Next, we summarize recent advances in approaches to enhance CAR T cell activity and persistence over the past 10 years, including bispecific CAR T cell design and early evidence of efficacy. Lastly, we examine mechanisms of T cell infiltration and tumour regression, and discuss the strengths and weaknesses of different strategies to investigate T cell function in murine tumour models.T cells play a key role in tumour surveillance, both identifying and eliminating transformed cells. However, as tumours become established they form their own suppressive microenvironments capable of shutting down T cell function, and allowing tumours to persist and grow. To further understand the tumour microenvironment, including the interplay between different immune cells and their role in anti-tumour immune responses, a number of studies from mouse models to clinical trials have been performed. In this review, we examine mechanisms utilized by tumour cells to reduce their visibility to CD8 As a part of the body’s immunosurveillance mechanism, all nucleated cells display major histocompatibility (MHC) class I molecules, enabling the routine presentation of cellular peptide fragments on their plasma membrane. Internal proteins are broken up by proteasomes to form peptides that are then transported by transporter associated with antigen processing (TAP) to MHC I molecules in the lumen of the endoplasmic reticulum. The peptide–MHC I complexes generated via these antigen processing and presentation pathways can be recognized by T cell receptors (TCRs) on Cytotoxic T lymphocytes (CTLs). Tumour-specific peptides are now known to exist, and to be capable of triggering spontaneous T cell responses in patients ,2,3,4,5.The microenvironment within solid tumours is often hostile to tumour-specific T cells, negatively affecting their function and survival. Metabolic alterations to tumour and stromal cells result in the overexpression of glucose transporters and key glycolytic enzymes, altered growth factor signalling, hypoxic conditions, increases in acidity, and abnormal angiogenesis. Furthermore, an abundance of suppressive immune cells, including regulatory T cells (Tregs), myeloid-derived suppressor cells, and tumour-associated macrophages, infiltrate into the tumour and deliver a host of negative signals to tumour-specific T cells and antigen-presenting cells including dendritic cells. As these features of the tumour microenvironment have been reviewed extensively elsewhere Interference with the expression level of single or multiple components of antigen processing and presentation pathways are common mechanisms used by malignant cells to reduce visibility to CTL. Examples include alterations in the expression of proteasome subunits involved in the processing of endogenous antigens, downregulation, loss, or imbalance in the expression of enzymes such as endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP involved in the loading of peptides into MHC I molecules, the loss of IFN-γ-signaling components resulting in the prevention of MHC I upregulation, and defects in MHC I expression itself, all of which result in changes to the repertoire of antigenic peptides presented to CTL ,16. The While defects in peptide-MHC I expression in tumours have been examined extensively, strategies to counteract these defects remain scarce and challenging. The majority of MHC I defects in human cancers such as melanoma, gastric, colorectal, and cervical carcinoma, belong to the category of “soft” lesions ,27. The Concurrently, “hard” MHC class I genetic aberrations exist in 30–40% of human cancers ,34 corre+ T cells identified by van Hall et al., may be able to recognise ‘hard’ lesions with impaired TAP, tapasin, or proteasome function [Subsets of CD8function ,41,42. Tfunction ,43,44. Vfunction . The potfunction , and thefunction . These sAnother way to overcome the problem of MHC I down-regulation on tumour cells is through the use of chimeric antigen receptor T cells (CAR T cells) . These T+ B-cell lymphoma and CD19− loss variants with equal efficiency, Zah et al. determined the structural parameters for dual-antigen recognition and created CD19-OR-CD20 CAR, an “OR-gate CAR”, to trigger robust T cell function by either antigen expression [− loss variant cells, OR-gate CAR T cells could efficiently target both. Their work presented the potential of CD19-OR-CD20 CAR as a powerful safeguard against antigen escape in CAR T treatments for wild-type and CD19− loss variant B-cell lymphomas. Bispecific CAR T cells that recognize CD19 and an additional antigen, have the potential to offset antigen-loss-associated tumour escape and improve long-term survival rates and are the subject of much interest in the field. In the last 5 years in particular, preclinical animal models have emerged to test the viability of CARs with dual targets, such as CD19/HER2 , CD19/CDpression . The in pression . While C−/CD22− loss variants edited by CRISPR-Cas9 technology [− or CD22− B-ALL with 99% wild-type cells (CD19+/CD22+), which mimics relapse from a small pre-existent clone as observed in clinical trials. Adoptive transfer of T cells expressing CD19/CD22 bivalent CAR eradicated CD19+/CD22− B-ALL xenografts and significantly inhibited the outgrowth of CD19−/CD22+ B-ALL populations. The potency of this bivalent CAR to reduce the risk of antigen escape was further investigated in the patient-derived xenografts: HMB15 (CD19+/CD22+) and HMB28 (CD19−/CD22+). Encouragingly, HMB28 was resistant to CD19 CAR but cleared by the bivalent CAR, while HMB15 was eradicated by either CAR. These clinically relevant models of CD19 CAR resistance have helped to confirm proof-of-concept in vivo activity of bispecific CARs and their potential applicability as adoptive T cell transfer therapies for cancer treatments.Another bispecific CAR, the CD19/CD22 bivalent CAR, was generated by Qin et al. and tested in a “spike-in” leukaemia relapse model utilising NALM6 cells and CD19chnology . The inoIt will be interesting to further utilize the design principles of bispecific CARs highlighted in the studies above to construct novel CARs targeting additional antigen pairs. Examinations of promising alternative targets are underway, such as CD30 in refractory Hodgkin’s lymphoma, BCMA in multiple myeloma, and CD33, CD123, and FLT3 in AML . It remaA current challenge for CAR T cell therapy is to overcome barriers to their persistence following adoptive transfer. Emerging research indicates that cell-intrinsic mechanisms affect CAR T cell exhaustion, activation-induced cell death, and lymphocyte contraction, suggesting that the production of CAR T cells from less differentiated T cells  is associated with superior functional properties (reviewed in Ref. ). A moreAn alternative to CAR T cell transfer for the treatment of tumours with down-regulated or defective MHC I expression is the adoptive transfer of γδ T cells. Like conventional αβ T cells, γδ T cells demonstrate cytotoxic activity via the granzyme-perforin axis, release cytokines such as TNF-α, IFN-γ, and IL-17, and can kill a wide array of tumour types . However+ T lymphocytes [+ lymphocytes are favourably associated with patient survival in several cancers  [+ T cells as the main therapeutic target in cancer treatments. However, tumour infiltrates can vary widely in composition, and studies have shown that while the infiltration of effector T cells, such as CD8+ lymphocytes and M1 macrophages, are associated with a favourable prognosis, the infiltration of Tregs and M2 macrophages are less favourable and may directly stimulate tumour growth [As a co-receptor for the αβ TCR, the CD8 molecule stabilizes TCR-MHC I complexes and provides downstream signalling for T cell activation. Detecting co-expression of CD3 and CD8a molecules can be used to identify the presence of tumour-infiltrating CD8phocytes . Studiescancers) ,68,69,70r growth ,72,73,74+ T cells as a critical factor in anti-tumour immunity. However, the over-activation of CD8+ T cells can potentially trigger autoimmune disorders, such as vitiligo when this occurs in the skin. Isolated auto-reactive T cells from vitiligo patients are able to recognise and trigger apoptosis of healthy melanocytes in vitro. Interestingly, this autoimmune tissue destruction has been reported to provide a hospitable niche for the residence of tumour-specific T cells, the necessary components of long-lasting protection against melanoma in peripheral tissue [+ T cell activation is tightly regulated through central and peripheral tolerance mechanisms, but the mechanisms that regulate memory/effector T cell function in the tumour microenvironment are still not well understood. It is of particular interest to elucidate how effector memory T cells are regulated in the context of anti-tumour responses. Furthermore, there is much to be learned about how cancers establish local immune tolerance to evade immune surveillance and how this may be exploited to develop more effective immunotherapies.Large quantities of literature have reported tumour-infiltration of CD8l tissue . Tumour-T cell-mediated tumour rejection involves several steps: (1) Effector cell activation/sensitization to tumour antigens in lymphoid organs, (2) Migration of antigen-specific T cells along with other effector cells to tumour sites, and (3) Target cell recognition and killing. Tumour rejection can potentially fail whenever any of these steps is suppressed. Above, we discussed the alterations made by tumour cells to interfere with the activation of antigen-specific T cells and strategies that could be utilized to break down corresponding barriers in immunotherapies. Here, the focus is on studies of tumour-infiltrating lymphocytes, and the cytokine/chemokine signalling networks that stimulate their trafficking to the tumour site. −/− mice, T cells were shown to fail to migrate to the tumour site [+ Th cells, CD8+ CTLs and memory T cells) as compared to naive precursors [−/− mice have indicated the importance of CXCR3 for CTL migration to the tumour site in melanoma [+ and CD8+ T cells in this process [+ T cell infiltration in melanoma patients. Thus, CXCR3 cognate chemokines can affect the recruitment of CTLs into the tumour mass and these effector cells can shape tumour immunity and anti-tumour therapy responses by antigen-specific or antigen-independent mechanisms. Recently, it has been proposed that tumour cells can reduce CXCL9/CXCL10 gradients by secreting galectin-3, a lectin that blocks IFN-γ diffusion [A key cytokine controlling lymphocyte extravasation is IFN-γ . The preour site . As the ecursors . Severalmelanoma ,82. Simi process . Clinica process ,84. Moreiffusion . Experimiffusion . Though lymphocyte infiltration is important for tumour control, blood vessels within tumours are often leaky with intense interstitial pressure effected by the density of the surrounding tumour mass, which may cause heterogeneous permeability and promote irregular blood flow . As a coTumour-specific infiltrates are necessary to eliminate tumours, but the mere presence of an infiltrate itself is not sufficient. The tumour microenvironment is known to contain many immunosuppressive factors suggesting immune regulation plays an active role in tumour progression . The dis−/− mice have a lethal autoimmune phenotype indicates the critical role of CTLA-4 in promoting self-tolerance [Further insight into the mechanistic differences that exist between major checkpoint pathways could help optimise combination therapies with checkpoint inhibitors. For example, CTLA-4 plays a role in regulation of T cell activation and in Treg-mediated suppression of dendritic cell function, but not in the control of NK-cell activities ,92. On tolerance . Similarolerance . Immunotherapy has shown substantial benefit in the treatment of multiple malignancies. Dramatic advances in our understanding of the interactions between the immune system and tumours have revolutionised the development of new immunotherapeutic approaches, aimed at harnessing the immune system to create effective anti-tumour responses. To evaluate the effectiveness of immunotherapeutic strategies in the context of a functional immune system, animal models are required to replicate human diseases. Mice become the first choice as they are relatively low cost, have a short reproductive cycle, can be easily genetically modified with specific characteristics, contain a functional immune system, and exhibit a high rate of tumour growth . However+ tumour-infiltrating lymphocytes were demonstrated to be over-activated and “exhausted” with notable co-expression of PD-1 and LAG-3, but not CTLA-4 [Syngeneic tumour models, obtained through transplantation of cultured tumour cell lines into immunocompetent mice, are the earliest and most commonly utilized murine models to investigate anticancer therapeutics  Figure . A majort CTLA-4 . Consistt CTLA-4 . The cavt CTLA-4 . Genetically engineered mouse models are produced based on transgenic technologies inducing systemic or tissue-specific expression or deletion of genes such as oncogenes and tumour suppressor genes . In geneIn carcinogen-induced tumour models, mice bearing tumours induced by carcinogens such as azoxymethane, ultraviolet light, or methylcholanthrene, are directly used to evaluate the efficacy of tumour treatments . These mHuman xenograft models are created to meet the requirement to have models of human tumours interacting with human immune cells . ImmunocThe main aim of cancer therapies is to destroy the tumour while preserving normal tissues and limiting toxicity as much as possible. Immunotherapy utilizing anti-tumour T cells greatly improves the potential for tumour specificity whilst minimizing off-target effects. The combination of immunotherapy with targeted therapies and chemotherapies may also induce stronger anti-tumour immune responses . Chemoth+ T cells are required to ensure the efficacy of a chemotherapy drug, doxorubicin, which is used to treat carcinogen-induced sarcomas [In spontaneous mouse models of fibrosarcoma, pro-B-cell leukemia, T cell acute lymphoblastic lymphoma, and gastrointestinal stromal tumours, it has been proven that the efficacy of drugs that target tumour oncoproteins depends on the activity of accompanying anti-tumour T cell responses ,127. Intsarcomas . Howeversarcomas ,130. In It is now well established from murine tumour models that T cells can play an important role in tumour control. However, as tumour models are continually refined to more clearly mimic human disease, it is also evident that tumours resist targeting by these T cells. Absence of tumour-specific antigens, regulation of antigen presentation pathways, interference with T cell trafficking and engagement of inhibitory receptors on T cells all contribute to progressive tumour growth. The broad challenge is to understand these immune escape pathways in the context of heterogeneous tumours and find new approaches to overcoming potentially multiple immune escape pathways simultaneously. CAR T cells were first engineered to overcome the single challenge of insufficient peptide/MHC complexes on tumours but now face the need to target multiple tumour antigens together with improved trafficking and survival in the tumour microenvironment. Overcoming multiple tumour-directed challenges requires a multifaceted approach to T cell activation, trafficking and survival in the future. This is increasingly evident with studies on combination immunotherapies and the immune interface with chemo/radiotherapy. Maintaining specificity for the tumour while broadly enhancing multiple T cell attributes will remain a key focus into the future.


Dear Editor,Patients diagnosed with metastatic colorectal cancer (mCRC) often suffer from unresectable metastases, which lead to a rather poor prognosis.We enrolled 26 patients diagnosed with mCRC and treated in Fudan University Shanghai Cancer Center (FUSCC) for CRC CTC organoid culture, and the baseline clinicopathological characteristics of the patients are shown in Table S1. To isolate CTCs, we used both filtration method and CD45 depletion method.After approximately 14 days, the first passage of CTC organoid was observed, which showed a solid subsphaeroidal shape Figure . After p® CTC detection system developed by Genovo (Dunwill Company), which uses wide field imaging system to capture fluorescence. EpCAM was used as a maker for CTCs, and CD45 was used as a marker for lymphocytes. Almost all the cells showed positive staining of EpCAM (orange), positive staining of DAPI (blue), and negative staining of CD45 (magenta), while a lymphocyte showed positive stainings of CD45 and DAPI, and negative staining of EpCAM  for the detection of CTCs and lymphocytes using ChimeraXM Figure –G. NotabM Figure , while tM Figure . In 100 M Figure . Thus, tIn contrast to the organoids derived from patients’ primary tumors Figure –C and J.In conclusion, we successfully cultured and identified CRC CTC organoids derived from the blood of mCRC patients, and this technique might serve as a primary basis for a model facilitating personalized therapy for mCRC patients suffering from unresectable metastases.Science and Technology Commission of Shanghai Municipality; Grant Number: 18401933402 (to Peng); Shanghai Sailing Program; Grant Number: 19YF1409500 (to Li); Shanghai Anticancer Association EYAS PROJECT; Grant Number: SACA‐CY1A05 (to Li); National Natural Science Foundation of China; Grant Number: 81672374 (to Cai); National Natural Science Foundation of China; Grant Number: 81972244 (to Hu); National Natural Science Foundation of China; Grant Number: U1932145 (to Peng).The authors declare that there is no conflict of interest.The research was approved by the Ethical Committee and Institutional Review Board of Fudan University Shanghai Cancer Center.Long Zhang, Yaqi Li, and Junjie Peng designed the study; Long Zhang performed most of the experiments; Long Zhang, Yaqi Li, and Junjie Peng analyzed the data and co‐wrote the manuscript. Shaobo Mo, Xiang Hu, and Xiaoji Ma participated in some experiments; Shaobo Mo and Xiang Hu co‐wrote the manuscript and organized the figures. Weixiang Jin and David Haixiang Peng participated in CTC identification experiments, Yu Zhou and Xiaofei Liang provided essential support to this study. Xinxin Rao, Xiaoya Xu, Zhen Zhang, and Guoqiang Hua provided support and suggestions for this study. Sanjun Cai and Junjie Peng conceptualized, designed, and directed the project. All authors read and approved the manuscript.SUPPORTING INFORMATIONClick here for additional data file.


A fluid warmer can prevent hypothermia during the perioperative period. This study evaluated the heating capabilities of Hotline and Barkey S-line under different flow rates and initial fluid temperatures, as well as after the extension line installation.We measured the temperature of a 0.9% sodium chloride solution at the fluid warmer outlet (TProx) and the extension line end  with three different initial fluid temperatures  and two flow rates (250 ml/hr and 100 mL/hr).At a 250 ml/hr flow rate, the TProx and TDistal values were observed to be higher in Hotline than in S-line when using room-temperature or cold fluid. Administering of the warm fluid at the same flow rate significantly increased the TProx and TDistal values in S-line more than the cold and room-temperature fluids. At flow rates of 100 ml/hr, TDistal values were significantly lower than TProx values in both devices regardless of the initial fluid temperature.Hotline outperformed S-line for warming fluids at a high flow rate with cold or room-temperature fluids. Administering warm fluid in S-line prevented a decrease in the fluid temperature at a high flow rate. However, at a low flow rate, the fluid temperature significantly decreased in both devices after passing through an extension line. Inadvertent perioperative hypothermia commonly occurs in patients undergoing surgery on account of a cold operating room, anesthetic agents that weaken thermoregulatory control, and administration of un-warmed fluid –8. Even It is recommended that physicians assess the risk factors associated with perioperative hypothermia to reduce hypothermia-related complications , 11. AftNumerous commercially available fluid warmers, such as Ranger, ThermoSens, Mega Acer Kit, FT800, and Hotline HL-90, have been investigated under different flow rates and room temperatures , 14–16. The purpose of this study was to evaluate the fluid heating capabilities of Barkey S-line and Hotline HL-90 according to different flow rates, fluid temperatures, and the presence of an extension line.This study was performed in the designated spot next to the nursing station in the post-anesthesia care unit (PACU). The PACU room temperature was maintained at 23 °C, and the humidity level was maintained at approximately 20%. Normal saline fluid  was used for all the experiments in this study. The fluid temperature was measured using a two-channel thermometer . For Barkey S-line , the operating temperature was maintained at 39.5 °C throughout the experiments. A standard infusion set was inserted in the 1.5-m heating profile of S-line according to the manufacturer instructions. For Hotline HL-90 , the operating temperature was set to 40 °C. The REF L-70 disposable tubing system  was installed according to the manufacturer instructions.Prox) and then at the end of the non-insulated extension line . After starting each trial, the temperatures at the two positions  were recorded every minute until the end of the trial , cold 9–10 °C), and warm (46–47 °C). The normal saline solutions were respectively maintained for 12 h in the operating room, in a refrigerator, and in a heating cabinet for each of the three different conditions. Two distinct flow rates were tested in this study: 100 ml/hr and 250 ml/hr. The roller clamp was fully open, and the flow rate was adjusted using a micro-flow regulator  attached to the infusion set at a flow rate of 100 or 250 ml/hr. The regulator tolerance ranged from − 10–20% according to the manufacturer instructions. The height of the infusion bag was the same in all experiments (1.8 m). One unit of a non-insulated extension set , which was 90 cm in length, was connected to the fluid warmer outlet. The fluid temperature was measured at two points, first at the fluid warmer outlet  and R software version 3.4.4 . A data chart was produced using Microsoft Excel 2007. All fluid temperatures were reported as a median (interquartile [IQR] range). The Wilcoxon signed-rank test was used to test for a statistical difference between TProx was higher in Hotline (38.7 [38.7–38.8]°C) than in S-line (28.2 [27.6–28.4]°C) based on the median difference of 10.5 °C (95% CI 9.6–12.3) at the flow rate of 250 ml/hr. At the flow rate of 100 ml/hr, the TProx value was higher in Hotline (40.3 [40.1–40.4]°C) than S-line (38.7 [38.6–38.7]°C) according to the median difference of 1.6 °C  than in S-line (27.8 [27.0-27.9]°C) based on the median difference of 9.8 °C (95% CI 7.6–11.5) at the flow rate of 250 ml/hr. However, the TDistal values were not significantly different between Hotline (25.6 [25.3–26.0]°C) and S-line (23.4 [23.4–23.5]°C) at 100 ml/hr . However, TProx in S-line was much higher at 100 ml/hr than at 250 ml/hr based on the median difference of 10.5 °C (95% CI 9.6–12.3).TDistal in Hotline was higher at 250 ml/hr than at 100 ml/hr by 12.0 °C (95% CI 9.8–14.2). TDistal in S-line was higher at 250 ml/hr than at 100 ml/hr by 4.4 °C (95% CI 2.6–5.3). The TDistal values were lower at 100 ml/hr than at 250 ml/hr. In addition, TProx was higher than TDistal in both devices regardless of the flow rate. However, the differences were more substantial at 100 ml/hr. At a flow rate of 250 ml/hr, the median differences between TProx and TDistal were 1.1 °C (95% CI 0.9–2.2) in Hotline and 0.4 °C (95% CI 0.3–0.6) in S-line. At 100 ml/hr, the median differences were 14.7 °C (95% CI 14.3–16.7) in Hotline and 15.1 °C (95% CI 13.9–16.0) in S-line.Furthermore, TProx was higher in Hotline (37.2 [36.3–37.4]°C) than in S-line (18.2 [17.6–19.5]°C) by the median difference of 19.0 °C (95% CI 16.7–20.2) at 250 ml/hr. However, the difference between Hotline (40.0 [39.8–40.0]°C) and S-line (38.6 [38.5–38.6]°C) was 1.4 °C (95% CI 0.3–4.3) at 100 ml/hr. TDistal at 250 ml/hr was also higher in Hotline (36.2 [35.6–36.3]°C) than in S-line (19.1 [18.5–20.0]°C) based on the median difference of 17.1 °C (95% CI 15.4–18.2). However, the TDistal values from Hotline (23.5 [23.3–24.1]°C) and S-line (24.5 [24.1–24.7]°C) were not statistically different at 100 ml/hr . In S-line, the TProx value was significantly higher at 100 ml/hr than at 250 ml/hr by 20.4 °C (95% CI 17.5–21.4). For TDistal in Hotline, the temperature at 250 ml/hr was higher than at 100 ml/hr, and the median difference was 12.7 °C (95% CI 11.4–13.6). In contrast, TDistal at 100 ml/hr was higher than at 250 ml/hr in S-line, and the median difference was 5.4 °C (95% CI 4.0–6.4). In both devices, the TDistal values at 100 ml/hr were similar to the ambient temperature. The TProx was higher than TDistal in both devices regardless of the flow rate. The only exception was observed in S-line at 250 ml/hr; TProx is 18.2 °C and TDistal is 19.1 °C. The difference between TProx and TDistal was greater at 100 ml/hr than at 250 ml/hr. At 250 ml/hr, the median differences between TProx and TDistal were 0.9 °C (95% CI 0.6–1.2) in Hotline and 0.7 °C (95% CI 0.5–0.9) in S-line. At 100 ml/hr, the median differences were 16.3 °C (95% CI 15.8–16.6) in Hotline and 13.9 °C (95% CI 11.2–14.6) in S-line. At 100 ml/hr, the extension line reversed most of the warming and substantially cooled the fluid as it traveled from TProx to TDistal.TProx value at 250 ml/hr in S-line (44.8 [44.7–45.4]°C) was higher than in Hotline (42.1 [42.1–42.1]°C) according to the median difference of 2.7 °C (95% CI 2.4–3.8). At 100 ml/hr, the TProx value was higher in Hotline (40.5 [40.5–40.5]°C) than S-line (37.8 [37.8–37.9]°C) according to the median difference of 2.7 °C (95% CI 1.6–3.7). TDistal in S-line (43.9 [43.5–44.2]°C) at 250 ml/hr was higher than in Hotline (41.2 [41.0-41.2]°C) according to the median difference of 2.7 °C (95% CI 2.2–3.2). At 100 ml/hr, no statistical difference existed between the TDistal values in Hotline (27.6 [27.4–27.9]°C) and S-line (26.6 [26.5–26.8]°C) . For the TDistal value, the temperature measured at 250 ml/hr was again higher, and the median difference was 13.6 °C (95% CI 13.1–14.9). Similarly, the TProx value in S-line was higher at 250 ml/hr than at 100 ml/hr; the median difference was 7.0 °C (95% CI 5.9–8.1). For the TDistal value, the measurement at 250 ml/hr was higher than at 100 ml/hr based on the median difference of 17.3 °C (95% CI 16.7–18.6). TProx was higher than TDistal at the two flow rates in both Hotline and S-line. A flow rate of 100 ml/hr produced larger differences between TProx and TDistal. At 250 ml/hr, the median differences between TProx and TDistal were 1.0 °C (95% CI 0.9–1.1) in Hotline and 1.2 °C (95% CI 0.8–1.5) in S-line. At 100 ml/hr, the median differences were 12.9 °C (95% CI 12.5–14.2) in Hotline and 11.3 °C (95% CI 11.1–12.1) in S-line. The slower flow rate correlated with a more substantial difference in the median values.In Hotline, the TThis study showed that Hotline was superior to S-line for warming fluids when using room-temperature or cold saline at a high flow rate. However, at a low flow rate, the fluid temperature significantly decreased in both devices after passing through an extension line.The difference in heating capacity between the two devices was likely due to the differences in their respective heating mechanisms, i.e., a coaxial circulating water bath (Hotline) versus a dry heating profile (S-line). The dry-heating system is expected to incur greater heat losses on account of the exposed portion of the extension line in ambient temperature. Thus, the Hotline coaxial warming system is apparently more effective than S-line in preventing hypothermia during rapid fluid administration. When administrating fluid rapidly, pre-warming fluid appears to be effective in maintaining fluid temperature warm especially in S-line. However, it would be important to check the warming temperature beforehand to avoid getting too hot fluid.Prox less in both Hotline and S-line. The slow flow rate would have provided sufficient time for both fluid warmers to affect the fluid temperatures. However, Tdistal of the warm fluid seem to be slightly higher than those of the room-temperature or cold fluids at 100 ml/hr in both Hotline and S-line. This suggests that using pre-warmed fluid may have some role in maintaining fluid temperature even at the low flow rate when the extension line is used. Warm fluid could have influenced TDistal through heating up the tube in which the fluid traveled down, however, the exact mechanism behind this finding remains to be further investigated.At 100 ml/hr, changing the initial temperature affected the TThere are several studies evaluating the performance of fluid warmers in literature , 15, 16.It is clinically important to determine whether warmed fluids can be delivered to the patient without heat loss as they pass through a non-insulated extension line. Interestingly, in this study, a statistically significant change in fluid temperature was observed after the extension line in every experimental condition regardless of the fluid warmer type, initial fluid temperature, or flow rate. The change in fluid temperature was more pronounced at the low flow rate than at the high one. The fluid lost a significant amount of heat as it traveled an additional 90 cm at a rate of 100 ml/hr. This finding warrants the utilization of additional measures for hypothermia prevention when using an extension at the low flow rate, especially when administering fluids to patients with a high risk of developing hypothermia such as neonatal and older patients . FurtherSeveral limitations of this study should be noted. In an actual clinical setting, the fluid administration rate, initial fluid temperature, and extension line length may differ from the conditions used in this study. For example, during rapid fluid resuscitation, the infusion rate can be as high as 60 to 80 mL/kg per hour . Also, iIn summary, Hotline outperformed S-line for warming fluids at a high flow rate with cold or room-temperature fluids. Administering warm fluid in S-line prevented a decrease in the fluid temperature at a high flow rate. Finally, using an extension line can reduce the final fluid temperature delivered to the patient at a low flow rate in both devices.


Patients were receiving BoNT/A injections as a treatment for movement disorders. Mean age 65.1 years (DM) and 61.9 years (AM). The mean jitter was abnormal in 13.9% (maximum 41.4 µs) of DM, and 40% (maximum 43.7 µs) of AM. Impulse blocking was sparse. We found no correlation of the mean jitter to age, BoNT/A most recent injection (days/units), number of muscles injected, total BoNT/A units summated, number of total BoNT/A sessions, beta-blockers/calcium channel blockers use, and cases with local spread symptoms such as eyelid drop/difficulty swallowing. Maximum mean jitter (41.4/43.7 µs) for DM/AM occurred 61 and 131 days since the most recent BoNT/A, respectively. The far abnormal mean jitter (32.6/36.9 µs) occurred 229 and 313 days since the most recent BoNT/A. We suggested that jitter measurement can be done after BoNT/A in a given muscle other than the injected one, after 8 (DM) and 11 (AM) months, with reference >33 µs and >37 µs, respectively.To study the jitter parameters in the distant (DM) and the adjacent muscle (AM) after botulinum neurotoxin type A (BoNT/A) injection in 78 patients, jitter was measured by voluntary activation in DM ( In 1977, Scott et at. ,3.Clostridium botulinum produces eight immunologically distinct serotypes (type A–H) that differ from each other by the nontoxic accessory protein (NAP) composition. For the clinical purpose, the BoNT/A is the most used and presented as onabotulinumtoxinA (ONA), abobotulinum toxin A (ABO), and incobotulinum toxin A (INCO) [A (INCO) . For phaA (INCO) .Botulinum neurotoxins enter the nerve terminals, where they cleave specifically to vesicle-associated membrane protein or synaptobrevin (VAMP), the synaptosomal associate protein of 25 kDa (SNAP25) or syntaxin .Blocking neuromuscular transmission with BoNT/A, despite no injury to the motor axon, induces chemodenervation with a massive axon sprouting and neuromuscular junction remodeling ,7. BotulIt is well known that the local effect of BoNT/A intramuscular injection either clinically  or through single-fiber electromyography (SFEMG) demonstrates very high jitter and impulse blocking ,12,13,14The reliability of the SFEMG jitter parameters in patients investigating neuromuscular junction disorders after receiving a BoNT/A injection is a challenge for the clinical neurophysiologists. From a practical perspective, there are no jitter reference values related to the time elapsed after the most recent BoNT/A injection obtained in a large patient cohort. The purpose here was to study the SFEMG jitter parameters in non-injected adjacent muscles and distant muscles from a large cohort of patients (78 cases) that have been in treatment with BoNT/A mostly for movement disorders. The objective is to find out the most reliable reference jitter parameters for those patients who had already been injected with BoNT/A and came to the SFEMG for a neuromuscular junction disorder suspicion.Frontalis) in 11/17 (64.7%) of those cases.One patient with Alzheimer’s disease was excluded from the study due to difficulties in getting spikes, remaining 78 cases. No patient complained of systemic symptoms like fatigue, blurred vision, or weakness, and they were very satisfied with the BoNT/A clinical results. Spread symptoms due to BoNT/A was far more common in cervical dystonia patients, where 57.1% complained of dysphagia  that lasted about 2 weeks; one case (7.1%) had associated dysphonia. In cases of hemifacial spasm, eyelid drop occurred in 28.9% extending for a few days, and dry eye occurred in 6.7% of patients. In patients with blepharospasm, just one case (7.7%) reported an eyelid drop. We found no relationship between cases with spread symptoms to the mean jitter values. Accordingly, the mean jitter was normal in the distant muscle (ED) in 5/7 (71.4%), and in the adjacent muscle (n = 70), followed by antidepressant drugs (n = 19), statins (n = 13), insulin, and metformin (n = 10), levothyroxine (n = 9), anticonvulsants (n = 7), and calcium channel blockers (n = 5). We checked specifically the relationship between the beta-blockers/calcium channel blockers to the mean jitter. From 22 patients that were on beta-blockers, 17 had normal and 5 an abnormal mean jitter. Of 5 patients in the use of calcium channel blockers, 4 had normal, and 1 had an abnormal mean jitter. So, we could not find any association that could act as a potential confusion factor.Most patients have comorbidities, and the mean daily pills used was 2.5, ranging from 0 to 11. Hypertension (35), depression (19), diabetes (10), hypothyroidism (9), and any arthritis (4) were the main associate disorders described by patients. History of atheromatous related-disorders, like chronic coronaropathy and mild stroke, were more difficult to obtain from patients, but some used antiplatelet medication. Any antihypertensive drug plus diuretics and beta-blockers represented the most used daily pills (Orbicularis Oculi (33), Risorius (21), Zygomaticus Major (21), Corrugator Supercilii (7), Sternocleidomastoideus (6), Splenius Capitis (6), Levator Scapulae (6), Orbicularis Oris (2), Procerus (2), Flexor Carpi Radialis (2), Trapezius (1), Masseter (1), Frontalis (1), Levator Nasalis (1), Platysma (1), Pectoralis Major (1), Biceps Brachii (1), Tibialis Posterior (1), and Extensor Pollicis Longus (1). In 2 patients, BoNT/A injections were made in the limb, but in these cases, the distant muscle tested was the contralateral one. In the remaining 41 patients, the muscles injected with BoNT/A were in the face or neck.The first group, to measure jitter parameters in a distant muscle, comprised 43 patients, age 65.1 ± 10.5 years (35 to 83), 70% female. The patients were in the regular outpatient schedule for the BoNT/A injections due to hemifacial spasm (22), blepharospasm (10), cervical dystonia (6), aesthetics (2), bruxism (1), focal dystonia in the upper limb (1) and spasticity (1). BoNT/A was injected into one muscle (7 patients), two muscles (4 patients), three muscles (30 patients), and five muscles (2 patients). The most frequent injected muscles were Orbicularis Oculi (28), Zygomaticus Major (24), Risorius (23), Sternocleidomastoideus (8), Splenius Capitis (8), Levator Scapulae (6), Corrugator Supercilii (4), Trapezius (3), and Masseter (1). In all cases, the muscles injected with BoNT/A were in the neck or the face.The second group, to measure jitter parameters in an adjacent muscle, comprised 35 patients, age 61.9 ± 12.1 years (30 to 79), 77% female. The patients were in the regular outpatient schedule for the BoNT/A injections due to hemifacial spasm (23), cervical dystonia (6), blepharospasm (3), segmental dystonia (1), generalized dystonia (1), and cervical dystonia plus blepharospasm (1). Botulinum neurotoxin type A was injected into two muscles (5 patients), three muscles (25 patients), four muscles (4 patients), and five muscles (1 patient). The most frequent injected muscles were The variables referring to the age, mean jitter, percentage of abnormal individual jitter values (“outliers”), the time elapsed (days) for SFEMG test since the first and most recent BoNT/A injection, the number of BoNT/A injections since the first one, the BoNT/A units injected per session, the total BoNT/A units summated since the first one, and the number of muscles injected per session are shown in The comparison between distant versus adjacent groups revealed no significant difference between age, the number of injections, the number of days from the first and the most recent injection of BoNT/A, BoNT/A units per injection, total BoNT/A units summed, and the number of muscles injected with BoNT/A per session .Extensor Digitorum (40), Deltoideus (2), and Tibialis Anterior (1). In the adjacent group (35 patients), the only muscle studied was the Frontalis. The comparison between distant versus adjacent groups revealed a significant difference between the percentage of abnormal individual jitter values (p = 0.0003), and the percentage of impulse blocking (p = 0.0383) (In the distant group (43 patients), the muscles studied were  0.0383) . The mea 0.0383) .The power of the correlation between the mean jitter values to some variables using a linear model and calculating the R-squared in percentiles were weak and ranged from 0% to 12.3% . The corFrontalis muscle, the mean jitter was increased by 40%. The maximum mean jitter obtained was 43.7 µs (56.1% above the reference). We found a statistically significant difference in impulse blocking between groups, with this being more frequent in the adjacent group. However, there was no correlation statistically significant for any variable-age, days from the most recent BoNT/A injection, BoNT/A units in the most recent injection, total number of injections, and total BoNT/A summated since the first injection to predict the mean jitter increase. We specify the extreme findings for the mean jitter (lowest/highest) in Our results revealed that a relatively mild mean jitter increased in 13.9% of patients from the distant group, in which the BoNT/A was injected mostly in facial or neck muscle, and the jitter was measured in a limb muscle, mostly ED. The maximum mean jitter obtained was 41.4 µs (38% above the reference). For the patients in the adjacent group, in which the BoNT/A was injected mostly in facial or neck muscle and the jitter was measured in the Even though both groups were composed of relatively older patients , with more comorbidities associated, and consequently more daily medicine use, we could not find any evidence of age bias for the jitter parameters. There was no difference in the mean jitter from those taking beta-blockers or calcium channel blockers. None of the patients presented a systemic effect that could be attributed to pseudo-botulism, nor any symptoms from BoNT/A intolerance. All patients were very satisfied with the clinical results. We could not find any association between the cases who presented symptoms from the local BoNT/A spread  with the mean jitter value in both groups.Abductor Digiti Minimi, Extensor Digitorum Brevis, Extensor Digitorum, and Tibialis Anterior) [Orbicularis Oculi) in a total of 18 muscles [We found fifteen papers related to the local or distant effect of BoNT/A in humans ,24,26,30nterior) ,14,21, a muscles ,12. The  muscles , high ji muscles . The finOrbicularis Oculi, glabellar region (Corrugator Supercillii and Procerus muscles), neck region, intragastric, detrusor, and various muscles in the face. The adjacent or distant muscle jitter studies were done mostly in Extensor Digitorum (87) and Orbicularis Oculi (32) [Orbicularis Oculi muscle, 25.9 to 31.3 µs, between 2 to 8 weeks. For the Biceps Brachii muscle, 38 to 66 µs, between 4 to 6 weeks. No or rare impulse blocking was described. We consider the findings of Garner et al. [Extensor Digitorum Brevis muscle, which frequently has neurogenic findings.We had selected reports in which the jitter parameters were studied in the adjacent or distant muscles from injected muscles or regions in healthy subjects or patients, provided that they do not have symptoms (fatigue or pseudo-botulism). The BoNT/A was injected into uli (32) ,24,26,30r et al.  probablyMasseter and the Orbicularis Oris muscles after BoNT/A injection in the Orbicularis Oculi muscle suggests local instead of spreading axonal diffusion [Orbicularis Oculi muscle. However, even in this well-controlled study, not all subjects had jitter values increased after two weeks since the injection. Garner et al. [The significant decline of CMAP amplitude, of both the iffusion . It alsoiffusion . Alimohaiffusion  emphasizr et al.  did not r et al.  concluder et al.  were morr et al.  The distOrbicularis Oculi BoNT/A injection), and dry eye. The unspecific fatigue symptom after BoNT/A should be seen with caution and clearly distinguished from the fluctuating weakness. If a given patient had a mean jitter greater than 37 µs for the Frontalis muscle (voluntary activation), and more than 11 months after the most recent BoNT/A injection, the increased jitter would probably be related to the MG. If a given patient had the mean jitter greater than 33 µs for the Extensor Digitorum muscle (voluntary activation), more than eight months after the most recent BoNT/A injection, the increased jitter would probably be related to the MG. If a given patient had a mean jitter higher than 43 µs (ED) and 45 µs  at any time, it will probably not be related to BoNT/A. The SFEMG test should never be done in the injected muscle, at any time.For the practical electrodiagnostic perspective for carrying out the SFEMG test for a MG suspicion, in a patient already injected with BoNT/A, we suggest reviewing the following rules. The major (and rare) systemic motor-autonomic symptoms related to the BoNT/A injection is the pseudo-botulism presented with diplopia, dysphagia, and dysarthria followed by a descending pattern of weakness affecting the upper limbs, then lower limbs, and in severe cases, respiratory muscle weakness, associated with blurred vision, constipation, dry mouth, postural hypotension, urinary retention, and pupillary abnormalities. The significant motor-autonomic symptoms related to the BoNT/A adjacent spread are dysphagia , eyelid drop  is the same as already reported. We found a jitter increase in a distant muscle from BoNT/A injection in 13.9% of 43 patients, the highest value being 41.4 µs (limit 30 µs). For the adjacent muscle from BoNT/A injection, we found a jitter increase in 40% of 35 patients, the highest value being 43.7 µs (limit 28 µs). There was sparse impulse blocking. There was no correlation between the mean jitter and several variables-days/units after the most recent BoNT/A injection, the number of muscles injected per session, the total number of BoNT/A sessions, total units summated since the first one, MUAP’s amplitude, daily use of beta-blockers/calcium channel blockers, cases with symptoms from the local spread, and age. We presume that personal factors could be involved in the distant group and operator injection precision for the adjacent group. For electrodiagnostic practice, we suggested that jitter measurement can be analyzed after BoNT/A in a given muscle other than the injected, after eight (distant) and eleven (adjacent) months, with reference limit >33 µs for and >37 µs, respectively.®/Ipsen Limited, Slough Berkshire, UK) was used in 77 patients. Only one patient has used three different BoNT/A injections over the years: Abobotulinum toxin A , Incobotulinum toxin A , and OnabotulinumtoxinA . The dilution was the same for all, 20U for every 0.1 mL.Between March 2018 to October 2019, 79 patients aged 30–83 years old, of both sexes, who routinely were injected with BoNT/A for movement disorders at the specific outpatient clinic of the Lucy Montoro Rehabilitation Center, Sao Jose do Rio Preto State Medical School (FAMERP), were recruited and invited to participate in the study. All cases were under the supervision of one of the authors (FO), the board-neurologist in charge of the diagnosis, and intramuscular injections of BoNT/A. Demographic data (sex and age), clinical data (diagnosis of movement disorder), comorbidities known to patients or described in the electronic medical records, drugs in continuous use, BoNT/A data , time in days from the most recent BoNT/A injection to the SFEMG test, time in days from the first BoNT/A injection until the SFEMG test, and total dose (summated units) of BoNT/A until the SFEMG tests were done. The BoNT/A dose (units) was related to the muscle size. Abobotulinum toxin A , a neurologist, and a clinical neurophysiologist who was board-certified. All patients were regularly scheduled for BoNT/A injections for movement disorders, mostly for hemifacial spasm, blepharospasm, and cervical dystonia ; other disorders included bruxism, focal dystonia, generalized dystonia, spasticity, and aesthetics . The Extensor Digitorum (n = 40), Deltoideus (n = 2), and Tibialis Anterior (n = 1). In this group, the BoNT/A was injected in the neck or facial muscles (41 cases), in the forearm muscle (1 case being the SFEMG test performed in the opposite ED muscle) and in multiple muscles in one side of the body . The choice of two other muscles instead of the ED was made when FPs were found, or the motor unit action potentials (MUAPs) revealed increased amplitude/duration  in the electromyography (EMG). In the second group , the SFEMG test was performed in the Frontalis muscle in all cases. In these patients, the BoNT/A injection was performed in the neck or facial muscles, and not in the Frontalis muscle directly. The measurement of jitter parameters was done using the Natus™ Keypoint-Net  or Natus™ UltraPro S100  machines, and an in-built software specifically developed for the SFEMG test. Before the SFEMG test, conventional non-quantitative EMG was performed in the chosen muscle to exclude neurogenic abnormalities.For the SFEMG test, patients were divided into two groups. In the first group , the analysis was performed in a limb muscle, as follows, ® Neuroline Concentric, 25 mm × 0.30 mm (30 G), recording area 0.02 mm2 and, Natus™ Dantec® DCN Disposable Concentric Needle Electrode, 25 mm × 0.30 mm (30 G), recording area 0.02 mm2. Both electrodes are routinely used and approved by the Brazilian Agency for Health Surveillance (ANVISA). The CNE was introduced into the chosen muscle. After minimal maintained voluntary contraction, the single fiber action potentials (SFAPs), or more appropriately, the “apparent single fiber action potentials” (ASFAPs) were recorded. Due to the use of disposable CNE, with a much larger recording area than the single fiber electrode, the probability of recording spikes from two or more muscle fibers increased; thus, the term ASFAP is better for defining these potentials. The low-frequency filter was set at 1 kHz to suppress the distant action potentials (APs) from the CNE recording area, passing only the APs with rapid rise time. Because the low-frequency filter reduces the amplitude of APs, we accepted only the ones with a minimum amplitude limit of 100 μV. Records were made after a minimum of three CNE insertions with radial advancement into the muscle. After slight movements of the electrode, a pair of ASFAPs was recorded, with both potentials belonging to the same motor unit; the trigger was put to the higher amplitude spike to get the temporal variation on the second smaller spike.The measurement of the jitter parameters by voluntary activation was performed using disposable concentric needle electrodes (CNE) of two brands: AmbuThe jitter value was calculated as the mean consecutive differences (MCD) for each pair, ideally for 100 pairs; in some circumstances, we accepted a minimum of 30 pairs. The MCD variation was calculated by the “amplitude level”, i.e., by the temporal variation of the ascending lines of depolarization of the ASFAPs. Acceptable potentials were based on the criteria established in the literature , as follDeltoideus and Tibialis Anterior muscles, and 28 µs for the Frontalis muscle [Extensor Digitorum, we use its reference for the Deltoideus muscle (use here in 2 cases) and the Tibialis Anterior muscle (used here in 1 case). The mean value of the sorted differences (MSD) was also obtained. MSD calculates the consecutive differences according to the frequency of discharge of the potential pairs. When the inter-discharge interval (IDI) variation is larger than 4 ms and not constant, there may be interference from the VRF effect. The mean jitter was calculated by the mean MCD or MSD, which had the lowest value for each pair. Mean jitter values higher than 150 μs were fixed at this value, to minimize the bias due to the VRF effect; the higher the jitter, the greater the probability of impulse blocking, consequently further artificially adding jitter. The other way for the test conclusion was calculated as the percentage of the individual MCDs above the reference limit (43 μs); when more than 10% of the ASFAP pairs, i.e., at least 3 out of 20, have values higher than 43 μs for the ED, Deltoideus and Tibialis Anterior muscles and, 38 µs for the Frontalis muscle, the test was abnormal. Impulse blocking was considered present or absent, regardless of the percentage in each pair. The skin temperature was maintained at more than 30 °C.The test conclusion was defined by the mean of 20 different MCDs for the reference value of 30 μs for the ED, s muscle . Althougt-test made the comparison of parametric variables with a significance of 0.05. The comparison of nonparametric variables was made by the Mann–Whitney nonparametric test (U-test) to verify the medians’ equality. Correlation of the mean jitter values (MCD) with variables was made with a linear model, calculating the R-squared in percentiles ranging from 0 to 100% to show the correlation power of jitter in the regression line.Descriptive statistics calculated the mean jitter (MCD) values of the 78 cases and all other parameters: mean and standard deviation for normal or Gaussian distribution or median/percentage for non-normal distribution. An Anderson–Darling normality test was used. The Student’s The study was in accord with the Helsinki Declaration of 1975 and approved by the ethics committee of the Faculdade de Medicina de São José do Rio Preto, São Paulo, Brazil, where the SFEMG tests were performed. Certificate of Presentation for Ethical Consideration (CAAE) approved number 83336818.0.0000.5415 on 22 March 2018. All patients signed informed consent.


Background: ISGylation is the conjugation of ISG15 with target proteins. ISGylation occurs through an enzymatic cascade, which is similar to that of ubiquitination. Through ISGylation, ISG15 can bind to proteins involved in cell proliferation and differentiation, thus promoting genesis and progression of malignancies. The present study aims to investigate expression of genes involved in ISGylation and ubiquitination in patients with hepatocellular carcinoma and to correlate gene expression with clinical laboratory parameters of these patients.Methods: mRNA expression of genes encoding enzymes involved in the ISGylation process  was evaluated by quantitative real-time PCR in 38 pairs of tumour and adjacent non-tumour tissues from patients with hepatocellular carcinoma and correlated with distinct clinical laboratory parameters.Results: Relative mRNA expression of EFP, HERC5, UBA1 and USP18 was significantly higher in tumour tissues compared to adjacent non-tumour tissues . The correlation pattern of mRNA expression between genes in the tumours differed from the pattern in adjacent non-tumour tissues. Relative expression of EFP, HERC5 and UBA1 in adjacent non-tumour tissues was positively associated with direct bilirubin levels  and relative expression of USP18 in adjacent non-tumour tissues correlated negatively with ALT levels .Conclusions: EFP, HERC5, UBA1, and USP18 genes are upregulated in tumour tissues of patients with HCC and, thus, may be associated with the pathogenesis of hepatocellular carcinoma. Hepatitis B virus (HBV) infection is responsible for up to 90% of hepatocellular carcinoma (HCC) cases, especially in regions with a high prevalence of HBV infection including Vietnam In response to HBV infection, immune cells secrete interferon (IFN) and other inflammatory cytokines. Subsequently, cellular signalling pathways are activated and IFN-stimulated genes (ISGs) are induced ISGylation has multiple functions, including inhibition of release of exosomes, downregulation of protein translation and regulation of distinct protein functions EFP, HERC5 and USP18 genes contribute to tumourigenesis through ISG15/ISGylation or other mechanisms. In addition, the exact role of ISG15 and other key enzymes involved in ISGylation in the formation and development of HBV-related HCC is still unclear. In the present study, we aim to investigate the expression profiles of genes encoding enzymes involved in the ISGylation process  in patients with HCC and their association with clinical outcomes.It remains, however, unclear so far whether the 0C until further use. Patients were further grouped as Child-A, Child-B or Child-C according to Child-Pugh scores The study included 38 patients with HCC who underwent surgery at the 108 Military Central Hospital in Hanoi, Vietnam. The patients were diagnosed according to the guidelines of the American Association for the Study of Liver Diseases (AASLD) for management of HCC All details of the study were explained to the participants and informed written consent was obtained before taking samples. All clinical procedures and experiments were performed in accordance with applying guidelines and regulations. The study was approved by the Institutional Review Board of the 108 Central Hospital, Hanoi, Vietnam.GAPDH  used as reference gene. Sequences of primers used in this study are presented in Table ® 480 real-time PCR system . Relative expression of target genes was normalized to the expression of GAPDH based on the ΔCt method.Total RNA was extracted from the 38 tumour and adjacent non-tumour tissue pairs by Trizol reagent  according to the protocol recommended by the manufacturer. Extracted total RNA samples were reverse transcribed into cDNA by using the QuantiTect Reverse Transcription Kit . cDNA was quantified by real-time PCR with P<0.05.Quantitative parameters including laboratory tests and relative gene expression values are given as means with standard deviation or medians with ranges where appropriate. The relative gene expression between tumour and adjacent non-tumour tissues was compared using the Wilcoxon signed ranks test. Spearman's rank correlation coefficient was used to analyze the correlation of relative gene expression between two genes or between expression of the genes with clinical parameters. The SPSS software version 22.0  was used for all statistical analyses; the significance level was set at EFP, HERC5, UBA1, UBC and USP18, which are related to ISGylation, was quantified and compared between HCC tumour and adjacent non-tumour tissues. Expression of EFP, HERC5, UBA1, and USP18 was significantly higher in HCC tumour tissues compared to the adjacent non-tumour tissues . No significant difference of UBC expression was observed between HCC tumour tissues and non-tumour tissues (P=0.91) . BCLC stages C and D did not occur among the study participants.We then examined whether gene expression was associated with progression of HCC by comparing their expression between different BCLC stages. However, expression of the genes did not differ between stage A and B HCC tissues as well as between the corresponding non-tumour tissues (P<0.0001). The strongest correlation was observed between expression of EFP and HERC5 , followed by expression of HERC5 and USP18  and of EFP and UBA1  . A similar result was observed when the correlation of HERC5 expression with that of UBA1 and USP18 was analysed  . A similar correlation was seen between UBA1 and USP18 . These results indicate that the expression pattern of the genes under study differed between HCC tumour and adjacent non-tumour tissues.We analysed the correlations of mRNA expression in both HCC tumour and non-tumour tissues. In HCC tissues, gene expression was strongly correlated with each other  Table . In non-1) Table . The corEFP, HERC5 and UBA1 in non-tumour tissues were positively correlated with direct bilirubin levels  , but positively correlated with albumin levels   , UBA1 and USP18 is significantly upregulated in HCC tumours compared to adjacent non-tumour liver tissues. In addition, the expression levels of the genes in non-tumour tissues is associated with several clinical parameters. Our results indicate that E3 ligases, UBA1 and USP18 are associated with HCC development and may possibly be considered as targets in the treatment of HCC.ISGylation plays a key role both in immunity against infections and in the formation and progression of tumours by affecting a wide range of target proteins EFP, HERC5, UBA1 and USP18 in HCC tissues and the different patterns of correlation of the genes in HCC tumour and in adjacent non-tumour tissues suggest that these genes may play a role in the pathogenesis of HCC. Studies have shown that EFP is closely associated with breast cancer EFP regulates metastasis of breast cancer through its E3 ubiquitin ligase activity rather than through estrogen signalling EFP is upregulated in breast and gastric cancers but downregulated in endometrial cancer EFP expression is associated with a poor prognosis of breast cancer EFP expression is a prognostic factor in prostate cancer EFP increases proliferation and survival of prostate cancer cells by affecting p53 signals EFP is also associated with HCC progression through degrading the metastasis-associated 1 protein (MTA-1) EFP is significantly upregulated in HCC tissue compared to non-tumour liver tissues and EFP expression in non-tumour tissues is correlated with the levels of direct bilirubin.The upregulation of in vitroHERC5 in HCC tumour tissues and a correlation of HERC5 expression with direct bilirubin levels in non-tumour tissues. Although UBA1 has been considered as a potential target gene for cancer therapy, especially for acute myeloid leukemia UBA1 expression in HCC tumour and adjacent non-tumour tissues and points towards its correlation with direct bilirubin levels.HERC5 is the dominant E3 enzyme for ISGylation and has been shown to be highly expressed in HCC tissues and USP18 is significantly upregulated in HCC tissues compared to corresponding non-tumour tissues. USP18 is an important factor in the immune response directed against HBV replication and its overexpression promotes growth of HCC cells USP18 mRNA in peripheral blood mononuclear cells (PBMCs) has been proposed as a predictive factor for monitoring treatment of HBV patients with interferon-alpha In agreement with a previous report UBC expression is upregulated in lung cancer UBC does not differ significantly between HCC tumour and non-tumour tissues, UBC expression in non-tumour tissues is correlated with AST levels, total bilirubin and albumin, indicating that UBC may not significantly contribute to the pathogenesis of HCC, but rather plays a role in liver functions. Limitations of this study apply to the rather small number of HCC patients, particularly the small number of HCC patients in BCLC stages C and D, implying the impossibility to perform association analyses of gene expression with HCC progression.EFP, HERC5, UBA1 and USP18 genes, upregulated in tumour tissues of HCC patients, is correlated with liver function parameters and, thus, may be associated with the pathogenesis of liver cancer.In conclusion, our study shows that expression of


Detarium microcarpum, Brachystegia eurycoma and rice husk, as fluid loss control additive in the water-based drilling fluid. The materials were prepared, ground and sieved to 125 microns. Four sets of water-based drilling muds were formulated using the local materials and CMC as fluid loss control additives. The mud formulation was based on the American Petroleum Institute (API) standard of 25g bentonite to 350mL of water. Also, the filtration test of the formulated muds was performed using API recommended practice for static filtration test at low temperature - low pressure (LTLP) condition. The results obtained showed that Detarium microcarpum and rice husk fluid loss volume and filter cake thickness were comparable with that of CMC from additive content of 10g, while Brachystegia eurycoma was comparable from additive content of 15g. Furthermore, the composite additive results indicated that Detarium microcarpum-rice husk at 95% Detarium microcarpum-5% rice husk performed better than Brachystegia eurycoma-rice husk of the same combination. Additionally, the fluid loss volume and filter cake thickness of Detarium microcarpum-rice husk additive were comparable with CMC from 10g content. Also, the results revealed that the fluid loss volume and filter cake thickness obtained from the locally sourced materials were within API specification for fluid loss control agents. The mud filter cake characteristics exhibited by these materials depicted that they have slippery, smooth and soft mud cakes; thus, the characteristics of a good mud cake that will prevent differential pipe sticking.In the exploration for hydrocarbons, a successful drilling operation to the desired depth hinges on the effective performance of the formulated drilling fluid. Apart from carrying drill cuttings to the surface, another major function of the fluid is to seal off the walls of the wellbore to prevent fluids from coming into and out of the wellbore while drilling a well. Numerous commercial fluid loss additives: carboxymethyl cellulose (CMC), polyanionic cellulose (PAC), among others have been in existence with their drawbacks and effect on the total drilling cost. This study evaluates the use of locally sourced materials:  Chemical engineering; Locally sourced materials; Fluid loss control additive; Fluid loss volume; Filter cake thickness; Mud cake characteristics. Udoh anIn a drilling operation, filtration control is the addition of materials to drilling mud to reduce filtration rate and improve mud cake characteristics . The twoDetarium microcarpum and Brachystegia eurycoma, and one cellulose-based product, rice husk as fluid loss control additive in water-based drilling fluid to establish their filtration capabilities.In recent times, there has been a shift in the use of commercial polymers to locally sourced materials as fluid loss control additives in water-based drilling fluids . Accordi1.1Detarium microcarpum and Brachystegia eurycoma in the local parlance are referred to as ‘Ofor’ and ‘Achi’ by the Igbos, and ‘Ogbogbo’ and ‘Akolodo’ by the Yorubas (Oryza sativa) is grown on every continent except in Antarctica, and the extent of paddy cultivation covers about one percent of the earth's surface  in the mud, while the mud sample number (e.g. SET-A1through SET-A4) depicts the fluid loss additive content (in gram) in the mud sample. Detarium microcarpum - 5% rice husk (MUD-A), 95% Brachystegia eurycoma - 5% rice husk (MUD-B), 5% Detarium microcarpum - 95% rice husk (MUD-C) and 5% a Brachystegia eurycoma - 95% rice husk (MUD-D). The formulated mud samples were kept to age for twenty-four (24) hours at room temperature. Some of these formulated drilling mud samples are shown in Sixteen (16) drilling mud samples were prepared (formulated) based on the American Petroleum Institute (API) standard of 25g–350 mL of water for non-treated bentonite. Using Hamilton Beach Commercial high-speed mixer , the var2.3The filtration test to evaluate the fluid loss control potential of the locally sourced material was based on the API recommended practice, API 13B-1. The standard recommends 100 psi (about 700 kPa) pressure and 30 min  for LTLP filtration test in the water-based drilling fluid. In this study, the LTLP filter press is a cylindrical cell of 3 inches internal diameter and 5 inches height to contain the drilling fluid samples. The API filter press consists of six (6) cylindrical cells on the stack . Whatman2.3.1Qualitative description of drilling fluid filter cake characteristics involves measurement of its thickness to establish whether the formed filter cake is either thick or thin. For the measurement of the qualitative characteristics of drilling fluid filter cake, 2.3.23)3)3)The permeability of the mud filter cake controls both static and dynamic filtration . It estaUsing conventional filter press data, 3The performance of the locally sourced materials as fluid loss control additives in water-based drilling fluid was evaluated based on the API specifications for standard polymer: CMC and PAC used in the industry. These specifications are presented in 3.13.1.1Detarium microcarpum at 10g content was comparable with 2g CMC content in the drilling fluid sample, while 15g Brachystegia eurycoma content was comparable with the same CMC content. Also, the results indicated that rice husk and Detarium microcarpum at 15g and 20g content had less fluid loss volume than CMC at 4g and 8g content, as Brachystegia eurycoma at 20g content could only have less fluid loss than 4g CMC content in the water-based drilling mud. Furthermore, comparing the filter loss performance of the local materials with API CMC and PAC specifications, this shows that the additives at 10g–20g content would perform favourably as CMC and PAC in the water-based drilling fluid. Thus, it can be said that the local fluid loss control additives have the potential to compare with CMC and PAC.Figures 3.1.2Detarium microcarpum and 5% rice husk (MUD-A), and 95% Brachystegia eurycoma and 5% rice husk (MUD-B) contents as fluid loss control additive in the drilling fluid samples. Detarium microcarpum and 95% rice husk (MUD-C), and 5% Brachystegia eurycoma and 95% rice husk (MUD-D) contents as fluid loss control additive in the mud samples. The results presented in Detarium microcarpum -5% rice husk) in MUD-A performed significantly to reduce the drilling mud filter loss volume than 95% Brachystegia eurycoma-5% rice husk control additive in MUD-B. Also, the results as presented in Detarium microcarpum-5% rice husk additive in the water-based drilling mud will compare favourably with API CMC and more than API PAC specifications from additive content of 10g. For the 95% Brachystegia eurycoma-5% rice husk content in the drilling mud sample (MUD-B), it will perform close to the API CMC specification from additive content of 15g and more than API PAC specification from additive content of 10g. The fluid loss results obtained further showed that 10g of the composite additive in MUD-A was comparable with 4g of CMC content, while 15g and 20g composite additive (MUD-A) resulted in less fluid loss than 6g and 8g CMC content in the drilling fluid. For composite additive in MUD-B, it is observed that only 20g content was near the fluid loss volume obtained for 8g CMC content in the drilling mud. On the other hand, Detarium microcarpum-95% rice husk (MUD-C), and 5% Brachystegia eurycoma-95% rice (MUD-D) as filter loss control additive will perform remarkable than API PAC specification and compare favourably with API CMC specification. This result implies that these composite additives at the mentioned content would be capable to control fluid loss in water-based drilling fluid as the CMC and PAC.The filter loss volume results for the composite drilling fluid samples are presented in Figures 3.1.3Detarium microcarpum and Brachystegia eurycoma muds , there was a noticeable decrease in the filter loss volume from their composite counterpart, that is MUD-A (95% Detarium microcarpum-5% rice husk) and MUD-B (95% Brachystegia eurycoma-5% rice husk) when compared to Detarium microcarpum and Brachystegia eurycoma muds  performed better than Brachystegia eurycoma composite (MUD-B) and was more comparable with the CMC mud as well as API specification for fluid loss control additive from 10g content. Further analysis of the results at 20g additive content showed composite additive (MUD-A) reduce fluid loss volume by 44.44% when compared to its non-composite complement (Detarium microcarpum mud). Also, composite additive MUD-B decreases the filter loss volume by 35.62% than its non-composite type (Brachystegia eurycoma mud). For the rice husk mud (non-composite) and its composite muds: MUD-C (5% Detarium microcarpum-95% rice husk) and MUD-D (5% Brachystegia eurycoma -95% rice husk), it is observed in Detarium microcarpum or Brachystegia eurycoma to the rice husk reduces its fluid loss control capability.Figures  Figures  and 15. 3.23.2.13.2.1.1Detarium microcarpum mud cake thickness at 15g additive content was relatively the same as that of 6g CMC content, while Brachystegia eurycoma 20g filter performance was equivalent to 4g CMC content in the drilling fluid. Also, the filter cake thickness obtained from rice husk and Detarium microcarpum at the 10g and 15g content respectively met the API specification (fication  for filtfication  and the fication  showed t3.2.1.2Detarium microcarpum-5% rice husk) filter thickness at additive content of 5g and 20g were closer to the CMC mud results at 2g and 8g content, while 10g and 15g MUD-A filter cakes were thicker than CMC mud cakes of 4g and 6g. Again, the result showed that 10g additive in MUD-A resulted in the same mud cake thickness with 6g CMC content in the drilling fluid sample. On the other hand, the mud cake thickness for composite MUD-B (95% Brachystegia eurycoma-5% rice husk) at additive content of 10g is comparable with 4g CMC mud. Also, it is noted that the filter cake thickness from MUD-B at additive content of 15g and 20g were thinner than 8g CMC content in the water-based mud. Furthermore, it is observed from the Figure that the filter cake thickness obtained for 20g MUD-B is close to 15g MUD-A. This observation means that composite MUD-A is more favourable than composite MUD-B when compared with CMC as a filtration control additive in the water-based drilling fluid.Figures Detarium microcarpum-95% rice husk) filter cake thickness at 5g additive content was the same as that of 6g CMC content in the drilling mud sample. Also, the results obtained for MUD-C and MUD-D (5% Brachystegia eurycoma-95% rice husk) at 10g additive content is relatively close. This is because the predominant additive material in the drilling fluid samples is rice husk, which is at the same quantity . Hence, the Figure revealed that the filter cake thickness of the composite muds MUD-C and MUD-D outperformed  the CMC mud at their equivalent comparable additive content  has the least mud cake permeability at the different additive content in the mud samples. This observation explains the reason for the favourable and comparable performance of the composite additive  as fluid control in the water-based mud with CMC and PAC, as presented in 3.2.3.3Brachystegia eurycoma-5% rice husk) than in non-composite Brachystegia eurycoma mud. Also, for the composite Detarium microcarpum mud and its non-composite counterpart (95% Detarium microcarpum-5% rice husk) mud, have about the same mud cake permeability values. This is because there was no much difference between these muds’ cake thickness values; as presented in From the results obtained, in Detarium microcarpum-95% rice husk and 5% Brachystegia eurycoma-95% rice husk, respectively), the results obtained depicts that mud cake permeability values of the composite MUD-D were higher than that of non-composite mud  form, iii.Detarium microcarpum - rice husk and Brachystegia eurycoma - rice husk reduce fluid loss by 44.44% and 35.62%, as filter cake increases by 40.57% and 77.56% over its non-composite counterpart;at 20g content, composite additives: iv.the mud cake characteristics of these locally sourced materials are acceptable for good mud filter cake requirement;v.the mud cake permeability depends on fluid loss additive contents in the drilling mud, thus, low additive content results in high mud cake permeability; andvi.fluid loss volume and filter cake thickness were mud cake permeability dependent, as high mud cake permeability gives high fluid loss volume and thin mud cake, while low mud cake permeability result in less fluid loss volume and thick mud cake.For successful drilling operation, drilling fluid “the blood of the operation” is expected to perform critical functions. One of these functions is to seal the walls of the formation being drilled to prevent filtration. Several commercial fluid loss control additives, namely, CMC, PAC, among others had been in use with their limitations and financial implications on the overall drilling cost. In this study, several locally sourced materials: It is recommended that further work be carried out on the locally sourced materials to establish their composite potential and/or effect on water-based drilling fluid viscosity  at different thermodynamic conditions.Anietie N. Okon: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.Julius U. Akpabio: Analyzed and interpreted the data; Wrote the paper.Kilaliba W. Tugwell: Performed the experiments.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


We report the case of a 75-year-old female patient with a big tumour in the lower rectum with intestinal obstruction and lower gastrointestinal bleeding history who underwent a tumour biopsy under laparotomy and end colostomy at another hospital in Peru. She came to our institution for clinical evaluation with a pathology result of a rectal gastrointestinal stromal tumour. An extra elevator abdominoperineal resection was performed with tumour-free margins. The histology confirmed a high-grade (G2) rectal gastrointestinal stromal tumour with a mitotic index of 27/50. DOC-1 (+) and CD117 (+) in immunohistochemistry. Genomic DNA was extracted from the paraffin-fixed tumour sample, and c.1504_1509dupGCCTAT (p.Ala502_Tyr503dup) mutation was detected in exon 9 of the KIT gene. Imatinib 400 mg per day for 3 years was indicated as adjuvant treatment. Currently, she has a disease-free survival of 12 months. The gastrointestinal stromal tumour (GIST) term was established by Mazur and Clark in 1983, in reference to non-epithelial tumours of the gastrointestinal tract without leiomyosarcoma features  and 0.1%This is about a 75-year-old Peruvian female with 4 months of abdominal pain associated with low gastrointestinal bleeding and faecal incontinence who had a laparotomy with tumour incisional biopsy and end colostomy at another hospital. She was referred to the National Cancer Institute with a pathology report of rectal GIST . A rectaAn extra elevator abdominoperineal resection of the primary tumour was performed on May 2019. Pelvic examination under anaesthesia showed a tumour at 3 cm from the anal verge with a wide base depending on the right and posterior walls of the rectum .The final pathology reported a tumour of 8 × 7 cm, distal margin of 4.5 cm and proximal margin of 30 cm, tumour-free margins. The histology confirmed a fusocelular subtype of rectal GIST with a mitotic index of 27/50, G2 and 30% of necrosis. No lymph node metastasis (0/53) was found. The final pathologic stage was pT3N0. Immunohistochemistry showed DOC-1 (+) and CD117 (+) .The patient began adjuvant treatment with Imatinib 400 mg per day for 10 weeks after surgery. Until the date of her last follow-up, there was no evidence of recurrent disease by imaging studies (12 months).Genomic DNA was extracted from the paraffin-fixed tumour specimen, and a Sanger sequencing reaction was performed from exons 9, 11, 13 and 17 of the KIT gene and exons 12, 14 and 18 of the PDGFRA gene. The mutation c.1504_1509dupGCCTAT (p.Ala502_Tyr503dup) was detected in exon 9 of the KIT gene.Rectal GIST is a rare condition, which is characterized by a large tumour mass with well-defined margins. Extraluminal location with an epicentre located outside the rectum is usually found in most cases and described as an adverse prognostic factor, regardless of the tumour size . In our MRI helps to determine the origin of the tumour and allows an adequate assessment of the surgical pelvic floor, the involvement of adjacent organs and CRM. T2-hyperintense foci with heterogeneous pattern of enhancement and T1-isointense image as skeletal muscle are frequently seen as in our case. Calcifications and areas of haemorrhagic foci can be present , 10. TheA biopsy is essential, ideally, with a transrectal core biopsy. Assessment of tumour behaviour by molecular analysis, especially in large tumours that require radical surgery or neoadjuvant/adjuvant treatment , and patA well-circumscribed mass of highly variable size (from <1.0 mm to > 20.0 cm) is usually shown macroscopically. The cut surface may show foci of haemorrhage, cystic change or necrosis in large lesions . TumoursInvasion into the mucosal and tumour necrosis are poor prognostic factors. The so-called ‘paediatric-type’, which is an SDH-deficient GIST with plexiform growth, commonly epithelioid with lymph node metastasis , is not Histologic grading is important in soft tissue sarcoma staging but not well suited for GIST. They usually have aggressive features despite having low mitotic rates . The gra2.Following the guidelines for risk assessment of primary GIST , 20, we Surgical resection is the treatment for all resectable rectal GIST patients , 25. TheLocal tumour resection (LTR) or sphincter-preserving procedures can be performed for small or low-grade tumours . DiffereBefore imatinib, surgical procedures were more radical and extensive for large and high-risk tumours with aggressive features. Low anterior resection or an abdominoperineal resection are still indicated for rectal tumours located below 5 cm from the anal verge with a high risk of positive CRM . Some au et al. [With the introduction of imatinib in the management of GIST, some absolute indications for surgery have changed. The benefit of a previous pathological study supports the use of neoadjuvant treatment in some cases even for dose resectable rectal GIST. The neoadjuvant treatment with imatinib is used when R0 surgery is not feasible or when a function-sparing surgery is planned . Wilkins et al.  reportedGastrointestinal stromal tumour of the rectum with intestinal obstruction emergency is a very rare situation. In this case, a diverting colostomy with a biopsy of the tumour was performed at a general hospital. An incisional biopsy is considered as a tumour rupture according to the Oslo criteria  and can Advantages reported for neoadjuvant treatment are tumour downsizing, mitotic activity reduction, high rates of organ and sphincter preservation, low rates of positive margins, low recurrence and less morbidity in patients with high-risk GIST tumours , 33; how et al. [Disease-free survival (DFS) depends on the risk stratification. Better results are achieved in patients with intermediate and high-risk tumours . Actuall et al.  showed 9 et al. .There are no differences in recurrence-free survival (RFS) and overall survival (OS) among LTR or more radical resections. The decision on the optimal surgical treatment mainly depends on the tumour size, mutational status and the surgeon’s capacity . The lacOptimal management of gastrointestinal stromal tumours of the rectal is challenging.A biopsy is essential for the molecular assessment by fine-needle aspiration cytology (FNAC) or trans rectal core biopsy.Multimodal treatment for GIST of the rectum should be managed in a specialized centre.Incisional biopsy must be avoided since tumour rupture and seeding could raise the risk of recurrence. In such cases, a more radical surgical procedure must be considered.The approach and surgical procedure with tumour-free resection margin have no significant impact on prognosis and survival.We know of no conflicts of interest associated with this publication.There has been no significant financial support for this work that could have influenced its outcome.


Combining positron emission tomography (PET) with MRI offers an opportunity to improve diagnostic accuracy. (2) Method: Our single-centre experience of  Tumours of the Central Nervous System (CNS) are the most common form of solid tumour in children and teenage/young adult (TYA) populations, accounting for more than 25% of all types of cancers and 15% of malignancies in the teenage and young adult population 15–24 years). According to the most recent statistics from Cancer Research UK, tumours of the CNS are the fourth most common cancer type in this age group with astrocytomas forming the largest sub-group followed by embryonal tumours, such as medulloblastoma and ependymoma ,4,5. years. Apseudoprogression, whereby an increase in tumour volume, oedema and enhancement shortly after completion of treatment is often difficult to distinguish from progressive tumour fluoro-D-glucose (FDG), and system L amino acid transport comprising 11C-methyl-methionine (MET), O-(2-[18F]-fluoroethyl)-L-tyrosine (FET) and 3,4-dihydroxy-6-[18F]-fluoro-L-phenylalanine (FDOPA) -methyl-L-tryptophan (AMT) and [18F]fluciclovie (FACBC) as well as glutamine-based amino acid PET tracers have also been evaluated in gliomas for tumour delineation, prognostication and differentiation of tumour recurrence from radiation injury, but currently, the number of examined subjects is low . Wh. Wh43]. Pre- and post-contrast T1-weighted sequences using identical MRI parameters allow voxel-to-voxel image subtraction, which may detect subtle residual enhancement and therefore provide a more accurate and reproducible assessment of the true extent . Whilst A variation of this method acquires two high-resolution 3D T1-weighted sequences in the same MRI session, and 3–5 and 60–75 min after the injection of the contrast medium and subsequent subtraction a colour-coded map known as a treatment response assessment map (TRAM) is produced. Areas where contrast accumulates correspond histologically to treatment-induced changes, whilst areas where contrast is cleared rapidly correspond to active tumour . TRAMs aDiffusion-weighted imaging (DWI) is sensitive to random microscopic (Brownian) motion of water molecules, which results in signal loss and consequent hyperintensity in areas of restricted diffusion. The apparent diffusion coefficient (ADC) reflects the magnitude of water motion with restricted diffusion having lower ADC values. ADC is inversely correlated with cell density due to reOne caveat to the application of DWI for identifying non-enhancing tumour is that lower ADC values do not always correlate with increasing tumour infiltration. Low and persistent diffusion restriction can be associated with non-viable tissue necrosis. In adults, these lesions tend to be periventricular, slowly change over several months and are thought to be a type of treatment toxicity, associated with better outcomes. Novel diffusion modelling techniques such as Vascular, Extracellular, and Restricted Diffusion for Cytometry in Tumours (VERDICT) have been developed at our institution that aim to identify diffusion parameters that more accurately reflect tumour microstructure  and willPerfusion-weighted imaging (PWI) exploits the neoangiogenic properties of proliferating gliomas, and is able to identify areas of high-grade tumour with high accuracy. PWI can be performed in a number of ways; the main techniques in clinical practice are dynamic susceptibility-weighted contrast-enhanced MRI (DSC), dynamic contrast-enhanced MRI (DCE) and arterial spin labelling (ASL).DSC-MRI rapidly acquires gradient echo or spin echo planar images during first-pass transit through the brain of an exogenous paramagnetic gadolinium based-contrast agent that transiently decreases signal intensity. Voxel-wise changes in contrast agent concentration are determined from time–signal curves and processed using kinetic tracer modelling and indicator dilution theory to estimate cerebral blood volume (CBV), cerebral blood flow (CBF) and mean transit time (MTT). Relative CBV (rCBV) is the most common DSC-MRI metric for evaluating brain tumours. To minimize the error caused by contrast extravasation, known to occur in brain tumours, preloading of the contrast agent along with model-based post-processing leakage correction can decrease both T1 and T2* effects ,55.trans) and rate constant. Ktrans is thought to reflect microvascular permeability but also representative of blood flow and vessel surface area, the most common metric in brain tumour studies  and 18fluoro-L-phenylalanine ad will have to be factored in for clinical use.Although the clinical situations presented do not necessarily require simultaneous acquisition in a PET–MRI scanner, the advantages, particularly in the paediatric population, are the requirement for only one examination, particularly if sedation or general anaesthetic is planned. Another advantage is the exact co-registration of PET and MRI, which is desirable post-surgery where anatomical distortion is likely to occur. However, there are several disadvantages in that PET–MRI is a lengthier examination process, at approximately 90 min acquisition, compared to MRI or PET alone. There is also likely to be decreased patient comfort due to smaller bore size causing claustrophobia and ear phones not being standard. Finally, the necessity of rigid head fixation for smaller head size can be a cause of increased imaging artefacts. However, next-generation PET–MRI patient scanners will hopefully improve patient comfort and realize the potential of a one-stop non-invasive imaging technique in neuro-oncology. It is important to stress that a one-off negative scan with PET–MRI during any phase of treatment does not equate to a complete metabolic response, and, hence, a baseline scan is mandatory. The ease of availability is a common limitation for both tracers— Is PET–MRI an effective tool for oncological imaging of primary brain tumours in children and TYA patients, especially in the delineation tumour extent, monitoring treatment response and post-treatment differentiation between tumour progression or recurrence versus treatment-related changes? The second question is, what is the optimal indication of PET–MRI imaging in brain tumours?Facilitating diagnostic neuro-surgical intervention, i.e., the site for pre-operative biopsy planning and navigation in children and TYA patients;The reassurance that metabolically inert post-therapy residual lesions in patients with intracranial germ cell tumours represent non-viable tissue;Defining the tumour and target volumes for radiotherapy by combining metabolic and radiological information of tumour extent and infiltrative margins;The detection of tumour recurrence during clinical and radiological surveillance;In distinguishing tumour recurrence from treatment necrosis/therapy-related changes;That PET–MRI alone is insufficient in providing accurate delineation and diagnostic sensitivity of observed lesions, as not all histological subtypes of brain tumours take up the radiotracers.Although conventional gadolinium-enhanced magnetic resonance imaging (MRI) is considered the primary imaging modality for patients with brain tumours, it has some limitations, primarily in differentiating tumour grade, its inability to provide metabolic or functional information of the biology of the tumour, and in distinguishing disease recurrence from post-therapy changes. This report corroborates the effectiveness as well as the limitations of PET–MRI in paediatric and TYA neuro-oncology in the following:There is limited published evidence of the use of PET–MRI as an imaging modality for diagnostic purposes, early treatment response assessment, end of treatment disease status or as an imaging tool for post-treatment radiological surveillance in a paediatric and TYA patients with brain tumours.This review highlights the importance of a multi-parametric imaging and that PET–MRI unequivocally is a promising imaging tool for children and TYA patients with a variety of brain tumours. It allows for the diagnosis and monitoring of morphological and metabolic response and changes during therapy and is an effective imaging tool in detecting viable residual tumour at the end of treatment.Overall, the conventional cross-sectional MRI combined with PET is a complementary modality for assessment of tumour burden. Combining and matching both imaging modalities is significantly superior to either imaging modality alone in assessing the presence of viable residual tumour and indicating the radiotherapy target volume. Additionally, PET–MRI generates quantitative measures that may add significant value to conventional SUV image-derived measures.While for primary tumour staging, MRI on its own is sufficient, the use of combined PET and MRI provides additional molecular and functional information for critical treatment decisions. This is highly relevant in clinical neuro-oncology, and the use of PET–MRI will increase in the years to come. Whilst mostly a research tool in paediatric/TYA neuro-oncology, there is enough evidence from the adult neuro-oncology literature to justify its clinical use. We have demonstrated a number of clinical situations where hybrid PET–MRI can improve diagnostic accuracy, and as improvements in PET–MRI scanners are made, the use in paediatric/TYA neuro-oncology is likely to increase.