Within the generated ST-seq datasets, immune cell types and states, termed here as exhausted/pro-tumour state or non-exhausted/anti-tumour state, were identified using multiple publicly available single-cell RNA and T-cell receptor sequencing datasets as references. HG TMEs revealed abundant exhausted/pro-tumour immune cells with no consistent increase in expression of PD-1, PD-L1 and CTLA4 checkpoints and angiogenic genes. Additional HG TME immunophenotype characteristics included: pro-tumour tissue-resident monocytes with consistently increased expression of HAVCR2 and LAG3 checkpoints; an exhausted CD8+ T cells sub-population with stem-like progenitor gene expression; and pro-tumour tumour-associated macrophages and monocytes within the recurrent TME with the expression of TREM2. Whilst limited by a modest sample size, this study represents the largest ST-seq dataset on human ccRCC. Our study reveals that high-risk ccRCC TMEs are infiltrated by exhausted/pro-tumour immunophenotypes lacking specific checkpoint gene expression confirming that HG ccRCC TME are immunogenic but not ICI favourable.Perioperative immune checkpoint inhibitor (ICI) trials for intermediate high-risk clear cell renal cell carcinoma (ccRCC) have failed to consistently demonstrate improved patient outcomes. These unsuccessful ICI trials suggest that the tumour infiltrating immunophenotypes, termed here as the immune cell types, states and their spatial location within the tumour microenvironment (TME), were unfavourable for ICI treatment. Defining the tumour infiltrating immune cells may assist with the identification of predictive immunophenotypes within the TME that are favourable for ICI treatment. To define the immunophenotypes within the ccRCC TME, fresh para-tumour (pTME,  Approved first-line systemic therapy for metastatic ccRCC includes immunotherapies such as immune checkpoint inhibitors (ICIs) that serve to reactivate anti-tumour immune responses10. However, the final results from adjuvant ICI trials for localised ccRCC have failed to demonstrate an improvement in survival outcomes13. The efficacy of perioperative ICIs within intermediate high-risk ccRCC patients has been assessed in randomised phase III clinical trials17. These perioperative (or neoadjuvant) ICIs are aimed at priming the anti-tumour immune response without delaying surgical intervention17. A recent neoadjuvant nivolumab trial for localised ccRCC reported no delays in surgery16. However, due to the short-term follow-up in this trial, it is uncertain if an anti-tumour immune response was initiated by earlier ICI treatment.Clear cell renal cell carcinoma (ccRCC) accounts for the majority of kidney cancer-related deaths due to the presence or development of metastatic disease+ T cells, tumour associate macrophages (TAM) and monocytes may clarify the precise immunophenotypes within the intermediate high-risk TME. The advancement within transcriptomics methodologies makes it feasible to profile the ccRCC tumour infiltrating immunophenotypes—defined here as the immune cell types, exhausted/pro-tumour or non-exhausted/anti-tumour states, and their spatial location within an individual patient’s TME  is an urgent unmet need for personalising oncology in high-risk ccRCC patients. Defining the TME-specific CD8ME Table . This stME Table . We hypop-value 2.9e-10) from para-tumour (pTME), low-grade (LG) and high-grade (HG) TMEs  and macrophages within LG TMEs  . Conversely, the highest exhausted CD8+ proliferative cells were found within HG TMEs . TAM and monocyte sub-typing  within the LG TMEs . However, the highest tissue-resident monocytes were found mainly within HG TMEs . Interestingly, the CD8+ T cells, TAM and tissue-resident monocytes identified within the LG TMEs were mirrored within the HG_2 TME. The proportion of immune cell sub-types was based on the immune cell types  and immunomodulatory marker ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1). Nonetheless, the TCF7 revealed the highest relative expression within sub-populations of CD8+ exhausted immediate-early genes (CD8+ exhausted IEG) and CD8+ exhausted T cells in HG_1.2 and HG_1.3 TMEs , a known marker for recurrence, was identified within a larger proportion of pro-tumour TAM sub-types in HG_3 TME; whilst an absence of immunosuppression markers human leucocyte antigen G (HLA-G) and cysteine protease cathepsin S (CTSS)19 was observed within TAM ISG high expression (TAM ISGhi) in HG_3 and HG_2 TME, respectively  and novel  IC genes. We determined the relative expression of targetable and novel IC genes within grouped CD8+ T cells  and novel  angiogenic genes within grouped CD8+ T cells , we show predominantly exhausted CD8+ T cells and pro-tumour tissue-resident monocytes within the tumour/immune regions. In line with previous studies, we show a sub-population of exhausted CD8+ T-cell sub-types expressing stem-like progenitor (TCF7). Both sub-populations of exhausted and non-exhausted CD8+ T-cell sub-types express immunomodulatory (ENTPD1) genes29. The pro-tumour TAM and monocyte sub-types express an anti-inflammatory gene signature30. Similarly, TREM2, an established marker for post-surgical recurrence of ccRCC30, shows higher expression in a large proportion of the pro-tumour TAM and tissue-resident monocytes for patient HG_3 with recurrent ccRCC. Furthermore, within distinct tumour/immune admixed regions, the tissue-resident monocytes surround the exhausted CD8+ T cells, suggestive of anti-inflammatory activity and/or an exclusion zone along the tumour margins in HG TME, as identified in other studies32. Importantly, we demonstrate a singular exhausted/pro-tumour or non-exhausted/anti-tumour immune cell states within individual specimens, suggesting that lymphoid and myeloid cells behave in unison, possibly through cellular interactions between the immune cell sub-types, as identified in previous studies33.In this study, we present the largest ST profile of immunophenotypes within para, low- and HG ccRCC TMEs. Consistent with previous bulk RNA-seq, single-cell RNA-seq (scRNA-seq) and single-nuclei RNA-seq (snRNA-seq) studies, we show abundant immune cell infiltration within the TME, dominated by T cells and monocytes+ T cells of the HG ccRCC TME, we show sub-populations expressing stem-like progenitor TCF7, suggestive of heterogeneity within the exhausted cell states and a capability of T cells to differentiate. It will be of interest to investigate within ex vivo studies, if these exhausted stem-like progenitors CD8+ T cells can be switched to an anti-tumour mode with ICI, as reported in other cancers39. The effectiveness of ICI is based on the expression of IC genes by infiltrating immune cells, as such we examined the expression of targetable and novel IC genes in grouped CD8+ T cells, TAM and monocytes across all samples. Targetable IC genes show no increase in expression within CD8+ T cells, TAM and monocytes, potentially explaining the limited efficacy of ICI40 observed in three adjuvant ICI trials13. However, these trial findings are in contrast with the Keynote-564 trial41. These conflicting trial outcomes of ICIs within intermediate high-risk ccRCC do not diminish the reported improved survival outcomes within the metastatic setting44. This further highlights a need for a precision oncological approach that integrates traditional histopathological and clinical information with systematic transcriptomics profiling.Within the exhausted CD8HAVCR2 (or TIM3) shows consistently increased expression within the tissue-resident monocytes which dominated the HG TMEs. Traditionally, tissue-resident monocyte populations are considered reservoirs for macrophages that play key roles in immune defence and inflammatory events45. However, it is uncertain if tissue-resident monocytes have the plasticity to differentiate into macrophages and later TAM within the ccRCC TME. Mouse model studies have demonstrated that tissue-resident monocyte- and macrophage-derived TAM respond very differently to cancer treatment46. Further exploration of the fate of these tissue-resident monocytes in the HG ccRCC TME is needed to elucidate their response to ICI treatment. As such, we are interested in the results of the phase I and II anti-HAVCR2 trial (NCT02817633) in advanced solid tumours. Looking beyond potential IC targets, we extended our investigation to also include the expression of targetable and novel angiogenic genes. However, immune cells show no consistent increased expression of angiogenic genes, suggesting that the promotion of angiogenesis within the ccRCC TME may involve non-immune cells.Novel IC Limitations in this study include minimal multi-region sampling at a single timepoint from a modest cohort of consenting ccRCC patients and variable tumour stages within both the LG  and HG  ccRCC groups. These limits may have restricted complete molecular profiling of the heterogeneity and complexity of the intermediate high-risk ccRCC TME. Nonetheless, we show intra- and inter-patient heterogeneity within the identified immunophenotypes of the limited TME regions  presenting at variable time points . Therefore, our decision to use minimal multi-region sampling from six ccRCC patients for ST-seq preserves surgical margins, delivers uncompromised patient management, and still captures transcriptomic heterogeneity within the immunophenotypes present in the ccRCC TME. In future, the use of non-clinical mouse models of ccRCC can overcome these limits by allowing sampling from multi-regions and timepoints at similar tumour stage or a large clinical human cohort.In summary, unbiased transcriptomic profiling identified heterogeneous immunophenotypes, without a consistent targetable IC expression profile, within intermediate high-risk ccRCC TMEs. Translating these findings in the future might result in identified immunophenotype characteristics in ccRCC that are favourable for ICI treatment and deliver precision oncology.+ T cells, TAM and monocytes within the TME due to their recognised effector and tolerogenic response, defined here as non-exhausted/anti-tumour and exhausted/pro-tumour states, respectively  presenting at a tertiary referral hospital from June 2021 to January 2022  on ten fresh frozen tissue sections collected in the operating theatre from six patients with ccRCC  and imaged on an Axio Z1 slide scanner (Zeiss). Afterwards, the same tissue sections were permeabilised to release mRNA. These mRNA were captured by underlying ST-spots, and complimentary DNA libraries incorporating the spatial barcodes were synthesised. All libraries were loaded at 1.8 pM and sequenced using a Mid output reagent kit (Illumina) on a NextSeq500 (Illumina) instrument. Sequencing was performed using the following protocol: Read1 - 28 bp, Index1 - 10 bp, Index2 - 10 bp, Read2 - 120 bp. After sequencing the genes were mapped to the H&E images to generate spatially resolved transcriptional profiles  involved the use of microarrayed glass slides with 55 µm spots (or ST-spots) containing oligonucleotides with a sequence of deoxythymine (oligo-dT) and unique spatial barcodes printed within capture arrays. Thin 8 µm cryosections were placed within a capture array overlaying the ST-spotsles Fig. 54.55 and Seurat (v4.1.0) R packages57. We confirmed the quality of the captured transcriptome using the following cut-offs: >50 genes per ST-spot, >100 unique molecular identifiers (UMI) per gene, ≥500 nCount per ST-spot, >500 nFeature per ST-spot and <30% mitochondrial genes per ST-spot. Then, we merged all the individual ST-seq datasets and removed batch effects due to individual patient samples using the SCTransformfunction in Seurat58. With this merged ccRCC ST-seq dataset, Louvain clustering was performed with the most stable cluster resolution (res 0.4). These clusters were then annotated as immune and non-immune using published kidney  and tumour immune atlas52 scRNA-seq datasets.The generated ST-seq datasets were processed and analysed using STUtility (v0.1.0)26 sequencing dataset from ccRCC patients that were ICI naïve or exposed. In brief, the scRNA/TCR-seq dataset contained curated transcriptome signatures for exhausted or non-exhausted lymphoid and pro- or anti-tumour myeloid cell sub-types. Based on these signatures, we annotated the cell states of the T cells, macrophages and monocytes using the Semi-supervised Category Identification and Assignment (SCINA) algorithm59. In brief, this method leverages previously established gene signatures in a semi-supervised model using an expectation–maximisation (EM) algorithm. Next, we focused on the CD8+ T cells, TAM and tissue-resident monocytes ; and non-exhausted state are: CD8+ tissue-resident and CD8+ NK-like. Myeloid cell sub-types with a pro-tumour state are: TAM with human leucocyte antigen DR (HLA-DR) high expression (TAM HLAhi), TAM with HLA-DR intermediate expression (TAM HLAint), TAM with interferon signalling genes high expression (TAM ISGhi) and CD14+/16+ monocytes termed tissue-resident monocytes; and anti-tumour state TAM ISG intermediate expression (TAM ISGint). This classification of exhausted/pro-tumour or non-exhausted/anti-tumour immune cell states is not static. Indeed, a spectrum of immune cell states is being recognised60. However, for brevity, here we utilised exhausted or non-exhausted states for five CD8+ T-cell sub-types and pro- or anti-tumour states for five TAM and tissue-resident monocyte sub-types identified within our ST-seq datasets es Table . CD8+ T-Further information on research design is available in the Supplementary MaterialReporting Summary


The architecture of inflorescence in crops is a key agronomic feature determining grain yield and thus has been a major target trait of cereal domestication.Spreading Panicle 9 (SPR9) locus, also has a significant impact on the resistance to rice false smut (RFS). Meanwhile, we mapped a novel spr9 mutant gene between markers Indel5-18 and Indel5-22 encompassing a genomic region of 43-kb with six candidate genes. Through gene prediction and cDNA sequencing, we confirmed that LOC_Os05g38520 is the target gene in the spr9 mutant, which encodes 60 S ribosomal protein L36-2. Further analysis showed that the spr9 mutant is caused by a 1 bp deletion in the first exon that resulted in premature termination. Knockout experiments showed that the SPR9 gene is responsible for the spreading panicle phenotype of the spr9 mutant. Interestingly, the spr9 mutant was found to improve resistance to RFS without affecting major agronomic traits. Taken together, our results revealed that the spr9 allele has good application prospects in rice breeding for disease resistance and panicle improvement.In this study, we show that a simple spreading panicle change in rice panicle shape, controlled by the SPR9, which encodes a 60 S ribosomal protein that regulates spreading panicles and affects the resistance to false smut in rice.We report the map-based cloning and functional characterization of The online version contains supplementary material available at 10.1186/s12870-023-04172-4. Plant inflorescence configuration is a key agronomic factor affecting mine grain yield and a major target for crop domestication and improvement . UnderstOryza sativa. L) is one of the most important food crops in the world, and its panicle morphological development and molecular regulation mechanism have been the focus of research. In recent years, some progresses have been made in the study of panicle development of rice, a monocot model plant, but it is far less detailed than that Arabidopsis thaliana [Gn1a/OsCKX2 [DEP1 [GNP1/GA20ox1 [IPA1/WFP/OsSPL14 [NPT1/OsOTUB1 [GS3[TGW6[GW8/OsSPL16 [GW7/GL 7 [GLW7/OsSPL13 [GS2/GL2/OsGRF4 [GW2 [GW5 [Rice  to the compact panicle of present cultivars (Oryza sativa L.) was a crucial event in rice domestication [spr1 [spr2 [spr3 [spr4 [spr5 [spr8 [OsLG1 [OsLG1 was successfully cloned as the SPR3 locus [OsLG1 is a squamosa promoter binding protein (SBP) domain transcription factor that controls leaf tongue development in rice, had a significant influence on seed florescence and pollination (self-cross or outcross mode) [OsLG1 gene resulted in a compact panicle type in cultivated rice during rice domestication, but it did not change its expression in the leaf tongue, resulting in a compact panicle type and normal leaf tongue development in cultivated rice [Transition from a spread panicle typical of ancestral wild rice  . Furtherted rice .Ustilaginoidea virens (U. virens), a unique flower-infecting fungal pathogen, has emerged as a serious grain disease in rice production worldwide. The disease not only causes a significant yield loss (up to 40%) but also produces various types of mycotoxins that contaminate rice grains and decrease the grain quality [Rice false smut (RFS) caused by  quality –30.SPR9, a novel gene associated with spike development, was identified in the spr9 mutant of R20-1 background. We analysed the genetic characteristics of the spr9 mutant, mapped spr9, and found that the spr9 gene contains a 1-bp deletion (T), leading to the loss of function of the SPR9 gene. Interestingly, the spr9 mutant altered the resistance to RFS. These results indicate that spr9 has excellent potential application prospects in rice disease resistance breeding and spike-type improvement breeding.In recent years, although some studies have speculated that RFS is related to rice panicle traits, especially to the traits of large panicle, erect panicle and dense panicle , 31, butIndica rice R20-1, which contains Pigm-1 [japonica Hui1586 [1 population treated by ethyl methanesulfonate (EMS) and approximately 10,000 plants of the M2 population, at the Sanya Experimental Station and Fuzhou Experimental Station, Fujian Academy of Agricultural Sciences, respectively. The M2 population was screened, and a spreading panicle mutant was obtained and named spreading panicle 9 (spr9).s Pigm-1  and japo Hui1586 , was culspr9 mutant was crossed with rice varieties R20-1 and Hui1586 as pollen donors. F1 seeds were sown in the spring at the experimental station in Sanya, Hainan Province, and F2 seeds were harvested in 2021. The F2 seeds, the spr9 mutant and R20-1 were planted at Fuzhou Experimental Station in Fujian Province in the summer of 2021. Hui1586 and the three knockout transgenic lines were planted at Fuzhou Experimental Station in Fujian Province in July 2022. The main agronomic traits, including plant height, panicle length, effective panicle number, spikelets per panicle, seed setting rate, 1000-grain weight, grain length and width, were investigated at the maturity stage. The segregation ratios of mutants versus the wild type were examined after maturity.In the summer of 2020, the All plants were grown according to standard commercial procedures with spacing between rows of 13.3 and 26.4 cm, and field management was carried out according to normal field management practices.spr9 mutant and the wild type R20-1, inoculation methods were performed according to Song et al. with minor modifications [U. virens hyphae/conidia (106 ml− 1) suspension in potato sucrose broth (PSB) was injected into a single rice panicle from the middle to the upper part at the late booting stage (3–5 days before heading) using an 18 gauge needle in a greenhouse. The liquid filled most of the panicle space. Three replications were carried out, three panicles of each individual plant were artificially inoculated. Inoculated plants were kept in a greenhouse at 27 °C with 90–100% RH for 7 days. Then, they were placed at 27 °C and 80% RH until RFS symptoms appeared.To evaluate the resistance to RFS disease between the ications . ApproxiAccording to the suggestion of a previous study , the incspr9 mutant (indica) was crossed with Hui1586 (japonica) to obtain a mapping population. The F2 population was constructed by selfing the F1 population, and the mutant phenotypes of 1452 individual plants in the F2 population were selected for accurate mapping.The japonica (cv. Nipponpare) and indica (cv. 93 − 11), and primers were designed to map the polymorphic regions of rice subspecies using Primer Premier 5.0.Insertion-deletion (InDel) markers were designed by manual comparison of genome sequences between Plant DNA and DNA amplification was performed by polymerase chain reaction (PCR) with minor modifications . The PCR2 population were used for construction of a mutant DNA library. SSR markers distributed in the rice genome were used to amplify the spr9 mutant DNA, and Hui1586 DNA was used as a control for linkage detection. The marker band of the mutant gene was the same as the marker band of the spr9 mutant.Markers of target genes were identified by bulked segregant analysis. Leaf DNA of 20 mutant plants randomly selected from the Fspr9 spr9) and wild type (SPR9 SPR9) were denoted as 1 and 3, respectively; 2 was used to represent a heterozygote (spr9 SPR9). Linkage analysis was performed using MAPMAKER version 3.0 software [The band types of the mutant , InDel markers in the open rice genome sequence were designed to predict the likelihood of polymorphism between spr9 and Hui1586.First, 326 SSR markers were screened from the rice molecular map to study the polymorphisms of http://rice.plantbiology.msu.edu/; http://www.tigr.org/). Clones were fixed on the target gene combination mark with the basic local alignment search tool (BLAST) in pairs (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) for sequence alignment. The DNA sequences of spr9 and SPR9 were used for a complete alignment with Clustal X version 1.81.Candidate genes were predicted based on existing sequence annotation databases  .A spr9 mutant with changes in spike traits in the R20-1 background, which displayed a spread panicle . Phenotype comparisons between the spr9 mutant and wild-type R20-1 are shown in Table spr9 mutant and the wild type , implying a single gene controlled the spr9 mutant phenotype. Together, these results indicate that the spr9 mutant is genetically controlled by a single recessive gene.To determine the genetic mode of spr9 mutant, we further conducted a gene mapping population by crossing the spr9 mutant with the japonica rice cultivar Hui1586. Total of 253 SSR markers showed polymorphism between spr9 and Hui1586 were obtained. Among these polymorphic markers, RM8211, on chromosome 5, showed a complete co-segregation with mutant phenotype in the selected 20 F2 plants with mutant phenotype and 20 plants with wild type phenotype. Furthermore, 193 recessive plants from the F2 population were genotyped by RM8211 and RM5970, recombinant plants were found, verifying the linkage relationship between the marker and spr9 mutant phenotype. Subsequently, we found one other polymorphic marker RM5970, located on chromosome 5, also showed linkage with spr9 mutant phenotype. The recombinants identified from RM8211 all showed homozygous genotype as spr9 mutant at the RM5970 locus, and the recombinants from RM5970 presented heterozygous or wild homozygous genotype at RM8211 locus, therefore, we preliminarily mapped the spr9 gene into the region of RM8211 and RM5970. Thus, spr9 was preliminarily located in a 16.5 cM region between SSR markers RM8211 and RM5970 on chromosome 5  in this 43-kb region  (LOC_Os05g38520) between wild-type R20-1 and the spr9 mutant  had three exons and two introns  would lead to the spread panicle phenotype. Using the CRISPR/Cas9 gene editing system, a sequence-specific guide RNA (sgRNA) was designed to knock out the SPR9 gene. We obtained a total of three homozygotes from three independent knockout events and confirmed their presence of insertion and deletion mutations at the target sites by Sanger DNA sequencing . Taking together, we concluded that spr9 gene was the causal gene for the spread panicle phenotype in the spr9 mutant.To determine the phenotype of ing Fig. a. We theing Fig. b, which SPR9, reverse transcription- quantitative PCR (RT‒qPCR) was used to detect the expression patterns of SPR9 at different developmental stages of rice  and in panicles (5–10 cm length) . The results showed the localization of SPR9 in the nucleus (Fig. In order to further analyze the localization of SPR9, we constructed 35 S: SPR9-pSuper1300-GFP vector (Primers are shown in Supplementary Table eus Fig. .spr1 [spr2 [spr3[spr4 [spr5[spr8 [OsLG1[spr1 and spr8 were controlled by a recessive nuclear gene, and other five sprouting trait genes were controlled by dominant genes. In this study, the spr9 gene was identified as a recessive mutation. Through further comparison, no cloned spread panicle gene was found in this region, so we speculated that spr9 was a new gene.The spread panicle trait is a typical mutant trait in rice, and there have been some reports on its gene mapping. To date, a total of 7 genes for spike traits have been reported, namely, spr1 , spr2 [2r1 [spr2 , spr3[24pr3[spr4 , spr5[26pr5[spr8  and OsLGSPR9 encodes the 60 S ribosomal protein L36-2, which is a ribosome associated protein. However, why does spr9 affect the disease resistance of rice? Studies have shown that posttranslational reprogramming is another fundamental regulatory pathway of plant immunity and specifically regulates translation in response to PAMP-triggered immunity induction [Pi21-silenced plants infected by Magnaporthe oryzae suggested that ribosomes were a third enrichment pathway compared to Nipponbare plants [GhRPL18A-6 can regulate the expression of cell wall synthesis, lignin synthesis and other disease-resistant pathway genes in upland cotton, and improve the Verticillium dahliae resistance of transgenic cotton at various growth stages [SPR9 acts as a negative regulator of the immune response in rice. However, further biochemical experiments are needed to verify how SPR9 is regulated by translation during immune activation in rice.nduction . Consiste plants . A recene plants . These rh stages . Based ospr9 mutants exhibit better disease resistance to RFS.In recent years, studies on different crops have found that plant panicle architectures are closely related to plant disease resistance , 46. In Many wild rice species have typical spread spikelets because the spread spikelets help them to improve the outcrossing seed setting rate and reproductive ability in the field to better adapt to the environment , 50. At spr9 mutant not only enhanced resistance to RFS but also did not affect the important agronomic traits of rice (Figs. SPR9 has good application prospects in future rice disease resistance and improved panicle breeding. For example, to improve the outcrossing rate of male sterile lines, we can transfer spr9 into sterile lines. At the same time, to improve rice smut resistance to a certain extent, we transferred spr9 into restorer lines.In this study, most importantly, our data indicated that the ce Figs. . TogetheSPR9 gene was mapped and identified as a ribosomal protein coding gene. Then, CRISPR/Cas9 knockout experiments confirmed that the SPR9 gene is responsible for the spreading panicle phenotype of the spr9 mutant. Importantly, the spr9 mutant was found to improve resistance to RFS without affecting major agronomic traits, indicative of potential applications of spr9 in broader breeding programs. Taken together, our results revealed that the spr9 gene has good application prospects in future rice disease resistance and improved panicle breeding.In this study, a novel Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3


Dear Editor,Tumour‐infiltrating lymphocytes (TILs) play important roles in the regulation of tumour growth and progression.p = .004). Importantly, the iTIL/sTIL ratio achieved the most significant association with DRFS . The advantage of iTIL/sTIL ratio over iTIL was more obvious after adjusting for age, tumour size, ER (oestrogen receptor) and HER2  status with multivariable Cox regression models . Moreover, when all three lymphocyte factors were considered simultaneously in a multivariable model, the iTIL/sTIL ratio was identified as the only prognostic factor . Prognostic analyses based on the overall survival (OS) of patients resulted in similar results , which provided iTIL and sTIL levels determined from H&E‐stained tumour slides as well as major clinical factors for 335 breast cancer patients.s Figure .n = 192), the ratio exhibited a more significant association with DRFS than iTIL according to the results from both univariable  and multivariable analyses  , similar results were observed  Figure . In the d Figure . Taken tp = .01) outperformed iTIL  and sTIL (p > .1) levels in predicting OS after adjusting for age, tumour stage and ER status .To validate our findings, we analysed the H&E‐stained images and clinical variables that were generated from the TCGA breast cancer (BRCA) data. We applied a deep learning model to separate tumour and adjacent stromal regions in each H&E image. By referring to the study by Abousamra et al.,s Figure . In the In conclusion, our study indicates the iTIL/sTIL ratio is more associated with prognosis compared iTILs and sTILs Figure . The iTIThe authors declare no potential conflicts of interest.Cancer Prevention Research Institute of Texas (CPRIT), Grant No.: RR180061 and National Cancer Institute of the National Institutes of Health, Grant No.: 1R01CA269764Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Microbial cell-free DNA (mcfDNA) sequencing is a promising potential tool in the detection of infectious pathogens when traditional methods fail to identify the causative agent. It may be particularly helpful in identifying pathogens that are present at low levels or that do not grow well in standard culture. The aim of this study was to determine whether plasma mcfDNA sequencing resulted in a change in therapeutic management.th, 2019, and January 4th, 2023, were included. Patient demographics, comorbid conditions, immunocompromised status, indication for testing, pre and post antimicrobial therapies from when the sample was collected, pathogen identified, ID consultation status, and ordering service at the time of testing were recorded. Each patient was analyzed by two independent reviewers for clinically significant microbial findings, treatment, and reviewer concordance with therapeutic management. If discordance between reviewers was present, a third reviewer analyzed the data.A retrospective cohort at UC Davis Medical Center was reviewed. All patients, both pediatric and adult, that underwent plasma mcfDNA (Karius) sequencing between December 11A total of 127 Karius tests were reviewed from 71 patients. Baseline characteristics included 62 (48%) pediatric and 65 (51%) adult tests. 25 (33%) tests were from female patients, and 37 tests (50%) were obtained from immunocompromised patients. The most frequent adult ordering services included the Medical Intensive Care Unit and Bone Marrow Transplant Unit. The most frequent pediatric ordering services included the Pediatric Intensive Care Unit followed by General Pediatrics. 69 (95%) of tests had a formal Infectious Diseases consultation associated with testing. 22 (30%) tests resulted in a change in antimicrobial therapy, and 35 (47%) of tests resulted in a clinically significant organism.In this retrospective cohort study conducted at a single center, Karius testing resulted in clinically significant pathogens in less than half of all tests conducted, and most test results did not lead to a change in therapeutic management. Testing in certain clinical scenarios or high-risk settings may be warranted, however further studies are needed.George R. Thompson, III, MD, Astellas: Advisor/Consultant|Astellas: Grant/Research Support|Cidara: Advisor/Consultant|Cidara: Grant/Research Support|F2G: Advisor/Consultant|F2G: Grant/Research Support|Mayne: Advisor/Consultant|Mayne: Grant/Research Support|Melinta: Advisor/Consultant|Melinta: Grant/Research Support|Mundipharma: Advisor/Consultant|Mundipharma: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support


CaTIFY4b contributes to drought adaptation in chickpea” by Barmukh et al. , Hyderabad, 502324, Telangana, India2Department of Information Technologies, Faculty of Economics and Management, Czech University of Life Sciences Prague, Kamýcká 129, Prague, 165 00, Czech RepublicIn the Acknowledgements section, the additional funding grant is:The Internal grant agency of the Faculty of Economics and Management from the Czech University of Life Sciences Prague, Life Sciences 4.0 Plus, grant no. 2022B0006.


In the process of drilling oil and gas wells, the shrinkage and falling of wellbore walls are often caused by the expansion of mud shale water. To date, conventional additives have been unable to plug the pore throats of shale rock with nanoscale pores and thus cannot effectively solve the problem of wellbore instability encountered in shale formation drilling. In view of this situation, the idea of using nanosilica to plug the nanopore throat of mud shale is proposed to reduce its permeability and to slow water intrusion. The influences of nanosilica on the properties of brine-base drilling fluid drilling fluid are evaluated by measuring the viscosity, filtration loss and swelling of the drilling fluid. The results of laboratory experiments show that the improvement in drilling fluid properties can only be achieved on the basis of salt resistance; that is, a salt-resistant soil slurry should be used. A concentration of 1–5% nanosilica can improve the viscosity of the drilling fluid by increasing the internal friction between particles. However, nanosilica materials are sensitive to salt concentration. Nanosilica particles can be deposited on the surface of a filter cake to block the pores of the filter paper, and the filtration loss reduction rate can reach 40.2%. Blocking the pores of the clay plays a role in preventing the clay from absorbing water and expanding. The optimal addition amount of silica is 3%, and its salt resistance can reach 16%. Considering the experimental results of filtration loss, swelling amount and cost performance, 3%NP + 4%NaCl + SWM-B is selected as the optimal formula. The results of this study can be applied to effectively improve the phenomenon of wellbore instability during drilling in shale formations, and it has important application value. Drilling operations require the right drilling fluid to stabilize shale formations near the borehole, which are prone to expansion and collapse due to interactions with water in the drilling fluid1. This problem is common when drilling in shale formations where water-based drilling fluids are used. This increased occurrence is because the water component of the fluid interacts with the shale formation minerals, resulting in the hydration, disintegration, and dispersion of the formation particles4 this phenomenon causes the clay minerals in the shale to hydrate and swell, leading to wellbore instability, such as wellbore collapse, necking, and pipe jamming6. One of the most important problems in drilling in shale formations is the instability of shale formations, which poses an enormous challenge to the oil and gas drilling industry8. Previous studies have focused on improving the stability of shale walls through the use of a variety of treatment agents with strong inhibition and collapsibility9. Existing studies have shown that nanoscale and microscale pores or fractures are developed in shale gas reservoirs in China11, the pore range is 5 ~ 300 nm, the main body is 80 ~ 200 nm12, and the pore throat size is 5 ~ 50 nm13. To date, conventional additives have not been able to plug nanoscale pore throats of shale, thus forming a thin and dense mud cake14. Drilling fluid intrudes shale formations through these microchannels, further exacerbating shale hydration5.In conventional oil and gas drilling, shale and clay formations account for approximately 75% of the total number of wells drilled, and in shale and clay formations, approximately 70% of borehole instability is associated with shale instability16. Nanoparticles have proven to be more stable than other chemicals during implementation, in addition to significantly improving drilling fluid quality. Nanomaterials have great potential to improve new fluids and devices in the oil and gas industry. Drilling fluid is one of the most challenging areas for nanomaterials to improve their properties. However, the role of nanoparticles in this field is still in its infancy and has therefore attracted considerable attention over the past few years17. Nanomaterials such as nano-SiO218 and laponite19 are beneficial for shale inhibition due to their strong adsorption ability and pore plugging performance, among other advantages20. Therefore, nanosized particles are introduced as bridging particles to successfully plug these orifice throats. By comparing nanosilica, nano-TiO2, and nano-Al2O3 as additives, we find that nanosilica is more effective in reducing liquid loss than nanotitanium and nanoalumina21.Nanofluids have been used in a wide range of oil and gas wells, from drilling to reservoir, cementing, enhanced oil recovery, completion, and processing22. By comparing the changes in viscosity of water-based drilling fluid under the action of nanosilica particles with different concentrations, it is believed that silica nanoparticles improve the rheological properties of water-based drilling fluid and increase the viscosity and yield point of water-based drilling fluid by more than 50%23. verified through laboratory experiments that the addition of nanosilica increases the viscosity of water-based drilling fluid, thus improving the sand carrying capacity and circulation capacity of mud. In addition, nanosilica water-based mud exhibits good mud stability and has a high tendency to prevent formation fluid intrusion. Al-Yasiri et al.24 developed and experimentally tested a novel drilling fluid formulation using a mixture of biopolymer nanosilica particles that can effectively improve the performance of water-based mud, enhancing the rheology, filtration and lubrication properties. Liu et al. reported a novel core–shell structure polymer (NS-DA) based on modified silica nanoparticles as a reducing agent for water-based drilling fluids. The experimental analysis shows that the thermal decomposition temperature of NS-DA is 293 °C. Moreover, the material has outstanding filtration reduction ability25. Sajjadian et al. researched the improvement in the properties of water-based drilling fluids by three nanomaterials: zirconia, multiwalled carbon nanotubes and titanium dioxide nanohybrids. The rheological properties, filtration properties and conductivity experiments show that the mixed nanomaterials and zirconia can effectively improve the rheological parameters of mud, such as plastic viscosity, yield point and gel strength26.Many experiments have been carried out to improve the properties of water-based drilling fluids with nanosilica materials1. Liu et al. synthesized nanosilica/cationic polymer composites (NS-DD) and evaluated them as novel shale inhibitors in water-based drilling fluids. The nanodioxide composite has significant advantages in terms of high temperature inhibition and cutting recovery27. Huang et al.5 described hydrophobic nanosilica for strengthening well walls when using water-based drilling fluids. The borehole strengthening performance of this material was studied through a linear expansion test, hot rolling recovery test and compressive strength test1. The inhibition effect, inhibition mechanism and advantages and disadvantages of different nanocomposites on shale were introduced. Wei et al. proposed a high-performance water-based drilling fluid suitable for the shales of a well support formation. The rheological property, filtration property, lubrication property, temperature resistance, corrosion inhibition property, pollution resistance and cost of this kind of water-based nanosilica drilling fluid were comprehensively evaluated. Nanosilica drilling fluid has advantages in terms of high temperature resistance, inhibition of shale hydration and operating cost28.In addition, when drilling projects are carried out in shale formations, conventional drilling fluids cannot effectively maintain the stability of shale. Adding the right number of nanoparticles to water-based mud can not only improve the rheological and thermal properties but also improve borehole stability of the mud29. We analyse and evaluate the effects of nanodioxide particles on the properties of brine-based drilling fluid by conducting viscosity testing, filtration loss testing and expansion experiments.To date, there are abundant studies on the effects of nanosilica materials on the properties of water-based drilling fluids and the inhibition of shale, and some achievements have been made. However, there are few studies on the effects of nanosilica materials on the properties of brine-based drilling fluids. Salt pollution has become the most serious challenge for water-based drilling fluids under extreme salinity conditions, which can lead to the deterioration of rheological and fluid loss properties, resulting in drilling accidents, such as leakage, formation collapse and borehole instabilityThe materials used in this experiment were saline-resistant soil produced in Anhui Province and bentonite produced in Shandong Province. Xanthan gum (XC) was used as the treating agent. The modified starch (DFD-140) was made in Beijing. The nanosilica material used in the experiment was the nanosilica dispersion liquid produced in Hangzhou. The model was VK-S01B, the average particle size was 15 ± 5/nm, the content of nanosilica was 30%, the solvent was water, and the pH value was 9–11.Two brine drilling fluids, brine-base drilling fluid A (SWM-A) and brine-base drilling fluid B (SWM-B), were formulated according to the slurry configuration method, combined with specifications and references to existing research results. The final test drilling fluid formulations were as follows:Brine-base drilling fluid A (SWM-A): 350 mL water + 10.5 g sodium bentonite + DFD-140 1.5% + 0.1% XC + 0.32 g NaOH.Brine-base drilling fluid B (SWM-B): 400 mL water + 16 g anti-salt soil + DFD-140 1.5% + 0.3% XC + 0.32 g NaOH.30 stated that when the slurry pH was adjusted to 9–10, the purpose was to allow sufficient hydration and dispersion of the bentonite. A total of 42 sets of experiments were performed. The density, plastic viscosity (PV), dynamic shear (YP), and water loss (API-FL) of the corresponding drilling fluid were tested.The base slurry was prepared according to the selected formula, and different contents of salt were added to the base slurry in six groups of 0%, 4%, 8%, 12%, 20%, and 30%, while four groups of different contents of nanosilica materials 0%, 1%, 3%, and 5% were added. Since the silica dispersion itself contained water, the same amount of water was added to the drilling fluid to eliminate the effect of water, which was represented by extra water. If the slurry pH was small, the pH was adjusted to 9–10 by adding an appropriate amount of sodium carbonate. Wu et al.In the test tube, 0%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 12%, 20%, and 30% saline solutions were prepared, and 1% and 6% nanosilica materials were added to investigate the colour, flocculation, and dispersion layer of the mixing system. The results showed that the salt resistance of nanosilica material was strong when the concentration was low, but the salt resistance decreased when the concentration was too high. The results showed that the salt resistance of the nanosilica material was 6%. Nanosilica materials had small particle sizes and large specific surface areas. Under the condition of high salt concentration, it was found that aggregation, flocculation, and stratification appeared. In addition, with the increase in the concentration of nanoparticles in the mixed system, the thermal motion was intensified, and the particles easily stratified and precipitated. The test results are shown in Fig. The NaCl concentration was adjusted from 0 to 30% by adding 1%, 3% and 5% nanosilica to the saline water-based pulp to explore the improvement effect of nanosilica on the properties of SWM-A.In the following figures, PV represents plastic viscosity, AV represents apparent viscosity, and FL represents water loss. The amount of water added to the base slurry should be equal to the amount of water contained in the nanosilica dispersion.Figures 3, the plastic viscosity was 11 mPa·s, the dynamic shear force was 6.5 Pa, the API filtration capacity was 17 mL, and the pH value was 11.To form a comparative experiment and obtain the improvement effect of nanosilica material on drilling fluid, it was necessary to conduct a performance test on the untreated drilling fluid. The density of the untreated drilling fluid was 1.03 g/cmBy changing the concentration of NaCl in brine drilling fluid B, the measured changes in the filtration and viscosity of SWM-B were determined, as shown in Figs. As seen from Figs. SWM-B was supplemented with 1% nanosilica, and the viscosity and filtration test results were obtained, as shown in Figs. As shown in Figs. The results of the viscosity and filtration loss experiments are shown in Figs. When the concentration of NaCl was lower than 20%, the viscosity of SWM-B showed an obvious increase with increasing salt concentration. With the salt concentration exceeding 20%, the increase rate of viscosity decreased and stabilized. Under the condition of a low concentration of NaCl, 3% nanosilica had a significant filtration loss reduction ability, and the maximum filtration loss reduction could reach 40.2% relative to the brine drilling fluid. When the salt concentration was greater than 16%, the nanosilica had a negative effect on the drilling fluid, probably because the high salt concentration not only inhibited the hydration of the soil but also caused the nanoparticles to lose their dispersion ability and exhibit aggregation, flocculation and stratification; they could not form a continuous and dense mud cake, leading to an increase in filtration.The viscosity and filtration experiments were conducted by adding 5% nanosilica to SWM-B, and the results were obtained as shown in Figs. Figures By integrating the filtration amounts of SWM-B brine drilling fluid under different concentrations of nanosilica, the trend of the variation in filtration properties of brine drilling fluid with the change in nanosilica content was obtained, as shown in Fig. As shown in Fig. The expansion amount experiments were conducted by adding different concentrations of silica nanomaterials to the salt-resistant drilling fluid, and since the silica dispersion itself contained water, an equal amount of water was added to the drilling fluid; this process was conducted to exclude the effect of water, which was called extra water. The results of the effect of silica nanomaterials on the expansion amount are shown in Figs. From Figs. Nanosilica has a significant improvement effect on the performance of saline drilling fluid with anti-saline soil configuration and a negative effect on the performance of bentonite brine drilling fluid configuration.In SWM-B brine-based drilling fluid, nanosilica particles can be deposited on the surface of filter cake effectively, the pores of filter paper can be sealed, and filtration loss can be significantly reduced. When the concentration of nanosilica is 1–5%, the optimal concentration of NaCl for its plugging capacity is 0–8%.The addition of nanosilica can effectively improve the inhibition performance of brine-based drilling fluid and reduce the amount of clay swelling. However, the amount of nanosilica does not easily exceed 5%; otherwise, it has a negative impact.1–5% concentration of nanosilica can effectively improve the viscosity of brine drilling fluid, but the improvement effect is sensitive to the change in salt concentration.Based on the experimental results, the optimal addition amount of silica is 3%, and the salt tolerance reaches 16%. In addition, considering the cost performance of the drilling fluid formula, 3% NP + 4% NaCl + SWM-B is selected as the best formula of brine-based drilling fluid. This formula can be used as a reference for drilling projects in shale or salt formations.


Perioperative fluid management during major abdominal surgery has been controversial. Postoperative pancreatic fistula (POPF) is a critical complication of pancreaticoduodenectomy (PD). We conducted a retrospective cohort study to analyze the impact of intraoperative fluid balance on the development of POPF.This retrospective cohort study enrolled 567 patients who underwent open pancreaticoduodenectomy, and the demographic, laboratory, and medical data were recorded. All patients were categorized into four groups according to quartiles of intraoperative fluid balance. Multivariate logistic regression and restricted cubic splines (RCSs) were used to analyze the relationship between intraoperative fluid balance and POPF.2, preoperative blood glucose < 6 mmol/L, long surgery time, and lesions not located in the pancreas were independent risk factors for POPF.The intraoperative fluid balance of all patients ranged from -8.47 to 13.56 mL/kg/h. A total of 108 patients reported POPF, and the incidence was 19.0%. After adjusting for potential confounders and using restricted cubic splines, the dose‒response relationship between intraoperative fluid balance and POPF was found to be statistically insignificant. The incidences of bile leakage, postpancreatectomy hemorrhage, and delayed gastric emptying were 4.4%, 20.8%, and 14.8%, respectively. Intraoperative fluid balance was not associated with these abdominal complications. BMI ≥ 25 kg/mThe study did not find a significant association between intraoperative fluid balance and POPF. Well-designed multicenter studies are necessary to explore the association between intraoperative fluid balance and POPF. From the perspective of anesthesiologists, perioperative fluid management is an important part of their anesthesia scheme. The fluid management strategies vary among anesthesiologists. In recent years, the classical perioperative fluid balance described in textbooks has been challenged. Fluid overload was reported to increase cardiopulmonary complications and impair the recovery of gastrointestinal function . RestricPancreaticoduodenectomy (PD), as one of the most complex and traumatic abdominal operations, is associated with many postoperative abdominal complications, including pancreatic fistula, bile leak (BL), postpancreatectomy hemorrhage (PPH), delayed gastric emptying (DGE), and intra-abdominal infection . PostopeWe used to hold the opinion that whether POPF occurred depended largely on the technique and experience of surgeons. Some previous studies have clarified multiple perioperative factors related to POPF –10. HoweAfter approval by the institutional ethics board of Peking Union Medical College Hospital , the study created a retrospective cohort of patients who underwent open pancreatoduodenectomy between July 2016 and July 2021 at Peking Union Medical College Hospital. The surgeries were performed by an experienced surgical team. Indications for the surgery were tumors and other disorders of the pancreas, duodenum, bile duct, and adjacent other organs. The standard procedure of PD involved the removal of the pancreatic head, duodenum, first 15 cm of the jejunum, common bile duct, gallbladder, partial gastrectomy, and the reconstruction of gastrointestinal continuity. An end-to-side anastomosis was adopted in pancreaticojejunostomy, biliary-enteric anastomosis or gastrointestinal anastomosis. Modified PD, pylorus-preserving pancreaticoduodenectomy (PPPD) which preserves the gastric antrum, pylorus, and proximal 3 to 4 cm of the duodenum has been gradually used in the hospital for gastrointestinal continuity. The inclusion criteria were as follows: 1) older than 18 years; 2) ASA I–III; 3) patients who underwent elective open PD . The exclusion criteria were as follows: 1) patients who underwent total pancreatectomy and duodenectomy and 2) patients who lacked fluid records.According to the Chinese conventional infusion regimen, the intraoperative fluid volume was calculated by subtracting the total fluid output  from the total fluid input . Specifically speaking, we used the “4–2-1” calculation to evaluate preoperative loss during fasting and physiological required volume which were essential for metabolism and fluid redistribution during the perioperative period. For example, A 60-kg patient would require . The grouping thresholds of the laboratory data were determined by the medical reference value. The intraoperative variables were comprised of the anesthetic mode, surgery option, surgery time, and intraoperative fluid management . PD and PPPD were two surgical options.We collected demographic, laboratory, and medical data via the electronic medical record system and surgical anesthesia system of Peking Union Medical College Hospital. The baseline characteristics included age, sex, BMI, and comorbidities. BMI was expressed as a continuous variable as well as a categorical variable, and BMI was categorized into two groups: < 25 kg/mAll participants were categorized into four groups according to quartiles of intraoperative fluid balance. Continuous variables were presented as the mean ± standard deviation or medians and interquartile ranges where appropriate. Categorical data were shown as numbers and percentages. Univariate and multivariate nonconditional logistic regression analyses were performed to assess the association between the fluid balance quartiles and primary outcome. Variables potentially associated with POPF or clinically important were selected for logistic regression. Odds ratios (ORs) and 95% confidence intervals (95% CIs) across the fluid balance quartiles (the lowest quartile as a reference) were calculated. Based on a multivariate logistic regression model, we used restricted cubic splines (RCSs) to explore the nonlinear dose‒response relationship between intraoperative fluid balance and POPF, with three knots defined at the 10th, 50th, and 90th percentiles of intraoperative fluid balance . Data weWe collected 595 patients who underwent elective open PD between July 2016 and July 2021 at Peking Union Medical College Hospital, and 567 patients were finally included for statistical analysis. Patient enrollment flowchart was shown in Fig. P value of the test for the overall association between intraoperative fluid balance and POPF was insignificant, which meant that regardless of the shape of this curve, there was no significant association between intraoperative fluid balance and POPF.Among the 567 patients, 108 were diagnosed with POPF, and the incidence was 19.0%. Among them, 80 patients were categorized into Grade B while 28 patients belonged to Grade C. There were 22 patients with POPF in the Q3 group of intraoperative fluid balance, which was lower than the other three groups. First, we used intraoperative fluid balance as a categorical variable and the lowest quartile as the reference to conduct a univariate analysis. The results showed no statistically significant relationship between intraoperative fluid balance and POPF. Then, multivariate logistic regression was performed after adjusting for potential confounding factors, including age, sex, BMI, abdominal surgery history, preoperative bilirubin, preoperative blood glucose, ASA classification, anesthetic mode, surgery option, and lesion site. The outcome did not change. Furthermore, we took into account the differentiation between grade B and grade C POPF, the outcome kept the same. The results are summarized in Table P < 0.1 in the multivariate regression analysis, and the results are shown in Table The incidences of BL, PPH, and GED were 4.4%, 20.8%, and 14.8%, respectively. We found that the association between intraoperative fluid balance and these abdominal complications was not statistically significant. For risk factors for POPF, after univariate regression analysis, we included the variables with Fluid management is complex and variable. Some studies tend to support restrictive fluid management, but the controversy over fluid management has never ceased. Zhang et al. enrolled 301 patients for a retrospective study, and the results showed that higher postoperative fluid balance appeared to be associated with morbidity after PD, systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), bleeding, and heart failure . HoweverThe relationship between intraoperative fluid balance and the specific abdominal complication of POPF is also ambiguous. Some studies have demonstrated that the incidence of POPF in the high fluid volume group was higher than that in the low fluid volume group during PD, and intraoperative fluid excess was also considered a risk factor for POPF –23. NeveThe study consecutively enrolled 567 patients who underwent PD surgery, and they were divided into four groups by the quartiles of intraoperative fluid balance to explore any difference in POPF between the four groups. Then, intraoperative fluid balance was regarded as a continuous variable, and we used restricted cubic splines to analyze whether a dose‒response relationship existed between intraoperative fluid balance and POPF. Finally, the relationship was found to be statistically insignificant. In addition, we did not find an association between intraoperative fluid balance and BL, DEG, or PPH. Identically, Braga et al. grouped patients undergoing PD according to whether they received a comprehensive ERAS protocol or standard perioperative care. A comprehensive ERAS protocol contained many items, such as a lack of bowel preparation and shortening of the preoperative fasting period, and the intravenous fluid infusion rate was lower in the ERAS group. The results showed that the incidence of POPF was similar in the two groups, as were BL, DGE, and PPH .2, preoperative hypoglycemia (blood glucose ≤ 6 mmol/L), long surgery time, and lesion not located in the pancreas were risk factors for POPF. Preoperative hypoglycemia, lesions not located in the pancreas, and BMI might lead to a softer pancreas, thereby influencing the occurrence of POPF [Sex, BMI, fasting blood glucose level, pancreatic texture, pancreatic duct, pancreatojejunostomy anastomosis technique, and tumor location were related risk factors for POPF. Our study found that BMI ≥ 25 kg/m of POPF –10. In a of POPF . Recentl of POPF .The definition of POPF is relatively uniform in many studies, but the calculation of perioperative fluid balance or fluid management varies. There is no uniform standard for the definition of perioperative fluid balance or fluid management, especially for the cutoff values, which can vary –23, and The anesthesiologists in our institution always formulate the intraoperative fluid management plans in advance based on the patient’s physiological needs. In fact, the intraoperative fluid management is affected by many factors, such as intraoperative bleeding, patient cardiovascular function, depth of anesthesia, and use of vasoactive drugs in a major surgery such as PD. Therefore, many anesthesiologists often use invasive monitoring methods in combination with other hemodynamic parameters to evaluate the patient's volume status and adjust their fluid management plan in a timely manner. As a result, the concept of goal-directed fluid therapy (GDFT) may be the most reasonable fluid management strategy available.The present study had some limitations. First, this study analyzed the association between intraoperative fluid balance and the incidence of POPF but did not further analyze the effect of intraoperative fluid balance on the severity of POPF. Second, confounding factors such as pancreatic texture and pancreatic duct played an influential role in whether pancreatic fistula occurred. However, we collected data retrospectively and the operative recordings in the electronic medical record system seldom documented these variables. The association between intraoperative fluid balance and the development of POPF could be more reliable if adjusted by these related factors. Third, all the patients were treated at a single center. To validate the effect of intraoperative fluid balance on POPF, a multicenter study with a larger number of patients is needed in the future.Our study failed to find a significant association between intraoperative fluid balance and POPF. Furthermore, the correlations between fluid balance and other abdominal complications were statistically insignificant. On the other hand, the findings of this study indicated that BMI, blood glucose, surgery time and lesion site were closely related to POPF. It is necessary to unify the definition of fluid balance and develop well-designed studies to validate the optimal fluid strategy and reduce abdominal complications such as POPF. Until then, the intraoperative fluid management plan of PD should be individualized and adjusted according to the actual situation.


Lupinus termis), and its methylated derivative (M11S), were investigated against seven pathogenic gram-positive and gram-negative bacteria. The MIC of 11S ranged from 0.1 to 4.0 μg/ml against 0.025 to 0.50 μg/ml for M11S, excelling some specific antibiotics. The MICs of M11S were 40–80 times lower than some specific antibiotics against gram-positive bacteria and 2–60 times lower than some specific antibiotics against gram-negative bacteria. One MIC of 11S and M11S highly reduced the liquid growth of all tested bacteria during 24 h at 37°C. They also inhibited biofilm formation by 80%−86% and 85%−94%, respectively (gram-positive), and 29%−44% and 43%−50%, respectively (gram-negative). M11S prevented biofilm formation by gram-positive bacteria at minimum biofilm inhibitory concentration (MBIC), 0.025–0.1 μg/ml against 0.1–0.5 μg/ml for gram-negative bacteria, i.e., 4–20 times and 4–7 times anti-biofilm inhibitory action compared with 11S, respectively. Biofilm formation of two bacteria revealed no adhered cells on glass slides for 24 h at 37°C, i.e., was entirely prevented by one MBIC of 11S and M11S. Scanning electron microscopy indicated microbial biofilm deformation under the action of 11S and M11S, indicating their broad specificity and cell membrane-targeted action.The antibacterial and anti-biofilm activities of the 11S globulins isolated from lupin seeds ( Pseudomonas aeruginosa and Staphylococcus aureus, are among the most virulent and problematic pathogens, causing life-threatening hazards. Biofilm can protect bacterial species from environmental challenges, e.g., nutrient depletion and the threat of antibiotics , obtained from a local market in Zagazig City, Sharkia, Egypt, were thoroughly cleaned and finely ground at the maximum speed with a Moulinex mixer for 3 min , to pass through a 1 mm2 sieve. Then, the powder was defatted with n-hexane for 8 h, and the defatted meal was dried and stored at 4°C till analysis.Lupine seeds  precipitated 11S globulin. Following dispersing and solubilizing the precipitate, the salts were removed from the solution by dialysis using the same buffer for 48 h.The 11S globulin was isolated from a defatted meal of lupine seeds following the procedure outlined by Abdel-Shafi et al. . To isolThe procedure mentioned by Sitohy et al. , 2001 waProtein pH–solubility curves were assayed using lyophilized 11S globulins and M11S from lupine seeds in the pH range of 2–10  as the solvent. Tetramethylsilane (TMS) was used as an internal standard to report the chemical shifts in δ (ppm).The potassium bromide (KBr) pellet method  was defined using agar well diffusion and disk diffusion methods. The MIC of an antimicrobial agent was taken as the lowest concentration (μg/ml) inhibiting the visible growth of a microorganism after overnight incubation.9 CFU ml−1 before spreading uniformly onto individual plates using sterile cotton swabs. Wells of 6 mm in diameter were created on BHI agar employing a gel-puncturing tool. Aliquots (40 μl) of tested 11S solutions  and M11S solutions  were transferred into each well. A transparent ruler measured the developed inhibition zone diameters after 24-h incubation at 37°C.The conventional well diffusion assay was used to determine the antibacterial activity against 11S globulin and the M11S against seven tested bacteria  of 1 MIC of each tested substance (11S and M11S) was added to 4-h-old bacterial cultures in BHIB, incubated at 37°C, and dispensed into the wells of a 96-well plate. The negative control was BHIB only, whereas the positive control contained cell cultures without adding treatment. Growth was determined by measuring turbidity  at 600 nm for 24 h using a microplate reader .The anti-biofilm activity of 11S and M11S was assessed according to the protocol mentioned by Saporito et al. . In brie−1) in BHIB supplemented with 1% (w/v) glucose were dispensed into each well of a 96-well plate and combined with another 100 μl of 11S  or 100 μl of M11S  for M11S. The negative control was BHIB only, whereas the positive control contained cell cultures without 11S or M11S. Following 24-h incubation at 37°C, the liquid layer of each well was decanted and gently rinsed twice with 300 μl of PBS (pH: 73 ± 03). The plates were air-dried for 30 min, stained with 0.1% (w/v) crystal violet for 30 min at room temperature  and K. oxytoca (gram-negative), were selected to compare the morphological appearance of the bacterial biofilm developed in the absence or presence of 11S and M11S using Jeol JSM-6510 L.V. Scanning electron microscopy (SEM) and following the standard protocols . Then, the two tested bacteria were treated with 1 MIC of 11S globulin and M11S, i.e., 0.5 and 0.025 μg/ml in the case of S. aureus and 0.5 and 0.1 μg/ml in the case of K. oxytoca, respectively. The control for each bacterium did not receive any treatment. All plates were placed in a shaking incubator for 24 h of incubation period, after which the disks were removed and gently washed three times with sterile PBS to eliminate the loosely attached bacterial cells, followed by a 2-h fixation with 2.5% (v/v) glutaraldehyde in PBS solution at 4°C. Finally, the plates were washed twice with PBS for 10 min before dehydration with graded ethanol concentrations: 30%, 50%, 70%, 90%, and 100% (v/v), followed by a 10-min subjection to serial dilutions of acetone  at 25°C. The samples were then dried to a critical point using an automated critical point dryer , before coating with gold–palladium and observation at 30 K.V.Two bacteria, L. monocytogenes, K. oxytoca, L. ivanovii, S. aureus, P. mirabilis, P. aeruginosa, and S. typhimurium), as well as the different concentrations, i.e., 0.05, 0.1, 0.3, 0.5, 1, 2, 4, and 8 μg/ml for 11S and 0.0125, 0.025, 0.05, 0.1, 0.3, and 0.5 μg/ml for M11S, and the interactions between them for inhibition zone diameter. The null hypothesis was rejected if the p-value of the ANOVA was <0.05, referring to the absence of significant differences between the means. P-values higher than 0.05 indicated the absence of significant differences. The two-way ANOVA test with a post-hoc test using Duncan's test was applied to make multiple comparisons between the averages of different groups. Means followed by the same letter were not significantly different at the 5% probability level (Duncan's multiple range tests). The results were presented as the means of three replicates ± SD.SPSS program version 23 statistically analyzed the mean and standard deviation data. Two-way ANOVA tests were performed to compare the different microorganisms , referring to the presence of aliphatic CH3 group, C=O stretching, and C=O stretching ester, respectively, according to I.R. Spectrum Table . Alternatively, the native form (11S) showed an I.R. absorption peak at 1,391 cm−1, absent from the modified form (M11S), referring to the free carboxylate groups. 1H NMR of native 11S globulin indicates the presence of main six δ signals at 1.51 , 5.09 , 5.52 , 7.48 , 7.50 , and 7.58–7.66 . The esterified product (M11S) indicated other 6 signals, 4 of them are shared with the native form (11S), while the other 2 are different  by the disk diffusion method, demonstrating antibacterial activity against all the tested bacteria, while the whole lupine seed protein isolate (LPI) did not produce any inhibition zones (data not shown).S. aureus and K. oxytoca, respectively) equally in terms of the specific MIC, detected at 0.5 μg/ml for both. However, the inhibition zone area data indicated that the gram-positive bacteria were more susceptible than the gram-negative ones. Chemically modifying 11S into the methylated form (M11S) enhanced the antibacterial action, where the effect on the gram-positive bacteria was much more pronounced than on the gram-negative bacteria. Regarding MIC, M11S recorded 0.025 and 0.100 μg/ml against the gram-positive and the gram-negative bacteria, respectively. Expanding the analysis to the whole bacterial collection (3 gram-positive and 4 gram-negative) confirmed that 11S and M11S were more inhibitory on gram-positive than gram-negative bacteria , respectively, and 0.1, 0.3, 0.5, and 0.1 μg/ml in the second case , respectively. The MIC of M11S was 4, 20, and 10 times less than the corresponding values of 11S against the three tested gram-positive bacteria, respectively, and 5, 3, 8, and 20 times lower than the corresponding values of 11S against the gram-negative bacteria, respectively.The data in bacteria . At the Listeria monocytogenes and K. oxytoca were the most inhibited organisms.The data in L. monocytogenes, L. ivanovii, and S. aureus, exhibiting MIC at 2, 7.5, and 1 μg/ml, respectively, while the four investigated gram-negative bacteria were explicitly susceptible to gentamycin and ciprofloxacin. The first bacterium (K. oxytoca) showed MIC at 6 μg/ml. In contrast, the three other bacteria  were susceptible to ciprofloxacin at 2, 1, and 4 μg/ml, respectively. Comparatively, the MICs of M11S were much lower than the specific antibiotics but slightly lower than 11S.L. monocytogenes while 1 and 0.1 μg/ml against L. ivanovii, respectively. Compared with the specific antibiotics, the MICs of M11S were 80, 75, and 40 times lower than those of the specific antibiotics against L. monocytogenes, L. ivanovii, and S. aureus, respectively. At the same time, they were 60, 2, 0.5, and 40 times lower than the respective values against the gram-negative bacteria, such as K. oxytoca, P. mirabilis, P. aeruginosa, and S. typhimurium.In the case of the three tested gram-positive bacteria, the MICs of 11S and M11S were 0.1 and 0.025 μg/ml against L. monocytogenes, L. ivanovii, and S. aureus). The anti-biofilm formation activity exerted by M11S against the gram-negative bacteria such as K. oxytoca, P. aeruginosa, P. mirabilis, and S. typhimurium registering 4%, 14%, 12%, and 48% increases over 11S.The data in L. monocytogenes, S. aureus, and L. ivanovii) at MBIC of 0.1, 0.5, and 1 μg/ml against 0.025, 0.025, and 0.1 μg/ml for M11S. Similarly, the MBIC against the gram-negative bacteria, such as K. oxytoca, P. mirabilis, P. aeruginosa, and S. typhimurium, recorded 0.5, 2, 4, and 2 μg/ml in the case of 11S against 0.1, 0.3, 0.5, and 0.1 μg/ml in the case of M11S. The MBIC and MIC of 11S and M11S , with their corresponding MICs , revealeand M11S  revealedS. aureus and K. oxytoca after overnight growth on BHIB in the absence or presence of 11S and M11S at 1 MBIC at 37°C under 150 rpm shaking condition followed by 24 h of incubation. Colony count was performed and calculated as cells per 1 cm2 biofilm. The data in S. aureus (gram-positive) and K. oxytoca (gram-negative) as 7.23 and 6.24 log CFU/cm2 in the absence of the two antibacterial agents. Both 11S and M11S, at 1 MBIC at 37°C under 150 rpm shaking condition followed by 24-h incubation, revealed no attached cells, confirming that they have equal potencies to completely counteract the formation of microbial biofilms developed by either gram-positive or gram-negative bacteria. This test indicated complete prevention of the biofilm formation in each case under low concentrations of the two antibacterial agents, i.e., 0.5 and 0.5 μg/ml in the case of 11S and 0.025 and 0.1 μg/ml in the case of M11S against S. aureus and K. oxytoca, respectively.The quantitative biofilm formation assay was conducted to enumerate both aureus (gram-positive) and K. oxytoca (gram-negative), on stainless-steel disks immersed in the wells of 6-well plates containing BHIB and 100 μl of an overnight culture (108 CFU ml−1) in the absence or presence of 11S and M11S (1 MIC) after 24-h incubation at 37°C. The SEM image of the control S. aureus (gram-positive) formed biofilm presents spherical bacterial cells dispersed on the adhered biofilm matrix. The bacterial cells lay on the extracellular polymer surface without fragmented or indented cells. The cells seem adherent to each other and confluent. The presence of 1.0 MBIC concentrations of 11S globulin (0.5 μg/ml) and M11S (0.025μg/ml) in BHIB media containing S. aureus and K. oxytoca reduced the relative content of the intact cells, thus destroying the biofilm after 2.5 h of incubation at 37°C. After treatment with 11S or M11S (1 MBIC), a few regular cells appeared, while most cells were either moderately or entirely deformed. The resulting apparent biofilm deformation was more observed in the case of M11S.The SEM images in The enhanced migration of the methylated form (M11S) toward the cathode proves its bigger net positive charges than the native globulin (11S), apparently due to the neutralization of the side-chain negative carboxylate groups on the protein molecule following the study by Mahgoub et al. . ElevatiComparing the FTIR spectrum data of M11S with the native form (11S) confirmed the formation of the ester groups and the disappearance of the free carboxylic groups on the modified protein molecules. The similarity of the other absorption peaks indicates that the modification reaction was mainly restricted to the regions rich in free carboxylic groups arising primarily from residual aspartate and glutamate groups on the surface protein molecules.The presence of two δ signals in the HNMR profile of M11S (2.68) is different from those of 11S (5.09), whereas the other four signals are similar, confirming that the esterification reaction induced only a few structural changes while maintaining most of the original structural features. Additionally, the shift of the signal appearing at 7.5 Hz in the native protein (11S) referring to the imine groups  to be at 8.56 Hz in the modified protein (M11S) may be due to the incorporated electron-donating methyl groups which enhance the electron intensity around the imine groups transforming them into a de-shielded status. Alternatively, the lower levels of proton resonance for most groups in the modified protein (M11S) may refer to a general shielding process by the incorporated methyl groups. Initially, these chemical changes predicted and supported outstanding biological activities of the methylated products as antibacterial and anti-biofilm formation agents.The values of both MIC and inhibition zones indicated that protein modification generally enhanced the antibacterial activity against the two bacterial types (gram-positive and gram-negative), following similar proteins , inhibiting bacterial colonization and preventing biofilm formation   may refer to a higher anti-biofilm potential. Calculative comparisons substantiated this conclusion as the anti-biofilm inhibitory action of M11S represented 4–20 times that of 11S against the gram-positive bacteria and 4–7 times against the gram-negative bacteria. The recorded lower values of the MBICs of M11S than 11S confirm its higher anti-biofilm capacity, probably due to the higher cationic nature following Sindelo et al.  and OpenS. aureus and K. oxytoca revealing no attached cells when subjected to 1 MBIC of 11S and M11S at 37°C indicated equal potencies to completely counteract biofilm formation by low concentrations of the two agents (0.5 and 0.5 μg/ ml). This action resulted probably from a quick adhesion of the tested proteins (11S and M11S) on the surface of Petri dishes due to their cationic nature, preventing the bacterial adhesion agreeing with Singh et al.  and gram-positive bacteria (S. aureus) is a significant result since the first one is known as an opportunistic and clinically dangerous pathogen  were nearly close, and the MBIC of M11S is always smaller than that of 11S. Both 11S and M11S excelled specific antibiotics in antibacterial efficiency and can be nominated as proper antibacterial substitutes. The more positively charged esterified product (M11S) is highly efficient as an anti-biofilm agent since its more considerable positive charges facilitate its attachment to the negatively charged solid surfaces, either metallic, glass, or plastic, thus preventing bacteria from forming biofilms. Moreover, M11S can be highly recommended as an anti-biofilm agent.Both 11S and M11S lupine seed globulins can be employed as effective antibacterial and anti-biofilm formation agents, both broad-specific and effective. The antibacterial activities of 11S and M11S considerably affected the growth of the pathogenic bacteria either on the solid agar or in liquid media. On the solid agar media, very minimal MIC values marked both 11S (0.1–4.0 μg/ml) and M11S , where the MICs of M11S were 4, 20, and 10 times lower than the corresponding values of 11S against the three tested gram-positive bacteria, The original contributions presented in the study are included in the article/GE: Resources, Supervision, Writing—original draft. SA-S: Resources, Supervision, Writing—review and editing. ME-N: Investigation, Methodology, Software, Writing—original draft. WS: Investigation, Methodology, Software, Writing—review and editing. AO: Methodology, Software, Conceptualization, Writing—original draft, Writing—review and editing. MS: Conceptualization, Writing—original draft, Writing—review and editing, Supervision. BS: Writing—review and editing, Funding acquisition, Validation.


The fluid and vasopressor regimes were characterized by an annual decrease in fluid balance  and an annual increase in the amount of norepinephrine of 0.002 µg·kg−1·min−1 . The interaction between the fluid balance and norepinephrine levels resulted in a U-shaped association with the risk of AKI; however, the magnitude and shape depended on the reference categories of confounders (age and BMI). We conclude that decreased intraoperative fluid balance combined with increased norepinephrine administration was associated with an increased risk of AKI. However, other potential drivers of the observed increase in AKI incidence need to be further investigated in the future.The use of norepinephrine and the restriction of intraoperative hydration have gained increasing acceptance over the last few decades. Recently, there have been concerns regarding the impact of this approach on renal function. The objective of this study was to examine the influence of norepinephrine, intraoperative fluid administration and their interaction on acute kidney injury (AKI) after cystectomy. In our cohort of 1488 consecutive patients scheduled for cystectomies and urinary diversions, the overall incidence of AKI was 21.6% (95%—CI: 19.6% to 23.8%) and increased by an average of 0.6% (95%—CI: 0.1% to 1.1%,  Radical cystectomies with urinary diversions are associated with a high postoperative complication rate up to 60%). In addition, preoperative impaired renal function due to patient comorbidities, preoperative tumour-associated postrenal obstructions or nephrotoxic medication is not uncommon. Postoperative acute kidney injury (AKI) is associated with increased morbidity, mortality and length of hospital stay (LOS) following many types of major cardiac and non-cardiac surgeries ,4,5,6,7.0%. In ad® protocols to avoid salt and water overload [During the last decades, intraoperative fluid administration has evolved from a liberal to a more restrictive regimen and has become a cornerstone of ERASoverload . While poverload ,12,13,14overload . In addioverload ,17,18.Therefore, concerns have arisen that restrictive perioperative hydration and the supportive use of vasopressors potentially cause hypovolemia, which in turn can lead to renal dysfunction . On one Postoperative AKI has been associated with increased morbidity, mortality and LOS in multiple major cardiac and non-cardiac procedures ,3,4,5,6.The goal of this study was to determine the association between intraoperative hydration and vasopressor administration with postoperative AKI in patients undergoing cystectomies and urinary diversions and particularly to investigate whether there is an interaction between these two entities with respect to AKI. In addition, we describe the trends in fluid and norepinephrine administration over 20 years in a high-case-load, single tertiary referral centre for cystectomy and urinary diversion and identify risk factors for AKI using a multivariable logistic regression.This observational study reports on a consecutive cohort from a single tertiary centre and is in accordance with the STROBE statement. Ethical approval for this study was provided by the Ethical Committee of Canton Bern, Switzerland  on 2 June 2016, and the need for informed consent was waived.All patient data were collected from a prospectively maintained cystectomy database, which fully complies with the federal act on research involving humans. Patients and procedures from 1 January 2000 to 30 June 2020 were extracted from the database and completed from the patients’ paper charts and anaesthetic protocols.At our institution, open cystectomies with urinary diversions and anaesthesia induction and maintenance have been performed following the same standardized surgical technique for the last 20 years as described previously ,25.−1 bolus, propofol 2 mg·kg−1 and rocuronium 0.6–0.9 mg·kg−1 intravenously to facilitate endotracheal intubation) and maintenance of anaesthesia with halogenics were performed according to our daily practice.After insertion of an epidural catheter at the low thoracic level, induction of anaesthesia . Packed red blood cells (PRBCs) were transfused if haemoglobin values decreased to <80 g·L−1. Assessment of urinary output was not feasible during surgery because of external derivation of the ureter.During the observation period, fluids administered were lactated Ringer’s solution and 6% hydroxyethyl starch 130/0.4 (starting from 2007) or 4% gelatin (before 2007). As of 2007, we aimed for a protocol-driven systematic restrictive fluid administration using a pre-emptive continuous administration of norepinephrine at around 1 to 2 µg·kgPostoperative hydration consisted of 1000 mL of Ringer’s solution and 500 mL of glucose 5% daily until resumption of normal food intake . In case−1·h−1). Likewise, continuous administration of norepinephrine (µg·kg−1·h−1) was recorded.The data collected included all relevant baseline characteristics, preoperative urine drainage due to postrenal obstruction and oral medication. Surgical factors recorded included the duration of surgery, type of urinary diversion, blood loss and the need for blood transfusion. Anaesthetic factors recorded included the type and total amount of fluid administered , intraoperative fluid balance  and fluid loss (OUT: blood loss in mL)) and differentiation in mL per kg bodyweight per hour  classification based on changes in plasma creatinine levels ,28. In at-test  or the Mann–Whitney test (skewed) for quantitative variables.In terms of summary statistics, categorical variables were presented by means of counts and percentages and numerical variables by means of mean and standard deviation in cases of normally distributed variables and by median and interquartile range otherwise. Unadjusted, exploratory landmark analyses with respect to the primary outcome AKI for baseline, intraoperative and postoperative variables were computed using the chi-square test or Fisher’s exact test for qualitative variables with either Student’s We used a data-driven approach to illustrate the various relationships between the variables in our dataset. First, all pairwise correlation coefficients  were computed. Second, univariable associations as expressed by the logarithmic odds ratio of each variable with respect to the primary outcome of AKI were computed using logistic regression. To facilitate the comparison between different numeric variables of different scales and value ranges, the numerical variables were scaled with respect to their mean and standard deviation for univariable logistic regression. The correlation matrix was visualized with a network graph and a directed force layout , in whicNetworks provide a versatile tool for visualizing and investigating patterns in patient- and treatment-related variables. Given the subtle shifts in the network over time particularly with respect to fluid management, we continued to quantitatively examine the temporal trends in the variables in more detail and quantify their relationship with the incidence of AKI over the observational period.Descriptive time series of different intraoperative fluids and norepinephrine administration were computed with a general additive model (GAM) to account for possible non-linear features over time. We adjusted for age, BMI and use of antihypertensives, beta-blockers, statins and platelet aggregation inhibitors as well as for hydronephrosis, nephrostomy, double-J stent (referred to as DJ stent in the figures and tables) and Charlson score.The adjusted associations of both intraoperative fluids and norepinephrine with AKI risk were computed using logistic regression, in which a cubic spline approach for each type of fluid was chosen to allow for possible non-linear associations; a fluid–vasopressor interaction was omitted. We further computed a multivariable logistic regression model accounting for the interaction between total fluid balance and norepinephrine interaction.The selection of variables used for confounder adjustment was based on clinical considerations and clinical practice; no variable selection procedure  was performed as this might lead to biases in the estimated regression coefficients .−1 min−1 for the norepinephrine administration was chosen to translate the non-linear fluid–vasopressor interaction to the nomogram. Model performance was examined using the area under the receiver operating characteristic curve (AUROC) and Brier score; model calibration was assessed using a calibration curve.To translate the associations into clinical risk prediction, a nomogram was computed based on a multivariable logistic regression model . Note, hIn terms of missing data, a complete case analysis was performed.rms package [ggraph package [psych package [emmeans package [Statistical significance was set at <0.05, and all computations were performed with R version 4.0.2 . The mul package . The net package . The com package . Estimat package .The patients with insufficient follow-ups  or who died ((N = 1) within 72 h after surgery) were excluded from the analysis, leaving 1483 (data missing: 0.34%) for the study.−1·h−1 (colloids), 1.1 (IQR: 0.8 to 1.7) mL·kg−1·h−1 (PRBCs) and 1.2 (IQR: 0.8 to 1.6) mLkg−1h−1 (FFP), respectively. Norepinephrine was administered in 69.7% of the patients with a median dose of 0.04 (IQR: 0.02 to 0.06) µg·kg−1·min−1. Overall, median amounts of 4.5 (IQR: 3.2 to 6.0) mL·kg−1·h−1 of crystalloids and 5.1 (IQR: 3.6 to 7.6) mL·kg−1·h−1 of total fluids were administered with a fluid balance (in–out) of 2.9 (IQR: 1.6 to 4.9) mL·kg−1·h−1, in which the quantity of each differed in patients with and without AKI .Anaesthesia-related details regarding the administration of intraoperative fluids and vasopressors are shown in The variables presented in During the years 2000–2007, there were strong associations between colloids, crystalloids, PRBC, FFPs and the fluid balance as can be seen by means of the arc widths. In addition, comorbidities and risk factors were strongly associated. In the second period (2008–2020), there was a noticeable shift in terms of the following associations: the comorbidities were grouped closer together, and the intraoperative fluids, for example the PRBCs and FFPs, were further away from the total fluid balance and are less correlated (they have thinning arc widths).Moreover, norepinephrine was further away from the total fluid balance than in the previous period.The time evolutions of the confounder-adjusted fluid management A,B and n−1·h−1, 95%—CI: −0.31 to −0.26 mL·kg−1·h−1, p < 0.001) and the fluid balance  was observed over the observation time. The administration of colloids decreased from 98.0% in all patients during the year 2000 to 35.9% in 2020; the administration of PRBCs decreased from 69.4% to 12.8% during the study period.The largest decrease in absolute terms was observed for colloids and crystalloids A, wherea−1·min−1 in 2000 to 0.05 µg·kg−1min−1 in 2020, corresponding to an average linear increase of 0.002 µg·kg−1·min−1  per year. In addition, the percentage of patients receiving norepinephrine increased annually by 5.1% . Note that the unadjusted values are shown here for illustration purposes because the adjusted values in In contrast, the median administered dose of norepinephrine increased from 0.02 µg·kgp = 0.025) over the following two decades. Importantly, the adjusted increase in the AKI incidence was lower when adjusted for confounders  featured values beyond these limits. We constrained the ranges of the figures in order to limit the amount of extrapolation in the total fluid balance vs. vasopressor parameter space.The confounder-adjusted predicted AKI risk as a function of fluid and vasopressor administration is shown in onounced B. We fouonounced C. Figure−1·h−1, the AKI risk only slightly increases with increasing norepinephrine doses, thus representing the trough in the “AKI risk valley”. For the lower (and negative) total fluid balance, the AKI risk strongly increases as a function of norepinephrine administration. For the total fluid balances higher than ~5–7 mL·kg−1·h−1, the AKI risk also increases with higher norepinephrine administration. The dependency of this risk increase on the total fluid balance was less pronounced than that for very low total fluid balances. Overall, We found that the AKI risk features a distinct U-shaped valley: for a total fluid balance in the range of about 3–5 mL·kg−1·min−1). The model demonstrates only a medium performance with an AUROC of 0.67 and a Brier score of 0.16. The independent risk factors were BMI (p < 0.001), the use of antihypertensives (p < 0.001) and the age-adjusted Charlson score (p = 0.012).To translate these results into clinical practice, we computed a nomogram based on a multivariable logistic regression model; In this monocentric retrospective cohort including a standardized high-risk surgical intervention over an observation period of two decades, AKI occurred in 21.6% of the patients undergoing cystectomies and urinary diversions. Almost 90% of the patients with postoperative AKI had AKIN grade 1. We found that a moderate fluid balance combined with a low rate of norepinephrine administration in the early 2000s was associated with a low risk of AKI.−1 (AKI patients) and 74 mL·min−1 are of clinical relevance, as both values qualify as CKD grade 2.During the observation period, we observed a shift to lower fluid balance combined with a moderate to high dosage of norepinephrine, resulting in an increased risk of postoperative AKI. This observation was confirmed after adjusting for confounding factors. Overall, the incidence of AKI was 12.2% in 2000 and increased by an average of 0.6% per year over the following two decades. In the patients with early postoperative AKI, their renal function remained impaired for more than five years compared to that in the patients without AKI. However, if assessed at 5 years postoperatively, it remains questionable whether the median GFR values between 63 mL·minOver the course of the two decades, we observed a more restrictive administration of intravenous fluid and an increase in the number of patients treated with norepinephrine, as well as an increase in norepinephrine administered intraoperatively. Of relevance, we found an association between decreased the intraoperative administration of intravenous fluids, number of patients treated with norepinephrine and amount of norepinephrine administered with AKI events. This is in line with numerous results from retrospective series and the conclusion of the RELIEF trial . Here, wBesides unadjusted fluid administration, fluid balance and norepinephrine, additional parameters associated with AKI were BMI, the severity of comorbidities as illustrated by the by age-adjusted Charlson comorbidity score, ischemic and valvular heart disease, hypertension, the duration of surgery and preoperative renal function.−1·h−1  as shown in −1·min−1. Here, once again, the U-shaped form could be detected which implies that the risk increase is either due to a change in the total fluid balance or norepinephrine administration depending on the specific values of the quantity that is held constant.In this cohort, the risk of AKI showed a U-shaped association with respect to individual fluid administration except for colloids: overall, the risk of developing acute kidney injury increased if the total intravenous fluid administration was less than or above the range of approximately 5–8 mL·kg−1·h−1 A. Howeve . Additionally, despite the use of a multiple regression analysis and risk adjustment, the potential for residual confounding factors could not be eliminated entirely.We explicitly emphasise here that large parts—in particular, extreme values—of the derived U-shaped risk curve that accounts for the total fluid balance–norepinephrine interaction are based on extrapolated values from a fitted multivariable logistic regression model and that these extreme regions might not be encountered in routine clinical practice.Another issue is that it is still unclear if and to what extent creatinine is reabsorbed by the ileum used for urinary diversions, especially in the early postoperative period, and whether this reabsorption is clinically relevant. It was also impossible to diagnose AKI based on urinary output for the following reasons: intraoperative diuresis cannot be assessed due to the externalization of the ureters during surgery and postoperative urine output can be biased due to polyuria . However, urine output is not mandatory for the assessment of AKI. Urine output is still used as a surrogate for renal function to guide perioperative fluid therapy. Additional fluid administration to reverse oliguria and maintain urine output above a specific predefined threshold is proposed in daily practice. However, oliguria does not always occur because of decreased renal perfusion pressure or glomerular perfusion and therefore cannot be prevented by fluid administration. Surgical-stress-induced perioperative neuro-hormonal regulation is probably fluid unresponsive, and administering fluids to increase urine output may lead to fluid overload without any renal benefits ,18. FinaOne additional limitation is the absence MAP data, as it has been shown that hypotension (having an MAP below 65 mmHg) could be associated with an increase in the AKI rate. However, Chiu et al. recently showed that a “cosmetic” correction of hypotension, for example using vasopressors, is not per se protective against AKI. It must be noted that an MAP is not the ultimate target for protecting organ function. Perfusion pressure might be a more accurate parameter; however, it is much more difficult to monitor in a real-life clinical setting. Blood flow, cardiac output and vascular resistance must also be considered for optimal protective organ perfusion.We observed a decreased administration of intravenous fluid, resulting in a decreased intraoperative fluid balance and the increased use of norepinephrine during cystectomies and urinary diversions over the last two decades. The decreased intraoperative fluid balance resulted in an increased incidence of AKI. However, as most AKI events are staged as AKIN grade 1, the clinical relevance of the observed AKI remains questionable.