Tumour size is considered a key oncological feature, as it reflects the primary tumour burden. In pancreatic cancer, tumour size also constitutes the defining criterion for staging of the primary tumour (T-stage), which provides essential information for patient treatment, risk stratification in clinical trials and cancer registries. While measurement of tumour size in pancreatic cancer resection specimens is considered accurate and reproducible, this has not been formally proven. This study investigated whether, and in how far, various approaches to tumour size measurement result in different tumour sizes and consequently different T-stages. The study findings show that tumour size and T-stage are different in a significant proportion of cases, depending on whether (i) the tumour was measured in one or two planes and (ii) the macroscopic tumour size was corroborated microscopically. Hence, a divergence in pathology practice may limit the comparability of tumour size and T-stage between institutions.p < 0.001). Compared to the most simple approach (macroscopic measurement in one plane), the comprehensive approach (macroscopic measurement in two planes with microscopic corroboration) resulted in a larger tumour size in 263 (83%) cases . T-stage assignment differed in 142 (45%) cases between the simple and comprehensive approach and affected 87%, 38% and 48% of the cases deemed to be stage T1, T2 and T3, respectively. In conclusion, tumour size and T-stage are highly approach-dependent. Consensus on an accurate method is required to ensure comparability of these basic data.In the eighth edition of the TNM classification for pancreatic ductal adenocarcinoma (PDAC), stages T1 to T3 are defined by tumour size, size measurement being deemed objective and accurate. This study investigated whether various, currently used approaches to tumour measurement result in different tumour sizes and differences in T-stage assignment. In a series of 315 resected PDAC, tumour sizes were measured as follows: macroscopically in a single or in two perpendicular planes and with or without microscopic corroboration. Comparison of the resulting tumour sizes showed that both macroscopic measurement in two planes and microscopic corroboration gave significantly different results ( For most solid cancer entities, including pancreatic cancer, the size of the tumour is considered of key oncological importance, as it reflects the primary tumour burden. Moreover, according to the eighth edition of the Tumour, Node, Metastasis (TNM) classification published by the Union for International Cancer Control (UICC) and the American Joint Commission on Cancer (AJCC) ,2, tumoulargest tumour size is recorded. This requires measurement in multiple planes, and current specimen dissection techniques allow for measurement in two planes: the plane in which the specimen is sliced and the plane perpendicular to that.Because tumour staging is key to both individual patient management, clinical research and cancer registries, tumour size is a core data item in national and international data sets for pancreatic cancer ,6,7,8,9.In practice, most pathologists record the tumour size during specimen grossing by measuring the tumour—usually in two dimensions—on the specimen slice in which the tumour appears at its largest expanse. Some will subsequently check the macroscopic size measurement during microscopic examination. Measurement of a third dimension, in the plane across specimen slices, may be performed by some pathologists for cancers in the pancreatic body or tail, for example in the context of a clinical study, but this approach is usually omitted for cancers located in the head of the pancreas .Given the importance of accurate measurement of the largest tumour size for correct T-staging, this study aimed at comparing the results of the above approaches, that is, two-dimensional versus three-dimensional measurement and macroscopic measurement with or without microscopic corroboration. Furthermore, the study aimed at investigating if, and in how far, different approaches to tumour size measurement affected assignment to T-stage.This observational study is based on a cohort of consecutive pancreatic ductal adenocarcinomas, for which data were prospectively collected and stored in a database at the Department of Pathology at Oslo University Hospital. Included were all ductal adenocarcinomas of the pancreas that were surgically resected at Oslo University Hospital, Norway, between 1 January 2015 and 31 December 2020. Excluded were tumours that had been treated neoadjuvantly, pancreatic ductal adenocarcinoma that had developed from intraductal papillary mucinous neoplasia and any other tumour entities in the pancreas, including mixed tumour entities, such as mixed neuroendocrine–non-neuroendocrine neoplasms.For each case in the study cohort, the following data were recorded: type of surgical resection, tumour site (head or body/tail of pancreas), T- and N-stage (according to the 8th edition of the AJCC/UICC TNM classification), presence or absence of lymphatic, vascular and perineural invasion, and resection margin status. Regarding the tumour dimensions, two sets of sizes were extracted from the database: (1) the maximum tumour size measured macroscopically in one specimen slice and the maximum tumour size measured across specimen slices and (2) the same tumour dimensions corroborated by microscopic measurement.Grossing of all specimens, undertaken by a pancreatic pathologist (CSV) or by residents and trained technical staff under the supervision of the pancreatic pathologist, was carried out in accordance with national guidelines [idelines  and a riidelines ,14. BrieThis (third) tumour size is hereafter referred to as “3D-macro”.Once the specimen slices were laid out in sequential order, the specimen slice in which the tumour was at its largest expanse was identified. The largest tumour dimension was measured on that slice (hereafter referred to as “2D-macro”). The second tumour dimension, which was measured on the same specimen slice, perpendicularly to the largest dimension, was not considered in the current study, as it did not represent the largest tumour size in that plane. Furthermore, the number of the axial specimen slices that were involved by tumour was recorded. The tumour dimension in the craniocaudal plane was then calculated based on the length of pancreatic head in craniocaudal direction , the total number of specimen slices and the number of tumour-bearing slices, according to the following formula:With this approach, the accuracy of the measurement in the craniocaudal direction was determined by the thickness of the specimen slices. In some cases, tumour tissue was visible only on one side but not on both sides of the specimen slice(s) containing the edge of the tumour. To increase accuracy on such occasions, only half of the slice thickness for that particular slice (or for both slices) was used in the size calculation. Given an average slice thickness of 3 mm, the accuracy of size measurement across specimen slices was 1.5 mm. Distal pancreatectomy specimens were dissected by serial slicing in the sagittal plane, that is, perpendicular to the longitudinal axis of the pancreatic body and tail. A similar procedure was followed to evaluate the maximum tumour size in three dimensions: by measuring the maximum tumour size on the slice with the largest tumour expanse (2D-macro) and by calculating the tumour size across sagittal specimen slices (3D-macro), as illustrated in Total pancreatectomy specimens were divided by the pathologist into a “pancreatoduodenectomy” and a “distal pancreatectomy” part, in which the tumour dimensions were measured as described above. To allow review of the macroscopic findings, each case was photo-documented by an overview image of all specimen slices as well as close-up images of individual slices.Tissue sampling was extensive in each case and included complete embedding of (i) the specimen slice deemed to contain the largest tumour cross-section and (ii) the slices that contained the craniocaudal or mediolateral ends of the tumour . In order to detect possible tumour extension in the craniocaudal or mediolateral direction that was invisible to naked-eye inspection, the slice (or multiple slices in cases with an unclear tumour boundary) flanking the presumed ends of the tumour were also embedded  representing the specimen slice that was identified as containing the tumour at its largest expanse. The largest tumour dimension, referred to as “2D-micro”, was measured in that tissue section. For evaluation of the largest tumour dimension across specimen slices, the number of specimen slices with microscopically detectable primary tumour growth was recorded and used for the calculation of this tumour dimension (hereafter referred to as “3D-micro”), as outlined above.For All other pathology data collected for this study  were assessed in line with international guidelines .p-value of <0.05 was considered significant. Statistical Package for the Social Sciences, Version 26  was used for all analyses.Descriptive statistics regarding the clinicopathological features of the study cohort are presented as numbers and percentages, both for the entire series and separately for cancers in the pancreatic head and body/tail. The measured tumour dimensions are presented both as means with standard deviations and as medians with interquartile range. Moreover, the overall range for each tumour dimension is presented. Group-wise comparisons between tumours in the pancreatic head and body/tail were performed with independent sample t-tests, while paired comparisons between the different measurement approaches were assessed with paired-samples t-tests. The N-stage distribution for tumours in each T-stage group was compared between the different measurement approaches with Mann–Whitney U tests. A two-sided A total of 315 eligible cases were included in the study cohort, of which 231 tumours were located in the head of the pancreas and 84 in the body/tail. Pathology data for the entire cohort are presented in For the purpose of the analysis, the four tumours that were removed by total pancreatectomy were considered as pancreatic head cancers, because these tumours were located mainly in the pancreatic head, with limited extension into the so-called pancreatic neck, that is, the transition to the pancreatic body.The results of tumour size assessment macroscopically in the plane of slicing (2D-macro) and across specimen slices (3D-macro) and following microscopic corroboration of both measurements  are shown in p < 0.001), both following macroscopic measurement and microscopic corroboration. In contrast, there was no statistically significant difference between locations for the tumour size measured in the plane of slicing.Comparison between both tumour locations revealed that the tumour size measured in the third dimension, that is, across specimen slices, was significantly larger for tumours in the body/tail than for cancers in the head region . The difference in size was particularly large for cancers in the pancreatic body/tail, with a mean size difference of 17 mm. The relative change in size, that is, the difference in size between both measurements divided by the tumour size obtained by 2D macroscopic measurement, was 61%. In 69 (82%) of these cases, the 3D-macro size was larger than the 2D-macro size, the mean difference between both sizes being 19 mm (range: 1–73 mm), representing a mean relative change in size of 72% (range: 4–280%). Only in a single case, the tumour size measured in both planes was identical, whereas in 14 (17%) of the body/tail cancers, the 3D-macro size was smaller than the one measured in 2D . As illustrated in Macroscopic measurement in the plane perpendicular to the plane of slicing  resulted in tumour sizes that were significantly different from those measured in the plane of slicing  than in tumours in the body/tail. In 117 (51%) cases, the 3D-macro size was larger than the one obtained by 2D-macro measurement, the mean difference in size being 6 mm (mean relative change in size: 22%). Tumour size was smaller in the 3D plane in 92 (40%) cases, and in 22 (9%) cases, tumour size was identical in both planes.p < 0.001; underestimation by 2D- and 3D-macroscopic measurement was both common (in 136 (59%) and 119 (52%) of the cases, respectively) and substantial , with up to 227% and 183% size change in individual cases, respectively. Size overestimation affected fewer cases (59 (25%) by 2D-macro, 35 (15%) by 3D-macro) to a lesser degree . For cancers in the pancreatic head, microscopic corroboration of the macroscopic measurement either on a specimen slice (2D) or across axial slices (3D) resulted in significantly different sizes  and occurred in 37 (44%) cases.p < 0.001) irrespective of tumour site (larger tumour size than the one obtained by 2D-macroscopic measurement only (In only 17 (5%) of all the cases, identical maximum tumour sizes were obtained by both a simple procedure, which was based exclusively on macroscopic measurement on a specimen slice (2D-macro), and a comprehensive approach, which consisted of measuring also across specimen slices with microscopic corroboration of both macroscopic sizes (combined 2D-3D-micro). The difference in tumour size based on either approach was statistically significant , was recorded separately for cancers in the pancreatic head and body/tail . When comparing measurement on a specimen slice without versus with microscopic corroboration (2D-macro versus 2D-micro), 57 (18%) cases in the entire study series changed T-category, with a shift affecting 19 of 38 (50%) cancers categorised as T1 based on 2D-macro assessment, 21 of 231 (9%) of those deemed to be T2 and 17 of 46 (37%) cancers that were categorised as T3. In 115 (37%) cases, T-stage assignment differed depending on whether tumour size was measured macroscopically in a single plane (2D-macro) or in two planes (3D-macro). This change in T-stage affected 22 of 38 (58%) of the cancers that were categorised as T1 based on 2D-macro assessment, 72 of 231 (31%) of the tumours deemed to be T2 and 20 of 46 (43%) cancers that were categorised as T3. Comparison between the simple approach (2D-macro) and the comprehensive approach based on measurement in two planes and microscopic corroboration (2D-3D-micro) revealed an even larger shift in T-stage, namely in 142 of all cases (45%). This change in T-stage affected the vast majority of cancers deemed to be T1 based on the 2D-macro approach  and more than a third  and nearly half  of the tumours in the categories T2 and T3, respectively. In the vast majority of cases with a change in T-stage, the T-stage shifted up , while in a smaller proportion , there was a down-shift. The shift was mainly by one T-stage, but in eight cancers  the shift was across two T-stages . The single case in the entire series that was assigned to stage T1b based on 2D-macro measurement shifted to T1c, that is, it remained within the T1-category and was therefore not regarded as a change in T-stage. T-stage changed somewhat more frequently for cancers in the pancreatic body/tail than for those located in the pancreatic head (40 of 84 (48%) versus 102 of 213 (44%), respectively). Otherwise, the distribution of changes in the three T-categories was similar irrespective of tumour site.The above-described shifts in T-stage resulted in a considerable change in T-stage distribution . Tumour Macroscopic measurement in the plane across specimen slices (3D-macro) introduced changes in T-stage distribution mainly for cancers in the body/tail, where it resulted in a T-stage distribution that was very similar to the one based on the comprehensive approach . In contrast, for cancers in the pancreatic head, T-stage distribution differed little from that based on 2D-macro measurement .Comparison with the T-stage distribution reported by other series shows that these are similar to the distribution based on the simple approach (2D-macro) but different from the one based on the comprehensive approach . Similar differences in N-stage were observed for T2 tumours (p = 0.03), whereas differences were not significant for T3 tumours.Finally, the N-stage distribution  was compared between tumours assigned to stage T1, T2 or T3 based on the simple versus the comprehensive approach . Based oTumour size is a key oncological data item, because it reflects the primary tumour burden. As for multiple other cancer entities, tumour size is the defining criterion of the T-stage (T1–T3) for ductal adenocarcinoma in the pancreas. The clinical interest in and importance attributed to the TNM system is reflected by the considerable number of studies that soon after the release of the eighth edition of the UICC/AJCC classification system validated the new T- (and N-) staging criteria in European, Asian and US data sets ,21,22,23This study aimed at investigating whether and to which extent different ways of measuring the tumour resulted in different tumour sizes and in how far this divergence affected T-stage.two dimensions, that is, the dimension in which the tumour size is largest and the perpendicular dimension . In contrast to this simple procedure, a comprehensive approach includes, in addition, (i) measurement in a third dimension, namely in the plane that is perpendicular to the plane of specimen slicing  and (ii) microscopic corroboration of both sizes that were obtained by macroscopic measurement in both planes  macroscopically on a specimen slice (referred to as 2D-macro) and (ii) across specimen slices (3D-macro) as well as  the size in either plane obtained by microscopic corroboration . Overall, the study findings reveal that measurement in two planes and microscopic corroboration results in considerable size differences as compared to exclusive macroscopic measurement in a single plane. p = 0.003 head, p < 0.001 body/tail). The size discrepancy was particularly large for tumours in the pancreatic body/tail: in 82% of cases, measurement limited to the plane of sectioning resulted in underestimation of tumour size by a mean of 19 mm or a 72% relative change in size. This marked difference in size between cross-sectional and longitudinal dimensions is explained by the fact that pancreatic cancers in the body/tail often have an oblong shape, as they grow extensively along the longitudinal axis of the organ without causing cross-sectional expansion to a similar extent . For cancers in the body/tail, size differences were not statistically significant, which is not surprising considering that the majority of these cancers already occupy the entire width of the pancreatic body/tail with—as outlined above—limited expansion of the latter. As recommended by (inter-)national guidelines, it is common practice to corroborate by microscopic examination the tumour size that is measured macroscopically. This is usually carried out only for the measurement taken on a specimen slice. The study shows that for pancreatic head cancers, tumour size was under- or overestimated in 59% or 25% of cases, respectively, if macroscopic measurement was not corroborated microscopically , with size underestimation in 52% (mean relative change in size 33%) if macroscopic measurement was not corroborated microscopically. For cancers in the body/tail, size differences were not statistically significant. However, in individual cases, large size discrepancies—overestimations of up to 29 mm or underestimations of up to 60 mm—were recorded. The reason for these large differences is the fact that pancreatic tissue upstream from the tumour is often severely affected by atrophy and fibrosis, which renders macroscopic distinction from the cancer difficult . In 83% of all cases, the comprehensive approach revealed a larger tumour size, which in 35% exceeded by more than 10 mm the size obtained by the simple approach , while T3 tumours more than doubled (from 15% to 36%). Of note, T1 and T2 tumours, according to the comprehensive approach, were associated with a lower N-stage than those deemed to be T1 or T2 according to the simple approach. This observation indicates that T1 and T2 tumours identified by the comprehensive approach truly represent an earlier cancer stage with, accordingly, a lower rate of lymph node metastasis.Taken together, the study demonstrates that various measurement approaches lead to significant differences in tumour size and consequently a different pT assignment in a considerable number of cases. The current most commonly used approach, which is based only on macroscopic measurement in the plane of specimen slicing, results in a different, incorrect T-stage assignment in 45% of pancreatic cancers as compared to the comprehensive approach.and substantial, they lead to a shift in T-stage in 45% of tumours, if the comprehensive approach is used instead of the simple approach. As such, the assumption that measurement of tumour size is accurate and reproducible—and results in correct T-staging—only applies if the comprehensive method is used systematically. To the best of our knowledge, this study is the first to show that different approaches to tumour measurement result in significantly different tumour sizes and consequently assignment to different T-stages. The study is also the first to reveal that identification of the largest tumour size is dependent on two key aspects of the pathology examination method: measurement in two planes, i.e., on a specimen slice and across slices and microscopic corroboration. While the latter is relevant irrespective of the tumour site, the former affects particularly (but not exclusively) the size assessment of tumours in the body/tail, resulting in a mean relative change in size of no less than 61%. Furthermore, the results show that because size discrepancies are both common The findings in this study have two important implications. First, given the current divergence in practice related to tumour size measurement, tumour size and T-stage assignment may not be comparable between institutions. While most guidelines acknowledge that microscopic corroboration is important ,7,8,9, pSince the ease of practice is an important factor to consider when deciding on which approach to follow, one could argue that universal adherence to the currently more commonly used approach, namely macroscopic measurement in the plane of specimen slicing without microscopic corroboration, would ensure comparability of data. However, while this would indeed improve reproducibility, it would not address the fact that—as outlined above—in a large proportion of cases both tumour size and T-stage are incorrect, with either under- or overestimation of a highly variable degree. A second implication of this study is that (inter-)national data sets for pancreatic cancer should not only recommend tumour size measurement in three dimensions but also provide practical guidance as to how this should be carried out, an important aspect that currently is lacking. Macroscopic measurement of the tumour dimension across specimen slices involves more detailed recording during macroscopic examination, and microscopic corroboration of this tumour dimension requires additional tissue sampling. In the experience of the authors, this required approximately 5 min of additional work. While more demanding, the comprehensive approach can be easily included in the standard operating protocol for specimen grossing  and microscopic assessment. The study has several limitations. First, the analysis is based on a single-institution cohort, which may have introduced bias. However, because clinical, surgical and pathology methods and decisions were standardised according to international guidelines ,24, the A third limitation is the relatively small size of the cohort n = 315). However, as discussed above, the mean and median tumour sizes observed in the current study for two-dimensional measurement are highly comparable with data from other studies . However,21,22,23Last but not least, the study did not investigate the correlation between tumour size and/or T-stage and patient survival. The reason for this omission is the fact that survival is determined by multiple factors other than tumour size and T-stage, in particular N-status and, for instance, adjuvant treatment. Indeed, the intention of the TNM system is in the first instance to allow unambiguous and precise communication of the tumour burden between multidisciplinary user groups, a goal to which the current study findings have contributed . The strengths of the study lie in the prospective data collection, the fact that the study series is recent and covers a short period of time (2015–2020) and the use of a detailed, fully standardised pathology examination protocol. A further strength is the extensive annotation of the study series, including macroscopic photo documentation of all cases, which enables review not only of the histology but also of the macroscopic findings. While tumour size measurement is generally considered accurate and reproducible, the study results show that different measurement approaches result in significantly different maximum tumour sizes and, consequently, significantly different T-stage assignment. Especially measurement in the plane perpendicular to the plane of specimen slicing, but also microscopic corroboration of the macroscopic measurements, has a significant impact on the resulting tumour size and T-stage assignment. As long as pathology practice varies, tumour size and T-stage may be neither accurate nor reproducible. This puts at risk the meaningfulness of comparing stage-related outcomes between institutions, to the detriment of patient management, clinical research and cancer registries. Even if, in the not-too-far future, artificial-intelligence-assisted tumour size measurement may overcome current obstacles, study cohorts with correctly measured pancreatic cancers will be invaluable for establishing a robust ground truth .


Paediatric brain cancer is the second most common childhood cancer and is the leading cause of cancer-related deaths in children. Despite significant advancements in the treatment modalities and improvements in the 5-year survival rate, it leaves long-term therapy-associated side effects in paediatric patients. Addressing these impairments demands further understanding of the molecularity and heterogeneity of these brain tumours, which can be demonstrated using different animal models of paediatric brain cancer. Here we review the use of zebrafish as potential in vivo models for paediatric brain tumour modelling, as well as catalogue the currently available zebrafish models used to study paediatric brain cancer pathophysiology, and discuss key findings, the unique attributes that these models add, current challenges and therapeutic significance. Brain and CNS tumours account for 25% of all paediatric cancers and is the second most common form of cancer in children ,2. ThesePaediatric brain tumours develop in children of ages ranging from 0–14 years, and have been accounted as one of the leading causes of cancer-related mortality in infants and adolescents ,11. The Medulloblastoma (MB) is the second largest group of brain tumours (18.8%) and is the most common paediatric embryonal malignant tumour originating from precursor cells in the cerebellum or dorsal brainstem . MBs areChildren with low-grade brain and CNS tumours have an overall survival rate of 80–90% , whereasAdverse effects imposed by these treatment modalities have raised the need for an alternative targeted approach for paediatric brain cancer treatment, which enhances efficiency with insignificant toxic effects. Advances in next-generation sequencing have led to the advent of an era of precision medicine that catalogues the genetic alterations in patient tumours, helping to understand the molecular mechanisms driving the tumour cell transformation ,34. FurtThe zebrafish is a valuable vertebrate model organism initially established for studying developmental biology . Recent Classic methods of cancer modelling in zebrafish involve genetic and transplantation approaches . Genetic−/− il2rgc.a−/−, which lacks B, T and NK cells, allowing xenotransplantation studies to be performed in adult animals and has reported ways to induce tolerance to higher temperatures in these animals [Zebrafish embryo xenotransplantation models have been widely used to understand the key steps of tumour progression, such as invasion ,59, extr animals . CollectThe advent of engineered nucleases has portended a booming growth in the field of reverse genetics in modelling cancer in zebrafish  Table 1Table 1. nf1a and nf1b, NF1 orthologues in zebrafish, which accelerated the tumour onset and increased the penetrance of high-grade gliomas and malignant peripheral nerve sheath tumours (MPNSTs), indicating the tumour suppressive role of nf1 in zebrafish [atrx in the abovementioned nf1/p53-deficient zebrafish background resulted in the onset of a broad spectrum of sarcomas and other carcinomas [Studies using MOs have been used to unravel the molecular mechanisms of glioblastoma pathogenesis and to identify potential therapeutics. The MO-mediated knockdown of Ephrin-B3 ortholog decreased tumour vascularization, facilitated EphA4-induced cell death and thereby decelerated the GBM growth in zebrafish . Similarebrafish . Furtherrcinomas .rb1 mutant CNS-PNET model to understand the epigenetic regulators driving oncogenesis in this zebrafish brain tumour model. An extended RNA sequencing analysis of rb1 somatic inactivation-induced brain tumours revealed the strong expression of oligo-neural differentiation markers olig2, sox10 and sox8b, consistent with those observed with human CNS-PNETs. The comparative transcriptome analysis of somatic rb1 tumours with germline rb1/rb1 homozygous mutant tissues revealed the overexpression of chromatin remodellers histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4 (rbbp4) in rb1 tumours. These epigenetic regulators are identified to be driving the embryonal brain tumour pathogenesis by facilitating neural stem cell/progenitor proliferation and survival [neurog1, atoh1a, atoh1b and atoh1c and the elevated expression of sox2 in rb1-deficient brain tumours [rb1 or cdkn2a/b tumour suppressor genes in zebrafish. The somatic inactivation of rb1 induced MB, such as PNETs, with tumour incidence accelerated from 23.3% to 57.5% when the rb1 gene was inactivated in the p53 mutant background. The molecular characterization of these tumours showed the upregulation of genes to be important for neuronal development, cell-cycle progression and protein synthesis. However, the somatic inactivation of cdkn2a/b-induced MPNSTs in zebrafish [mlh1, msh2 and msh6, in addition to neurofibromas, some of the animals developed PNETs by 6 months of age [The TALEN-mediated somatic inactivation of zebrafish retinoblastoma 1 (rb1) tumour suppressor predominantly induces CNS-PNET tumours at a high frequency. The histological analysis of these tumours revealed a lack of neuronal differentiation markers (HuC/D and SV2) indicating the highly infiltrating feature of the tumour . The usesurvival . Althoug tumours . Along tebrafish . In anots of age . fli1:GFP) is one of the first stable zebrafish transgenic lines generated, which labels their entire vasculature system with green fluorescence [mpx:GFP), which marks neutrophils, and Tg(mpeg:GFP), which marks macrophages, have been used for innate immunity studies as well as to understand these immune cell interactions with the tumour microenvironment [Gal4/UAS system and site-specific recombinases, such as Cre-loxP, Flp/Frt, Dre/rox and phiC31 systems [The transparency of zebrafish embryos and the optically clear adult casper lines have escalated the diverse potential of this model in generating transgenic zebrafish lines that express fluorescent tissue-specific proteins or express mammalian oncogenes under a zebrafish tissue-specific promoter ,126,127.rescence . These frescence ,137,138.ironment ,139,140.ironment ,143,144. systems ,148,149. systems . An alte systems . Once in systems ,153. Gal systems . Gal4 is systems . This sy systems ,157. Cre systems ,162,163. systems ,157. Alo systems ,164. Dre systems . Whereas systems . Tempora systems ,174,175. systems ,177. How systems . The tet systems . Tet-On  systems . Finally systems . Optogen systems . AlthougGal4VP16-UAS system, which co-expressed an oncogenic zebrafish Smoa1 with a constitutively active human AKT1, which induced serval types of tumours with a higher incidence of glioblastoma and astrocytoma [cytokeratin 5 (krt5) gene promoter using a similar Gal4VP16-UAS approach. These animals developed various retinal tumours and optic pathway gliomas [G12V in putative neural stem and progenitor cells driven by krt5 and gfap promoters by employing a doxycycline-inducible Tet-On/Gal4VP16-UAS system, which resulted in the formation of canonical Ras and mTOR pathway-driven malignant brain tumours in the cranial cavity and parenchyma, respectively [G12V (DARac1) under the ptf1 promoter. The induction of DAAkt1 alone induced glioma in the cerebellum at a higher frequency of about 36.6–49% over the period of 6 to 9 months. Although DARac1 alone did not induce gliomagenesis, co-expression of DARac1 with DAAkt1 induced gliomagenesis at an accelerated rate with higher incidence (62% at 6 months and 73.3% at 9 months), progressed histological grade and highly invasive tumours. Results indicated that DARac1 accelerates gliomagenesis by enhancing epithelial-to-mesenchymal transition (EMT), proliferation and survival [G12V or YAPS5A, KRASG12V, AKT, EGFRvIII and BRAFV600E under the control of zic4 enhancer, which activates MAPK and PI3K signalling pathways. Oncogenic RAS induced aggressive brain tumours and/or heterotopias in zebrafish, with persistence in signal determining the occurrence of benign or aggressive tumours. These aggressive tumours resembled the human mesenchymal GBM with a strong YAP component, validating YAP activation as a critical hallmark for determining malignant brain cancer. This model provides a strong platform for performing pre-clinical studies and drug screening that could prevent malignant transformation of the GBM subtype [Recent years have witnessed the generation of a variety of zebrafish paediatric tumour models that have been exploited for unravelling the molecular mechanisms driving tumour formation. A transgenic model of zebrafish glioblastoma was generated by Ju et al. in 2009, by employing a binary transgenic approach rocytoma . The sam gliomas . Furtherectively . Jung IHsurvival . In anot subtype . This GB subtype . Further subtype . Recentl subtype .SOX10 and OLIG2. Based on this, a zebrafish model of CNS NB-FOXR2 was generated using an I-SceI-mediated transgenesis system by activating NRAS in Olig2 and Sox10 expressing OPCs in homozygous p53M214K; mitfaw2 embryos (lacking p53 activity and deficient in pigment (melanophore)), which displayed brain tumour onset by 6 weeks post fertilisation (wpf). Genomic analysis and histological studies on the tumour sections showed similar histopathology and cell morphology to human CNS NB-FOXR2. The drug response of zebrafish CNS NB-FOXR2 tumours to MEK inhibitor (AZD6244) showed promising effects on tumour burden decline and increase in overall survival [Based on comparative genomic analysis, CNS-PNETs are classified into three distinct sub-groups, namely primitive-neuronal, oligoneural and mesenchymal. Oligoneural subtype, CNS NB forkhead box R2 (NB-FOXR2) is characterised by the elevated expression of oligodendrocyte precursor cell (OPC) genes survival . Furthersurvival . G12V in cerebellar GABAergic neurons. The expression of oncogenic KRASG12V in exocrine pancreas induced pancreatic adenocarcinoma in zebrafish. Zebrafish MB model displayed the dysregulation of Smad3/TGFβ, Shh and Notch pathways during MB pathogenesis, with the inhibition of Notch signalling during the early stages of MB development and the upregulation of TGFβ and Shh pathways during MB development and carcinogenesis. This study elevated the potential of coupling zebrafish cancer models with fluorescent reporter lines in the in vivo tracking of hallmark signalling pathway components contributing to tumorigenesis [The Mebulloblastoma (MB) model of zebrafish was developed based on a transgenic Gal4-UAS system using the promoter and enhancer elements of ptf1a to drive the expression of KRASigenesis . The transplantation of tumour cells into zebrafish has emerged as a robust platform for unravelling the mechanisms driving tumour cell initiation, progression, invasion, angiogenesis, metastasis, interaction with tumour microenvironment and therapy response. Zebrafish xenografts have been proven to engraft and develop tumours that share similar histopathology and molecular features to human cancers ,183. The−/− prkdc−/− zebrafish line [fli1:GFP) zebrafish line with GFP-labelled vasculature, in particular, helps in understanding the mechanisms of neovascularisation, a crucial step in the tumour progression process that supports the growing tumour, as well as allowing therapeutic testing of antiangiogenic agents to inhibit tumour neovascularisation and thereby prevent tumour progression [mpx:GFP) and Tg(mpeg:GFP), have been used to determine the influence of neutrophils and macrophages in driving tumour metastasis [Zebrafish embryos are extensively used for xenotransplantation studies as they develop adaptive immunity, including the generation of T, B and NK cell repertoires after 7 dpf, which are not fully functional by 3 weeks, providing a short window of up to 7–10 days for studying the tumour invasion and metastasis of human cancer cells, including those that are derived from patients ,197,198.ish line . These aish line . Transgegression ,138,211.gression ,219,220.tastasis ,57,221. tastasis . CollectSOX10 and OLIG2 [fli1:GFP) casper embryos and validated the pharmacological disruption of glioma cell-vascular interactions by inhibiting the Wnt signalling pathways [SMARCB1 deletion for the therapeutic evaluation of epigenetically targeted drugs as an efficient treatment option for ceasing tumour growth and progression [Investigations in paediatric brain cancer biology and associated novel therapeutics have marked a significant milestone with the help of zebrafish xenograft models. Several studies have modelled human brain tumours, such as glioblastoma and medulloblastoma using zebrafish embryonic xenografts to study tumour growth, angiogenesis, cellular interactions and response to anticancer therapies . Althougnd OLIG2 . Casey and OLIG2 . Furthernd OLIG2 . On the nd OLIG2 . A recenpathways . Other sgression . To examgression . An orthgression .Overall, zebrafish xenograft models of various paediatric tumours, which address diverse questions in brain cancer biology, have been reported here. These studies validate zebrafish xenografts as a unique and robust model in understanding key mechanisms driving paediatric brain tumour pathogenesis as well as serve as a high-potent drug screening platform for evaluating novel therapeutics.The earlier examples of zebrafish paediatric cancer models demonstrate their suitability to recapitulate patient tumour histopathology and reveal their direct clinical implications in developing diagnostic markers and potential therapeutics. Cancer modelling in zebrafish is based on employing multiple genetic tools to replicate patient tumour pathology allowing to understand the molecular drivers and signalling pathways involved in tumour pathogenesis. This is mainly achieved through the cell-type specific expression of human oncogenes using transgenesis techniques, creating the loss-of-function mutations of tumour suppressors facilitated by genome editing or by performing molecular or functional analysis on xenografted human cancer cells. Although a myriad of genetic tools or approaches are available for these techniques, each has its own advantages and limitations. Although ZFNs and TALENs were used initially for paediatric cancer modelling in zebrafish, both these techniques are inefficient, costly, labour-intensive and impose off-targeting effects. Efforts have been made to improve the efficiency and off-targeting effects by generating FokI variants with improved dimerization and cleavage. However, recent advancement in CRISPR/Cas9 system has opened its unprecedented opportunities for precise genome editing in zebrafish, including in the generation of paediatric cancer models. Custom-made CRISPR gRNAs have facilitated the rapid screening of genetic modifiers and tumour suppressors in cancer. Further, the development of CRISPR/Cas9 system for efficient biallelic gene targeting has facilitated the rapid screening of potential identifiers in F0 generation. Furthermore, the CRISPR/Cas9 system has been used for generating tissue-specific knockouts in zebrafish, facilitating the spatial control of gene disruption in somatic cells . HoweverDespite the significant advances and clear benefits that zebrafish tumour models have brought to further our understanding of cancer and its molecular heterogeneity, there still remains a large number of pre-clinical in vivo studies relying on other animal models, such as mouse and rats, as the primary species to study paediatric brain tumours pathophysiology. Similar to zebrafish, the major techniques used in murine models include PDX and transgenic and, in some rare cases, carcinogen-induced models and although these models are used frequently, each of them has its own advantages and limitations ,233.PDX is the most widely used method in murine models, which involves transplanting established cancer cells, patient-derived cancer cells or brain tissues extracted directly from the patient into the animals. With all three methods, host animals are primarily immunodeficient , thus ensuring the success of the transplantation and development of the tumour. Using either orthotopic or heterotopic xenograft administration techniques, the growth, progression, invasive, metastatic and response to therapies can be easily monitored in this model by examining changes in tumour volume and spread. Although PDX murine models are popular in paediatric brain cancer studies, it is accompanied by significant limitations, such as the reduced rates of tumour engraftment, which depends greatly on the integrity of the tissue and its handling. Another major limitation includes the time the tissue is obtained , as it has been shown that molecular features and response to therapies differ depending on the time of tissue collection , and a fPaediatric brain cancers are rare and differ from adult brain tumours in many aspects, including their tumour type, incidence and treatment . Pre-cli−/−; prkdc−/−il2rgc.a strain takes about 7–10 days for tumour engraftment. Zebrafish metastatic heterotopic models and intracranial orthotopic models have added great value in understanding the key processes in the tumour progression as well as in defining the therapeutic inclination of these tumours. The suitability of zebrafish embryos for high throughput drug screening and their unique pharmacokinetic profile further adds to this in vivo model’s arsenal of skills [Owing to the high physiological and genetic homology to humans, the zebrafish serves as a powerful alternate in vivo model to decipher the molecular mechanisms driving paediatric brain cancers. Unlike mouse models, zebrafish are well-suited for performing the functional characterization of multiple genetic mutations, tumour heterogeneity and for determining the synergy of mutations observed in human tumours. The optical clarity of zebrafish embryos and the transparent adult casper line facilitates in vivo real-time visualisation and monitoring of tumour initiation, tumour cell morphology, motility, metastasis, tumour cell interactions and response to drugs at a single cell resolution. Transgenic zebrafish lines expressing vascular-specific fluorescence facilitate investigations on neo-vascularisation, a pivotal step underpinning the process of tumour growth and proliferation. Progress in the zebrafish cancer modelling approaches from mutagenesis or transgenesis to transplantation techniques has expanded the potential of this in vivo model in paediatric brain tumour studies. Unlike rodent models, paediatric xenograft brain tumour modelling in zebrafish is rapid, with engraftment in embryos observed at 3 dpf following transplantation on 2 dpf embryos, whereas adult Casper f skills . Studiesf skills ,239. Del−/− il2rga−/−prkdc zebrafish line that lacks T, B and natural killer cells and which has been shown to tolerate temperatures as high as 37 °C [Despite the plethora of studies on paediatric brain tumour models, thus far there is no single in vivo model that meets all the essential criteria. Zebrafish, similar to other animal models, present some limitations. Genetic differences between humans and zebrafish add a level of complexity to efficient cancer modelling, as teleost-specific genome duplication in zebrafish results in the presence of more than one ortholog for human genes . Gene duas 37 °C . Tumour as 37 °C . Furtheras 37 °C . In conc


The intramolecular or intermolecular associations between the macromolecular chains of the graft copolymers form different forms of supramolecular network structures, and they interact with nanoparticles to form stable structures to enhance their related properties. The structure of the obtained graft copolymer was characterized by Fourier transform infrared spectroscopy (FT-IR) and laser particle size analysis. Thermogravimetric analysis (TGA) showed the thermal stability of the graft copolymer, and the results showed that the graft copolymer obtained thermally decomposed after 370.86 °C, indicating that it has good thermal stability. Scanning electron microscopy (SEM) revealed the mechanism of the graft copolymers in drilling fluids. The fluid loss control performance and rheology of the filtration reducer were evaluated before and after hot rolling at 180 °C for 16 hours. The results showed that the graft copolymer has excellent fluid loss reduction performance, and it has good fluid loss reduction performance in fresh water, brine and saturated brine. The API fluid loss was only 6.4 mL after hot rolling at 180 °C for 16 h in the brine base slurry. Moreover, the obtained graft copolymer is easily biodegradable, has EC In this research study, we modified hydroxyethyl cellulose to obtain hydrophobically associating hydroxyethyl cellulose, and grafted it onto the surface of nano-calcium carbonate to obtain a graft copolymer. Under such drilling conditions, the coordination of the filterability and environmental protection of the drilling fluid has become more important. Thus, the research and application of high-performance environmentally friendly filter-reducing agents have received extensive attention. At present, many countries have made some progress in drilling fluid technology research, but they often fail to take into account environmental protection and drilling fluid performance. In addition, there are problems such as high cost and difficulty in field promotion.2–4 The high-performance environmentally friendly filtration reducer can take into account both environmental protection and drilling fluid performance.With the continuous development of oil and gas field exploration in deep reservoirs, there are more deep and ultra-deep wells, and the demand for high-performance environmentally friendly drilling fluid treatment agents is more urgent. In recent years, people have paid more attention to the environmental protection performance of drilling fluids.5 In the past few decades, natural high molecular weight polymer filtration reducers have developed rapidly. According to previous studies, some modified starches have API fluid loss between 10 and 20 mL after aging at 150 °C.6 Saudi Arabia uses a filtrate reducer produced by the local plant date palm, which is suitable for freshwater and saltwater drilling fluids. It also has good environmental performance, and its HTHP filter loss is 30 mL after the filtrate reducer is added to the clay-free freshwater drilling fluid. Furthermore, its HTHP filter loss is 20 mL after the filtrate reducer is added to the clay-free seawater drilling fluid.7 A filtrate reducer synthesized from a low-viscosity polyanionic cellulose polymer is suitable for freshwater, saltwater and seawater conditions, can effectively control fluid loss, and has little effect on rheology.8Natural plants such as starch, xanthan gum, tannin, lignin, vegetable gum, and cellulose have the advantages of non-polluting and biodegradable properties. In addition, high molecular weight polymers based on natural plant synthesis have been widely used in petroleum exploration.9 Cellulose has the advantages of wide distribution, high yield and easy biodegradation.10,11 Cellulose is alkali-treated from refined cotton and reacted with ethylene oxide as an etherifying agent in the presence of acetone to produce hydroxyethyl cellulose (HEC), which has strong water absorption and is a kind of nonionic cellulose.6,12,13 HEC can be modified by attaching a hydrophobic group to a hydroxyethyl group to obtain a hydrophobically modified HEC (HMHEC).14,15 The hydrophobic associating hydroxyethyl cellulose has a high aspect ratio, high specific surface area, good mechanical properties and biocompatibility,16–20 and is composed of a long-chain hydrophilic main chain, but its molecular structure contains a small amount of hydrophobic side chains or end groups.21,22 In addition, their skeleton structure will make the polymer dissolve in the aqueous medium. The intermolecular or intramolecular association of the hydrophobic groups also forms a reversible three-dimensional network structure. Furthermore, the intermolecular hydrophobic association region has high affinity, and has a significant impact on its performance.15,23–25 With the emergence of new nanomaterials, the application of polymer nanocomposites is rapidly developing. Due to the size effect, specific surface effect, selective adsorption and other properties of the nanoparticles,26 the introduction of nanoparticles into a single polymer matrix can enhance the relevant properties of the polymer and give it other excellent properties. This can reduce the application of other materials and save costs, and the biopolymer nanocomposites have better performance and environmental protection characteristics.27 Nanomaterials are used in water-based drilling fluids to improve the lubricity of drilling fluids, and to improve the rheology and fluid loss of drilling fluids.11 In this study, we proposed the branching of the short link with hydrophobic groups to the main chain of hydroxyethyl cellulose to obtain hydrophobic polymerized hydroxyethyl cellulose by carrying out a chain transfer reaction to the polymer. The hydrophobically associating hydroxyethyl cellulose is adsorbed on the nano calcium carbonate to form a micro–nano environmentally friendly filtration reducer.28,29Although various cellulose-based graft copolymers have been synthesized and applied to water-based drilling fluids, the application of nanocomposite polymers synthesized by the introduction of nano-materials into cellulose-based polymers in water-based drilling fluids has been reported less.2.2.1When preparing a high-performance environmentally friendly filtration reducer, the reagents and materials used are: hydroxyethyl cellulose, which can be obtained from Feicheng Yutian Chemical Co., Ltd.; and 1-bromododecane, which was purchased from Macklin. The remaining chemical reagents were purchased from the Guo Yao Group Chemical Reagent Co., Ltd. All reagents and materials were stored at room temperature and under anhydrous conditions. The calcium-based bentonite used for the preparation of the drilling fluid base slurry was purchased from Weifang Huawei Bentonite Group Co., Ltd. China.Preparation of base slurry: 0.8 g sodium carbonate was added to 400 mL water. Then, 16 g of bentonite was added on the high-speed mixer while stirring. The mixture was stirred continuously for 20 minutes, and attention was paid to scrape off the bentonite adhering to the wall of the cup during the stirring. The mixture then underwent static hydration for 24 h at room temperature.2.2Solid sodium hydroxide (2.5 g) and absolute ethanol were put into a three-necked flask and magnetically stirred for 20 minutes to dissolve the mixture evenly. Nano-calcium carbonate (10 g) and 1-bromododecane were dissolved in a trace amount of absolute ethanol and added to the solution, magnetically stirred for 20 minutes, and then ultrasonically shaken for 30 minutes. The above suspension was heated to 65 °C, while magnetically stirring. After the temperature became constant, hydroxyethyl cellulose (10 g) was added and the mixture was stirred for 5 h. The liquid obtained above was then centrifuged at a high speed of 3000 rpm for 10 min to get the lower solid. The obtained solid was put into a thermostat at 80 °C for drying and grinding to obtain a hydroxyethyl cellulose graft copolymer .2.3−1 to 4000 cm−1 to obtain its infrared spectrum  Co., Ltd.) was used to measure the fluid loss volume of the base pulp before and after aging at 160 °C/16 h. The obtained filter cake was dried naturally, and then gold was sprayed on the filter cake. Then, an electron microscope, JSM 7900F,  was used to scan the filter cake.2.5API). The GGS42 type high temperature and high-pressure filter device  was used to measure the filtration loss (HTHPAPI) of HTHP at a pressure of 4.2 ± 0.03 MPa and a temperature of 180.0 °C. The measurement was performed within 30.0 minutes, according to API standards. The rheological parameters were calculated as follows:30,31The rheology of the drilling fluid added with the filtration reducer was tested using a MOD ZNN-D6 six-speed rotary viscometer . Its apparent viscosity (AV), plastic viscosity (PV) and yield point (YP) were measured according to API standards. Under the conditions of temperature of 25.0 °C and pressure of 0.69 ± 0.03 MPa, a SD3B triple medium pressure filter loss instrument (Kense Instrument (Shanghai) Co., Ltd.) was used to measure the static filter loss  of filtrate reducers were added to the 4% fresh water base slurry and stirred at high speed for 20 minutes. The samples were then heated at 180 °C for 16 hours. Then, its medium pressure filter loss, high temperature and high-pressure filter loss and various rheological parameters, and the results are shown in 2.62.6.1.5/CODcr ratio evaluation method. The biodegradability of organic matter can be evaluated by the BOD5/CODcr ratio. The higher the ratio, the easier it is to be biodegraded.Biodegradability refers to the possibility of environmental pollutants being degraded by microorganisms. It is an important indicator for evaluating the acceptance of organic matter by the environment. At present, the commonly used evaluation method at home and abroad is the BOD5 and chemical oxygen demand CODcr of the decomposed fluid loss control agent, and the BOD5/CODcr ratio was then calculated. The chemical oxygen demand CODcr is the amount of oxidant consumed to oxidize the reducing substances in 1 L of water sample. 20 mL of 1% filtrate reducer aqueous solution was oxidized by adding 10 mL of potassium dichromate with a concentration of 0.25 mol L−1, and then titrated with (NH4)Fe(SO4)2 to calculate the chemical oxygen demand of the fluid loss agent.The biodegradability of the fluid loss control agent was analyzed by measuring the biochemical oxygen demand, the BOD5 was collected and domesticated by surrounding bacteria after adding a certain amount of filtrate reducer solution was added, incubated for 5 days and measured by BOD5 tester.The biochemical oxygen demand BOD2.6.2.50 value of the biological toxicity of the drilling fluid with the fluid loss control agent, this experiment used the luminescent bacteria method to determine the biological toxicity of the drilling fluid, and used EC50 (when the relative luminescence rate is 50%) to characterize the biological toxicity of the test object. The larger the EC50 value, the lower the biological toxicity of the test substance; the smaller the EC50 value, the greater the biological toxicity of the test substance.Testing the EC3.3.13.1.1.−1. The peak at 1599 cm−1 represents the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C tensile vibration in the cellulose molecule. There is no peak position between the peaks of 1449 cm−1 and 1058 cm−1 in the FTIR spectrum of hydroxyethyl cellulose. Compared with the FTIR spectrum of the fluid loss agent, the peak position has decreased to 1327.45 cm−1 for the reactive –OH bending vibration. The analysis shows that the more reactive hydroxyl group on the hydroxyethyl cellulose molecular chain undergoes a Williamson etherification reaction with halogenated alkanes,32 and the grafting reaction introduces long-chain alkanes into the hydrophilic molecular chain of hydroxyethyl cellulose. In addition, the infrared spectrum of the filtrate reducer increased the peak at 878.68 cm−1. The bending vibration at 878.68 cm−1 is formed by the long chain of the introduced alkane. It can be seen from the infrared spectrum of the copolymer that the copolymer contains all of the molecular groups originally designed, which indicates that the hydroxyethyl fiber bundle and 1-bromododecane has formed a graft copolymer.As shown in 3.1.2.33,343.1.3.2. The nano-calcium carbonate begins to decompose rapidly at about 800 °C. Most of the mass residues at 949 °C are CaO solids and a very small amount of nano-calcium carbonate that has not been completely decomposed. When the temperature reaches 949.3 °C, the mass retention rate is 26.29%. In summary, the thermogravimetric curve shows that the filtrate reducer has good thermal stability at high temperatures.As shown in 3.2via intramolecular association, in which the macromolecular chain curls, the hydrodynamic volume decreases, and the intrinsic viscosity decreases. When the graft copolymer concentration exceeds a certain value, the macromolecular chain will form a dynamic physical cross-linked network structure dominated by intermolecular association, and the solution viscosity will be greatly increased. The second aspect is different from the irreversible shear degradation of general high molecular weight polymers. Under the action of a higher shear force, the cross-linked structure formed by the hydrophobic association of the graft copolymer is destroyed and the solution viscosity decreases. When the shearing effect is eliminated, the cross-linked network structure formed by the association between the macromolecules is reformed and the viscosity is restored. On the other hand, in addition to the van der Waals force, hydrogen bonding force and electrostatic force between HMHEC and nano calcium carbonate particles, hydrophobic association also plays a major role. HMHEC is adsorbed on the surface of nano-calcium carbonate and forms a larger spatial network structure with it using the bridging mechanism, which improves the degree of cross-linking of the entire inorganic/organic system. Moreover, the hydrophobic association is an endothermic effect.35 In summary, the filtrate reducer has a certain temperature resistance and viscosity increase.36,37 In addition, the electron pair on the carbonyl group on the graft copolymer long chain easily adsorbs calcium ions, reducing the influence of the calcium ions on the clay particles, thereby enhancing the stability of the drilling fluid system. However, as the amount of salt increased, the fluid loss of the drilling fluid did not increase significantly after aging at 180 °C. This is because the copolymer macromolecular chain can effectively shield the influence of salt on the clay particles, protect the clay particles from aggregation, help improve the stability of the drilling fluid system, and reduce the fluid loss under high salt conditions. In summary, the filtrate reducer has obvious salt and calcium resistance.It can be seen from 3.338–40 On the other hand, the fluid loss additive is a macromolecular copolymer, which has a certain viscosity increase and dynamic shearing effect in the drilling fluid base slurry. It can reduce the fluid loss to a certain extent, and the macromolecular polymer filtration reducer can also block the smaller pores in the drilling fluid, which can effectively reduce the fluid loss.41–43As shown in 3.45/CODcr can indicate the biodegradability of the fluid loss agent. If BOD5 represents the non-biodegradable part of CODcr, the proportion of the non-biodegradable organic matter in the base pulp can be represented by BOD5/CODcr. When the ratio exceeds 0.45, it means that the non-biodegradable organic matter accounts for less than 20% of all organic matter. When the ratio is lower than 0.2, the non-biodegradable organic matter accounts for more than 60% of all organic matter. Using the dichromate method to test the value of CODcr. The determination of BOD5 adopts the GB7488-87 water quality five-day biochemical oxygen demand determination method.44Testing the biodegradability of the drilling fluid base slurry added with a fluid loss agent, BOD50 value of the biological toxicity of the drilling fluid base slurry with added fluid loss agent, this experiment uses the luminescent bacteria method to test the biological toxicity of the fluid loss agent. Using EC50 (when the relative luminescence rate is 50%) to characterize the biological toxicity of the test substance, we found that the biological toxicity of the test substance decreased with larger EC50 value. In addition, a greater biological toxicity of the test substance resulted in a smaller EC50 value.45To test the ECY = BOD5/CODcr = 17.42%, which is a type that is easily biodegradable. A Microtox-type biological toxicity tester was used to test the biological toxicity of the filtrate reducer. The EC50 value of the filtrate reducer was 3.41 × 104 mg L−1, indicating that the filtrate reducer was non-toxic ,47 which are commonly used in oil fields. It can be seen that the fluid loss of the micro–nano environment-friendly fluid loss control agent synthesized in this paper before aging at 180 °C is 5.6 mL, and the fluid loss after aging at 180 °C is 6.4 mL. The micro–nano environment-friendly fluid loss reducer synthesized in this paper is better than other similar products.We compared the synthetic fluid loss agent with sulfonated lignite (SMC)4.In this paper, the hydrophobically associating hydroxyethyl cellulose was obtained by modifying the hydroxyethyl cellulose, and the graft copolymer was obtained by grafting it onto the surface of the nanometer calcium carbonate. Finally, the micro–nano environment-friendly filtration reducer was successfully synthesized. The structure of the filtration reducer was characterized by particle size analysis and infrared spectroscopy, and the mud cake was characterized by scanning electron microscope SEM. Its thermal stability was characterized by TGA, and the results showed that the filtration reducer has strong thermal stability. This is because the synthetic filtration reducer belongs to the micro–nano level, has a high specific surface area, and its contribution to contrast heat is higher. Moreover, the filtration reducer is a macromolecular copolymer, which has the effect of increasing the viscosity and dynamic shear force in the drilling fluid base slurry, and can reduce the fluid loss to a certain extent. In addition, the macromolecular polymer filtration reducer can block the micro–nano pore size in the drilling fluid by itself, stabilize the well wall, and can effectively reduce the fluid loss. The results showed that when the added amount of filtration reducer was 2.0 wt%, the fluid loss could be reduced to 6.4 mL after aging at 180 °C for 16 h. The results show that the filtration reducer has good thermal stability, salt and calcium resistance. In addition, from the scanning electron micrograph analysis of the mud cake, it can be seen that the surface of the mud cake formed by adding the filtration reducer to the base slurry is smooth and compact, which indicates that the filtration reducer inhibits the coalescence of the clay particles and maintains the dispersion of the clay particles. This makes it more difficult for water to enter the formation, and improves the performance of reducing fluid loss. Finally, through its environmental performance test, the results show that the filtration reducer has no biological toxicity and is easily biodegradable.There is no conflict to declare.


Zoster vaccines generate antibody responses against varicella-zoster virus (VZV). We compared antibody-dependent cell cytotoxicity (ADCC) elicited by zoster vaccine live (ZVL) and recombinant zoster vaccine (RZV). ADCC mediated by antibodies against VZV lysate (VZV-ADCC) and recombinant glycoprotein E (gE-ADCC) was measured using plasma from 20 RZV- and 20 ZVL-recipients, including half 50–60-years-old and half ≥70-years-old. Solid phase-bound anti-VZV antibodies stimulated TNFα in NK cells as measured by flow cytometry or ELISA. VZV-ADCC pre- and post-immunization was higher in younger vaccinees. ZVL did not appreciably increase VZV-ADCC, whereas RZV increased VZV-ADCC in older vaccinees. ELISA-measured gE-ADCC was similar across groups pre-immunization; significantly increased after ZVL; and RZV and was higher in younger RZV than ZVL recipients. IgG3 antibodies increased after RZV and ZVL, with greater anti-gE than anti-VZV responses. Moreover, gE-ADCC strongly correlated with anti-gE antibody avidity, but there were no appreciable correlations between VZV-ADCC and avidity. NK cells stimulated by anti-gE antibodies showed increased IFNγ and CD107a expression, which was not observed with anti-VZV antibodies. In conclusion, anti-gE antibodies generated more robust ADCC than anti-VZV antibodies. RZV induced higher ADCC antibodies than ZVL depending on the antigen and age of vaccinees. Older adults had lower ADCC antibodies before and after vaccination than younger adults. Abundant clinical and immunologic data indicate: that VZV-specific cell-mediated immunity (CMI) is necessary and sufficient to prevent HZ4; that the frequency and severity of HZ correlates inversely with these responses4; and that there is no such correlation with VZV-specific antibody6. However, such antibody(ies) could contribute to the prevention or recovery from HZ. For example, antibody responses after live HZ vaccine  correlated with protection2. In another study with ZVL the fold-rise in the antibody response qualified as a surrogate marker for protection7. However, a mechanism involving antibody in protection against HZ remains undefined. For example, it is unlikely that neutralizing antibodies play a role in protection against VZV, which spreads readily from cell-to-cell and which circulates within immune cells9. Two other mechanisms have been proposed by which VZV-specific antibody might limit VZV infection in general, and HZ in particular. The first proposal is that the antibody attaches to and interferes with the function of a virally encoded fusogen within the cell membrane, thus inhibiting virion egress and cell-to-cell spread or infectivity10. This mechanism is based on in vitro model that has not been universally reproduced. A second, more plausible role for VZV-specific antibodies, is antibody-dependent cellular cytotoxicity (ADCC). It is well established that individuals with defects in NK cells, which are central to ADCC, suffer from severe and recurrent, often fatal VZV infections12. ADCC may also contribute to the partial clinical success achieved using high titer VZV hyperimmune globulin for passive immunization of naïve individuals14. Investigations of ADCC mediated by VZV antibodies were undertaken several decades ago with the methods then available18. In this report we apply contemporary methods to determine if ADCC antibodies are stimulated by the two licensed HZ vaccines – the live attenuated vaccine, ZVL and the adjuvanted recombinant glycoprotein E (gE) zoster vaccine (RZV). This is critical in understanding the basis of the striking differences in efficacy and applicability of these vaccines to an elderly population19. Differences in CMI in response to each vaccine have been demonstrated20. The current report investigates whether functional differences in the humoral response to the two vaccines might contribute to differences in their efficacy.Herpes zoster (HZ), which is caused by reactivation of varicella-zoster virus (VZV) latent in cranial nerve and sensory ganglia following varicella, is prevented by the VZV-specific immune responses that develop during childhood varicellap = 0.02, single sample Wilcoxon test; Fig. The assays were developed using a convenience sample of sera from 7 VZV-seropositive and 9 VZV-seronegative individuals characterized at the CDC. The ADCC methods were first validated for VZV and subsequently for gE as described in the Methods. For flow cytometry, NK cells in PBMC exposed to sera from VZV-seropositive donors bound to wells coated with VZV showed significantly higher expression of TNFα compared to PBMC exposed to VZV-seropositive sera in wells coated with mock-infected control antigen , we determined the amount of TNFα produced by PBMC in the conditions described above. PBMC exposed to VZV-seropositive sera generated significantly higher concentrations of TNFα in wells coated with VZV compared with wells coated with mock-infected antigen . Similar age differences were observed for peak responses after vaccination . Neither ZVL nor RZV generated significant increases in VZV-ADCC in younger adults from pre- to post-vaccination. However, older ZVL recipients showed a trend toward an increase from pre-vaccination to peak response , while older RZV recipients showed significant increases . The comparison of post-immunization VZV-ADCC between the two vaccines did not reveal significant differences.nts Fig. . VZV-ADCp = 0.07, Mann-Whitney test). The comparison of responses between vaccines showed significantly higher peak responses in younger RZV recipients compared with younger ZVL recipients (p = 0.002).gE-ADCC prevaccination was similar across age and vaccine groups , to HZ (reactivation infection), and to varicella vaccination23. Before and after HZ vaccination, anti-VZV and anti-gE IgG1, IgG3 and IgG4 were similar in recipients of each vaccine and both age groups (not depicted). Figure . There were no significant differences in IgG3 anti-gE responses by age or vaccine group.To define the differences between anti-gE and anti-VZV ADCC responses to vaccination, we measured the IgG subtypes composing the antibody response. IgG3 antibodies are deemed to be most effective in mediating ADCC due to their long hinge that allows optimal flexibility to accommodate both target and effector cellsure Fig. . There w24. gE-ADCC strongly correlated with anti-gE antibody avidity at peak response after administration of either HZ vaccine . gE-ADCC weakly correlated with neutralizing antibody titers  before vaccination, VZV-ADCC, similar to avidity were already close to peak, whereas gE-ADCC and avidity were below their peak capacity to expand in response to vaccination; (2) gE contains more ADCC antibody binding sites per molecule than the average contained in VZV glycoproteins; and (3) the purified recombinant gE preparation used in our assays exposed antibody binding sites better than the inactivated whole virus VZV preparation.19. Although previous studies showed similar total IgG responses to HZ vaccines in younger and older adults, we observed a significant effect of age on ADCC, consisting of lower levels of VZV-ADCC antibodies in older compared with younger adults pre- and post-administration of either HZ vaccine. Thus, older age had similar effects on ADCC and vaccine efficacy. The investigation of IgG3 anti-VZV or anti-gE responses between younger and older adults pre-vaccination did not reveal any differences, while post-vaccination only RZV recipients showed higher levels of anti-VZV IgG3 in younger than older adults. Thus, the levels of IgG3 did not correlate with the difference in ADCC magnitude between younger and older vaccine recipients. Additional information about the physicochemical properties of the antibodies generated by the vaccines in different age groups may be required to define the age effect on ADCC. For example, the pattern of antibody glycosylation was shown to play a role in ADCC, whereby afucosylated antibodies were most likely to elicit ADCC38. Adjuvants may also affect the pattern of antibody glycosylation generated by vaccines39. In addition, our observations should be expanded with studies of other vaccines to determine if lower ADCC responses to vaccination contribute to the lower efficacy of vaccines in older than younger adults.We analyzed the effect of age on ADCC antibodies because older age is associated with decreased efficacy of many vaccines, including ZVLOur study was limited by the relatively low number of participants for the comparison of responses in younger versus older adults. Because chronological and biological age may differ in the geriatric population, hundreds of participants may be necessary to tease out the effect of age. The strength of our study was that participants were randomized to ZVL and RZV in the parent study. We also developed a new ELISA assay for ADCC based on the correlation with NK activation examined by flow cytometry. This assay is technically easier to perform and is more sensitive than the flow cytometry assay.In conclusion, both zoster vaccines generated robust anti-gE antibodies, including IgG3. The increase in gE-ADCC post-vaccination may contribute to the protection conferred by ZVL and RZV in all age groups.20. The study was approved by the Colorado Multiple Institution Review Board. All participants signed informed consent. All participants had prior varicella or had resided in the US at least 30 years, and none had prior HZ. Twenty participants were 50–59 years old; 10 received ZVL and 10 received RZV. The remaining 20 participants were 70–85 years old and were also equally divided between ZVL or RZV recipients. We used samples collected before vaccination in all participants, 30 days after ZVL administration, and 30 days after the second dose of RZV (day 90).For assay development, we used a convenience sample of sera with known VZV-seropositive or negative status. The effect of vaccination was evaluated using archived samples from 40 participants in a previously published study (NCT02114333)2 atmosphere, Brefeldin A , Monensin , and anti-CD107a  were added for the last 4 hours. At the end of the incubation, PBMCs were removed, washed and stained with Zombie Yellow Viability Stain ; antibodies against CD3 , CD19 , CD14 , granzyme B , CD56 , CD16 ; HLA-DR , CD107a , and perforin . Then cells were fixed with FACS Lysing Solution (BD) and permeabilized with Permeabilizing Solution 2 (BD) and intracellular staining was performed with antibodies against granzyme B , TNFα , perforin  and INFγ . PBMC  were acquired with the Gallios instrument (Beckman Coulter). Analysis used FlowJo (Becton Dickinson) software. Gating strategy to identify activated NK cells by expression of TNFα in the ADCC assay is shown in Fig. Adapted from Gonzalez-Gonzalez et al., Immulon II 96-microwell ELISA plates were coated overnight either with VZV antigen obtained from VZV Oka strain-infected human lung fibroblasts at a multiplicity of infection of 1, harvested and UV-irradiated after 4 days of culture (VZV); similarly treated mock-infected lung fibroblasts (mock) diluted 1:20 in PBS; recombinant gE glycoprotein at 1ug/ml in PBS ; or PBS control. After overnight incubation at 4 °C, plates were washed and blocked with 5% fetal calf serum in PBS for 2 h at room temperature. Plasma or serum samples at the pre-optimized dilution of 1:100 in PBS were added at 100 µl/well to duplicate wells for 2 h at room temperature and plates were washed with PBS containing 0.5% Tween. PBMC from a Leukopack dedicated to this research were added at a preoptimized concentration of 200,000 cells/well in 200 µl of RPMI 1640 (Corning) containing 10% fetal bovine serum , 1% glutamine (Gemini Bio-Products), 1% penicillin-streptomycin (Gemini Bio-Products), and HEPES buffer (Corning). After overnight incubation at 37 °C in a humidified 5% CO41. PBMC stimulation was performed in Immulon II plates coated with relevant antigens as described above, at 50,000 cells/well. After overnight incubation cells were lysed with 1% v/v TritonX-100 solution in 150 mM NaCl and 20 mM Tris 7.5. Supernatants were collected and assayed for the presence of TNFα using a commercial ELISA kit as per manufacturer’s instructions .The assay was performed as previously described with modifications96-well strips  were coated with 100μL gE at 1μg/mL (courtesy of GlaxoSmithKline) or VZV Oka strain lysate at 1:40. Plates were incubated at 4 °C overnight with 500 rpm shaking. Wells were washed 4x with PBS and blocked with 200μL PBS/.05% Tween-20/5% FBS for 2 hours. Wells were washed 4x with PBS/.05% Tween-20. Plasma samples were diluted in PBS/.05% Tween-20/5% FBS at the following preoptimized ratios: IgG1 assay 1:100; IgG3 assay 1:10; IgG4 assay 1:50. 100 μL sample/well was incubated for 2 hours at room temperature on a shaker at 500 rpm. After washing wells 4x, the following detection antibodies diluted in PBS/.05% Tween-20/5% FBS were added: Mouse anti-human IgG1 hinge-AP (Southern Biotech) 1:2000; Mouse anti-human IgG3 hinge-AP (Southern Biotech) 1:500; Mouse anti-human IgG4 Fc-AP (Southern Biotech) 1:1000. Wells were washed 4x and 100 μL pNPP 1-component AP substrate (ImmunoChemistry Technologies) were added. Color development was read at 405 nm on a MultiSkan FC (Thermo) using SkanIt software. Results were interpolated using Prism (Graph Pad).P value of <0.05 defined statistical significance.Statistical analyses and graphs were performed using Graphpad Prism 9 . Analyses were performed using nonparametric tests. A two-tailed Further information on research design is available in the Supplemental materialReporting Summary


The combination of CZA-ATM is frequently used to treat patients with metallo-β-lactamase (MBL)-producing Enterobacterales (EB) infections, but its safety has not been established in controlled trials. This phase 1 study evaluated the safety of the optimal CZA-ATM regimens identified in the hollow fiber infection model of MBL-producing EB (PMID: 32464664).Table 1). Study safety was monitored by assessments of adverse events (AEs), vital signs, and clinical laboratory safety tests.The phase I, open-label, single center study enrolled healthy adults aged 18-45 years (NCT03978091). Subjects were sequentially assigned to 1 of 6 Cohorts and administered investigational product(s) (IP) for 7 days . The most common IP related investigation AEs were ALT/AST elevations (35%) with 94% occurring in subjects who received ATM alone or in combination. The incidence of ALT/AST elevation AEs in the combination Cohorts were comparable to the ATM alone Cohorts. In the ATM single IP Cohorts, 3 subjects experienced severe ALT/AST elevation AEs, which halted the study. All subjects who experienced ALT/AST elevations were asymptomatic, had no other clinical findings suggestive of liver injury, and all resolved without intervention. Most other IP related AEs were of mild severity and similar across Cohorts except prolonged prothrombin time (PT) AEs, which was more frequent in combination Cohorts.Of 48 subjects enrolled, 50% were female and 60% were Black. The mean (SD) age was 33.5 (6.2) years and mean (SD) weight was 75.7 (12.1) kg. The number of subjects who had ≥ 1 AE and experienced ≥ 1 IP related AE was 46 (96%) and 41 (85%), respectively. Frequency of IP related AEs by MedDRA system organ class, severity, and Cohort are shown in Clinicians should only consider using CZA-ATM when the benefits outweigh the risks. If CZA-ATM is prescribed, clinicians are advised to monitor for hepatic injury. Close monitoring of coagulation parameters may also be prudent with CZA-ATM. Future comparator-controlled randomized clinical trials are required to better define the safety and efficacy of the CZA-ATM regimens.Thomas Lodise, Jr., Pharm.D., PhD, BioFire Diagnostics: Grant/Research Support|cidara: Advisor/Consultant|cidara: Honoraria|Entasis: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Paratek: Advisor/Consultant|Shionogi: Advisor/Consultant|Spero: Advisor/Consultant|Venatrox: Advisor/Consultant J Nicholas O'Donnell, Pharm.D., Merck & Co, Inc: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support Stephen Balevic, MD, Purdue Pharma: Grant/Research Support|UCB: Advisor/Consultant Jeffrey Guptill, MD, argenx: Stocks/Bonds Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds henry chambers, MD, Merck: DSMB member|Merck: Stocks/Bonds|Moderna: Stocks/Bonds.


The replacement of dermatan sulfate chains on decorin proteoglycan with chondroitin sulfate chains is proposed to lead to the disorganization of collagen networks in the skin. However, the pathogenic mechanisms of mcEDS-CHST14 are not fully understood, partly due to the lack of in vitro models of this disease. In the present study, we established in vitro models of fibroblast-mediated collagen network formation that recapacitate mcEDS-CHST14 pathology. Electron microscopy analysis of mcEDS-CHST14-mimicking collagen gels revealed an impaired fibrillar organization that resulted in weaker mechanical strength of the gels. The addition of decorin isolated from patients with mcEDS-CHST14 and Chst14−/− mice disturbed the assembly of collagen fibrils in vitro compared to control decorin. Our study may provide useful in vitro models of mcEDS-CHST14 to elucidate the pathomechanism of this disease.Loss-of-function mutations in  Ehlers–Danlos syndrome (EDS) comprises a clinically and genetically heterogeneous group of heritable connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility ,2. CurreMusculocontractural EDS-CHST14 (mcEDS-CHST14) is a recently delineated subtype of EDS caused by biallelic loss-of-function mutations in CHST14 ,7,8,9. CCHST14 and mice model of mcEDS-CHST14 deficient in Chst14 (Chst14−/−) shows misoriented collagen fibrils, disorganized collagen fibers, and decreased skin tensile strength ;c.88A>G,p.[PGAGs in the cultured medium of fibroblasts were analyzed as described previously . Briefly6 dermal fibroblasts were suspended in 4 mL of DMEM containing FBS, sodium bicarbonate, penicillin, and streptomycin. Then, 4 mL of the cell suspension was mixed with 2 mL of bovine type I collagen acidic solution . Then, 5 mL of the mixture was placed in a 6-well plastic dish and incubated, at 37 °C, for allowing gelation of collagen. The resultant collagen gels containing fibroblasts were detached from the surrounding brim of plastic dishes and maintained, at 37 °C, in air supplemented with 5% CO2. The gel contraction was evaluated by measuring the diameter of the gels at the indicated time. We used the 0.1% collagen gels for gel contraction assay and transmission electron microscopy analysis and 0.22% collagen gels for measuring the breaking strength and scanning electron microscopy analysis.The collagen gels were prepared as described previously with minor modifications . BrieflyTransmission electron microscopy was performed as described previously ,17. BrieScanning electron microscopy was performed as described previously . BrieflyMechanical strength of the collagen gel was analyzed using a creep meter  as described previously . The breHuman decorin was isolated from the cultured medium of normal and the patient’s fibroblasts. Serum-free cultured medium (700–900 mL) was collected, freeze-dried, and dissolved in the extraction buffer , 0.1 M NaCl, and protease inhibitors). The extract was applied to a DEAE-Toyopearl 650 M column  equilibrated with the extraction buffer. Decorin was eluted with stepwise increases of 0.3, 0.5, and 1.0 M NaCl. Fractionated samples were precipitated with ethanol and dissolved in 0.4 mL of water. For mouse decorin, skin powder was delipidated by mixing with ethanol and extracted with 4 M guanidine hydrochloride solution containing 50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 5 mM benzamidine hydrochloride, and 10 mM EDTA, at 4 °C, for 72 h with rotating. After centrifugation, the supernatant was dialyzed against the extraction buffer and applied to a DEAE column as described. To detect decorin, we separated samples by acrylamide gel electrophoresis, transferred them onto a polyvinylidene difluoride membrane , and incubated them with the following primary antibodies: anti-human decorin , and anti-mouse decorin . After washing, the membranes were incubated with the appropriate peroxidase-labeled secondary antibodies and developed using a chemiluminescent peroxidase substrate (Millipore). For human decorin, the 0.5 M NaCl fraction was collected, and for mouse decorin, the 0.3 M and 0.5 M NaCl fractions were pooled and used for subsequent experiments.Chst14+/+ and Chst14−/− mice were digested with chondroitinase ABC (Seikagaku Co.) or chondroitinase B . The digested and undigested lysates were subjected to immunoblotting to detect decorin.Skin lysate obtained from w/w)) for 2 h in a neutral solution. The resultant collagen gels were analyzed by scanning electron microscopy as described.Isolated decorin was quantified by a direct enzyme-linked immunosorbent assay using the anti-decorin antibodies. Decorin was incubated with bovine type I collagen acidic solution ) for 2 h in a neutral solution, and the resultant collagen gels were analyzed by scanning electron microscopy (Type I collagen was incubated with or without decorin (collagen:decorin = 500:1 (croscopy . In the croscopy . GAG chacroscopy d. We alscroscopy e. The dicroscopy f. These CHST14 model mouse lacking functional CHST14 protein has been established recently. We examined the effect of fibroblasts from Chst14-deficient mice on the fibrillar organization of type I collagen gels in vitro. Scanning electron microscopy analysis showed the assembly of collagen fibers composed of multiple fibrils (Chst14−/− mice compared to those from Chst14+/+ and Chst14+/− mice (Chst14-deficient condition (A mcEDS- fibrils . The dia fibrils a,b. We f+/− mice c. Higherondition b.Chst14+/+ mice was shifted to lower molecular weight after digestion with chondroitinase ABC and chondroitinase B (Chst14−/− mice was partially resistant to chondroitinase B but was digested by chondroitinase ABC. This result indicated that DS chains on decorin were markedly reduced and replaced by CS chains in Chst14−/− mice, as previously reported in patients with mcEDS-CHST14 [Chst14−/− mice had significantly smaller fibril diameters compared to decorin from Chst14+/+ and Chst14+/− mice (We digested the skin lysate with two chondroitinases to investigate GAG modification on decorin. Chondroitinase ABC degrades both DS and CS chains, whereas chondroitinase B acts only on DS. Decorin from tinase B a. In conS-CHST14 . We isol+/− mice c,d. ThesCHST14 are dispersed in the reticular dermis, in contrast to the regularly and tightly assembled fibrils observed in healthy individuals [CHST14 pathology. Electron microscopic analysis revealed impaired fibrillar organization in mcEDS-CHST14-mimicking gels, similar to the abnormal collagen network observed in patients with mcEDS-CHST14.Previous electron microscopic analyses have shown that collagen fibrils in patients with mcEDS-ividuals ,16. In aividuals . In the CHST14 or DSE [DSE than in patients with mcEDS-CHST14 [Dse-deficient fibrils have a larger diameter than wild-type fibrils, while Chst14-deficient fibrils do not show such an increase [DSE but also other EDS pathologies.There are two forms of mcEDS resulting from loss-of-function mutations in 4 or DSE ,3,4. BotS-CHST14 . In miceincrease . These sChst14−/− mice, we indicated that decorin is responsible for pathological collagen fibrinogenesis. The decorin preparation used in this study may contain proteoglycans other than decorin. However, since decorin is the major carrier protein of DS chains in the dermis, the impaired collagen fibrillar organization in the mcEDS-CHST14-mimicking gels may be due to the abnormal glycosylation of decorin. Further studies using recombinantly expressed decorin may clarify the exact contribution of decorin in mcEDS.Using decorin isolated from cultured skin fibroblasts of patients and CHST14 models. Furthermore, an in vitro study also shows that the inclusion of decorin during collagen fibrillogenesis increases the elasticity and tensile strength of resulting collagen gels [CHST14 models is due to a functional defect of decorin.Decorin, a member of small leucine-rich proteoglycans, localizes to the surfaces of collagen fibrils and plays pivotal functions in fibrillogenesis ,27. A cegen gels . These rThe amino-terminal domain of decorin contains a single DS chain. Since the protein core of decorin mainly mediates the interaction with collagen, it is not fully understood how the loss of DS chain affects collagen fibrinogenesis. However, emerging evidence has indicated a link between DS chains on decorin and the collagen network. In the shape module model, the protein core of decorin is associated with collagen fibrils, while DS chains interact with each other and form antiparallel duplexes that bridge adjacent collagen fibrils ,34,35. OCHST14 patient [Chst14-deficient mice. DS chains show unique conformational flexibility because IdoA residues are in an equilibrium of 1C4, 2S0, and 4C1 conformations. In contrast, the structure of CS chains is rigid due to the fixed 4C1 conformation of GlcA in CS chains [CHST14-deficient humans and mice, CS chains on decorin linearly extend from collagen fibrils resulting in a disrupted ring mesh-like surrounding collagen fibrils and spatial disorganization of collagen networks. The present study also demonstrates that the alternation of GAG chains on decorin from DS to CS chains causes impaired collagen fibrinogenesis using in vitro reconstitution models.The DS chains on decorin are replaced with a CS chain in the mcEDS- patient ,16. We aS chains . TherefoS chains . In CHSTCHST14 models suggesting that DS supports the initial phase of collagen gel contraction. DS impacts cell proliferation, migration, and tissue morphogenesis through interactions with secreted molecules such as fibroblast growth factors and hepatocyte growth factors [CHST14 models may result in delayed gel contraction during the early phase of collagen network remodeling. It has been reported that the initial interaction between fibroblasts and collagen leads to rearrangement and increased density of the collagen fibrils ,40. Coll factors ,42,43,44CHST14 and revealed that the alternation of GAG chains on decorin from DS to CS chains leads to abnormal collagen assembly. This study may provide useful in vitro models of mcEDS-CHST14 to elucidate the pathomechanism of this disease.In conclusion, this study established in vitro collagen network formation models of mcEDS-