Further, we identified two target enzymes as novel biomarker enzymes for discriminating between tumour and non-tumour tissues. This library-based approach is expected to be an efficient tool to develop tumour-imaging probes and to discover new biomarker enzyme activities for various tumours and other diseases.Fluorescent probes that can selectively detect tumour lesions have great potential for fluorescence imaging-guided surgery. Here, we established a library-based approach for efficient screening of probes for tumour-selective imaging based on discovery of biomarker enzymes. We constructed a combinatorial fluorescent probe library for aminopeptidases and proteases, which is composed of 380 probes with various substrate moieties. Using this probe library, we performed lysate-based  Efficient methodology to develop tumor-imaging fluorescent probes based on screening with our newly constructed probe library for aminopeptidase/protease (380 probes) and clinical samples has been established. We and other groups have focused on cancer-associated proteases as imaging targets,9–13 since they exhibit altered expression levels in the pathological context, and some of them are known to be related to malignant tumour phenotypes such as reconstruction of extracellular matrixes,14 generation of peptide messengers,15 and utilization of amino acids from degradation of proteins.16 For example, fluorescent probes have been developed targeting γ-glutamyl transferase (GGT),17,18 which has been shown to visualize breast,19,20 hepatic21 and oral cancers.22 However, available biomarker enzymes are still limited, and finding new biomarker enzymes is important to broaden the applicability of intraoperative tumour imaging.Complete surgical resection of tumour lesions is critical for the patients' prognosis. However, the ability to detect tiny tumour foci and to accurately delineate the border between tumour and non-tumour tissues with the unaided human eye is quite limited, and thus recurrence due to tumour cells left behind after surgery is a serious problem. In order to ensure complete resection of tumour lesions, intraoperative fluorescence imaging has attracted considerable attention, because of its high sensitivity, high spatiotemporal resolution, low cost and real-time capability.23,24 we thought that an efficient strategy would be to prepare a combinatorial library of fluorogenic probes for proteases that would be suitable not only for in vitro screening, but also for ex vivo screening of patients' specimens to directly evaluate altered enzyme activities under pathophysiological conditions. So far, aminocoumarin-based fluorogenic substrate libraries for proteases have been developed for in vitro screening of specific substrates or optimal inhibitors.25–29 However, their use is mainly limited to in vitro biochemical assay due to the relatively short wavelengths of the fluorescence emission, and especially they cannot be applied for imaging-based screening on living samples.To uncover new biomarker enzymes, it is important to evaluate altered enzyme activities between tumour and non-tumour tissues, rather than just altered protein expression levels, since the activities are required for fluorescence activation. Considering that the enzyme activities in living systems are dynamically modified by many factors, including protein–protein interactions and posttranslational modifications,30,31 Thus, it is suitable for both in vitro screening and ex vivo screening using patients' specimens. Further, the selected probes can be directly used to provide intraoperative fluorescence guidance for tumour-selective imaging. Here, we used solid-phase synthesis to construct a fluorescent probe library composed of 380 HMRG-based probes for aminopeptidases and proteases, and we performed in vitro/ex vivo screening with resected specimens from lung or gastric cancer patients. We found promising probes for tumour-selective visualization of lung and gastric cancers, and identified two main target enzymes as novel biomarker enzymes for discriminating between tumour and non-tumour tissues in the local tumour environment.In this study, we aimed at developing new fluorogenic substrate libraries for proteases making use of our previously reported scaffold fluorophore, HMRG (hydroxymethyl rhodamine green) Fig. S1. HMRG is32 and included TBDMS protection on the hydroxyl group to reduce side reactions. This compound was linked to 2-chlorotrityl chloride resin, and coupled with Fmoc-amino acid (Fmoc-AA-OH). However, the condensation reaction between compound 3 on the resin and Fmoc-AA-OH did not proceed efficiently under any of the conditions we tried, probably due to insufficient reactivity of the amino group on the resin (data not shown). Thus, we next tried the approach illustrated in p-chloranil to form a xanthene fluorophore form, second amino acids were incorporated, followed by cleavage from the resin with TFA/TES/H2O and simple purification by ether precipitation . After optimizing each step, we synthesized 380 peptidyl-HMRGs with various substrate moieties as a probe library for aminopeptidases and proteases , N-methylglycine (MeGly) and methionine sulfoxide (MetO)) as P1 amino acids. As P2 amino acids, we selected six representative natural amino acids with different properties (glycine (no side chain), glutamate (acidic), lysine (basic), tyrosine (aromatic), leucine , and proline (cyclic)), together with the above-mentioned unnatural amino acids except methionine sulfoxide. In addition, we synthesized N-terminally acetylated derivatives of a part of the probe library to target endopeptidases  . We focuroteases . The aveptidases . The pre33 Therefore, fluorescent probes targeting other biomarker enzymes are needed.With the constructed libraries in hand, we next performed screening of probes suitable for detecting lung cancer , which hex vivo lung cancer fluorescence imaging study. The Research Review Board at our institution examined and approved the research protocol, which was in accordance with the Declaration of Helsinki. We prepared five pairs of lysates of tumour and adjacent non-tumour tissues from five patients with lung adenocarcinoma (the most common subtype of lung cancer) and examined their reactivities with the probe libraries by measuring the time-dependent fluorescence increase after application to the 384-well plates. In order to perform tumour-selective imaging, we thought it important that the reactivity of probes in tumour lysate should be sufficiently strong and higher than that in non-tumour lysate. Thus, we set two criteria for selecting candidate probes: (1) more than 10% of the probe was converted to the hydrolysis product HMRG after one-hour incubation ; (2) the tumour-to-normal ratio of the conversion rate was more than 1.5. Considering tumour heterogeneity, we picked up 16 probes that met the criteria in at least three pairs of lysates out of the five. Among these 16 candidate probes, we further selected 7 probes for the second screening from the viewpoints of similarity of substrate peptide sequences: KK-, GP-, EP-, PP-, KR-, KA- and KM-HMRG  and non-tumour tissues, and monitored the fluorescence increase ex vivo. After testing at least 7 specimens from different patients, we found 5 probes  having the ability to distinguish tumour tissues with relatively high accuracy . Puromycin strongly inhibited the fluorescence increase of KK-HMRG with tumour lysate in a concentration-dependent manner  assay4 Fig. S7. In the 4 Fig. S7 and for 4 Fig. S7, and the4 Fig. S7. To ident manner , while tt manner , suggestt manner  and S10†a tissue , and thaa tissue . These rex vivo imaging-based screening. To evaluate the reactivities of a few probes simultaneously on one specimen, we used Tetra-PEG gels39 or medical gauzes that were pre-soaked with probe solutions, and measured the fluorescence increase at tumour regions and non-tumour regions , fluorescence activation was observed with both HT1080 and HEK293 cells  specimens from gastric cancer patients. In contrast to the case of lung cancer specimens, it was impossible to prepare tissue lysate of ESD specimens due to their small size. Therefore, we selected several probes as a focused library, and applied them to ESD specimens to perform  regions  and S12† regions  and S15† to HMRG . Further Fig. S22. In addi Fig. S22, which m11,31,43,44 that are altered at tumour lesions, and these could also be determined by the devised library-based screening. In addition, this methodology should be applicable not only for finding imaging probes for other diseases,45 but also finding probes to detect a specific subset of cells.46 Therefore, we believe that our approach will accelerate research into characteristic enzyme activities in specific environments, with many potential medical and biological applications.We have established a library-based approach for efficient screening of fluorescent probes suitable for tumour imaging and novel biomarker enzyme activities. To construct the fluorescent probe library based on the HMRG scaffold, we devised a simple synthetic scheme using SPPS to prepare 380 peptidyl-HMRGs with various substrate moieties. With the library in hand, we performed screening of probes for lung and gastric cancer detection. For lung cancer, we found several probes with good ability to distinguish tumour and non-tumour tissues, and among them, KK-HMRG targeting PSA exhibited particularly high accuracy. For gastric cancer, we identified KH-HMRG targeting APN as a negative-staining probe due to its selective fluorescence increase in non-tumour regions. The reactivities of these probes were similar or superior to those of the aminomethyl coumarin-based substrates (Fig. S24, Tables S4 and S5Y. K., M. K. and Y. U. co-wrote and reviewed the manuscript. Y. K., A. N. and K. T. synthesized and analysed the compounds. Y. K. and T. Y. performed biochemical experiments. T. Y., H. H., J. N., Y. K., H. T., A. S. and Y. S. performed cancer imaging and analysed the data. Y. A. prepared tetra-PEG gels and supervised the related experiments. K. F. and R. H. performed histological analyses. T. K., R. K., T. U., and K. H. discussed the results and reviewed and commented the manuscript. M. K. and Y. U. supervised the entire project.There are no conflicts to declare.SC-013-D1SC06889J-s001


Canis lupus) with a history of severe facial swelling was submitted for necropsy. Clinical and radiological examination demonstrated an expansile neoplastic mass in the nasal and frontal sinuses. On necropsy, an amorphous neoplastic mass and extensive necrosis were observed in the nasal turbinate. Microscopic examination revealed a tumour principally composed of obvious clear tumour cells characterised by small hyperchromatic nuclei and abundant clear cytoplasm. These clear cells were positive for mucin with PAS, PAS‐D reaction, and alcian blue (pH 2.5) staining, but negative for PTAH staining. Immunohistochemically, some of tumour cells were strongly positive for mesenchymal cells (vimentin), whereas they were negative for myoepithelial antigen  and cytokeratin. Based on the histopathological and immunohistochemical features, the present case was diagnosed as high‐grade clear cell variant mucoepidermoid carcinoma (MEC). This is the first description of clear cell variant MEC in a wolf.A 14‐year‐old male grey wolf ( Canis lupus). Extensive osteoblastic and osteolytic changes of the frontal bone and maxillary sinus were observed in radiography. Variously‐sized and ‐shaped clear cells had small hyperchromatic nuclei and abundant clear cytoplasm in histopathological examination.Mucoepidermoid carcinoma in gray wolf ( The glands, as the name implies, produce and secrete saliva containing digesting enzymes as well as antibodies. Salivary glands are divided into major and minor salivary glands and are widely distributed over the face , salivary gland tumours are classified into 10 and 24 specific benign and malignant epithelial tumours in human, respectively .Mucoepidermoid carcinoma (MEC) is the most common type of malignant epithelial salivary gland tumour , periodic acid‐Schiff , PAS with diastase , alcian blue  and phosphotungstic acid‐haematoxylin . In addition, immunohistochemical staining with antibodies against vimentin , cytokeratins , and alpha smooth muscle actin  were performed for differential diagnosis. Immunohistochemistry was performed with standard protocol. Antigens were retrieved and heated in buffers at pH 6.0. HRP‐conjugated secondary antibody was applied , and 3,3′‐diaminobenzidine  was used as a chromogen. All antibodies were diluted in antibody diluent in ratio with manufacturer's instructions .Microscopic examination revealed that the tumour was principally composed of obvious clear tumour cells. Histologically, these variously sized and shaped clear cells had small hyperchromatic nuclei and abundant clear cytoplasm. Some of the tumour cells were round to oval in shape and contained small, dark‐staining nuclei and eosinophilic cytoplasm Figure . They weNormal salivary glands are composed of several types of cells, and tumours can arise from multiple cell types. Moreover, the histological characteristics of the salivary duct epithelium rapidly change in non‐neoplastic conditions such as inflammation as well as in tumours   along with a malignant tumour, but if the veterinarians suspected the tumour at early stage, they would have focused on reducing the pain. Animals kept in zoos have a longer average life expectancy compared to those in wild conditions because they do not struggle for survival and are constantly monitored and treated (Müller et al., Besides, data on tumours in wolf are limited, and until now, a squamous cell carcinoma of the tonsil and pulmonary neuroendocrine tumour were reported (Shiraki et al., In summary, we reported an extremely rare case of high‐grade clear cell variant MEC in a wolf. This is the first report in a wolf in the veterinary literature.Bumseok Kim: Conceptualisation; data curation; formal analysis; funding acquisition; investigation; project administration; supervision; writing – review & editing. Myeon‐Sik Yang and Hyejin Yun: Data curation; formal analysis; investigation; methodology; visualisation; writing – original draft; writing – review & editing. Ki Chang Lee: Data curation; formal analysis. Chae Woong Lim: Supervision; writing – review & editing.The authors declare no conflict of interest with respect to the publication of this manuscript.This case and procedures were reviewed and approved by the Animal Ethics Committee of Jeonbuk National University.https://publons.com/publon/10.1002/vms3.909.The peer review history for this article is available at


To investigate the role of chemokines in Wilms tumours, especially their chemotaxis to immune cells and the role of DNA methylation in regulating the expression level of chemokines.RNAseqV2 gene expression and clinical data were downloaded from the TARGET database. DNA methylation data were downloaded from the GEO and cBioPortal database. The difference analysis and Kaplan-Meier(KM) analysis of chemokines were performed by edgeR package. Then predictive model based on chemokines was constructed by lasso regression and multivariate COX regression. ROC curve, DCA curve, Calibration curve, and Nomogram were used to evaluate the prognostic model. MCPcounter and Cibersort algorithm was used to calculate the infiltration of immune cells in Wilms tumour and para-tumour samples. Then the difference analysis of the immune cells was performed. The relationship between chemokines and immune cells were calculated by Pearson correlation. In addition, DNA methylation differences between Wilms tumour and para-tumour samples was performed. The correlation between DNA methylation and mRNA expression was calculated by Pearson correlation. Western blot(WB)and immunofluorescence were used to confirm the differential expression of CX3CL1 and T cells, and the correlation between them.A total of 16 chemokines were differentially expressed in tumour and para-tumour samples. A total of seven chemokines were associated with survival. CCL2 and CX3CL1 were positively correlated with prognosis, while high expression of CCL3, CCL8, CCL15, CCL18 and CXCL9 predicted poor prognosis. By lasso regression and multivariate COX regression, CCL3, CCL15, CXCL9 and CX3CL1 were finally included to construct a prediction model. The model shows good prediction ability. MCPcounter and Cibersort algorithm both showed that T cells were higher in para-tumour tissues than cancer tissues. Correlation analysis showed that CX3CL1 had a strong correlation with T cells. These were verified by Weston blot and immunofluorescence. DNA methylation analysis showed that various chemokines were different in para-tumours and tumours. CX3CL1 was hypermethylated in tumours, and the degree of methylation was negatively correlated with mRNA expression.1. There is low T cell infiltration in nephroblastoma. 2. Chemokines such as CX3CL1 indicate a favourable prognosis and positively correlate with the number of T cells. 3. chemokines such as CX3CL1 are negatively regulated by DNA hypermethylation. Wilms tumour is a common abdominal malignant tumour in children, accounting for 90% of renal tumours in children aged 0-14 years . HoweverImmune checkpoint inhibitors have been successful in various adult tumours but are rarely reported in children with solid tumours. The causes of poor immunotherapy in children with tumours are related to ‘ cold tumours’, which are known as tumours that lack infiltration of effector CD8+ T cells or include massive accumulation of Tregs that suppress their activities , 6.Despite this immune deficiency, this does not imply that immunotherapy is useless for ‘cold tumours’. There are two complementary solutions in response to this part of the patients with ineffective immunotherapy. One focuses on accurate identification of immune therapy sensitive patients. Another is committed to converting immune-insensitive patients into immune-sensitive patients or combining them with traditional treatment methods . For theChemokines are a class of secretory inflammatory cytokines with the directional movement of chemokine cells. The primary function is to manage the migration of white blood cells to their respective positions during inflammation and homeostasis . AccordiChemokines play an essential role in tumour metastasis, homing and regulating the tumour microenvironment , 12. In The nephroblastoma (TARGET-WT) mRNA expression and clinical data were downloaded by the TCGAbiolink package of R software (R4.0.1) from the TARGET database. Screening criteria are as follows: 1. From primary tumours and para-tumour samples. 2. There are both mRNA expression data and clinical data. A total of 130 samples were obtained, including 124 tumour samples and 6 para-tumour samples. The clinical data collected included gender, age at diagnosis, stage, survival status and survival days. We downloaded the corresponding DNA methylation-related data from the cBioPortal database. DNA methylation data and mRNA expression data came from the same cohort of patients. A total of 118 samples both had DNA methylation data and mRNA expression data. In addition, another DNA methylation data were downloaded from the GEO database (GSE163372), including a total of 7 pairs of primary tumours and para-tumour.We used raw counts for the difference analysis.The genes with an average expression level of less than 3 were removed. And finally, we obtained 32 chemokines that were expressed in Wilms tumour. These chemokines are used for differential analysis. | Log2FC | > 1 and p < 0.05 were considered significant. Peatmap and ggplot2 packages were used to draw a pheatmap.Survival and Survminer R packages were used for KM analysis, and the samples were divided into high and low groups according to the median expression value. p < 0.05 was considered statistically significant.To find the chemokines most related to prognosis and solve the collinearity problem, all chemokines were included in the lasso regression, and the parameters were set as family = ‘cox’, nlambda = 100. The variables screened by lasso were used for multiple stepwise regression. The R packages used here were Glmnet and Survminer. To verify the model’s prediction ability, the ROC curve, Calibration curves, and DCA curve were used. In addition, to verify whether the prediction model is independent of clinical information, we further conducted multiple stepwise regression based on the risk scores, gender, age at diagnosis and stage, then we draw a nomograms through the rms package.There are mainly two algorithms to evaluate immune infiltration based on gene expression data. One is the ssGSEA-like algorithm based on marker genes, represented by MCPcounter . The othThe correlation between immune cells and chemokines were analyzed by Pearson correlation. The Ggcorrplot package was used to calculate correlation, and p < 0.05 was conspicuous.To further understand the reasons for the change of chemokines, the differences analysis of DNA Methylation was performed by limma package between the para-tumour and tumour tissues. In addition, we analyzed the correlation between the degree of methylation and the expression of this mRNA by Pearson. p < 0.05 and |r| >0.3 were considered significant.A total of 24 Wilms tumour tissues and 24 para-tumour tissues from patients undergoing urological surgery in the children’s Hospital of Chongqing Medical University were collected. This study has got the approval of the Ethics Committee of Children’s Hospital of Chongqing Medical University, and all patients and their parents signed informed consent before joining this study. Immediately, specimens were placed in liquid nitrogen and stored at -80°C for further examination.Protein samples were lysed in RIPA buffer (Beyotime) supplemented with 1% proteinase inhibitors (Beyotime). The protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Beyotime). Equal amounts of proteins (10 µg) were separated on an 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel and transferred onto polyvinylidene fluoride  membranes. The membranes were then blocked in rapid blocking solution and incubated with the primary antibodies overnight at 4°C. The following antibodies were used: CD4 , CD8 , CD3, CX3CL1. Finally, the membranes were incubated with HRP−conjugated secondary antibody  for 2 h at room temperature. The immunoblots were visualized using the Immobilon Western Chemiluminescent HRP Substrate, and the bands were quantified, relative to GAPDH, by densitometric analysis .We co-stained CX3CL1 with T cell markers CD3, CD4 and CD8. The details are as follows. tumour and para-tumour samples were fixed with 4% paraformaldehyde (PFA), dehydrated overnight at 4°C. Paraffin embedding, sectioning, routine deparaffinization, 0.5% BSA blocking and incubated with primary antibody overnight. The primary antibodies were rabbit anti-CD4 (1: 200), rabbit anti-CD8 (1: 200), rabbit anti-CD3 (1: 200), and mouse anti-CX3CL1 (1: 200). CY3 – coupled and FITC - coupled fluorescent secondary antibodies (1:200) were incubated at 25°C for 30 min, and the nucleus was stained with DAPI for 15 min. At least three optical fields were selected for each section for morphological evaluation.Difference analysis was undergoing by edgeR package and limma package. The KM method was performed to evaluate the expression of patients’ chemokines and immune cells and OS. A log-rank test was used to compare the survival differences between the groups. Lasso and multi-factor Cox regression were used to constructing the model, and the ROC curve and correction curve was used for evaluation. The difference of immune cells between tumour tissues and para-tumour was compared by Kruskal.test. Pearson correlation analysis was used. The above statistics were performed using R software (version 4.0.1). Bilateral P < 0.05 was considered statistically significant.Of all 32 chemokines, 16 were differentially expressed between Wilms tumour and para-tumour sample  can be found in the article/The studies involving human participants were reviewed and approved by The Institutional Review Board, Children’s Hospital of Chongqing Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.Conceptualization: ; Data curation: ; Formal analysis: ; Funding acquisition: (DH); Methodology: ; Writing—original draft: ; Resources: (DH); Supervision: (DH). All authors contributed to the article and approved the submitted version.The present study was supported by the Special Project of Science and Technology Innovation for Social Undertakings and Livelihood Guarantee of Chongqing (Grant Nos. cstc2019jscx-tjsbX0003 and cstc2017shmsA130103).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Participants were monitored for clinical, laboratory and electrocardiographic side effects and adverse events. Urine and blood radioactivity was measured. Spherical volumes of interest were drawn over tumour, blood pool and organs to determine biodistribution and calculate dosimetry. In one participant, tumour specimens were analysed for cell death using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining.Five participants with at least one extracranial site of solid malignancy > 2 cm and no active cancer treatment in the 8 weeks prior to the study were enrolled. Participants were administered 205 ± 4.1 MBq  of [68Ga]Ga-CDI is safe and well-tolerated with no side effects or adverse events. [68Ga]Ga-CDI is renally excreted, demonstrates low levels of physiologic uptake in the other organs and has excellent imaging characteristics. The mean effective dose was 2.17E − 02 ± 4.61E − 03 mSv/MBq. It images constitutive tumour cell death and correlates with tumour cell death on histology.[68Ga]Ga-CDI is a novel cell death imaging radiopharmaceutical that is safe, has low radiation dosimetry and excellent biodistribution and imaging characteristics. It has potential advantages over previously investigated radiopharmaceuticals for imaging of cell death and has progressed to a proof-of-concept trial.[ACTRN12621000641897 The online version contains supplementary material available at 10.1007/s00259-022-05880-z. Assessment of response in oncology ultimately aims to measure tumour cell death following treatment. Cell death may be directly assessed in vitro and ex vivo by light microscopy, histochemical  and immunohistochemical (e.g. anti-caspase 3 antibodies) techniques. However, these require invasive and potentially morbid sampling procedures which may be difficult to perform repeatedly for serial dynamic assessment and such samples are small and may not be representative. Presently, imaging response assessment in oncology relies on indirect measurements of tumour cell death, most often assessing changes in tumour size using x-ray computed tomography (CT) assessed by Response Evaluation Criteria in Solid Tumours (RECIST)  or chang99mTc was demonstrated to bind to and image apoptotic tumour cells [99mTc-labelled annexin V has been observed in response to chemotherapy [99mTc-labelled annexin V has high levels of physiologic uptake particularly in the kidneys and liver [18F]F-ICMT-11, underwent a first-in-human study which demonstrated high levels of hepatobiliary excretion that makes imaging of the abdomen and pelvis sub-optimal In DTPA GSAO Ga-Cell Death Indicator ([68Ga]Ga-CDI). In vivo studies demonstrated favourable biodistribution and imaging characteristics and predicted dosimetry similar to clinical 68Ga radiopharmaceuticals suitable for human use [68Ga]Ga-CDI.Initial in vivo studies of radionuclide conjugates of GSAO demonstrated that  kidneys . GSAO waTPA GSAO . Subsequ(NODAGA) , hereaftuman use . This stThis open label, single arm interventional study recruited participants with histologically or cytologically confirmed solid malignancy with at least one measurable lesion > 2 cm. The study was approved by the South Eastern Sydney Local Health District Human Research Ethics committee (2019/ETH04821) and all participants signed an informed consent form. The study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12621000641897) and is registered with the Therapeutics Goods Administration of the Department of Health, Australian Government under the Clinical Trials Notification scheme (CT-2018-CTN-00827–1). Within 28 days of undergoing the study, participants were screened to ensure they met inclusion and exclusion criteria and written consent obtained. The full inclusion and exclusion criteria are shown in Table 68Ga]Ga-CDI was prepared as previously described. In summary, CDI was first prepared by dissolving GSAO in 0.1 N sodium bicarbonate. 2,2′-oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid  was dissolved in anhydrous dimethylformamide and added to the reaction mixture dropwise over 1 h. The reaction mixture was stirred for 4 h, acidified by the addition of 1 N hydrochloric acid, shock-frozen in liquid nitrogen and freeze-dried. The residue was redissolved in de-aerated water and purified by high performance liquid chromatography (HPLC). Purity was confirmed by liquid chromatography–mass spectroscopy (LC–MS) and frozen in 100 µL aliquots (54 µg).[68 Ga]Cl3 from the Ge/Ga generator (Eckert and Ziegler-IGG-100) onto a cation exchange cartridge (Agilent Bond Elut SCX). 68 Ga (III) was then eluted from the cation exchange cartridge using a mixture of hydrochloric acid (HCl) and sodium chloride (NaCl) (12.5 µL of 5.5 N HCl and 500 µL of 5 N NaCl) into a reaction vial containing 54 µg of CDI, ascorbic acid (4.4 mg in 100 µl), sodium acetate buffer  and 3.5 mL water (Ultrapur). The reaction vial was incubated at room temperature for 10 min. Phosphate buffer  was added and the mixture filtered through a 0.22-µm filter (Millipore Millex-GV 33 mm). Quality control was performed with HPLC and thin layer chromatography (TLC) [CDI was radiolabelled by eluting [hy (TLC) .68Ga]Ga-CDI administration; otherwise, there was no preparation required. Two intravenous cannulas were placed (one for administration of [68Ga]Ga-CDI and the other for blood sampling during the study). Immediately prior to [68Ga]Ga-CDI administration, clinical assessment of participants was performed, vital signs  measured and a 12-lead electrocardiogram (ECG) obtained and QTc calculated according to Bazett’s formula to confirm eligibility. [68Ga]Ga-CDI was administered as an intravenous bolus. The mean and standard deviation of the administered mass of [68Ga]Ga-CDI was 40.1 ± 5.8 µg . The mean and standard deviation of administered activity was 205 ± 4.1 MBq (range 200–211 MBq) of [68Ga]Ga-CDI.Participants were encouraged to be well orally hydrated prior to [68Ga]Ga-CDI, with subsequent scans commencing at approximately 7, 17, 29, 47, 64, 120 and 180 min after administration (+ / − 5 min). All scans were performed on a Phillips ingenuity TF 128 PET CT scanner  with acquisition times increasing for each subsequent scan ranging from 30 (immediately after administration) to 240 s/bed (at 180 min after administration) to compensate for excretion and decay. All PET scans were reconstructed using CT attenuation correction with a time of flight, list-mode, blob-based, ordered subsets maximum likelihood expectation maximisation algorithm (BLOB-OS-TF) .Eight serial PET scans were then performed from skull vertex to the proximal femora. The first PET scan commenced immediately following administration of Ga-CDI administration. Participants were asked to void between 1.5 to 2 h and 2.75 to 3 h after [68Ga]Ga-CDI administration, urine collected, the volume measured and an aliquot counted in a cross calibrated gamma counter to determine activity concentration. Following completion of the scanning protocol, ECG was recorded, and blood was collected for biochemical and haematological assessment.During and immediately following completion of the scanning protocol, participants were clinically assessed for adverse reactions and vital signs measured. Blood was collected for venous radioactivity measurements at 1, 2 and 3 h following [68Ga]Ga-CDI PET CT, participants were clinically assessed for adverse reactions and vital signs, ECG was obtained, and blood was collected for biochemical and haematological assessment. Clinical follow-up was also performed 7 days later for assessment of adverse reactions.On the day following the [68Ga]Ga-CDI-PET emission data on a workstation  by an experienced nuclear medicine physician over clearly tumour-free regions of organs and the left ventricular and aortic blood pool (the average of which was used for blood pool activity). The imaging-derived blood pool activity was used for biodistribution and dosimetry calculations . VOI diameter varied depending on organ size but where possible was at least 10 mm. Spherical VOIs were also drawn over all tumour deposits greater than 10 mm in diameter. These VOIs were replicated across all time points and the mean, maximum and standard deviation of the activity concentration (Bq/mL) of all VOIs at each time point was recorded. Activity concentration was used to calculate the % injected activity (%IA) for organs and tissues based on the masses of source organs for standard phantom male and female models Ga-CDI PET study, histological correlation of the tumour deposits was undertaken using haematoxylin and eosin staining and TUNEL staining to specifically identify dead and dying cells. Representative samples of the formalin fixed surgical specimens  from the right axillary and right cervical nodal masses were selected and provided by the reporting clinical pathologist. The tissues were fixed, rehydrated with ethanol and xylene and embedded in paraffin. A series of 4 µm thick sections were cut and stained with haematoxylin and eosin. Apoptotic cells were stained using the TUNEL assay kit HRP-DAB  according to manufacturer’s protocol. Briefly, 4-µm sections of paraffin embedded tumour were deparaffinised in xylene and rehydrated in decreasing concentrations of ethanol. Tumour sections were permeabilized with Proteinase K for 20 min at room temperature. The endogenous peroxidase activity was quenched with 3% H2O2 for 5 min. Apoptotic cells were labelled with biotinylated Terminal deoxynucleotidyl Transferase (TdT) at 37 °C in a humidified chamber for 2 h followed by a 30-min incubation with streptavidin-HRP conjugate. HRP-positive cells were developed using diaminobenzidine (DAB) and specimens were counterstained with methyl green . The entire tumour section was imaged using PowerMosaic scanning at 10 × magnification on a Leica DM6000D microscope.In one participant, who proceeded to surgical excision of the tumour deposits 1 day after the Ga-CDI PET study. Representative sections of the right axillary nodal metastases demonstrated many more TUNEL-positive cells than the right cervical nodal metastases. Corresponding haematoxylin and eosin–stained sections demonstrated that the TUNEL-positive cells corresponded to squamous cell carcinoma and adjacent normal lymphoid tissue did not demonstrate TUNEL staining. The number of TUNEL-positive cells correlated with the intensity of [68Ga]Ga-CDI uptake , the treating surgeon considered that this was likely to represent metastatic disease from a different cutaneous primary to that responsible for the right axillary nodal metastatic disease. The patient proceeded to right lateral neck dissection and right axillary dissection 1 day after the [ake Fig. .Fig. 4Me68Ga]Ga-CDI, for imaging of cell death that is safe, well-tolerated, has dosimetry similar to clinical 68 Ga radiopharmaceuticals and demonstrates excellent biodistribution and imaging characteristics. [68 Ga]Ga-CDI overcomes most of the limitations encountered with previous attempts at molecular imaging of cell death and creates opportunities for personalised treatment based on near real-time imaging of treatment-induced cell death in cancer.This first-in-human study reports on a novel radiopharmaceutical, [68Ga]Ga-CDI were an expected observation. Although [68Ga]Ga-CDI contains an arsenic atom, the active phenylarsenous targeting moiety of CDI, GSAO, has been previously assessed in a phase I study in patients with advanced solid tumours as a mitochondrial toxin. GSAO was infused 5 days per week for 2 weeks in every three and found to have a maximum tolerated dose of 22 mg/m2/day, which is almost 1000-fold greater than the administered mass in this study Ga-dotatate in 6 participants and found that the mean effective dose was 2.57E − 02 mSv/MBq and renal dose was 9.21E − 02 mSv/MBq [68Ga]Ga-PSMA had a mean effective dose of 2.3E − 02 mSv/MBq, renal dose of 2.6E − 01 mSv/MBq and a urinary bladder dose of 1.3E − 01 mSv/MBq [68Ga]Ga-PSMA dosimetry which included 5 participants reported an effective dose of 2.37E − 02 mSv/MBq, renal dose of 1.21E − 01 mSv/MBq and urinary bladder dose of 1.64E − 01 mSv/MBq [68Ga]Ga-dotatate and [68Ga]-PSMA, [68Ga]Ga-CDI has a slightly lower effective dose and renal dose but a slightly higher urinary bladder dose and is therefore well-suited for routine and repeated human use.This study utilised dosimetry methodology employed for other  mSv/MBq . A study mSv/MBq . In summ68 Ga]Ga-CDI is low. In comparison, the activated caspase 3/7 agent, [18F]ICMT, has high levels of uptake in the hepatobiliary system and bowel as it undergoes both hepatobiliary and renal excretion [68Ga]Ga-CDI translate to ideal imaging characteristics with minimal or no interference from physiologic uptake, whereas high physiologic uptake observed with other cell death imaging radiopharmaceuticals especially in the hepatobiliary system and bowel makes abdominal and pelvic imaging suboptimal. Furthermore, as CDI is labelled with 68 Ga, it enables short-interval serial PET for near real time, quantitative dynamic assessment of treatment-induced changes in tumour cell death.Aside from the renal tract, which is the route of excretion, physiologic uptake of 6 GaGa-CDI68 Ga]Ga-CDI detected constitutive tumour cell death with levels of [68 Ga]Ga-CDI uptake varying between different tumour types but also within tumours of the same histological subtype. The variations in intensity of uptake in different tumours is likely due to varying rates of constitutive tumour cell death. Tumour constitutive necrosis correlates with tumour aggressiveness [68Ga]Ga-CDI uptake in their tumour deposits. In addition, [68Ga]Ga-CDI tumour uptake correlated with histological tumour cell death assessed by TUNEL on operative specimens. Tumour uptake generally peaks at approximately at 60 min post injection and remains stable thereafter. However, tumour:blood pool ratios continue to rise beyond this due to progressive clearance of activity from the blood pool. Taken together and considering the half-life of 68Ga, it is likely that the optimal imaging time is between 1 and 2 h after injection.Whilst acceptable dosimetry, good biodistribution and imaging characteristics are important, for a cell death imaging agent to be successful, it must have the sensitivity to quantitively detect tumour cell death. In vitro assessments of fluorophore conjugates of GSAO demonstrated rapid and specific uptake in dying/dead tumour cells. Half-maximal uptake in cultured tumour cells occurred in ~ 2 min and the specificity for dying/dead versus viable cells was at least 1000-fold [siveness . Partici68Ga]Ga-CDI for imaging tumour cell death. In contrast, previous radiopharmaceuticals for imaging cell death did not reliably detect tumour cell death even after treatment. For example, the caspase ligand [18F]ICMT did not detect an increase in cell death in a proof-of-concept study. This was attributed to the low abundance and spatial and temporal heterogeneity of the target and that response may occur by mechanisms independent of caspase 3/7 activation [68Ga]Ga-CDI resolves these limitations. Firstly, hsp90 is highly abundant, constituting up to 1–2% of total cellular protein content and is often upregulated in malignancy [68Ga]Ga-CDI to image even small numbers of dead and dying tumour cells such as occurs with constitutive tumour cell death. Furthermore, even in tumours with low rate of constitutive cell death (and hence low [68 Ga]Ga-CDI uptake prior to treatment), [68 Ga]Ga-CDI is still likely to be able to detect treatment response as even a small increase in tumour cell death following treatment should be detectable with [68Ga]Ga-CDI PET. Secondly, it is a spatially and temporally stable target, as once a cell is committed to death, the target is accessible and stable until the cellular debris is cleared by physiologic mechanisms, which may take several weeks. The optimal time for imaging tumour cell death with [68Ga]Ga-CDI after commencing treatment remains to be determined. Until now, effective methods for imaging tumour cell death in vivo have not been available to image the kinetics of tumour cell death following therapy and it is possible that this may vary between different treatment classes and modalities. Additional preclinical and clinical kinetic studies are warranted to further characterise this. However, the combination of preclinical studies with fluorophore-labelled GSAO demonstrating an increase in treatment induced tumour cell death 1 day following a single dose of chemotherapy, and the likely slow clearance of dead and dying cells from the tumour bed suggests that optimal imaging is likely to be within days to a few weeks after commencing treatment. Finally, in contrast to previous cell death imaging radiopharmaceuticals which have largely targeted aspects of apoptosis, [68Ga]Ga-CDI detects different forms of cell death and there is increasing evidence of the importance of non-apoptotic cell death in malignancy [The ability to detect constitutive tumour cell death demonstrates the sensitivity of [tivation . Gammon tivation . [68Ga]Glignancy , providilignancy .The development of optimal and widely accessible molecular imaging of tumour cell death creates many new opportunities to improve outcomes in oncology. Most obviously, cell death imaging has the potential to assess treatment response more accurately and rapidly. Although current imaging modalities such as CT, FDG PET and MRI have proven utility for assessing treatment response, in many circumstances they are suboptimal. For example, following radiotherapy treatment, related effects such as fibrosis and post radiotherapy inflammatory change make anatomic and molecular imaging inaccurate . AccuratImaging-guided response–adapted individualised treatment provides the opportunity to improve outcomes in oncology but is predicated by imaging that provides timely and sufficiently accurate response assessment. With the possible exception of FDG PET in Hodgkin’s and non-Hodgkin’s lymphoma, current imaging modalities are inadequate. For example, in breast carcinoma being treated with neoadjuvant systemic therapy, MRI and FDG PET are moderately accurate for prediction of pathological response; however, neither are sufficiently accurate to avoid unnecessary surgery in patients who have a complete pathological response following neoadjuvant therapy . Similar68Ga]Ga-CDI is a novel radiopharmaceutical for imaging of cell death. It is safe, has radiation dosimetry similar to clinical 68Ga radiopharmaceuticals and demonstrates optimal biodistribution and imaging characteristics. It is able to detect constitutive tumour cell death and uptake correlates with histological cell death. It is now entering a proof-of-concept trial and has the potential to improve treatment response assessment and enable cell death imaging–adapted personalised treatment approaches.[Supplementary file1 (DOCX 8486 KB)Below is the link to the electronic supplementary material.


Details of correction: correct grant no. in acknowledgementExisting text: “The Deanship of Scientific Research (DSR) at King Abdulaziz University (KAU), Jeddah, Saudi Arabia, has funded this Project Under grant no (G: 683-253-3443).”Corrected text should read: “The Deanship of Scientific Research (DSR) at King Abdulaziz University (KAU), Jeddah, Saudi Arabia, has funded this Project Under grant no (G: 683-253-1443).”


Lymphatic vessels serve as a major conduit for the transport of interstitial fluid, immune cells, lipids and drugs. Therefore, increased knowledge about their development and function is relevant to clinical issues ranging from chronic inflammation and edema, to cancer metastasis to targeted drug delivery. Murray's Law is a widely-applied branching rule upheld in diverse circulatory systems including leaf venation, sponge canals, and various human organs for optimal fluid transport. Considering the unique and diverse functions of lymphatic fluid transport, we specifically address the branching of developing lymphatic capillaries, and the flow of lymph through these vessels. Using an empirically-generated dataset from wild type and genetic lymphatic insufficiency mouse models we confirmed that branching blood capillaries consistently follow Murray's Law. However surprisingly, we found that the optimization law for lymphatic vessels follows a different pattern, namely a Murray's Law exponent of ~1.45. In this case, the daughter vessels are smaller relative to the parent than would be predicted by the hypothesized radius-cubed law for impermeable vessels. By implementing a computational fluid dynamics model, we further examined the extent to which the assumptions of Murray's Law were violated. We found that the flow profiles were predominantly parabolic and reasonably followed the assumptions of Murray's Law. These data suggest an alternate hypothesis for optimization of the branching structure of the lymphatic system, which may have bearing on the unique physiological functions of lymphatics compared to the blood vascular system. Thus, it may be the case that the lymphatic branching structure is optimized to enhance lymph mixing, particle exchange, or immune cell transport, which are particularly germane to the use of lymphatics as drug delivery routes. The lymphatic vascular system is a major transport network, responsible for critical tasks including the trafficking and maturation of immune cells as they respond to infection and inflammation and absorbing lipids from food , 2. In aThe lymphatic system is comprised of a network of vessels that develop outward from a central location to the extremities . Both lyMurray's Law has been shown to describe vascular branching structures in many tissue types in many organisms, including human coronary  and retip raised to some power is equal to the sum of the N radii of the daughter vessels raised to that same power (or that the radius of the parent vessel rme power  18). To. Top raime power , 18.−1 m2, the optimal value of D2/D1 decreased to 0.6 such that the daughter vessels are smaller relative to the impermeable case. Solving for the Murray's Law exponent in this case results in values that range from 1.4 to 3, with lower values corresponding to more permeable vessels. On the other hand, a study by Miguel  knockout mice, which exhibit pathological hyper-permeability of lymphatics and systemic lymphatic insufficiency  knockout mice (n = 16) and Calcrl−/− knockout (n = 14) mice were stained for lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), an extracellular link domain, to detect lymphatic endothelial cells and imaged with a fluorescence microscope. Tissue samples were taken from the ear, back, forelimb, hind limb, and chest. We evaluated samples from both E14.5 (embryonic day 14.5) and adult mice (3–6 months old). Wild type mice (n = 8) were also stained for the surface protein platelet endothelial cell adhesion molecule (PECAM) to detect blood vasculature and imaged with a fluorescence microscope.Both wild type and calcitonin receptor-like receptor  (p < 10−4) than the x = 3 observed in larger veins and arteries  (mean ± standard error), with the peak of the right-tailed distribution occurring at x ≈ 1 . This in × 10−4) . These barteries  but stilarteries . Figuresarteries  and dermarteries . Note thCalcrl−/− knockout mice are not significantly different with regard to their mean exponent , which is consistent with previous reports of functional lymphatic insufficiency in this model  is significantly larger than wild type dermal capillary diameter (41.8 ± 1.8 microns) (p < 10−4)  . This is−7 and as high as 10−3m/s (The physical impact of vessel geometry on the flow of lymph through the system can be examined through a computational fluid model using an actual lymphatic junction from our data . Figures 10−3m/s , 32. The 10−3m/s .−2 N/m2, are consistent with the knowledge that dermal lymphatic capillaries operate in a low-pressure environment (As expected, a negative pressure gradient develops as the lymph flows out of the parent vessel, presumably into a larger network that drains into a collecting vessel see . This drironment .We also measured the tortuosity of the lymphatic capillaries as a proxy for uneven shear along the vessel wall. Tortuosity was measured as the actual vessel length between two bifurcation points divided by the shortest distance between the branches. Tortuosity values >1 imply that surface area is increased in this system. We found tortuosity values for dermal lymphatic capillaries to have a mean of 1.07 ± 0.04. Higher values of tortuosity result in increased surface area which could allow for greater fluid and immune cell exchange between the vasculature and the interstitium. This potential increase in exchange is consistent with reported values for enhanced exchange of 5% in blood vessels of comparable size . AdditioUsing an empirical dataset that we generated from wild type and genetic models, we demonstrate that murine dermal lymphatic capillaries do not follow Murray's Law for x = 3. In other words, the lymphatic network does not follow a minimization rule for volumetric cost that assumes impermeable vessels with fully developed, laminar flow. The best fit mean exponent is approximately 1.45. This is within the range of a volumetric cost minimization rule for permeable T-junctions. We note that lymphatic capillaries are permeable, and Murray's Law exponents for optimal transport in permeable vessels will differ from the optimal value of 3 for impermeable vessels. This may be particularly relevant to larger lymphatic vessels, such as pre-collectors ad collectors, which have lymphatic smooth muscle cells and tighter cellular junctions. Indeed, the physiological functions and regulation of these larger, less permeable lymphatics differs significantly from the dermal lymphatics modeled in this study, such that Murray's Law exponents for pre-collectors and collectors might be predicted to differ as well.Unfortunately, the permeability of dermal lymphatic vessels has not been well characterized, and it is not currently possible to determine whether or not the branching structure is optimized accordingly. However, considering the unique fluid absorptive and mixing properties of dermal lymphatics, it is also likely that the branching structure is representative of an advantageous design for fluid uptake, lymph mixing or immune cell trafficking. The later hypotheses would require an optimization rule outside of Murray's Law for ideal fluid transport. The derivation of such a rule would need to account for other features of vascular structure and function, such as the vessels' ability to dilate, exchange chemicals, traffic large immune cells and respond to bother intrinsic and extrinsic pumping forces , 36.Calcrl−/− mice have a similar mean exponent but with greater variation. These mice display hyperpermeable lymphatic vessels which are insufficient for maintaining lymphatic transport in numerous organs (We also found that relative to wild type mice, s organs . TherefoIn addition to efficient lymph transport, it is necessary for the structure of the lymphatic capillaries to ensure well-mixed fluid in the daughter vessels. As the lymph is pulled upstream into the network, mixing is critical for avoiding a buildup of chemokines, cytokines, or other inflammatory agents and chemicals, like drugs and nanoparticles. While mixing ability in the daughters may appear to be an important feature of lymphatic structure, computational fluid simulations using impermeable vessels suggests that this is not necessarily the case. On the other hand, the observed increased surface area of permeable vessels suggests that immune cell transport may play a greater role in the optimized function of dermal lymphatic capillaries. This is supported by the calculated tortuosities which are representative of increased transport in analogous blood capillaries . It is aCalcrl−/− mouse model, or a gradient buildup in the case of targeted drug delivery via lymphatic routes.Future work and extensions of the numerical model include the incorporation of elasticity or porosity to account for vessel permeability, as well as the incorporation of spatiotemporal regulators of extrinsic and intrinsic forces on lymphatic capillaries , 36. TheThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of UNC-CH.AT designed the study, collected data, performed experiments, analyzed data, and wrote the manuscript. RD prepared animal tissues for the study, advised on study design, and supervised data analysis. ND and EG collected and analyzed data. LM and KC conceived the research hypothesis, designed the study, analyzed data, supervised data analysis and interpretation, edited the manuscript, and provided funding for the study. All authors contributed to the article and approved the submitted version.This work was supported in part by grants from the National Institutes of Health, NHLBI R01 HL129086 and NIDDK R01 DK119145 to KC and the National Science Foundation IOS #1558052 and DMS #1151478. This material was based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650116.Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our aim was to quantify the decrease in Hb after rapid fluid administration.Rapid fluid administration may decrease hemoglobin concentration (Hb) by a diluting effect, which could limit the increase in oxygen delivery . We searched PubMed, the Cochrane Database, and Embase from inception until February 15, 2022. We selected studies that reported Hb before and after rapid fluid administration (bolus fluid given over less than 120 min) with crystalloids and/or colloids in adults. Exclusion criteria were studies that included bleeding patients, or used transfusions or extracorporeal circulation procedures. Studies were divided according to whether they involved non-acutely ill or acutely ill  subjects. The mean Hb difference and, where reported, the DOp < 0.001; I2 = 96.88] after fluid administration: in non-acutely ill subjects, the mean decrease was 1.56 g/dL  and in acutely ill patients 0.84 g/dL . The decrease in Hb was less marked in patients with sepsis than in other acutely ill patients. The DO2 decreased significantly in fluid non-responders with a significant decrease in Hb.Sixty-five studies met our inclusion criteria (40 in non-acutely ill and 25 in acutely ill subjects), with a total of 2794 participants. Risk of bias was assessed as “low” for randomized controlled trials (RCTs) and ‘low to moderate’ for non-RCTs. Across 63 studies suitable for meta-analysis, the Hb decreased significantly by a mean of 1.33 g/dL [95% CI − 1.45 to − 1.12; 2 and thus on tissue oxygenation.Hb decreased consistently after rapid fluid administration with moderate certainty of evidence. This effect may limit the positive effects of fluid challenges on DOThe online version contains supplementary material available at 10.1186/s13054-022-04191-x. Additionally, a decrease in Hb after a fluid bolus might be (mis)interpreted as anemia, resulting in an unnecessary blood transfusion ) than in the non-acutely ill (ΔHb − 1.56 g/dL [95% CI − 1.69 to − 1.42])   and in non-acutely ill and acutely ill groups   . When the baseline Hb was < 14 g/dL, the decrease in Hb was less marked in acutely ill than in non-acutely ill patients, but similar in acutely ill patients and in non-acutely ill when the baseline Hb was > 14 g/dL ); 2 studies . There was a significant difference in the change in DO2 between fluid responders (67.76 [95% CI 46.11–89.40]) and non-responders (− 16.30 [95% CI − 31.52 to − 1.09]) (p < 0.001)  after fluid administration in all groups of acutely and non-acutely ill subjects, despite marked heterogeneity across studies as evidenced by the high Under physiological conditions, a decrease in Hb after rapid fluid administration is both intuitive, through a hemodiluting effect, and counterintuitive, as the kidneys should eliminate the excessive fluid and there may be a fluid shift toward the extravascular space. Interest in this concept was initially raised with the observations by Greenfield and colleagues  that rapThe effects of fluid administration vary according to a number of factors, including type and amount of fluid –76, renaThe amount of fluid given is an important factor in the likely degree of hemodilution induced. The degree by which the Hb decreased was directly related to the amount of fluid given, up to 1000 mL, during the first hour in the non-acutely ill population. However, this trend was not evident in acutely ill patients, likely because of the altered physiological mechanisms in acute illness described previously, notably the fluid extravasation.In acutely and non-acutely ill populations, the Hb decrease was greatest when the baseline Hb was > 14 g/dL; this may have been due to initial hemoconcentration in some patients . In someIn the surgery/trauma subgroup, the type and duration of the procedure , 87, the2 and tissue oxygenation. In subjects in whom Hb decreased, DO2 either increased [In the presence of hypovolemia, fluid administration may result in an increase in cardiac output via the Frank-Starling mechanism , althougncreased , 64, 65 ncreased , 66 whenncreased , 60 whenOf note, hemodilution may also have beneficial effects. For example, hemodilution has been shown to promote cerebral blood flow in preclinical cardiac arrest studies , 93. TheStrengths of this study are the exhaustive review of clinical studies in different settings. We excluded major confounders, such as acute bleeding and transfusion, and classified the risk of bias –98.However, we acknowledge that there was considerable heterogeneity in the included patient populations, in terms of the underlying fluid status of the patients, the fluid tonicity, the timing of the fluid bolus in relation to resuscitation status, the type, amount, and duration of fluid administration, and the timing of observations, which might create bias and limit the interpretation and application of any aggregate quantification. Moreover, there may have been some overlap among groups; for example, some of the patients in the sepsis studies may have had septic shock. Despite our exclusion criteria, in some cases  unrecognized bleeding may have influenced the results. We were also unable to investigate the longer-term effects of rapid fluid administration. None of the studies was specifically designed to evaluate hemoglobin decrease, so we were unable to assess patient-centered outcomes. We extracted some data from graphs, which may have reduced the accuracy of these values, although the participation of two reviewers in this process reduced any observation bias. When needed, we assigned a standard weight and height to calculate fluid amounts and indexes. Finally, the clinical implications of the magnitude of pooled decrease in hemoglobin are not clear, as our study was not designed for this purpose.2 in fluid non-responders, i.e., when cardiac index does not increase during the fluid challenge. These observations add a cautious note to the enthusiastic performance of fluid challenges and remind us how important it is to stop the test when cardiac output does not increase.Notwithstanding the study heterogeneity and moderate certainty of evidence, our observations were consistent across studies, showing a systematic decrease in Hb after rapid fluid administration and raising uncertainty about the effects of fluid on DO Additional file 1. Online supplementary data.


Analysis of routine biochemical levels of cerebrospinal fluid (CSF), distribution of pathogenic bacteria, and risk factors in patients with intracranial infections secondary to brain tumour surgery.  A total of 208 patients admitted to our hospital for brain tumour surgery from January 2020 to May 2022 were selected. Fully automated biochemical analyzer was employed for CSF routine and for measuring biochemical parameters such as white blood cell (WBC), micrototal protein (M-TP), glucose (GLU), and chlorine (CI). Double antibody sandwich assay for CSF procalcitonin (PCT), heparin-binding protein (HBP), and matrix metalloproteinase-9 (MMP-9) was performed. Fully automated microbiological analyzer for pathogen identification was utilized. Based on the above results, we determined whether the patients had secondary intracranial infections after surgery and analyzed the risk factors for secondary intracranial infections after brain tumour surgery by univariate and multifactorial logistic regression. P < 0.05), and GLU was significantly lower than that of uninfected patients (P < 0.05), and the levels of PCT, HBP, and MMP-9 in infected patients were significantly lower than those before treatment after 3, 7, and 10 d and tended to decrease over time (P < 0.05). A total of 62 pathogenic strains were isolated from 65 intracranial infections, of which 41 (66.13%) were Gram-negative bacteria, mainly resistant to amikacin and ciprofloxacin and sensitive to meropenem and imipenem; 19 (30.65%) were Gram-positive bacteria, mainly highly resistant to penicillin and erythromycin and sensitive to vancomycin. Univariate analysis showed that age, gender, tumour type, history of glucocorticoid application, and prophylactic application of antibiotics were not associated with secondary intracranial infection after brain tumour surgery (P > 0.05); tumour site, operation time, postoperative indwelling drainage time, postoperative cerebrospinal fluid leakage, and history of diabetics were all associated with secondary intracranial infection after brain tumour surgery (P < 0.05). Multivariate logistic regression analysis showed that infratentorial tumour, operation time ≥4 h, postoperative indwelling drainage time ≥24 h, and postoperative cerebrospinal fluid leakage were independent risk factors for secondary intracranial infection after brain tumour surgery (P < 0.05).  Among 208 patients with brain tumour surgery, 65 cases (31.25%) had secondary intracranial infection and 143 cases (68.75%) had no secondary intracranial infection. The levels of WBC, M-TP, CI, PCT, HBP, and MMP-9 in the CSF of intracranially infected patients were significantly higher than those of uninfected patients ( Patients with intracranial infections secondary to brain tumour surgery have abnormal levels of CSF routine and biochemical parameters, and the detection rate of Gram-negative bacteria is higher than that of Gram-positive bacteria in patients. Treatment should be based on the characteristics of pathogenic bacteria and risk factors with targeted interventions to reduce intracranial infections. Brain tumours are the most common intracranial tumours in neurosurgery, accounting for approximately 45% of all intracranial tumours, with slightly more men than women . In the A total of 208 patients admitted to our hospital for brain tumour surgery from January 2020 to May 2022 were selected. Our goal is to determine whether the patient had a secondary intracranial infection after surgery by CSF routine and biochemical tests and identification of pathogenic bacteria. Diagnosis of intracranial infection was based on the following: (1) the patient's body temperature continued to rise, >38°C, and there were symptoms or signs such as fatigue, high fever, headache, vomiting, and meningeal irritation; (2) the bacterial culture of intracranial drainage fluid or CSF was positive; and (3) routine and biochemical tests of CSF samples suggested that WBC count in CSF increases; CSF turbidity and M-TP quantitatively increase; or GLU quantitatively decrease, etc., and intracranial infection could be determined if the above one was met . InclusiA lumbar puncture was performed to collect 10 ml of CSF from the patients 2 d after surgery  and on the 3, 7, and 10 days after drug treatment. AU5800 fully automatic biochemical analyzer (Beckman Coulter Inc) was employed for CSF routine and for measuring biochemical parameters such as WBC, M-TP, GLU, and CI levels. Double antibody sandwich assay was carried out for the determination of PCT, HBP, and MMP-9 levels in CSF, and the kits were purchased from Abcam. Vitek32 fully automated microbiological analyzer  was utilized for pathogen identification. Based on these findings, we determined whether the patient had a secondary intracranial infection after surgery.The clinical data of patients were collected by querying electronic cases, including age, sex, tumour type, tumour site, operation time, postoperative indwelling drainage time, postoperative cerebrospinal fluid leakage, history of diabetics, history of glucocorticoid application, and prophylactic application of antibacterial drugs. The case data collected were entered and checked centrally using EpiData, which was required to ensure the accuracy of the data in the entry process.Treat with broad-spectrum antibacterial drugs or select sensitive antibacterial drugs based on the patient's drug sensitivity test results. Antibacterial drugs that could cross the blood-brain barrier such as amikacin, meropenem, and vancomycin should be chosen.t-test was performed for both groups. Statistical data were described in terms of cases and percentages (%), with the χ2 test. Univariate analyses that were statistically significant were included in multivariate analyses, and risk factors for secondary intracranial infection after brain tumour surgery were analyzed by multivariate logistic regression. P > 0.05 was considered statistically significant.SPSS 22.0 was employed for statistical analysis. The measurement data were expressed as Among 208 patients with brain tumour surgery, 65 cases (31.25%) had secondary intracranial infection and 143 cases (68.75%) had no secondary intracranial infection .P < 0.05), and GLU was significantly lower than that of uninfected patients (P < 0.05). . .P < 0.05), and the levels of PCT, HBP, and MMP-9 in infected patients were significantly lower than those before treatment after 3, 7, and 10 d and tended to decrease over time (P < 0.05).  were Gram-negative bacteria, mainly resistant to amikacin and ciprofloxacin and sensitive to meropenem and imipenem; 19 (30.65%) were Gram-positive bacteria, mainly highly resistant to penicillin and erythromycin and sensitive to vancomycin (Tables 2P > 0.05); tumour site, operation time, postoperative indwelling drainage time, postoperative cerebrospinal fluid leakage, and history of diabetics were all associated with secondary intracranial infection after brain tumour surgery (P < 0.05). . .P < 0.05). . .μg/L, the sensitivity of its differential diagnosis between acute bacterial meningitis and viral meningitis can reach more than 90%. MMP-9 is a novel marker of infection that relies on the assistance of metal ions such as Ca2+ and Zn2+ to exert extracellular matrix degradation [During craniotomy, patients may present with clinical symptoms similar to those of intracranial infections due to irritation by the implant or other factors, making the differential diagnosis of intracranial infections difficult. CSF's laboratory-related tests are of great interest because of their ease of operation and rapidity of detection. The results of CSF routine and biochemical parameters in this study showed that the levels of WBC, M-TP, and CI in the CSF of those with intracranial infection secondary to brain tumour surgery were significantly higher than those of without infection, and the GLU was significantly lower than those of without infection. This suggests that CSF routine and biochemical tests can go some way to diagnosing intracranial infections. Nevertheless, as intracranial infections have many causes, the main common ones being bacterial, viral, and tuberculosis infections , 13, thiradation . Animal radation  have shoKlebsiella pneumoniae being the most distributed, and these two bacteria were mainly highly resistant to amikacin and ciprofloxacin and sensitive to meropenem and imipenem, so we presume that intracranial infections caused by Gram-negative bacteria should be treated with beta-lactam antibiotics; Gram-positive bacteria accounted for 30.65%, with coagulase-negative staphylococci and Staphylococcus aureus being the most distributed, and these two bacteria were mainly highly resistant to penicillin and erythromycin and sensitive to vancomycin, suggesting that vancomycin could be the antibacterial drug of choice for intracranial infection caused by Gram-positive bacteria.Brain tumours occur in a confined environment formed by the tissues of the meninges, skull, and scalp, and when patients need to undergo craniotomy, the disruption of the anatomical structure caused by the surgery and the placement of postoperative drains can open up the brain tissue to the outside world; thus, causing germs to invade the skull and increasing the probability of infection. CSF pathogenic cultures are the gold standard for the diagnosis of intracranial infections, and their epidemiological surveillance is important in guiding the empirical clinical use of drugs. In this study, 62 strains of pathogenic bacteria were isolated from 65 cases of intracranial infections, of which Gram-negative bacteria accounted for 66.13%, with Acinetobacter baumannii and Intracranial infection after craniocerebral surgery often coexists with encephalocele, intracerebral hypertension, brain edema, and hydrocephalus, and they interact with each other to aggravate the condition; thus, directly affecting the prognosis. At present, it is believed that there are some differences in the related factors of secondary intracranial infection after brain tumour surgery at home and abroad. In this study, multivariate logistic regression was used to adjust for confounding factors. It was found that the independent risk factors of intracranial infection after brain tumour surgery include the following: first, infratentorial tumour: according to the report , the incBrain tumour surgery often requires intracranial or subcutaneous indwelling drainage tubes in order to successfully drain cerebrospinal fluid or hematocele, but if the drainage device is not managed properly and the position of the drainage tube is too high, it can lead to poor drainage and even the drainage fluid flowing back to the brain; in addition, the risk of pollution exposure is correspondingly increased due to the long time of tube placement. Fourthly, postoperative cerebrospinal fluid leakage: cerebrospinal fluid leakage can be divided into rhinorrhea and otorrhea, and pathogenic bacteria can enter the brain along the channel of cerebrospinal fluid leakage, resulting in intracranial infection. It can be seen from the above that for patients with brain tumour surgery, adequate preparation and detailed operation plan should be made before operation, the operation time should be shortened as far as possible, aseptic operation should be strictly implemented, the wound should be closely sutured after operation, cerebrospinal fluid leakage should be prevented, drainage tubes should be placed to avoid reflux and blockage caused by improper placement, and drainage time should also be controlled. For patients with brain tumour surgery with high-risk factors, prophylactic drugs should be used and monitoring should be strengthened during the perioperative period. Once there are suspected symptoms of intracranial infection such as high fever and meningeal irritation, diagnosis and treatment should be carried out as soon as possible without any delay.In summary, patients with intracranial infections secondary to brain tumour surgery have abnormal levels of CSF routine and biochemical parameters, and the detection rate of Gram-negative bacteria is higher than that of Gram-positive bacteria in patients. Treatment should be based on the characteristics of pathogenic bacteria and risk factors with targeted interventions to reduce intracranial infections. This study also has shortcomings. There are many factors that contribute to cerebrospinal fluid infection after craniotomy, and the clinical symptoms of each type of infection lack specificity. Studying the routine and biochemistry of the cerebrospinal fluid in different types of infections may further strengthen the diagnosis and differentiation. However, given the inadequate sample size and technical support in this study, further classification analysis is pending for future studies with larger samples.


Phase retrieval wavefront sensing methods are now of importance for imaging quality maintenance of space telescopes. However, their accuracy is susceptible to line-of-sight jitter due to the micro-vibration of the platform, which changes the intensity distribution of the image. The effect of the jitter shows some stochastic properties and it is hard to present an analytic solution to this problem. This paper establishes a framework for jitter-robust image-based wavefront sensing algorithm, which utilizes two-dimensional Gaussian convolution to describe the effect of jitter on an image. On this basis, two classes of jitter-robust phase retrieval algorithms are proposed, which can be categorized into iterative-transform algorithms and parametric algorithms, respectively. Further discussions are presented for the cases where the magnitude of jitter is unknown to us. Detailed simulations and a real experiment are performed to demonstrate the effectiveness and practicality of the proposed approaches. This work improves the accuracy and practicality of the phase retrieval wavefront sensing methods in the space condition with non-ignorable micro-vibration. The imaging performance of space-based large-aperture astronomical telescopes is susceptible to space thermal/mechanical perturbations, which can gradually lead to mirror misalignments or deformations during long-term on-orbit observation and operation . Mirror Image-based wavefront sensing (WFS) represents a class of WFS methods that directly utilize image plane intensity measurements to recover the pupil plane wavefront phase of the optical system. This class of methods mainly includes iterative-transform methods  ,4,5,6,7,However, the accuracy of the image-based wavefront sensing methods for space telescopes is inevitably influenced by space perturbations. Meanwhile, line-of-sight jitter is one of the most common forms of disturbances due to micro-vibration of the platform which can be induced by a refrigerator or reaction wheel . Line-ofMany researchers have studied how to reduce the effect of jitter on imaging quality. Sun et al. proposed a new mathematical method named DIROEF to estimate the true value of the jitter, and the SVBM and VACM methods were used to compensate for the radiometric and geometric effects of the jitter on the image . Liu et While the effect of jitter presents stochastic property, it still obeys some statistics laws. In the presence of jitter, the position of the intersection of the line-of-sight with the image plane at any moment within the exposure time obeys normal distribution. Based on this fact, Gaussian convolution is introduced to describe the effect of jitter on image ,32. ThenThe remainder of this paper is organized as follows. In This section first discusses how to describe the intensity distribution of the point spread function (PSF) in the presence of jitter. On this basis, two kinds of jitter-robust image-based wavefront sensing algorithms are proposed, which can be categorized into iterative-transform algorithms and parametric algorithms, respectively.Specifically, the first jitter-robust algorithm involves iterative Fourier transformation back and forth between the object and Fourier domains and application of the measured data and established imaging model in the presence of jitter. The second jitter-robust algorithm recovers the parameterized wavefront aberrations by minimizing the objective function which includes the effects of jitter with nonlinear optimization methods.We will first discuss how to describe the intensity distribution of the point spread function (PSF) in the presence of jitter. Let us suppose that the object is illuminated with noncoherent quasi-monochromatic light, and the imaging system is a linear shift-invariant system. According to Fourier optics, the point spread function in the image plane can be expressed asG is a Gaussian convolution kernel, which is used to represent the effect of jitter on the intensity distribution of the PSF. To accurately describe the effect of jitter, the element in G should be carefully determined.While the effect of jitter presents stochastic property, it still obeys some statistics laws. In the presence of jitter, the position of the intersection of the line-of-sight with the image plane at any moment within the exposure time obeys normal distribution. Therefore, Gaussian convolution can be introduced to describe the effect of jitter on the image during the exposure time. In other words, in the presence of jitter, the intensity distribution of a PSF can be modeled as:G can be determined according to the two-dimensional Gaussian normal distribution, which can be expressed asu and v are coordinate variables , G is the Gaussian kernel which is used to perform convolution and g is the Gaussian function which is used to discuss the property of the Gaussian function.The value of the element in In general, we can further supposeThe value of On this basis, we can obtain the value of each element in the Gaussian kernel, G. Specifically, we can first draw grid lines in a plane, and the intervals between grid lines are equivalent to the pixel size. Then, we assume the center of the Gaussian function is in consistent with the center of the central square in the grid, as shown in According to the convolution theorem, Equation (2) can be rewritten asThis equation can further be rewritten asOn this basis, we can propose an iterative-transform jitter-robust image-based wavefront sensing algorithm. When only one focal image is available, this algorithm is illustrated in The algorithm illustrated in Since the relationship between PSF and aberration coefficients in the presence of jitter has been established, we can present the following equations after collecting two PSFs with a detector,It is hard to directly obtain the analytical solution of the complicated nonlinear equations ∂F⊗The Simulations are performed to demonstrate the proposed jitter-robust phase retrieval algorithms. Here we first discuss how to simulate the PSF in the presence of jitter. We suppose the position of the intersection of the line-of-sight with the image plane at any moment within the exposure time obeys normal distribution. A large series of PSFs with random amount of transverse translation  is generated for certain aberration coefficient sets. The values of translation obey normal distribution with a certain value of variance . These PSFs with different amounts of image translations are superimposed and a new PSF is obtained after normalization. On this basis, a certain amount of noise can be added to this image to simulate the true noisy condition.Suppose the aperture of the optical system is 2000 mm, the focal length of the system is 28,000 mm, the wavelength is 632.8 nm, the pixel size is 5.5 μm, and the defocusing distance between two PSF images is 1 mm. Three pairs of simulated PSF images in the presence of different jitter parameters for the same wavefront error aberration coefficients and noise level are presented in We can see from Traditional phase retrieval algorithms that do not take jitter into consideration and the two jitter-robust phase retrieval approaches are applied to those three pairs of simulated PSF images. The results are presented in In (1)While jitter-robust phase retrieval algorithms cannot thoroughly eliminate the effects of jitter on phase retrieval accuracy, they can effectively reduce its effects and maintain wavefront accuracy.(2)The accuracy of C4 (defocus aberration) and C5, C6 (astigmatism) are more susceptible to the effects of jitter for traditional phase retrieval algorithms.(3)It seems that the accuracy of the parametric jitter-robust phase retrieval algorithm is a little higher than the accuracy of the iterative-transform jitter-robust phase retrieval algorithm.The following conclusions can be drawn from Then we take the case of sigma = 0.3 pixels as an example to test the convergence of these four algorithms, and the results are presented in Monte Carlo simulations are further presented to demonstrate the effectiveness of the proposed jitter-robust approaches. Here the number of aberration coefficients considered is increased to 36. A total of 100 sets of aberration coefficients are randomly generated within the range of , and 100 pairs of PSF images are then generated according to the simulation process presented in To simulate noise, we model each image to have Gaussian CCD read noise with a standard deviation of The peak pixel SNR is defined as(1)We can see from (2)We can see from (3)Comparing The following conclusions can be drawn from We can see that, compared to traditional phase retrieval algorithms, the accuracy of jitter-robust phase retrieval algorithms is insensitive to the presence of jitter.Real experiments are performed to demonstrate the effectiveness and practicality of the proposed jitter-robust algorithms. A physical map of the optical path used to generate PSF images including the effects of jitter is shown in (1)The reconstructed images for A2 and B2 are very similar to the original images, and RMSEs for A2 and B2 are smaller than RMSEs for A1 and B1, which demonstrates the effectiveness and practicality of the proposed jitter-robust phase retrieval algorithms.(2)The reconstructed images for A1 are also similar to the original images. However, the RMSE for A1 is quite large. This fact indicates that traditional phase retrieval algorithms attribute image vague to aberration, not jitter. Therefore, while the reconstructed images are similar to the original ones, the accuracy is low.(3)We find that in this experiment, the accuracy of B2 is a litter higher than A2. The possible reason may be that the parametric jitter-robust phase retrieval algorithm is more robust to image noise than the iterative-transform jitter-robust phase retrieval algorithm. This conclusion is consistent with the results presented in The results are presented in In the previous sections, we consider that the magnitude of jitter is known to us. In this section, we further discuss the case where the magnitude of jitter is unknown to us (we suppose that the magnitude of jitter is isotropic). In this case, the iterative jitter-robust phase retrieval algorithms are no longer applicable. For an unknown magnitude of jitter, the procedure of deconvolution in On the other hand, the parametric jitter-robust phase retrieval algorithm presented in In other words, in the optimization process of searching the sets of aberration coefficients, the magnitude of jitter can also be solved. At the same time, aberration coefficients are more accurate since this optimization process has taken the unknown jitter parameter into consideration.Monte Carlo simulations are also presented to demonstrate the accuracy of the proposed jitter-robust approaches for an unknown magnitude of jitter  (we no longer perform experiments to demonstrate its effectiveness). A total of 100 sets of aberration coefficients are randomly generated within the range of , and 100 pairs of PSF images are then generated according to the simulation process presented in Comparing (a) and (b) in To conclude, this paper proposes a framework for jitter-robust image-based wavefront sensing algorithm by including the effects of jitter and proposed two kinds of jitter-robust phase retrieval algorithms, which can be categorized into iterative-transform algorithms and parametric algorithms, respectively. The effect of the jitter shows some stochastic properties and it is hard to present an analytic solution to this problem. This paper utilizes two-dimensional Gaussian convolution to describe the effect of jitter on the image, keeping in mind that the variance of this Gaussian function should be consistent with the magnitude of jitter. Further discussions are presented for the cases where the magnitude of jitter is unknown to us, and the parametric jitter-robust phase retrieval algorithm can be extended to this case. Detailed simulations and a real experiment are performed to demonstrate the effectiveness and practicality of the proposed approach.Overall, we find that the parametric jitter-robust phase retrieval algorithm is superior to the iterative-transform jitter-robust phase retrieval algorithm. This method can be extended to those cases where the magnitude of jitter is unknown to us, which is of significance for the application of the jitter-robust phase retrieval algorithm in practical situations.