π2A/8B, where A is the area of each chamber and B is the width of the BM, as discussed below. Assuming that the wavelength of the slow wave is small compared with the length of the elements, then the second spatial derivative in equation Assuming 1D fluid coupling, the equation for the fluid mass and momentum conservation can be combined to provide a differential equation relating the longitudinal distribution of the complex pressure difference across the BM, v1 is the stapes velocity when p1 is equal to 0, i.e. when the middle ear in unloaded, as described below. Using the finite difference approximation for the spatial derivative in equation The boundary condition at the base of the cochlea can be written in terms of the momentum equation for the complex pressure aspN = 0.The boundary condition at the apex of the cochlea, at the helicotrema, is generally taken to be pressure release, i.e. the pressure difference across the BM is equalised in the two fluid chambers so that F is the matrix of finite difference terms above. Assuming that this matrix is not singular then we can write the vector of pressures asThese finite difference equations can be written in matrix form asΔ2/h2 but discussed more fully below.The need to be able to invert this matrix requires that a finite value be entered into its bottom right element, taken here as If the vector of complex pressures is written in the form of equation N-th, element of vs is equal to the inner product of the bottom row of the matrix [1/iωF + YBM] and the vector p. Since all the elements in the bottom row of YBM are zero and only the final element in the bottom row of F is finite, then the final element of p, p(N), is proportional to the final element of vs, vs(N), which is set to zero, thus imposing the helicotrema boundary condition.The final, 2.3YBM(n) is the mechanical admittance of the BM and the negative sign has been discussed above. If the passive BM dynamics are approximated by a single degree of freedom system, thenω is the excitation frequency and mn, sn and rn are the mass, stiffness and damping, per unit area, of the BM at the position of the n-th element, i.e. at a distance of xn = nΔ along the cochlea.The mechanical coupling along the length of the BM is relatively weak and longitudinal coupling in the cochlea is dominated by that due to the fluid in the chamber, so that a reasonable first approximation is to assume that the BM is locally reacting. The BM is said to be locally reacting if the velocity of an element at one position depends only on the pressure difference at that point, so thatmn, is constant along the cochlea, and is denoted, m0. The BM stiffness is chosen to match the distribution of natural frequencies along the cochlea, which is assumed to take the formfB is the natural frequency at the base and l is a characteristic length for the frequency variation, so thatIt is assumed in the example below that BM mass, Q factor of the micromechanical elements is constant along the cochlea , and the BM and cochlear partition width, B(x) and W(x) respectively  is the effective area, given by the harmonic mean of the upper and lower areas of the fluid chambers. It should be noted that equation x0 gives rise to a far-field pressure that is constant basal to this point, since the fluid is uniformly accelerated in this region. The integral in equation x. In contrast, the pressure difference apical to x0 in this simplified (uniform) case falls away linearly with x, until it goes to zero at x = L. Thus the integral in equation x to L to give such a variation in pressure.It has been assumed above that the two fluid chambers have a uniform area along the length. This restriction can be removed by modifying equation ectively . GeneralN discrete sections, as for the BM, the integrals in equations n-th element asn0 = x0/Δ.If the areas of the fluid chambers in the cochlear models are divided up into 3.2ZFC in this equation are that they give the pressure distribution along the cochlear due to a single element of the BM vibrating, with all the other elements being fixed. If ZFC is calculated assuming 1D fluid coupling in a uniform cochlear, so that ZFC is given by equation The vector of pressures in the elemental model is related to the vector of velocities by the fluid coupling matrix, equation 1D fluid coupling assumes that the pressure is uniform across the cross-section of the cochlea, but as the wavelength of the slow wave becomes smaller there are significant variations of the pressure close to the BM, in the cross-section of the fluid chambers, and the fluid coupling is said to be 3D. The classic analysis of fluid coupling in this case, by x0 is the position of vibrating element, ω is angular frequency, v0 is the linear velocity of the BM element, Δ is its width, B is the width of the BM, W is the width of the cochlear partition, and a and b are fitting coefficients  is the vector of time histories of the M pressures in the elemental model, t) is the vector of accelerations of the elements and t) is the vector containing the stapes acceleration as the first element, with all the other zero.The elemental model can also be formulated in the time domain, to give a state space description of the cochlear mechanics . It is mn-th element, for n = 2 to N−1, are now described in terms of individual state variable models, asxn(t) is the vector of state variables for the n-th element, which has a pressure difference pn(t) acting on it, resulting in a velocity vn(t).The micromechanics of the x11(t) and x12(t), corresponding to the displacement and velocity of the element, and the equations above can be written asFor example, if the passive micromechanics are described by a single degree-of-freedom system as above, then there are two state variables per element, p1(t) to v1(t).The dynamics of the middle ear can be similarly formulated to relate The uncoupled micromechanics of all the elements can thus be gathered together in a combined matrix equationp(t) above into this and noting that CE are real, the state space model for the coupled elemental model can be written asSubstituting the expression for Again, an example MATLAB code can be found in the 6This paper describes an elemental approach to modelling the mechanics of the cochlea, particularly the interaction between the BM dynamics and the fluid coupling. The elemental model reduces to the classical transmission line formulation for a box model if the BM dynamics are locally-reacting and the fluid coupling is 1D, but is seen to be valid even without these restrictive assumptions. In particular, modifications are described for the model to incorporate non-uniform scala and BM dimensions, 3D fluid coupling and both symmetric and feedforward forms of longitudinal coupling along the BM.Elemental formulations are described in the frequency domain, for linear models, and in the time domain, with the potential for extension to nonlinear models, and MATLAB programmes are included for these models in the


Transarterial chemoembolisation (TACE) is well established in the treatment of primary hepatocellular carcinoma and of metastatic disease from colorectal and neuroendocrine tumours. There are few published studies on the effectiveness of treating hepatic metastases from medullary thyroid carcinoma with chemoembolisation and none to our knowledge utilising bland particle transarterial embolisation (TAE).Here we describe the management of multifocal hepatic metastases from medullary thyroid cancer in a 39-year-old woman who underwent bland particle TAE with a biochemical and radiological response and discuss the potential for a wider scope of clinical application for bland TAE in hepatic metastases. Prior reports have evaluated TACE in metastatic medullary thyroid cancer.Bland particle TAE is a potentially useful treatment for metastatic medullary thyroid cancer.Serum calcitonin is useful in evaluating the response to treatment.Medullary thyroid cancer (MTC) is a well differentiated neuroendocrine tumour of the parafollicular or C cells of the thyroid and accounts for 3–5% of all thyroid cancers . DistantThere are only a few published reports of the treatment of metastatic MTC with transarterial chemoembolisation (TACE) and none to our knowledge with transarterial embolisation (TAE) –4. ThereIt is important to note that approximately 25% of MTC is related to a mutation in the RET proto-oncogene. If considering hepatic embolisation, it is crucial to exclude hepatic metastases from a pheochromocytoma in patients with a background of multiple endocrine neoplasia type 2. Unwitting embolisation of pheochromocytoma metastases has been resulted in death secondary to an ensuing hypertensive crisis .A 39-year-old woman presented with a left neck swelling which underwent sonographic evaluation and fine needle aspiration. Cytology demonstrated medullary thyroid carcinoma with positive immunostaining for calcitonin and carcinoembryonic antigen in an adjacent lymph node confirming local spread. Baseline positron emission tomography/computed tomography (PET/CT) and magnetic resonance imaging (MRI) revealed innumerable metastases within both hepatic lobes Fig. . Serum cThe patient was treated with vandetanib to downstage the tumour prior to resection. Vandetanib is an oral small-molecule tyrosine kinase inhibitor that targets vascular endothelial growth factor receptors 2 and 3, RET, and, at higher concentrations, epithelial growth factor receptor. It has been shown to improve progression-free survival and tumour response in patients with unresectable, locally advanced or metastatic disease . Three mVandetanib was held in anticipation of surgical resection of the primary tumour and within four weeks the serum calcitonin had risen to to 663 pg/ml. Six weeks following Vandetanib cessation, the patient underwent total thyroidectomy and bilateral central neck dissection with a left modified radical neck dissection. The resection specimen revealed a 3-cm tumour with extra thyroidal extension and lymphovascular invasion, with a total 14 out of 43 nodes positive for tumour involvement (American Joint Committee on Cancer staging pT3 pNb1).On serial monitoring after thyroidectomy, calcitonin steadily increased from 3,131 pg/ml on the fifth postoperative day to 17,764 pg/ml 100 days later, a doubling time of six weeks.The patient remained asymptomatic and was reticent to restart systemic therapy in consideration of its associated toxicities. However, given the significant rise in tumour markers, a decision was taken to perform embolisation of the hepatic metastases.TAE was performed in two stages: first the right hepatic lobe followed by the left hepatic lobe eight days later. Both procedures were carried out through 5Fr right femoral access. Using a Mikaelsson catheter, selective catheterisation was performed of the coeliac and superior mesenteric arteries and arteriography performed. The proper hepatic artery was selectively catheterised using a microcatheter and selective arteriography was performed.During the first procedure, selective right hepatic arteriography revealed a significant disease burden within the right lobe of the liver. The right hepatic artery was embolised with 100-μm microspheres . During the second procedure, repeat right hepatic arteriography revealed a small volume of residual disease; therefore, further subselective right hepatic artery embolisation using 100-μm microspheres was performed. This was followed by selective embolisation of the left hepatic artery and of a large accessory left hepatic artery arising from the left gastric artery Fig. . The patWhile the pre-treatment metastases were relatively occult on CT, a CT one day following the second embolisation procedure showed multiple hypoenhancing foci throughout both hepatic lobes consistent with devascularised metastases Fig. . A folloPrior studies have described treatment of MTC hepatic metastases with TACE utilising Lipiodol and doxorubicin in a similar manner to other forms of metastatic neuroendocrine tumour, albeit with a small number of patients –4. FromiSerum calcitonin is a highly sensitive and specific tumour marker for MTC . At leasIn the current case, the doubling time was six weeks, placing the patient in a high-risk category for further disease progression. The serum calcitonin levels had decreased by 65.9% to 6,065 pg/ml at four weeks after TAE, a finding consistent with a clinically relevant reduction in the volume of viable tumour. Post-treatment liver MRI also demonstrated decreased enhancement of the hepatic metastases.While there is at least a conceptual logic for adding a chemotherapeutic agent to which the tumour is sensitive, there is currently no specific chemotherapeutic agent that can be bound to an embolic particle that has been shown to have a significant chemotherapeutic effect in metastatic MTC. The purported benefit of drug-eluting beads is based on a low peak plasma concentration of the chemotherapeutic drugs resulting in exposure of the tumour to the therapeutic agents with less exposure of healthy liver tissue . Even alIt is also debatable whether the addition of a chemotherapeutic agent provides any additional benefit over bland particle TAE alone. It remains unclear whether the treatment effect seen is primarily the result of the ischaemic effect of the embolic agent and to what extent, if any, the addition of chemotherapeutic agent contributes to this effect. Bland TAE has previously been described as effective in the management of liver metastases from multiple tumour types, including metastatic sarcoma and gastrointestinal stromal tumour , 14. BonGiven this uncertainty, and taking into account the added cost and potential side effects associated with TACE compared with TAE, we propose that TAE may be an effective treatment in cases of metastatic MTC.To summarise, while a small number of previous studies have shown promise in treating metastatic MTC with TACE, this case demonstrates that bland particle embolisation alone may achieve a similar radiological and biochemical response. Serum calcitonin may be useful as a tumour marker in evaluating the response to treatment, although how this relates to progression-free survival needs to be further elucidated. We believe that TAE may be considered as a potentially useful adjunctive therapy in achieving stabilisation of disease in patients with hepatic metastases from MTC.


H3F3A mutations have been described in 92% of GCT cases. GCTs involving the Clivus are extremely rare lesions and less than 15 cases are described in the literature. They represent a surgery challenge and are easily misdiagnosed. Our aim was to reveal if the genetic bases underlying Clival GCTs were the same of GCTs of long bones to improve the diagnosis and treatment.Giant Cell Tumour of Bone (GCT) is a locally aggressive primary bone tumour that usually occurs at the epiphyses of the long bones of the appendicular skeleton with a tendency to recurrence. Recurrent somatic H3F3A, H3F3B, IDH1, IDH2 and ZNF687) was performed on Clival GCT biopsies of two different cases. Histological analyses on the same tissues were used to detect the neoplastic population and its expression profile.The targeted somatic sequencing of GCT-related genes  in these surgically challenging cases. Moreover, H3F3A genetic screening can be combined to the histological analysis to differentiate GCTs from morphologically similar giant cell-rich sarcomas, while the histological diagnostic algorithm could help the differential diagnosis of other clival lesions.We conclude that the Clival GCT is genetically defined by somatic mutation in the  H3F3A gene only restricted to the stromal cell population, rather than to cells of the osteoclast lineage, demonstrating that GCT is a mesenchymal neoplasm [H3.3 alterations was observed among different bone tumours, emphasizing the importance of genotyping tumours for diagnostic purposes [ZNF687 gene [Giant cell tumour of bone (GCT) is a primary intramedullary neoplasm that accounts for 5% of skeletal tumours, composed by numerous multinucleated osteoclast-like giant cells evenly scattered throughout the mass and ovoid or spindle mononuclear stromal cells . GCT is Even though giant cell tumour shows a low potential for metastasis, it may locally recur at high rate . TherefoGCT typically occurs when the growth plate has closed and therefore is frequently observed in skeletally mature individuals with its peak incidence in the third and fourth decade of life . The majH3F3A gene in tumour biopsies of two patients, highlighting that Clival GCT can be considered like GCT of long bones.In the present article, we defined the genetic basis of giant cell tumour of the clivus, demonstrating the presence of somatic mutation in the The patient material comprises primary giant cell tumours of the clivus. Tissue samples were obtained as Formalin Fixed Paraffin Embedded (FFPE) specimens from 2 patients surgically treated in Università Vita-Salute San Raffaele, Milan, Italy (patient 1) and Department of Neurosurgery, Umberto I General Hospital, Ancona, Italy (patient 2).Sections (7-μm-thick) were cut from FFPE tissue blocks and subjected to DNA extraction with GeneRead DNA FFPE Kit (Qiagen), following the manufacturer’s instructions.H3F3A, H3F3B, IDH1, IDH2, and ZNF687) was conducted by PCR followed by Sanger sequencing, as recently reported [Mutation analysis of GCT-related genes , and subjected to Sanger sequencing.The molecular cloning of escribed . BrieflyTumour sections 7-μm-thick) of paraffin blocks were cut using a microtome and collected on Superfrost glass slides (Thermo Fisher Scientific). FFPE tumour tissues were then deparaffinised and rehydrated as we recently reported -μm-thick. ImmunofWe recently recruited a 55-year-old female (hereafter referred to as “patient 1”), diagnosed in 2014 with giant cell tumour of the clivus. Since 2013, she complained holocranial headache associated with vomiting, tongue numbness and difficulty with swallowing and speech. Computed tomography (CT) showed a lytic mass 5 cm in size involving the clivus. Magnetic resonance imaging (MRI) revealed a lobulated mass  originating from the clivus and extending into the sella and the epistropheus. In 2014, she underwent surgical removal of the mass through suboccipital approach and from the postoperative histopathology the diagnosis of Clival GCT was made. The tumour was highly vascularized and composed of numerous osteoclast-like giant cells that were bigger than those usually described in GCTs, with 50 to 70 nuclei, scattered on a background of mononuclear cells Fig. . HoweverIn 2013, the patient 2 was operated through endoscopic endonasal approach to remove the giant cell tumour of the clivus, responsible for his progressive headache and diplopia. After surgery, the patient was asymptomatic and did not undergo radiation therapy. Brain MRI, regularly performed up to six years after the operation, revealed that he did not present any relapse of the disease . The haeH3F3A, H3F3B, IDH1 and IDH2) as well as of the ZNF687 gene that we recently identified as gene responsible for giant cell tumour associated with Paget’s disease of bone [H3F3A gene; whereas the coding regions of the other analysed genes resulted negative for any mutations , hepatocellular carcinoma , and thyroid follicular carcinoma . Although they are more uncommon than primary clival tumours, they should be considered in the differential diagnosis that should be made using the histological markers specific for the primary cancers –69.Giant cell tumours of the skull account for 0,51% of all GCTs and tend to affect the sphenoid and the temporal bone. The selective and preferential occurrence of the tumour in these bones rather than other calvarial bones may depend on their embryologic origin: as in the case of long bones, also the sphenoid and the temporal bone are generated through endochondral bone formation; whereas the other skull bones are produced by an intramembranous formation . In factH3F3A, gene mutated in most cases of giant cell tumour of long bones. Indeed, since 2013, H3.3 mutations have been identified in more than 90% of all GCT cases analysed, with the p.Gly34Trp as the most frequently harboured mutation [H3F3B, IDH1 and IDH2 that, though more rarely, have been associated to GCT [ZNF687 gene that we recently identified in giant cell tumour complicating Paget’s disease of bone [H3F3A gene and demonstrated that the change was somatically acquired, as it was not found in the peripheral blood. Moreover, our immunofluorescence analysis further confirmed the restriction of the H3F3A mutation to the stromal cell population that was abundant in both tumour biopsies. Consequently, even though in a relatively low number of samples, we demonstrated that two cases of GCT affecting the clivus are genetically defined by mutation in the H3F3A gene and hence, Clival GCT can be considered a typical giant cell tumour with a peculiar anatomical localisation. Therefore, the mutational analysis of the H3F3A gene is an accurate tool for the diagnosis of Clival GCT that can be combined with the histological analysis of the tumour sample in order to distinguish the giant cell tumour from other osteoclast-rich tumours. In agreement, it has been recently demonstrated that H3F3A mutations were not detectable in neither giant cell-rich sarcomas nor giant cell-rich benign lesions, whereas almost all GCT tissues carried H3F3A mutation [H3F3A genetic screening allows to distinguish GCT from other giant cell-rich lesions, the histology-based diagnostic algorithm that we generated in this study may be useful to differentiate other morphologically overlapping lesions arising within the clivus.In this study, we unveiled the genetic basis of the giant cell tumour of the clivus, analysing the tumour specimens of two patients affected by this aggressive bone lesion. We analysed the coding regions of mutation , 70–72. d to GCT , 28. Bes of bone . Considemutation . While tWe also found a strong Tenascin-C immunoreactivity in the two Clival GCT biopsies, indicative of an aggressive tumour with a tendency to recur in situ, as this extracellular glycoprotein is highly expressed in the microenvironment of most solid tumours to promote migration and epithelial-mesenchymal transition . As a maOverall, these results demonstrate that Clival GCTs are caused by the same genetic defect of GCTs of long bones and allowed us to hypothesize that the two tumour types can be managed using the same approaches. The gold standard for management of this tumour contemplates the surgical resection, followed by adjuvant treatment with chemotherapy or radiotherapy , 20, 24.H3F3A gene. Our data also suggest that the GCT of the clivus can be treated on the same principles as that of GCT of long bones and that the relatively more aggressive phenotype of Clival GCTs should only be addressed to their anatomical location.We conclude that the Clival GCT is genetically defined by somatic mutation in the