Prostate-specific antigen (PSA) is a critical part of prostate cancer (PCa) screening, diagnosis, staging, and prognosis. However, elevated PSA levels can also be caused by several external factors. To improve the specificity and sensitivity of PSA in clinical practice, we explored whether markers or parameters may be used as prognostic targets for PCa by long-term follow-up.A total of 121 PCa patients who underwent laparoscopic radical prostatectomy (LRP) were included in our study, all of whom underwent imaging and preoperative pathological diagnosis. Endocrine therapy has long been applied to treat postoperative patients. The prognosis of enrolled patients was followed, and statistics were collected. Spearman's correlation analysis was applied to examine the relationship and clinical parameters. Kaplan–Meier analysis was used to process the clinical variables of PCa patients. Cox proportional hazards regression analysis was applied to examine univariate and multivariate variables.P < 0.05). The GS, PSA, clinical stage, organ confined, Ki67, nerve infiltration and ADC were included in the multivariate analysis . Ultimately, Ki67 and the ADC were found to provide meaningful predictive information (both P < 0.05).The Gleason score (GS), PSA, clinical stage, nerve infiltration, organ confinement, Ki67 and apparent diffusion coefficient (ADC) were significantly associated with prognosis (all Ki67 and the ADC may be clinically and analytically valid prognostic biomarkers and imaging parameters in PCa. They may be useful for predicting the prognosis and risk of PCa patients undergoing postoperative routine endocrine therapy. Prostate cancer is one of the most common urological malignancies in elderly men. Its morbidity and mortality rank second and fifth, respectively, among all malignant tumors worldwide . CurrentKi67, a nuclear DNA-binding protein expressed in all vertebrates, is connected to cell proliferation activity . Ki67 isOver the past decade, the efficacy and safety of LRP have improved, thus providing a minimally invasive alternative to open surgery with shorter recovery times and hospital stays , 16. MorCurrently, most studies have been conducted on the predictive and clinical effectiveness of Ki67, but few studies have utilized the characteristics of Ki67 and the ADC in combination. We strongly believe in the importance of ADCs in PCa and hold the opinion that Ki67 can be applied in combination with postoperative PSA testing and imaging parameters  to improve specificity and sensitivity , 20, 21.This study protocol and informed consent form were approved by The Affiliated Hospital of Guizhou Medical University , and written and verbal informed consent was obtained from all patients. Data from the retrospective study were collected from 2013 to 2022, and 149 patients who underwent radical prostatectomy were included in our research. All patients with PCa were included in this study based on the following criteria: (1) Written and verbal informed consent was obtained from all patients before participation in the study. (2) Prostate cancer was pathologically confirmed by prostate biopsy. (3) Those patients' physical conditions were relatively good, and the patients' life expectancy was greater than 10 years. (4) Prostate cancer is usually localized (up to T3b). The exclusion criteria for all PCa patients were as follows: (1) severe cardiopulmonary insufficiency or crucial organ dysfunction; (2) uncorrected coagulation disorders; (3) severe mental disorder or cognitive impairment; (4) poor nutritional status; and (5) history of previous radiation or immunomodulation therapy. All patients were followed up until June 30, 2022, and the end point event was the death of patients due to PCa. During a median 56 months of follow-up, a total of 28 patients were excluded from our study, including 7 patients who were lost to follow-up due to telephone changes or address changes, 11 patients who died of other causes, 6 patients who developed castration-resistant prostate cancer, and 4 patients who received additional chemotherapy and immunotherapy.2). The apparent diffusion coefficient (ADC) value was calculated by monoexponential regression, and ADC mapping was automatically generated pixel by pixel using all b values. The mean ADC was 1.038 × 10−3 mm2/s in this study  in the supine head-first position combined with a phased-array surface coil. T1-weighted (T1W) imaging was performed in the axial plane with the following parameters: repetition time (TR), 500.00 ms; echo time (TE), 9.00 ms; matrix 225 × 200; and slice thickness, 3.50 mm. Axial T2-weighted (T2W) imaging was performed with the following parameters: TR, 3,500 ms; TE, 108 ms; matrix, 225 × 200; and section thickness, 3.50 mm. Axial T2W imaging enhancement was observed with the following parameters: TR, 3.94 ms; TE, 1.48 ms; matrix, 200 × 230; and section thickness, 2.00 mm. Diffusion-weighted imaging (DWI) was acquired using the single-shot echo-planar imaging technique with three different b values , and sections were stained with goat anti-rabbit secondary antibody . Hematoxylin and 3,3-diaminobenzidine (DAB) were utilized to stain tissue sections separately. After immersing the sections in various concentrations of graded ethanol, they were covered with coverslips. The sections were observed and photographed using a microscope.The expression of Ki67 in prostate cancer samples was assessed independently by two pathologists. IHC slides were photographed, and the positive cells were formally counted as the Ki67 labeling index. The results were divided into four groups based on the percentage of Ki67-positive cells .The endocrine therapy of lutein hormone-releasing hormone (LHRH) agonist coupled with nonsteroidal antiandrogen drugs was acceptable by all patients. Goserelin sustained-release implants , one of the LHRH agonists, were injected subcutaneously at 28-day intervals. Bicalutamide  is recommended as one tablet daily (50 mg/day). We conducted long-term follow-up with patients once every month to ensure therapy compliance, evaluate effectiveness, and detect adverse events. The mean duration of endocrine therapy was 41 months, with a maximum duration of 72 months.P values <0.05 were considered statistically significant, and P values were two-tailed. Survival curves were drawn using GraphPad Prism 9.0 .Statistical analysis was performed with Statistical Product and Service Solutions . The quantitative data were expressed as the mean ± standard deviation (SD), and the quantitative data were statistically classified as either qualitative or ordinal data. Spearman correlation was applied to test the relationship between Ki67 expression and clinical parameters. Kaplan‒Meier analysis was used to process univariate variables of prostate cancer patients. Cox proportional hazards regression analysis (Forward:LR) was applied to examine multivariate variables. A Cox proportional hazards regression model was used for proportional hazards analysis. All r = 0.514, P < 0.01), PSA , clinical stage , organ confinement , and nerve infiltration . Spearman correlation analysis revealed that the ADC was significantly correlated with GS , PSA , nerve infiltration , and surgical margin . Ki67 expression was positively correlated with GS  and PSA   (The correlations of PCa patients are listed in  < 0.01)  and sign < 0.01) .P < 0.05), which were significantly identified as more adverse variables. In univariate analysis, significant differences were discovered by Cox proportional hazards regression analysis. GS, PSA, clinical stage, organ confined, Ki67, nerve infiltration and the ADC were included in the multivariate analysis (P < 0.05). In multivariate analysis, Ki67  and the ADC  may provide meaningful predictive information (P < 0.05).As shown in Cox regression multiplicative interaction analysis was applied to analyze three variables. Ki67 and ADC revealed that clinical variables satisfied proportional hazards.The World Health Organization (WHO) most recent research estimates that PCa in men is diagnosed in nearly 1.4 million new cases and 375,000 deaths worldwide , 5. EnhaRecent studies have discovered that Ki67 can be used as a diagnostic and prognostic biomarker in renal cancer , 22, ovaThere remains a lack of clarity regarding the clinical application and health care of PSA. Indeed, PSA screening has a variety of benefits for the elderly population and gives them the opportunity to detect disease in its early stages , 26. On Over the past two decades, multiparametric MRI (mp-MRI) containing T1W imaging, T2W imaging and DWI has gained widespread acceptance in the management of PCa patients because of its high accuracy , 30. EviMore recently, minimally invasive techniques have taken the place of surgical treatments for prostate cancer . In partKi67, a nuclear DNA binding protein originating from its city of name (Kiel) and the 67th original clone in the 96-well plate, is a biomarker of proliferation to evaluate the proportion of dividing cells to grade tumors . PreviouAlthough the follow-up duration was long in the current study, more patients should be included in future studies. Potential or relative variables may not have been identified due to small sample sizes. Additionally, a few variables are included because some data are either missing or inadequate. In addition, the single-center design in our study was a limitation, and multicenter studies are required for further clarification.via IHC and the assessment of ADC via MRI may provide prognostic biomarker and imaging parameters with analytical and clinical validity in PCa. For PCa patients with postoperative routine endocrine therapy, Ki67 and the ADC are prognostic factors that bring new approaches.Ki67 and the ADC are highly associated with PCa patients who undergo laparoscopic radical prostatectomy. The assessment of proliferation-associated nuclear protein Ki67


We conclude that p53 expression could be a beneficial marker for primary ET resistance in locally advanced luminal B HER2-negative BC.Luminal B HER2-negative breast cancer (BC) is the most common type in Indonesian BC patients, and frequently manifests with locally advanced staging. Recurrence often occurs within two years of the endocrine therapy course (primary endocrine therapy (ET) resistance). p53 mutation often exists in luminal B HER2-negative BC, but its application as an ET resistance predictor in those populations is still limited. The primary purpose of this research is to evaluate p53 expression and its association with primary ET resistance in luminal B HER2-negative BC. This cross-sectional study compiled 67 luminal B HER2-negative patients’ clinical data during their pre-treatment period until they completed a two-year course of endocrine therapy. They were divided into two groups: 29 patients with primary ET resistance and 38 without primary ET resistance. Pre-treatment paraffin blocks from each patient were retrieved, and the p53 expression difference between the two groups was analyzed. Positive p53 expression was significantly higher in patients with primary ET resistance [odds ratio (OR) of 11.78 (95% CI: 3.72–37.37,  Breast cancer (BC) is the number one leading cancer case in Indonesia, according to GLOBOCAN 2020 data, both in incidence and mortality rate . From seThe long-term prognosis of luminal B BC depends on the success of endocrine therapy (ET), in which the patients will receive suppression of estrogen production and/or estrogen receptor (ER) blockade or ER degradation for years . NeverthAlthough ET resistance has been a paramount problem for patients with HR (+) BC and clinicians, no established biological marker related to ET resistance occurrence has been distinguished . A biolop53 has been studied in many studies for its role in luminal BC ,16,17,18The primary purpose of this research is to evaluate p53 expression and its association with primary ET resistance in luminal B HER2-negative patients. Our main hypothesis is that p53 protein expression, as a surrogate marker for p53 mutations, will be expressed higher in patients with primary ET resistance than in the group without primary ET resistance.This research is a retrospective cross-sectional study conducted from April to November 2022 in four hospitals located in three provinces in Indonesia: Hasan Sadikin General Hospital and Santosa General Hospital in Bandung City, West Java Province; Siloam Hospital Karawaci in Tangerang City, Banten Province; and Mochtar Riady Comprehensive Cancer Center (MRCCC) Siloam Hospital Semanggi in the Special Region of Jakarta, Indonesia’s capital city.The study protocol was approved by the Institutional Review Board of Padjajaran University  on 14 March 2022. All ethical review boards reviewed and agreed to the study protocol. We followed the Strengthening of Reporting Observational Studies in Epidemiology (STROBE) guidelines [We collected data from the Pathology Anatomy Laboratory and Medical Records in those four hospitals for newly diagnosed locally advanced stage (IIIA–IIIC) luminal B HER2-negative patients from 2016–2022; all patients were treated according to the Indonesian guidelines for breast cancer treatment published by the Indonesian Health Ministry in 2018 [All patients underwent the pre-treatment biopsy procedure (open or core needle biopsy) and neoadjuvant chemotherapy. Radiotherapy was not mandatory; it was only accomplished if indicated. All patients underwent a mastectomy procedure  and received ET for five years or more. However, all patients included in this research must complete at least two years of ET.2), bilateral breast cancer, metastatic disease when pre-treatment staging was performed, being unresponsive to neoadjuvant chemotherapy, and patients with any missing data. Confounding variables in the study were the patient’s age, stage and histological grade, ER expression level, chemotherapy, and radiotherapy usage.The inclusion criteria for our study were locally advanced (stages IIIA–IIIC) luminal B HER2-negative BC, complete clinical data, and acceptable quality of paraffin block to be tested. The exclusion criteria for our study were obesity  staining was first performed on one 4 μm section of each paraffin block and checked by two pathological anatomy experts to verify that the presentable number of carcinoma cells was present and the fixation quality was acceptable for immuno-histochemistry IHC analysis . See theExpression of p53 was immunohistochemically evaluated (LSAB method) using a mouse monoclonal antibody to p53 . All staining was carried out in a single lab: Hasan Sadikin Pathological Anatomy Laboratory in Bandung, West Java. See the Data Availability Statement for the full link to the IHC staining protocol for p53 expression conducted in this study.Expression of p53 was scored by assigning the proportion of the stained nuclei of the cells; any intensity of nuclear staining in breast carcinoma epithelium counted for the positive proportion. The two pathological anatomy experts read a positive proportion of the tumor to avoid bias.Ten percent of positive cell nuclei was considered positive for p53 expression and thus was a surrogate marker for p53 mutations in the patient’s tissue ,19. The The sample size was calculated by taking power at 95%, and the confidence level was 95%. We calculated the sample using data from a previous study, in which the percentage of control subjects exposed was 0.88, and p53 positive expression is associated with relapse during endocrine therapy with an odds ratio of 4.82 . We defiIn this study, we analyzed the IHC expression of p53 and clinical data in 29 luminal B HER2-negative cases with primary ET resistance and 38 cases without primary ET. (See Data Availability Statement).We excluded 309 patients, with the majority reason (179 patients—58.68% of the excluded population) being incomplete clinical data (pre-treatment and/or follow-up data). Other reasons for exclusion were diagnosed as a metastatic disease in the pre-treatment period (76 patients—24.60%), lost or poor quality of paraffin block (14 patients—4.53%), obesity (16 patients—5.17%), bilateral diseases (11 patients—3.60%), and unresponsive to neo-adjuvant chemotherapy and/or radiotherapy (six patients—1.94%).The mean age of patients was 48.68 ± 9.43, with the youngest being 25 and the oldest 69 years old. All ER statuses of the patients were positive. Further, we carried out a bivariate analysis between p53 and primary ET resistance, as depicted in Based on the statistical analysis results, we found no clinicopathological difference between the groups with and without primary ET resistance . This mep-value < 0.0001). Two pathological anatomy experts interpreted these p53 expression results with an inter-observer agreement of 94.03% and a kappa-score of 0.88. .We found that positive p53 expression is significantly higher in patients with primary ET resistance (22 patients—73.33%) than without primary ET resistance (eight patients—26.67%), OR 11.78 , compared with luminal A BC. These features are prominent hallmarks in this group, thus making it more prone to ET resistance ,25,26,27Many studies and clinical trials have been conducted in the hope of finding the proper treatment for patients with ET resistance, but to no avail until this article is written ,31. The Our previous published review has noted that p53 roles are entangled in several prominent pathways involved in ER and PR molecular mechanisms, such as the NF-kB pathway, PI3K/Akt/mTOR pathway, and other ER target genes, thus making p53 mutations a plausible predictor for ET resistance, especially in the luminal B group . p53 is p-value < 0.0001). This finding was consistent with other studies in the hormonal receptor-positive HER2-negative BC group [For the p53 expression cut-off in IHC staining, we decided on 10% as a positive expression because it already correlates significantly with clinicopathological characteristics, specifically in the luminal B Her2-negative population . With biBC group ,24,28.p-values of 0.0049 and 0.024, respectively) [p-value < 0.0001) [A study by Yamashita et al. (2006) in 73 BC patients proved that p53 protein accumulation and high Ki67 expression are more resistant to aromatase inhibitors in metastatic disease patients ( 0.0001) . But the 0.0001) . It is w 0.0001) ,39.An extensive study by Anh et al. (2013) in 15,598 BC patients noted that positive p53 expression in hormonal receptor (HR)+/Her2-negative patients is significantly associated with the patient’s response to hormonal therapy and, therefore, could affect overall survival (OS) and breast cancer-specific survival (BCSS) . AlthougIt is also well known that IHC is affected by subjectivity. Anh et al. stated that their study is conducted retrospectively and just based on the BC registry  and not centrally validated; therefore, it could have a bias in their result . In this2), bilateral breast cancer, and any unresponsiveness to neoadjuvant chemotherapy, beside metastatic disease [Those aforementioned three studies also did not homogenously collect the locally advanced BC patients, which could affect the study result as, e.g., the metastatic disease could have more significant mutations and more aggressive biological behavior and, therefore, could have a higher risk for recurrence than early BC. Besides metastatic disease, in an effort to minimize bias in our study, we also tried to exclude several risk factors that could affect ET resistance, such as obesity  are not significantly different in both groups, and thus, we cannot proceed to multivariate analysis. But these findings could be due to our limited sample. For instance, patients with young BC (age < 35 years old at diagnosis) were only four, and patients admitted for radiotherapy were only six. Therefore, these findings should be explored more in future studies because they are inconsistent with previous research with a larger sample ,16,43,44p-Value: 0.685).This study is distinct from other studies as we differentiate the ER expression into five categories: negative . This is our effort to further investigate the difference in ER expression level, as many studies have discussed the gradation of ER expression level as an essential factor for endocrine therapy resistance . A studyThis study has several limitations. As we mentioned before, we realized that we have a limited number of samples, and our study design was conducted in a retrospective and cross-sectional fashion due to the study’s feasibility. A prospective cohort design could provide a more accurate depiction of p53 association with primary ET resistance in luminal B HER2-negative patients. Therefore, we noted that the results and conclusions should be interpreted cautiously.We acknowledged that patients included in our study used various ET drugs with diverse mechanisms of action; therefore, this caused bias. Undeniably, individual variability in drug efficacy will result in different outcomes. For instance, the inactivity of cytochrome p450 enzymes caused by gene polymorphism may cause relative resistance to Tamoxifen . A more Another limitation of our study was that we investigated p53 expression, ER expression, and PR expression using immuno-histochemistry (IHC) in a paraffin block. However, a more accurate result could be achieved by using genomic testing with fresh tissue . MoreoveIn addition, we only conducted a single p53 expression analysis during the pre-treatment period. It is known that BC in its locally advanced stage could undergo genetic changes throughout treatment and the disease course ,50. ThusLast but not least, we did not analyze several fundamental biomarkers in ET resistance, such as NF-kB and PIK3CA mutations. Loss of ET usage due to ET resistance drives clinicians and scientists to explore further alternative therapies for luminal BC with ET resistance . Both biHowever, despite all the study limitations, we could show in this research that p53 protein expression as a surrogate marker for p53 mutations is significantly associated with primary ET resistance. As we mentioned above, this is the first study to do so, and we do believe this topic is important, relevant to the daily problems encountered by clinicians in locally advanced luminal B HER2-negative BC in Indonesia, and could be researched in the future with some of the considerations above.p53 expression could be a beneficial marker for clinicians to determine which luminal B HER2-negative patients with locally advanced disease would likely have primary ET resistance, so they could receive more scrutinized monitoring to improve prognosis. However, this finding requires further studies with larger sample sizes using a prospective cohort design with patients who only use one type of ET drug. We also recommend serial analysis of genomic testing for p53 mutations using fresh specimens throughout patients’ treatment periods and analyzing their association with immunologic markers, especially TILs.


Perturbation of osmolality is associated with increased mortality in adults and children in critically ill conditions. However, it is still unclear whether osmolality imbalance impacts the prognosis of critically ill infants. This study aimed to investigate the relationship between plasma osmolality and prognosis in critically ill infants within 24 h of admission.This retrospective study enrolled 1,042 infants who had plasma osmolality data from 2010 to 2018. The initial plasma osmolality (within 24 h after admission) was extracted from the pediatric intensive care database (PIC V1.1). The locally weighted scatter-plot smoothing (LOWESS) and restricted cubic splines (RCS) methods were used to explore the approximate relationship between plasma osmolality and in-hospital mortality. Univariate and multivariate logistic regression analyses were used to further analyse this relationship. Kaplan–Meier analysis was applied to estimate the probability of hospital mortality within 90 days of admission. Subgroup analysis was employed to assess the impact of potential confounders .P < 0.05; OR 2.52; 95% CI, 1.15–5.06). Plasma osmolality >300 mmol/L  was also significantly associated with mortality . This association remained even after multivariable adjustments. The 90-day survival rate was lower in the abnormal plasma osmolality group  than in the intermediate group . The abnormal plasma osmolality group had a significantly higher incidence of all-cause mortality in the 0–7 postnatal days subgroup . Infants with abnormal osmolality had a significantly higher mortality rate in the female group (P < 0.05). High osmolality was associated with a higher mortality rate in the preterm group (P < 0.05).An approximately“U”-shaped relationship between plasma osmolality and mortality was detected. In the logistic regression model, plasma osmolality <270 mmol/L  was significantly associated with in-hospital mortality (Both hypoosmolality and hyperosmolality were shown to be independently associated with increased risk of in-hospital infant mortality in NICUs. Serum osmolality measures the concentration of all the dissolved particles in body fluids. It reflects bodily fluid balance, electrolytes, and renal function and is strongly affected by the concentrations of sodium (Na+), potassium (K+), glucose, and urea , 2. ChanDisturbance of osmolality is associated with increased risks of adverse clinical outcomes in adults and children in critical care. Prior studies have shown that plasma osmolality is correlated with the prognosis of patients with diabetic ketoacidosis and myocardial infarction, elderly hemodialysis patients, and emergency room patients –8. PlasmNeonatal patients are more vulnerable to disturbances in the internal environment due to their unique physiological characteristics. It is not yet known whether osmolality imbalance affects the prognosis of critically ill infants in neonatal intensive care units (NICUs). This study aimed to examine the relationship between plasma osmolality and prognosis in the first 24 h after NICU admission.http://pic.nbscn.org/)  database. The PIC database is a large and freely accessible Chinese-English bilingual pediatric-specific critical care database containing the clinical data of all children admitted to multiple ICUs at the Children's Hospital of Zhejiang University School of Medicine, China, from 2010 to 2018. The PIC database was established based on the success of the widely used Medical Information Mart for Intensive Care (MIMIC) database. It can be downloaded free after registration, application, and certification (cn.org/) .Inclusion criteria: (1) Patients admitted to the NICU ≤28 days old. (2) Only the first hospitalization was considered if a patient had multiple hospitalizations. (3) Data collection of laboratory results was defined using the first-time examination at admission (within 24 h after admission).Exclusion criteria: (1) Patients without plasma osmolality data. (2) Patients who did not have demographic data were excluded. Only the initial demographic record was used for patients with multiple demographic observations. The extracted data included demographics, comorbidity, laboratory test results, and treatment. The International Classification of Diseases (ICD)-10 was used to define, diagnose, and classify neonatal illness and its complications. The formula used to calculate plasma osmolality was 2 (Na + K) + (Glu/18) + (Urea/6), and the unit was mmol/L . AccordiThe primary outcome was all-cause in-hospital mortality, defined as death during hospitalization. Secondary outcomes included 30-day mortality, 90-day mortality, NICU stay duration, and hospital stay duration.U-test. Nominal variables were analysed using the chi-square test. Infants’ plasma osmolality values were grouped based on the relationship between in-hospital mortality and plasma osmolality using the Local Weighted Scatter Plot Smoothing (LOWESS) and restricted cubic splines (RCS) methods. Univariate and multivariate logistic regression analyses were used to identify the associations between hospital mortality and plasma osmolality. These confounders were chosen a priori based on previous research, clinical relevance, and experience . All analyses were fit in RStudio 1.0.44  using R v.4.0.2.Kaplan–Meier analysis was applied to estimate the probability of hospital mortality within 90 days of admission, which was then analysed with the log-rank test. Subgroup analysis was performed according to postnatal days, sex, and gestational age using logistic regression. To verify the interaction between plasma osmolality and these variables, multiplicative interaction terms were included in the regression model. A We analysed data from 1,042 infants. The overall population's mean age was 1.5  days, and the mean weight was 1.87  kg. Males comprised 59.9% of the population. The mean plasma osmolality level was 281  mmol/L. The relationship between plasma osmolality levels and the risk of mortality was approximately U-shaped . We diviP < 0.05), were not significantly different between the intermediate and low osmolality groups. There were significant differences in Anion. Gap, creatinine, urea, plasma osmolality, and sodium between the low or high osmolality and intermediate osmolality groups (P < 0.05). A statistically significant difference was seen between the low and intermediate osmolality groups for albumin and lactate (P < 0.05). For bicarbonate and base excess, there was a statistically significant difference between the high and intermediate osmolality groups (P < 0.05)  .P > 0.05). The abnormal plasma osmolality group  had a lower 90-day survival rate than the intermediate group (P < 0.05)  . The ove < 0.05) .P < 0.05; OR 2.52; 95% CI, 1.15–5.06). When plasma osmolality was >300 mmol/L, the OR of in-hospital mortality remained significant . The extended multiple logistic regression model confirmed the significant association between plasma osmolality in the high or low osmolality group and in-hospital mortality (P < 0.05).Univariate logistic regression analysis (Model 1) determined the association between in-hospital mortality and plasma osmolality in critically ill infants . When plP < 0.05; OR 5.25; low osmolality group: P < 0.05; OR 3.01). The same pattern was observed in the female subgroup . Patients in the high osmolality group had a higher mortality rate than those in the intermediate group in the preterm infant subgroup   .The primary objective of this study was to ascertain whether infants experience adverse outcomes if they have plasma osmolality imbalance within 24 h of admission. The above data suggest that the correlation between plasma osmolality and mortality in NICU patients may be “U-shaped.” Higher and lower plasma osmolality levels were significantly associated with increased in-hospital mortality in critically ill neonates compared with the intermediate group. This association remained significant after adjustments for other variables. In addition, subgroup analyses based on gender, postnatal days, and gestational age revealed that plasma osmolality levels that are too high or too low are associated with an increased in-hospital mortality rate. In the 90-day infant mortality rate, the survival rate of the abnormal plasma osmolality group was lower than that of the intermediate group. Therefore, serum osmolality could be considered as a potentially valuable risk factor.In this study, the LOWESS and RCS models showed that plasma osmolality between 270 and 300 mmol/L had a good prognosis for NICU patients. This result was inconsistent with the previous reports on pediatrics and adults in intensive care units (ICUs) , 15. TheOur findings suggest that elevated plasma osmolality is associated with increased in-hospital mortality among infants in NICUs, which is consistent with previous adult and pediatric research findings , 20, 21.P < 0.05). Second, low osmolality can also affect the body's balance of water and electrolytes, which is essential for maintaining normal physiological functions . However, no significant difference emerged between groups in the late phase. Optimizing thermoregulation, body water metabolism, and electrolyte balance during the early phase is critical for high-risk neonates' long-term outcomes. This highlights the importance of osmolality balance in early life stages. Infants with abnormal osmolality had a significantly higher mortality rate in the female group (P < 0.05). High osmolality was associated with a higher rate of mortality in the preterm group (P < 0.05). Further experiments will be necessary to clarify the mechanisms involved. It was found that the interaction test was not statistically significant for gender, postnatal days, and gestational age (P for interaction >0.05).Furthermore, subgroup analysis was carried out by postnatal days, gestational age, and gender. The neonatal period comprised early (first seven days) and late (remaining 21 days) phases, with most neonatal mortality occurring in the early phase , 31. InfThis is the first study investigating plasma osmolality and prognosis in NICU infants. The osmolality thresholds of critically ill newborns differed significantly from healthy newborns, reflecting only those with severe plasma osmolality perturbations in the NICU , 17. As This study suggests an approximate “U-shaped” relationship between plasma osmolality and in-hospital mortality. The intermediate serum osmolality group (270–300 mmol/L) had a lower mortality risk than both the hypoosmolality and hyperosmolality groups, which were independently associated with increased in-hospital mortality in NICUs. This study provides a potential target for therapeutic intervention, namely, correcting abnormal plasma osmolality may improve the prognosis of infants in NICUs. Future studies with adequate sample sizes are needed to further understand the effects of plasma osmolality on mortality in critically ill infants.


Despite the high prevalence of chronic low back pain (CLBP) and osteoarthritis (OA), few estimates of the economic cost of these conditions in England have been published. The aim of the present analysis was to characterise the economic burden of moderate-to-severe pain associated with CLBP + OA and CLBP alone compared with general population-matched controls without CLBP or OA. The primary objective was to describe the total healthcare resource use (HCRU) and direct healthcare costs associated with the target patient populations. Secondary objectives were to describe treatment patterns and surgical procedures.  This was a retrospective, observational cohort study of patients receiving healthcare indicative of moderate-to-severe chronic pain associated with CLBP, with or without OA. We used linked longitudinal data from the Clinical Practice Research Datalink GOLD and Hospital Episode Statistics (HES). Patients (cases) were matched 1 : 1 with controls on age, sex, comorbidity burden, GP practice, and HES data availability. p < 0.0001). By 36 months after indexing, the mean (SD) per-patient total direct healthcare cost in the CLBP-alone cohort was £5081 (£5905) for cases and £1809 (£4451) for controls (p < 0.0001); in the CLBP + OA cohort, the mean (SD) per-patient total direct healthcare cost was £8819 (£7143) for cases and £2428 (£4280) for controls (p < 0.0001).  The CLBP-alone cohort comprised 13 554 cases with CLBP and 13 554 matched controls; the CLBP + OA cohort comprised 7803 cases with both OA and CLBP and 7803 matched controls. Across all follow-up periods, patients with CLBP alone and those with CLBP + OA had significantly more GP consultations, outpatient attendances, emergency department visits, and inpatient stays than controls (all  Moderate-to-severe chronic pain associated with CLBP—with or without OA—has a substantial impact on patients and healthcare providers, leading to higher HCRU and costs versus controls among people with CLBP alone or together with OA. Lower back pain and osteoarthritis (OA) are common musculoskeletal conditions and major causes of chronic pain and disability in the UK. Chronic pain was reported by 34% of respondents in the 2017 Health Survey for England, with the most commonly reported being musculoskeletal, including the back, hips, and legs , respectAn estimated one in 10 adults in the UK has symptomatic, clinically diagnosed OA.  Between Co-occurrence of low back pain (LBP) and OA is common. Gore and colleagues analysed data from patients who were newly prescribed selected pain medicines , cyclooxygenase-2 inhibitors, paracetamol, tramadol, and weak or strong opioids) in The Health Improvement Network UK database. They identified OA in 9.2–24.7% of patients with a diagnosis of CLBP and LBP in 7.4–21.9% of patients with a diagnosis of OA . In a stManagement of pain due to CLBP or osteoarthritis is complex and generally includes a combination of pharmacological and nonpharmacological approaches, with varying results , 14. Phast January 2007 to 31st December 2009  [Despite the high prevalence of CLBP and OA, to our knowledge, there are few recent published estimates of the economic cost of these conditions in the UK, and existing evidence predates published guidance for the care and management of this patient population , 20. The < 0.05) . General < 0.05) . Histori < 0.05) . ExistinThe aim of the present analysis was to characterise the economic burden of patients in England receiving healthcare indicative of moderate-to-severe pain associated with CLBP + OA, and those with CLBP alone, compared with general population-matched controls without CLBP or OA.This retrospective, observational cohort study of patients presenting to primary care with an episode of moderate-to-severe chronic pain associated with CLBP, with or without OA, and matched controls without CLBP or OA was conducted using linked data from the CPRD and Hospital Episode Statistics (HES). The study design is shown in Patients with an existing diagnosis  code) of LBP, either with or without OA, were indexed between December 2009 and November 2017 on a moderate-to-severe pain event occurring within a period of chronic pain. To be included, patients were required to be aged ≥18 years at index, to be eligible for linkage to HES data, to have ≥12 months of data before indexing and ≥12 months of data after indexing, and to have data of acceptable research quality as defined by the CPRD .A chronic pain episode was defined as a series of pain-related GP consultations relating to pain symptoms associated with CLBP or OA and/or a pain-related specialist consultation  in the secondary care setting, where gaps between visits were ≤12 months. The patient's first/earliest date of a moderate or severe pain event documented within the CPRD determined the index date.Moderate-to-severe pain events were defined as any of the following: a referral to/attendance with a pain specialist; a surgical or nonsurgical invasive procedure relating to the treatment of CLBP or OA ; two or more prescriptions for NSAIDs, including at least two different NSAIDs or opioids within a 3-month period; or a pain-related emergency department (ED) visit, with a pain-related GP follow-up within 14 days.Each patient within the study cohort (cases) was index matched 1 : 1 to a general population control within CPRD  on year of birth , sex, lifetime Charlson Comorbidity Index (CCI) score, GP practice, and linkage eligibility. The CCI is a score calculated based on the presence of a number of prespecified chronic conditions; the lifetime CCI score is the score calculated across the patient's entire medical record. Inclusion criteria and medical codes were reviewed for clinical relevance and appropriateness by RDK, AJD, and DAW. Each matched control patient was assigned a pseudoindex date equal to the index date of the case patient with whom they were matched.The CPRD GOLD primary care database is a longitudinal, anonymised research database of computerised medical records held by GPs across the UK. Over 650 primary care practices in the UK, covering 11 million people, participate in the CPRD, with clinical records for over 12 million individuals; an estimated 5.5 million people are actively registered. Data are broadly representative of the UK population . AvailabApproximately 75% of English practices contributing to CPRD are linked to the HES dataset, which provides data on all inpatient and outpatient contacts, including ED visits, occurring at NHS hospitals in England, with diagnoses recorded using ICD-10 codes.The primary study objective was to describe total healthcare resource use (HCRU) and direct healthcare costs associated with the target patient populations . Secondary study objectives were to describe CLBP/OA treatment patterns and surgical procedures.The key study outcomes were HCRU and direct healthcare costs. Components of resource use included GP appointments, outpatient appointments, hospitalisations, ED attendances, medicine use, and total direct healthcare costs . Use of physiotherapy and other outpatient services was limited to services provided in the secondary care setting. Pharmacological management included nonopioid analgesics , opioid analgesics , and adjuvant medicines . Pharmacological treatments assessed in this study were driven by the scope of NICE guidelines (NG59 and CG177) , 20, witUnit cost data were transformed into direct healthcare costs using appropriate unit cost data. GP consultation costs were sourced from Unit Costs of Health and Social Care, compiled and provided by the Personal Social Services Research Unit . For medThe study was conducted in accordance with legal and regulatory requirements and followed generally accepted research practices described in the Guidelines for Good Pharmacoepidemiology Practices issued by the International Society for Pharmacoepidemiology  and Goodst, 2019). Institutional review board approval was not required; no study participants were put at risk during the study, and confidentiality was maintained by use of data from deidentified electronic medical records provided by the CPRD.This study was approved by the Independent Scientific Advisory Committee of the Medicines and Healthcare Products Regulation Agency , 25th and 75th percentiles, minimum, and maximum values were reported for numeric variables.p < 0.05 was used for comparison of cases and controls. No corrections were made for multiple comparisons.All statistical tests were two-sided in nature; a significance level of Analyses were performed using Stata .st, 2008 and November 30th, 2018 , and an equal number of matched controls . Not all of these comorbidities were included in the CCI, upon which cases and controls were matched. Comorbid mental health conditions, including anxiety and depression, were also more common in cases compared with controls . The mean length of follow-up was 43.6 months in the CLBP-alone cohort and 39.9 months in the CLBP + OA cohort.Patient demographic and clinical characteristics are shown in p < 0.0001) (p < 0.0001); 26.7% of cases used pain management outpatient services during the same period compared with 0.2% of controls . Similar findings were observed during 0–6, 0–24, and 0–36 months of follow-up , patients with CLBP alone had significantly more GP consultations, outpatient attendances, ED visits, and inpatient stays compared with matched controls  . Orthopaollow-up .p < 0.0001). The mean (SD) length of stay over the 36-month follow-up was 0.82 (2.86) days in cases and 0.58 (4.49) days in controls (p < 0.0001).By 36 months after indexing, cases had a mean (SD) of 35.09 (26.94) GP consultations, 13.12 (14.44) outpatient attendances, 1.44 (2.91) ED visits, and 2.22 (4.57) inpatient stays compared with 14.32 (17.61), 4.15 (8.69), 0.56 (1.17), and 0.66 (5.62), respectively, in controls  number of treatment lines received was 9.0 (5−18), and the mean (SD) was 13.1 (12.3). Among cases, prescribing of paracetamol (9.9–20.9%) and topical NSAIDs (4.9–8.8%) increased substantially from line 1 to 10  had fusion surgery, 248 (2.1%) had disc replacement surgery, 1692 (14.0%) had medial branch block, and 334 (2.8%) had radiofrequency denervation.p < 0.0001) , patients with CLBP + OA had significantly more GP consultations, outpatient attendances, ED visits, and inpatient stays compared with matched controls  .p < 0.0001); 18.8% of cases used the pain management outpatient service during the same period compared with 0.3% of controls .The cumulative inpatient length of stay was almost three times longer for cases compared with controls in the first 12 months of follow-up (mean (SD) 2.52 (9.03) vs 0.90 (7.13) days; Similar findings were observed during 0–6, 0–24, and 0–36 months of follow-up . By 36 mth and 75th percentiles) number of treatment lines received was 11.0 (6–20), while the mean (SD) was 14.8 (12.5). Prescribing of paracetamol (19.2–32.3%), topical NSAIDs (9.1–15.6%) increased substantially from line 1 to 10 in cases with OA + CLBP, whereas the prescribing of systemic NSAIDs (23.8–27.9%), weak opioids (11.3–11.3%), and strong opioids (26.3–30.3%) remained relatively consistent. Use of antidepressants (12.3–26.5%), antiepileptics , and anxiolytics (6.5–13.3%) increased substantially from line 1 to 10 , 197 (2.6%) had fusion surgery, 188 (2.5%) had arthroscopy , 122 (1.6%) underwent osteotomy , and 70 (0.9%) had disc-replacement surgery. A further 698 cases (9.3%) had a medial branch block, 150 (2.0%) had radiofrequency denervation, and 2044 (27.2%) had an intraarticular injection.Among the 7520 incident cases, i.e., those without any moderate-to-severe chronic pain associated with CLBP prior to indexing, 1844 (24.5%) had a total joint replacement  (p < 0.0001). Individual cost elements for each healthcare resource used were also significantly higher for cases compared with controls. The contribution of inpatient stays to total costs was higher for cases (49–55%) versus controls (41–43%) across all time periods. GP consultations and outpatient attendances were other substantial contributors to the total costs .NHS England total healthcare costs incurred through cases during the 0–6, 0–12, 0–24, and 0–36 months of follow-up were 4, 3.5, 3, and 2.8 times higher, respectively, compared with matched controls  . By 36 mMedication costs were numerically lower in cases versus controls. They accounted for 2-3% of total costs in cases (£26 of £1271 at 0−6 months to £131 of £5081 at 0−36 months) and 7-8% of total costs in controls (£24 of £319 at 0−6 months to £127 of £1809 at 0−36 months).p < 0.0001) (p < 0.0001). Individual cost elements for HCRU were significantly higher for cases compared with controls.Total costs incurred by cases during the 0–6, 0–12, 0–24, and 0–36 months of follow-up were 5, 4.4, 3.8, and 3.6 times higher, respectively, compared with matched controls  . By 36 mp < 0.0001). GP consultations and outpatient attendances were other substantial contributors to the total costs .Inpatient stay costs accounted for approximately 60−66% of total costs for cases and 42−44% for controls  versus 6–8% of the total cost in controls (£24 of £415 at 0–6 months to £123 of £2428 at 0−36 months).This retrospective, longitudinal cohort study with matched controls is, to our knowledge, the first to provide a detailed analysis of HCRU and costs incurred by patients in England who have CLBP, with or without OA, and who experience moderate-to-severe pain. A total of 13734 patients with moderate-to-severe chronic pain associated with CLBP were identified, of whom 7803 (57%) also had a diagnosis of OA. Moreover, we identified 21357 patients with moderate-to-severe chronic pain associated with CLBP, of whom 7803 (37%) also had a diagnosis of OA. These estimates of OA as a comorbidity in patients with CLBP are higher than reported elsewhere , 12, mosPatients with moderate-to-severe pain associated with CLBP alone used significantly more healthcare services and incurred greater direct healthcare costs than their age-, sex-, geography-, and comorbidity-matched controls. The total direct cost of treating this patient cohort over 36 months was approximately £35 million (mean direct cost per patient of £5081 over 36 months), a considerable cost bearing in mind the relatively small size of the patient cohort (6724 patients with data at 36 months). Importantly, these direct costs represent a small proportion of the overall cost associated with CLBP, as our analysis did not take into consideration indirect costs, such as sickness absence and reduced productivity, and the cost of informal care, such as self-care and family care, not provided by the NHS. Inpatient stays were the main drivers of costs incurred in this patient population.Similarly, patients with moderate-to-severe pain associated with CLBP with concomitant OA used significantly more healthcare services and incurred greater direct healthcare costs than their matched controls. The total direct cost of treating these patients over 36 months was almost £31 million, with each patient incurring costs that were almost twice as much as those incurred by a patient with CLBP alone, primarily driven by inpatient stays and outpatient appointments. Almost one-quarter of patients in the CLBP + OA cohort underwent total joint replacement during the follow-up period, most commonly knee and hip replacement, considerably adding to the cost of their management. A small proportion of patients in both the CLBP-alone and CLBP + OA cohorts underwent procedures that were not recommended in the most recent guidelines for CLBP management (NICE low back pain and sciatica treatment guidelines published in 2016 and updated in 2020 ). Use ofOthers have reported similar HCRU trends in patients with CLBP compared with matched controls. In a US study of data from a managed care system, Mapel and colleagues observed that patients with CLBP were three times more likely to be hospitalised and had longer average lengths of stay—incurring greater costs—than their matched controls . In an aThe use of some analgesic medicines, including paracetamol and topical NSAIDs, increased across treatment lines in both cohorts of cases. Use of strong opioids remained consistent across lines in the CLBP cohort, at approximately one-third of patients. Since 2016, NICE guidelines for managing pain in people with chronic LBP advise against the use of most opioids, whereas the 2020 update to the OA management guidelines recommend opioids use with caution , 20. NotSome limitations of the study, many of which are inherent in real-world data analyses, should be considered. Diagnoses were identified using ICD-10 and Read codes, which are subject to potential miscoding. Secondary data sources are liable to have incomplete, missing, or low-quality data; the absence of a specific diagnosis or drug code was required to be interpreted as the absence of disease and medicines, respectively, resulting in potential misclassification bias. The CPRD is, however, widely considered a gold standard in healthcare event reporting, and missed diagnoses or prescriptions are likely to be rare. The requirement for 12 months of follow-up data following the index date introduces potential survivorship bias to the study. The chronic pain end date may not truly indicate the date when the chronic pain episode ended, as some patients may instead have decided not to use the NHS further and/or self-manage despite minimal improvement (if any) in their chronic pain.Not all HRGs have a national tariff, as some prices are negotiated locally; therefore, direct healthcare costs are likely to have been underestimated in this analysis as missing costs were not imputed. Our data were captured before changes in the recommended pharmacological treatment for people with LBP, specifically the use of opioids, and therefore might not reflect current pain management practice. Medicines bought over the counter, such as paracetamol and some NSAIDs, which have been reported to account for an annual spend of £600 million, and those administered through hospital pharmacies are not captured, and there is no national individual-level hospital prescribing database for England . This maSome analgesic medications have multiple indications; therefore, it is unknown if the medicine was prescribed to specifically treat CLBP or OA pain, or other pain and nonpain conditions. The definition of the study cohort and the involvement of clinical experts in the development of the protocol and statistical analysis plan meant that analgesics were likely to have been prescribed to treat the chronic pain associated with CLBP. The costs described in this study represent direct costs only; additional self-care and private care costs, plus societal and indirect employment costs, were not captured in this analysis but are likely to substantially increase the overall cost of chronic pain associated with CLBP.A key study strength is the real-world nature of the data. The linked CPRD and HES datasets have broad coverage, encompassing 75% of patients in English GP practices. Consequently, our findings are likely to be generalisable as an accurate representation of adults with CLBP alone and adults with OA + CLBP in England.Moderate-to-severe chronic pain associated with CLBP with or without OA has a substantial impact on patients and healthcare providers. We have shown that CLBP and OA are commonly comorbid and that healthcare utilisation and costs are increased for people with CLBP alone or comorbid with OA. This increase in healthcare utilisation and costs is largely associated with hospital admissions and outpatient attendances. Current costs for analgesic medicines are relatively low, but improvements in medical management have substantial potential to reduce resource utilisation if they can abrogate the need for more intensive care. Future research may look to describe resource use in patients with mild chronic pain and CLBP, to explore the indirect economic burden in this patient population. Further, with the establishment of integrated care boards (ICBs) and as the NHS becomes more data-driven, opportunities for similar research to use data derived from a broader breadth of aspects of care, such as from hospital pharmacies, may arise.


Crocus sativus L.). Due to its high price, economically motivated adulteration occurs relatively often. The presented study aimed to develop an effective strategy for the detection of the following potential botanical adulterants used for a saffron substitution or dilution: safflower (Carthamus tinctorius L.), calendula , turmeric (Curcuma longa L.), achiote (Bixa orellana L.), red pepper (Capsicum spp.), mountain arnica (Arnica montana L.), beet (Beta vulgaris L.), and pomegranate (Punica granatum L.). A non-target screening strategy based on ultra-high performance reverse-phase liquid chromatography coupled to tandem high-resolution mass spectrometry (UHPLC-HRMS/MS) was employed for the analysis of an aqueous ethanol plant extract. By using multivariate statistical methods, principal components analysis (PCA), and partial least squares discriminant analysis (PLS-DA), for processing the generated “chemical fingerprints,” metabolites unique to the investigated plants could be identified. To enable routine saffron authenticity control by target screening, an internal spectral database was developed; currently, it involves 82 unique markers. In this way, the detection addition as low as 1% (w/w) of all analyzed botanical adulterants in admixture with saffron was possible. The developed method was used to control 7 saffron powder samples from the Czech market, and none of the monitored adulterants were confirmed.Saffron is a unique spice obtained by drying stigmas of saffron flowers (The online version contains supplementary material available at 10.1007/s00216-023-04853-x. Crocus sativus L., which belongs to the Iridaceae family [Saffron has always been one of the world’s most precious spices. It is obtained by drying the stigma of e family , 2. Thise family . In addie family , 4.Carthamus tinctorius L.) [Calendula officinalis L.) [Curcuma longa L.) [Gardenia jasminoides J. Ellis) [Arnica montana L.) [Bixa orellana L.) [hrysanthemum morifolium L.) [Hemerocallis sp.) [Crocus vernus (L.) Hill) [Punica granatum L.) [Buddleja officinalis Maxim) [Beta vulgaris L.) [For food flavoring, saffron is sold in various forms. Stigmas are of the highest quality, but ground stigmas (saffron powder) are often sold for easier use. Existing data show that this form is prone to adulteration . For insrius L.) –8, 10–23alis L.) , 17, 20,onga L.) , 23, ext. Ellis) –27, petatana L.) , 17, dyelana L.) , 17, petlium L.) , 16, 17,lis sp.) , 17, sti.) Hill) , dye fros Maxim) , 7, dye The quality and classification of saffron are defined by the ISO 3632-1:2011 and ISO 3632-2:2010 standards, where the criteria for determining taste, color, aroma, moisture, minerals, foreign substances, and exogenous dyes are listed , 29. The1H NMR) [In addition to spectrophotometry, methods based on the combination of liquid chromatography (LC) and mass spectrometry (MS) , 16, 24 1H NMR) , 14, 18 1H NMR) , 19 and As shown in Table In the presented study, liquid chromatography coupled with mass spectrometry technique as a tool for saffron adulteration was selected, which is nowadays commonly used in food/natural product composition studies. The ambition of this research was not only to involve a broad range of botanical adulterants most often used for powdered saffron adulteration, but the study also aimed at finding unique markers which could be used for a simple and fast routine analysis by UHPLC-MS/MS.v/v) was obtained from Lach-Ner (Czech Republic), HPLC grade methanol from Honeywell (Germany), formic acid from VWR Chemicals (Great Britain), ammonium formate from Merck (Germany), and APCI Positive and Negative Calibration Solutions for the SCIEX X500 System from SCIEX (Canada).Deionized water was obtained from a Milli-Q purification system supplied by Millipore (USA); ethanol  and 7 samples of saffron powder were analyzed. Regarding geographic origin, 12 samples of saffron stigmas were from Iran, 4 from Spain, and 1 from Greece. The declaration of the geographic origin of other samples was unknown. In addition, potential adulterants were analyzed. The following samples of botanicals were available: 4 safflower petals (Carthamus tinctorius L.), 9 calendula petals , 6 turmeric rhizomes (Curcuma longa L.), 3 achiote seeds (Bixa orellana L.), 6 dried fruit of red pepper (Capsicum spp.), 3 mountain arnica petals (Arnica montana L.), 3 bulbs of red beet (Bea vulgaris L.), 2 pomegranate petals (Punica granatum L.), and 3 pomegranate seeds. The samples of saffron stigmas and potential adulterants were obtained at the Czech market by credible traders with spices.In this project, stigmas of 23 different samples of saffron (v/v) was added. The content of the tube was sonicated for 20 min, and then the sample was centrifuged . The aliquot of the supernatant was transferred into a 2-mL vial and analyzed. All samples were prepared in duplicate.The extraction procedure was based on an earlier published study . Brieflyw/w ratios 90/10, 95/5, 97/3, and 99/1) were prepared for analysis. These mixtures were prepared separately for each saffron-adulterant pair. For the preparation of these mixtures, all samples of saffron’s stigmas and separately individual adulterants that were available in this study were mixed. This step was taken to cover possible sample variability. The total weight of the prepared mixture was 100 mg, so twice the volume of solvent was added, i.e., 10 mL of ethanol/water . Subsequent extraction was performed under the conditions described above. All mixtures of saffron and adulterants were prepared in triplicate.In the next phase, mixtures of saffron and individual potential adulterants (v/v) formic acid. The mobile phase flow rate was 0.4 mL/min, and the gradient was as follows, 0–1 min 95% (A), 1–8 min 95–0% (A), 8–13 min 0% (A), 13–13.1 min 0–95% (A), and 13.1–15 min 95% (A); the sample injection volume was 2 µL.The chromatographic separation of sample extracts was performed using a Dionex UltiMate 3000 RS UHPLC system  with a reversed-phase Acquity UPLC HSS T3 column  held at 45 °C. The mobile phase consisted of water (A) and methanol (B), and both containing 5 mM ammonium formate and 0.1 % .Mass spectrometric detection was performed using a TripleTOF 6600 instrument . The ion source was a Duo Spray with a separated ESI ion source and atmospheric pressure chemical ionization (APCI). ESI was used for the measurement of extracts of the sample, and APCI was used for the exact mass calibration of the instrument. The parameters of the ESI ion source were as follows: capillary voltage, + 5000 V in ESI+ and − 4500 V in ESI−; collision energy, 35 ± 15 V in ESI+ and − 35 ± 15 V in ESI−; declustering potential, 80 V in ESI+ and − 80 V in ESI−; desolvation temperature, 480 °C; curtain gas, 35 psi; drying gas pressure, 55 psi; and nebulizing gas pressure, 55 psi. TOF MS method and information-dependent acquisition (IDA) method were employed to record full MS and MS/MS spectra at the same time. The To check the absence of carry-over effects and to control the stability of recorded fingerprints, blank and quality control (QC) matrix samples were analyzed within UHPLC-HRMS/MS sequences. The in-batch sequence of tested samples was random to avoid any possible time-dependent changes during analysis, which could result in false clustering. To control the overall performance of the instrumental system, QC samples were inserted into the sequence, always after a set of ten tested samples, and analyzed under the same conditions. The QC sample was prepared as a mixture of all analyzed samples. The good instrument performance was documented by a tight clustering of these QC samples  in the principal component analysis (PCA) score plot. The instrument control was carried out with the Analyst TF 1.7.1 software  and PeakView 2.2 software .Q2) and by the parameter known as “a goodness of fit” (R2(Y)), which was calculated by 7-fold internal cross-validation .The software MarkerView 1.2.1  was used for pre-processing raw data generated by the UHPLC-HRMS/MS instrument, separately for ESI+ and ESI−. For the detection of peaks in the range of retention time 0.5–10.5 min, the following parameters were applied: retention time tolerance 0.1 min; mass tolerance 0.01 Da; and data deisotoping. Also, the total area sum normalization  of the data set was performed, and before the principal component analysis (PCA), the Pareto scaling and logarithmic transformation were applied to all datasets. Different multivariate analysis techniques, such as PCA and partial least squared discriminant analysis (PLS-DA) for the identification of the most significant features, were employed by SIMCA software v. 13.0.3 . The model performance was expressed by the predictive ability parameter , which was used for receiver operating characteristics (ROC). The area under the ROC curve (AUC), t-test false discovery rate (FDR) adjusted p-value, and fold change (FC) parameters were determined for the created binary models of saffron stigmas and potential plant adulterants. The most important variables with VIP scores > 1 and AUC > 0.8 were chosen.For ranking the marker ions according to their importance, the VIP (variable importance in the projection) plots were used. To illustrate the role of these ions as markers, trend plots were constructed. Another filtration was performed by MetaboAnalyst open-source software v 5.0  were used. This software was also used to determine the lowest detectable amount of potential adulterant in a mixture with saffron when using respective marker ions. The detectability of individual marker ions in extracted ion chromatograms was carefully controlled; the lowest detectable amount of respective adulterant in a mixture of saffron stigmas and individual adulterants corresponded to signal-to-noise ratio of 10:1.m/z values in MS1 were considered) and isotopic pattern in MS spectra. To confirm the suggested identification of markers ions, their product ions were investigated in MS/MS spectra. For compound identification, online databases such as ChemSpider (http://www.chemspider.com/), mzCloud (https://www.mzcloud.org/home), PubChem (https://pubchem.ncbi.nlm.nih.gov/), or METLIN (https://metlin.scripps.edu/index.php) were employed.The tentative identification of markers was based on the calculation of the elemental formula  and was subsequently imported into the SCIEX OS 1.5 software . The formula or exact measured mass, retention time, and MS/MS spectra of all markers are saved in this database. This software was also used to evaluate unknown samples in which markers from the database were searched.– Relative abundance of saffron markers in samples of stigmas and saffron powder in Supplementary materials. A sample would be considered adulterated supposing more than 80% of the markers of one adulterant were detected. The limit of 80% is based on the Pareto principle.To control the authenticity of unknown saffron samples in terms of their dilution by some plant adulterants, a targeted screening of 82 markers shown in Table S2Untargeted analysis in combination with chemometrics was employed for the search of specific markers enabling the detection of saffron dilution by particular plant adulterants. UHPLC-HRMS/MS-based approach, rather similar to that earlier developed for the identification of saffron geographic origin , was useAfter peak detection and their alignment  within the sample set, using MarkerView software, a list of 5736 molecular features for the ESI+ and 5511 molecular features for the ESI− was obtained . These final datasets were exported into SIMCA software for statistical analysis. In the first step, the data were processed by an unsupervised tool, PCA, to give a first view of the data structure. The PCA score plot  were very high, which indicates excellent models.In the follow-up phase, only stigmas of saffron were included in the analysis of the principal component analysis together with the individual tested adulterants. Further, partial least squares discriminant analysis was performed for pairs of saffron and other individual potential adulterants. In Table The models created in this way  were also used to the selection of the characteristic markers of individual potential adulterants and saffron. Variable importance plots were constructed. For this purpose, only ions with VIP score > 1 and simultaneously with AUC > 0.8 were investigated as potential “markers.”m/z 235.1694  was not present in any of the saffron samples. Due to the unavailability of fragmentation spectra in the used databases, it is not possible to obtain unbiased identification of the marker. Based on the exact mass and isotopic pattern in MS spectra, it is probably one of the following substances: dihydrocostunolide, isocurcumenol, or curcumenol.For the demonstration of marker ion specificity, a trend plot for each marker was used. An example is shown in Fig. In the next phase, UHPLC-HRMS/MS data were assessed using SCIEX OS software to check the specificity of saffron markers, i.e., whether they cannot be detected also in other adulterants. As far as was the case, they were removed from the list of markers. A similar approach was employed to control whether compounds identified as markers in adulterants are not present in saffron. On the other hand, their presence in more than one plant species was not the reason for excluding them from the markers list.w/w) addition level detectable in at least three marker ions with the signal-to-noise ratio of at least 10:1. It is worth to mention that comparable results were achieved only in studies [The verification of the suitability of adulterant markers for fraud detection continued by testing their signal increase by increasing the amount of respective plant in a mixture with saffron. An example of a relevant turmeric marker is shown in Fig.  studies , 19 usin p-value, FC a VIP score, are summarized in Table As soon as marker ions were selected, at least tentative identification was suggested. Some substances were identified as known secondary metabolites — like safranal, crocetin, hydroxysafflower yellow A, and capsaicin. The highest mass error between the predicted and measured values was −4.8 ppm. All marker information, including mode of ionization, retention time, exact mass, MS/MS spectrum, predicted formula, adduct, mass error, tentative identification, confidence level, the lowest detectable amount in mixture with saffron using this marker, and statistical parameter like AUC,For target screening, the internal spectral database was created using the LibraryView software, which was then inserted into the SCIEX OS 1.5 software. The database currently contains 82 markers, 53 detectable in ESI+, and 29 in ESI−. The formula or exact measured mass, retention time, and the MS/MS spectra of markers were saved in the internal database, and these information were used for evaluating the sample.The knowledge of such markers enables significant simplification of saffron authentication, as the processing of data obtained by target screening is fairly less time demanding; moreover, a triple quadrupole mass analyzer can be used instead of a high-resolution one, which is needed for metabolic fingerprinting.This library of markers was tested to evaluate 7 samples of saffron powder from the Czech market. These samples were previously analyzed together with the set of samples of saffron stigmas, as shown in the PCA score plot Fig. . Target  – Relative abundance of saffron markers in samples of stigmas and saffron powder in Supplementary materials. As can be seen, the results in both saffron stigmas and powder were similar.The relative abundance of saffron markers in samples of stigmas and powdered saffron was also compared. This was determined by calculating the sum of the areas of all saffron markers in one sample (separately for positive and negative electrospray ionization modes), and each area was divided by this sum. The value was converted to percentages and shown in Figs. S2Carthamus tinctorius L.), petals of calendula , rhizomes of turmeric (Curcuma longa L.), seeds of achiote (Bixa orellana L.), dried fruits of red pepper (Capsicum spp.), petals of mountain arnica (Arnica montana L.), bulbs of red beet (Beta vulgaris L.), and petals and seeds of pomegranate (Punica granatum L.). Metabolomic fingerprinting (non-target screening) of aqueous ethanolic saffron/plant extracts obtained by UHPLC-HRMS/MS followed by multivariate statistical processing of generated data sets resulted in the selection of 82 unique marker ion characteristic for tested plant species. Targeted screening of these markers can be used for unbiased authenticity evaluation of samples labeled as “powdered saffron.” Addition as low as 1 % (w/w) of botanical adulterant to saffron powder is possible to detect, which is “sensitivity” comparable to that achievable by fairly more demanding methods based on molecular biology. It is worth to notice that while searching for markers requires the use of an instrumental platform with a high-resolution mass analyzer, the knowledge of concrete markers could allow a simple targeted analysis by widely available instruments equipped with a common triple quadrupole mass analyzer.Within this study, a simple and fast method was developed, which enabled the detection of powdered saffron dilution/substitution by any of the eight most often used botanical adulterants represented by petals of safflower (Supplementary file1 (XLSX 34 KB)Supplementary file2 (DOCX 20 KB)Below is the link to the electronic supplementary material.


Immune checkpoint inhibitors (ICIs) are widely used in metastatic melanoma and dramatically alter the treatment of these patients. Given the high cost and potential toxicity, a reliable method for evaluating treatment response is needed. In this study, we assessed tumor response in patients with metastatic melanoma treated with ICIs using three modified response criteria: PET Response Evaluation Criteria for Immunotherapy (PERCIMT), PET Response Criteria in Solid Tumors for up to Five Lesions (PERCIST5), and immunotherapy-modified PET Response Criteria in Solid Tumors for up to Five Lesions (imPERCIST5).18F]FDG PET/CT scans performed before and after ICI therapy. Responses at the follow-up scan were evaluated according to PERCIMT, PERCIST5, and imPERCIST5 criteria. Patients were classified into four groups: complete metabolic response (CMR), partial metabolic response (PMR), progressive metabolic disease (PMD), and stable metabolic disease (SMD). To assess the “disease control rate,” two groups have been defined based on each criterion: patients with CMR, PMR, and SMD as “disease-controlled group ” and PMD as the “uncontrolled-disease group ”. The correspondence between metabolic tumor response defined by these criteria and clinical outcome was assessed and compared.Ninety-one patients with non-resectable stage IV metastatic melanoma who received ICIs were retrospectively enrolled in this study. Each patient had two [P < 0.001), whereas it was not significant between PERCIMT and imPERCIST5. Overall survival was significantly longer in the metabolic responder groups than in the non-responder groups based on PERCIMT and PERCIST5 criteria . However, according to imPERCIST5 criterion, this difference was not observed (P = 0.12).The response and the disease control rates were 40.7% and 71.4%, 41.8% and 50.5%, and 54.9% and 74.7% based on the PERCIMT, PERCIST5, and imPERCIST5 criteria, respectively. PERCIMT and imPERCIST5 showed significantly different disease control rates from that of PERCIST5 (Although the appearance of new lesions can be secondary to an inflammatory response to ICIs and indicative of pseudoprogression, given the higher rate of true progression, the appearance of new lesions should be interpreted deliberately. Of the three assessed modified criteria, PERCIMT appear to provide more reliable metabolic response assessment that correlates strongly with overall patient survival.The online version contains supplementary material available at 10.1007/s00259-023-06247-8. Melanoma is a common skin tumor with increasing incidence, rapid progression, and a high rate of metastasis –3. In reDespite these dramatic advances, approximately 40–45% of patients do not respond to immunotherapy , 10. In Different patterns of response to treatment with ICIs have been described . PseudopGiven these atypical patterns of responses, the efficacy of conventional criteria such as Response Evaluation Criteria in Solid Tumors (RECIST) or PET Response Criteria in Solid Tumors (PERCIST) in the therapeutic evaluation of ICIs is limited , 5.These challenges affect all imaging modalities of treatment response assessment. At least four modified criteria have already been proposed for morphologic imaging: immune-related Response Criteria (irRC) (2009) , immune-18F]FDG PET/CT, in combination with brain MRI, is the preferred imaging strategy for staging metastatic melanoma FDG PET/CT imaging, comprising Prediction of Response to Immune Checkpoint Inhibitor Therapy (PECRIT) (2017) FDG PET/CT for progression-free survival (PFS) and overall survival (OS) of patients with advanced melanoma treated with ICIs.In this study, we evaluated the predictive value of PERCIMT, PERCIST5, and imPERCIST5 criteria in response to immunotherapy on [18F]FDG PET/CT between January 2015 and July 2021, were analyzed. Patients with stage IV metastatic melanoma treated with ICIs according to ESMO guidelines  or MAP/ERK kinase (MEK) inhibitors], second cancer, resectable metastasis, and inadequate imaging parameters at baseline and/or therapy monitoring. A diagram flowchart summarizing the patient selection process is provided in Fig. In this retrospective study, clinical and imaging findings of 426 consecutive patients with advanced melanoma, who underwent baseline and follow-up 8FFDG PET18F]FDG PET/CT scans were performed based on a standard protocol [18F]FDG injection. An amount of 4 MBq/kg body weight for each patient was administered. The mean interval between [18F]FDG administration and the start of the acquisition was 60 ± 12 min. A non-contrast-enhanced low-dose CT scan  was performed for attenuation correction and precise anatomical location. Slice measuring 3-mm iterative algorithm (Siemens: PSF + TOF and Philips: BLOB-OS-TF) were used for reconstruction in both scanners. PET data were corrected to account for scattering, attenuation, random coincidence, and scanner dead time.Whole-body [protocol . ImagingBoth PET/CT scanners were EANM Research Ltd./European Association of Nuclear Medicine (EARL/EANM) accredited.3 sphere voxel within the selected VOI and was determined automatically using a commercial software provided by scanner manufacture. Lesions with greater uptake than the reference threshold defined on the healthy liver or mediastinal blood pool without a pattern of inflammation or infection were defined as target lesion(s). A preset SUV cut-off window of 0–5 was defined for semi-quantitative and metabolic diameter measurements.SUV normalized to lean body mass (SUL) was measured by manually drawing a volume of interest (VOI) around the target lesion(s). SULpeak was defined as the highest mean SUL in a 1.0-cm18F]FDG PET/CT reports and clinical features.A board-certified nuclear medicine physician reviewed the images while blinded to the patient’s previous [18F]FDG PET/CT findings: complete metabolic response (CMR), partial metabolic response (PMR), progressive metabolic disease (PMD), and stable metabolic disease (SMD) according to PERCIMT, PERCIST5, and imPERCIST5 . Functional size is the maximum metabolic diameter of pathologic lesion (in centimeters) measured on the fused PET/CT images FDG PET/CT scans. CMR was defined as complete resolution of [18F]FDG uptake in the metastatic lesions without appearance of new metastasis on PET. PMR was defined as a decrease of more than 30% in the sum of SULpeak. PMD was defined as an increase of more than 30% in the sum of SULpeak in up to 5 prominent lesions or the appearance of new lesions. All other metabolic changes of [18F]FDG-avid lesions that did not fit into the definitions of CMR, PMR or PMD, were considered SMD.According to PERCIST5 criterion , the sum18F]FDG-avid lesion was detectable, and all metastases disappeared. PMR included a decrease of more than 30% or an absolute rate of more than 0.8 in the sum of the SULpeak, regardless of a new lesion. PMD included an increase of more than 30% or more than 0.8 absolute rates in the sum of the SULpeak, independent of a new lesion. SMD is classified when the definitions for CMR, PMR, and PMD were not met  . Using iWe calculated the “response rate” for each criterion separately. For this, the number of patients with CMR and PMR was divided by the number of all patients in the study and multiplied by 100. To assess the “disease control rate,” we also considered two groups based on each criterion: the “disease-controlled group”  and the “uncontrolled-disease group” (PMD). A comparison between the three criteria was performed using these classifications. The predictive value of PERCIMT, PERCIST5, and imPERCIST5 criteria for PFS and OS was assessed. For this purpose, patients with CMR, PMR, and SMD were classified as “responders,” and cases with PMD were classified as “non-responders”.p values < 0.05 were considered statistically significant. All statistical analyses in this report were performed using NCSS , STATISTICA 13 .Data were checked for consistency and normality. Fisher’s exact test or Pearson’s test was used to analyze cross-tabulations. Generalized linear models with log-normal distributions were used for continuously distributed variables, and LSD tests were used for pairwise comparisons. McNemar’s test compared dependent proportions with 95% confidence intervals. Kaplan–Meier analyses were done using log-rank tests based on randomization tests. All reported tests were two-sided, and 18F]FDG PET/CT with a median interval of 104 days. There were no therapeutic interventions for the patients between the baseline scans and the initiation of treatment and between the last treatment sessions and the follow-up scans. None of the baseline and follow-up scans was performed outside the recommended uptake time range (50–70 min after the injection). In all patients, the uptake time difference between the baseline and follow-up scans was less than 15 min. At the time of follow-up scans, patients had received a minimum of 2 and a maximum of 10 ICI doses. The number of doses received was unknown for eight patients at the follow-up scan. The last follow-up for calculating OS was performed on July 31, 2021. Overall, patients received a minimum of 2 and a maximum of 56 doses of ICIs at the end of the study (median of 9 doses).Overall, 91 patients with stage IV melanoma referred to our department between January 2015 and June 2017 were eligible to include in this study. The patient’s baseline characteristics are summarized in Table Patients were divided into four groups: CMR, PMD, SMD, and PMR, based on each criterion. The CMR patients were the same in all three response criteria. The number and percentage of different classifications in the three approaches are presented in Table P = 1.0) were found concerning the response rates , while it was significantly different (P < 0.001) from that of imPERCIST5 . PERCIMT and imPERCIST5 criteria showed disease control rates  of 71.4% and 74.7%, respectively (P = 0.57), which were significantly different (P < 0.001) from that of PERCIST5 criterion . The details of these findings are shown in Table Using the PERCIMT and PERCIST5 criteria, comparable results . One of the 91 enrolled patients had unclear clinical status at the last follow-up. Overall, concordant findings were observed between modified response criteria and clinical status at the end of follow-up in 48% (43/90), 53% (48/90), and 41% (37/90) of patients by PERCIMT, PERCIST5, and imPERCIST5 criteria, respectively.P = 0.001) and age ≥ 75 years with OS (P = 0.004). No significant correlation was found between BRAF mutation and a history of treatment with BRAF/MEK inhibitors and PFS or OS, despite the fact that 33% (30/92) of patients had BRAF mutation and 27% (25/92) of them were being treated with BRAF/MEK inhibitors. More details have been tabulated in Table The median PFS time was 0.51 years (95% CI: 0.41–0.76). The median OS time was 2.01 years (95% CI: 1.87–2.51), and the mean OS was 2.19 ± 1.52 years. Among the type of ICI regimen and clinical risk factors, a meaningful relation was only reported for the previous history of chemotherapy with PFS , Fig. P < 0.001). The OS was also higher for CMR compared to PMD using PERCIMT and PERCIST5 criteria . However, according to imPERCIST5, no significant difference in OS was observed between response groups .The PFS values showed a statistically significant difference between responders and non-responders based on all modified response criteria . However, based on the imPERCIST5 criteria, this difference was not observed (P = 0.12). Table On the other hand, the OS was significantly longer in the metabolic responder groups as compared with the non-responder groups based on PERCIMT and PERCIST5 criteria  FDG PET/CT imaging in immunomodulatory treatment recommend the use of spleen-to-liver ratio as a sign of the systemic immune response [One of our limitations was the relatively small population used to develop these criteria. It seems more comprehensive studies are needed for verification of the modified criteria. Additional larger studies with prospective designs can serve as a good guide to achieving consensus criteria for response assessment in melanoma patients treated with ICIs. It is also necessary to apply the modified criteria in standardization approaches of clinics. In addition, the PET/CT acquisitions were performed with two different scanners. This may result in non-identical SUV values between two scanner types. Furthermore, the recent EANM/SNMMI/ANZSNM joint practice guidelines for [response ; howeverThe results of this study indicate that although the appearance of new lesions may be secondary to an inflammatory response to ICIs and indicative of pseudoprogression, given the higher rate of true progression, the appearance of new lesions should be interpreted deliberately. Of the three evaluated modified criteria, PERCIMT appears to provide more reliable assessment of metabolic response that correlates strongly with overall patient survival.Supplementary file1 (DOCX 12 kb)Below is the link to the electronic supplementary material.


Salmonella enterica, a foodborne and human pathogen, is a constant threat to human health. Agricultural environments, for example, soil and plants, can be ecological niches and vectors for Salmonella transmission. Salmonella persistence in such environments increases the risk for consumers. Therefore, it is necessary to investigate the mechanisms used by Salmonella to adapt to agricultural environments. We assessed the adaptation strategy of S. enterica serovar Typhimurium strain 14028s to agricultural-relevant situations by analyzing the abundance of intermediates in glycolysis and the tricarboxylic acid pathway in tested environments , as well as in bacterial cells grown in such conditions. By reanalyzing the transcriptome data of Salmonella grown in those environments and using an independent RT-qPCR approach for verification, several genes were identified as important for persistence in root or leaf tissues, including the pyruvate dehydrogenase subunit E1 encoding gene aceE. In vivo persistence assay in tomato leaves confirmed the crucial role of aceE. A mutant in another tomato leaf persistence-related gene, aceB, encoding malate synthase A, displayed opposite persistence features. By comparing the metabolites and gene expression of the wild-type strain and its aceB mutant, fumarate accumulation was discovered as a potential way to replenish the effects of the aceB mutation. Our research interprets the mechanism of S. enterica adaptation to agriculture by adapting its carbon metabolism to the carbon sources available in the environment. These insights may assist in the development of strategies aimed at diminishing Salmonella persistence in food production systems. Salmonella enterica is an important foodborne pathogen, causing an estimated 1.35 million and 91,000 human cases in the United States and the European Union every year , tomato (Solanum lycopersicum), cabbage (Brassica oleracea), and onions (Allium cepa L.), have been reported to cause outbreaks in the past years by the CDC , for invasion, reproduction, and colonization. Type III Secretion Systems (T3SSs) and effectors encoded on diverse SPIs assist Salmonella in overcoming the host's innate immunity. In atypical hosts, for example, plants, the use of T3SSs and effectors remains ambiguous, and corresponding reports are sometimes conflicting. SPI1 and SPI2 were shown to be implicated in the colonization of Arabidopsis  and lettuce  cycle , fructose-bisphosphate aldolase (fba), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogenase (gapA), and triosephosphate isomerase (tpiA) are deficient in survival in macrophage cells   actively adapts to agricultural environments as ecological niches and changes its metabolism accordingly. In the first step, GC-MS analysis was applied to assess the availability of intermediate compounds in central carbon metabolism in diverse ecological niches related to agricultural systems, which included diluvial sand (DS) soil suspension, tomato leaf-based medium (TM), and lettuce leaf-based medium (LM). In parallel, the same compounds were assessed in correspondingly cultured Salmonella cells. Furthermore, genes advantageous for Salmonella grown in root- or leaf-related environments were identified by the comparison of transcriptome data  and malate synthase A (aceB). Mutated and complemented strains in aceE and aceB were subjected to persistence assay in tomato leaves in order to validate our observations in vivo. In addition, major intermediates of central carbon metabolism were compared between S. Typhimurium 14028s and the mutants. Our results suggest that fumarate accumulation contributed to the enhanced persistence of the aceB mutant. The downregulation of fumarate catabolism genes in the aceB mutant, compared to S. Typhimurium 14028s, suggests a possible route for fumarate accumulation. Taken together, our research provides new insights into how Salmonella modifies its own carbon metabolism in order to thrive in agricultural environments.In this study, we hypothesized that Salmonella enterica serovar Typhimurium strain 14028s (S. Typhimurium 14028s) with spontaneous rifampicin resistance (50 μg/mL) used in this study was constructed by Fornefeld et al.  with kanamycin resistance (50 μg/mL) were provided by Michael Hensel . Since aceB and aceE are the first genes in corresponding operons, the complete sequences of aceBAK and aceEF were cloned and introduced to ΔaceB and ΔaceE, respectively. Complemented strains (ΔaceB::aceBAK and ΔaceE::aceEF) are both kanamycin (50 μg/mL) and carbenicillin (50 μg/mL) resistant. Briefly, sequences were cloned to the backbone of pWSK29. The vectors (PaceB aceBAK and PaceE aceEF) were thereafter transformed into Escherichia coli strain NEB5α , respectively. Single colonies picked from medium-added carbenicillin (50 μg/mL) were verified by sequencing. Afterward, vectors of PaceB aceBAK and PaceE aceEF extracted from E.coli NEB5α were electroporated using the Ec2 program in the MicroPulser Electroporator  into ΔaceB and ΔaceE competent cells, respectively. After 1 h of recovery in SOC medium [SOB  + 20 mM glucose ] at 37°C with a shaking speed of 150 rpm, the bacterial suspension was spread on LB agar containing kanamycin (50 μg/mL) and carbenicillin (50 μg/mL) and incubated at 37°C overnight. Single colonies were picked, streaked onto novel plates, and verified by PCR. Primers for verification are listed in d et al. . S. TyphSalmonella was cultured in LB medium  with the required antibiotics for inoculums. Minimal medium (MM) was prepared from 12.3 mM glucose, 1 × M9 salts , 2 mM MgSO4 , and sterile deionized water  proportion  bag, which was then sealed and immersed in 30 mL of the respective medium in a 50 mL centrifuge tube. The tube was shaken at 28°C for 24 h. In the metabolism experiment, part of the samples was separated for bacterial enumeration, while the rest was pelleted at 4°C in a precooled centrifuge tube. After being rinsed on ice with 10 mM MgCl2, the pellets were centrifuged again and instantly frozen in liquid nitrogen. Salmonella cultures designated for RNA extraction were immediately vortexed with double the volume of RNAprotect Bacteria Reagent . After 5 min of incubation, the bacteria were centrifuged at 5,000 × g for 10 min at room temperature. Bacterial pellets for both metabolism and RT-qPCR assays were stored at −80°C until further processing. Four biological replicates were prepared for each treatment.A dialysis membrane system was used to collect 13C-ribitol solution to re-suspend the cells. Thereafter, the sequential addition of 100 μL of H2O and 150 μL of chloroform was performed with vortexing for 5 min in between. After centrifugation, 150 μL of the polar phase was dried in a vacuum concentrator. For extracellular metabolites, 10 μL of cell-free culture supernatant was mixed with 10 μL of methanol/13C-ribitol solution and then dried in a vacuum concentrator. Derivatization and GC-MS measurement (splitless and split 1:10) of metabolites were performed as described in a previous study . MetaboAnalyst 5.0 , a web-based tool, was used for analysis. The principle component analysis (PCA) was performed using the statistical analysis (one factor) module. In the heatmaps, the cluster method is Ward.D and the distance is Euclidean distance. The relative abundance of metabolites in S. Typhimurium 14028s and ΔaceB grown in TM was compared to that in MM using log2 fold change. Four biological replicates were prepared for each treatment.Metabolites were extracted from wet biomass through the methanol–water–chloroform extraction process using 100 μL of methanol/Salmonella total RNA was extracted using the RNeasy Mini Kit , DNA was eliminated, and cDNA was synthesized using the Maxima H Minus First Strand cDNA Synthesis Kit  according to the manufacturer's instructions. Thereafter, 20 μL of the cDNA generated from 1 μg DNA-digested RNA was diluted to 100 μL for qPCR. The qPCR reaction system was 20 μL, consisting of 10 μL of LUNA Master Mix , 4 μL of nuclease-free water, 0.5 μL of forward or reverse primers (10 μM), and 5 μL of diluted cDNA. The Bio-Rad CFX Connect cycler  was programmed with an initial heating of 95°C for 5 min, followed by 39 cycles of 95°C for 15 s and 60°C for 30 s (+plate read). Melting curves were measured for quality control. The rfaH gene  seeds germinated in Substrate 1  were transplanted to 9 cm diameter pots in the greenhouse with 18 h of daylight at 20°C. To avoid contamination of the leaves, plants were irrigated and fertilized from the bottom of the trays as needed. Plants were ready for treatment 3 weeks after transplantation.Three weeks after sowing, tomato . The most marginal two leaflets of the plant's second and third leaves were chosen. When the inoculated leaflets recovered from their water-stained appearance 3 h post-infiltration, as well as 7 and 14 days post-inoculation (dpi), four 5-mm diameter leaf disks were sampled and pooled in a 2 mL centrifuge tube, homogenized with 200 μL of 10 mM MgCl2 using a motor handpiece , and then replenished to 1 mL. Two technical replicates of homogenized leaf slurry were serially diluted in a 96-well plate. Ten μL of the dilution was dropped on XLD agar supplemented with appropriate antibiotics in duplicate. After overnight incubation at 37°C, Salmonella colonies with easily identifiable black coloration were enumerated. The persistence was evaluated by calculating the log10 CFU in each leaf disk. One-way ANOVA with the Tukey HSD test was used to verify differences. The error bars represented the standard deviation among the four biological replicates.To evaluate the persistence of Salmonella enterica serovar Typhimurium strain 14028s (S. Typhimurium 14028s) was reported to persist in diluvial sand (DS) soil for several weeks  cycle. To determine whether S. Typhimurium 14028s used a carbon metabolism adaptation strategy to persist in agricultural environments, the abundance of the central carbon metabolism intermediate products was evaluated both in the mimicking media  to imply the availability for bacteria and in the correspondingly incubated S. Typhimurium 14028s cells.2 and M9 salt-based minimal medium (MM) were used. MgCl2 solution, which was used to pre-suspend the bacteria before inoculation and to wash pellets during sampling, displayed a clean background for all metabolites discussed below  provided supplementary information on their transcriptional adjustment to root-related niches  catabolizes glyoxylate into malate.On the one hand, the upregulation of S. Typhimurium 14028s, aceE and aceB mutant strains (ΔaceE and ΔaceB), and genetically complemented strains (ΔaceE::aceEF and ΔaceB::aceBAK) were used. Gene expression of aceE or aceB in the complemented strains was similar to the level in S. Typhimurium 14028s cultured in TM, while no signal was detected in the corresponding mutant strains , while complementation recovered that deficiency , abundances of multiple metabolites were altered, albeit to variable degrees , which had lower persistence ability in tomato leaves, was significantly lower than that in S. Typhimurium 14028s when grown in TM  to succinate. To investigate the possible origin of the fumarate accumulated in ΔaceB, we evaluated the expression of frdD and frdA in ΔaceB and S. Typhimurium 14028s using RT-qPCR. Compared to the control (S. Typhimurium 14028s grown in MM), frdD and frdA expressions in S. Typhimurium 14028s were upregulated in TM , spinach (Spinacia oleracea), mugwort (Artemisia), green tea (Camellia sinensis), Phalaenopsis, and rubber tree (Hevea brasiliensis) , seemed related to carbon metabolism as well  affecting PPP and KDPGP, displayed impaired virulence in vivo when using the mice model , acting as an electron donor, was also reduced compared to S. Typhimurium 14028s, although not significantly (fumB) or C4-dicarboxylate transporter (dcuB) in both S. Typhimurium 14028s and ΔaceB grown in TM was comparable to that grown in MM (S. Typhimurium 14028s. However, the association between the aceB mutation and diminished fumarate catabolism still needs to be investigated in the future.While analyzing the metabolite abundance, fumarate accumulation was identified as one of the potential reasons for enhanced Δn leaves . The bion leaves . Fumaratand frdD , rather dD (sdhC  in the TdD (sdhC  and RT-qdD (sdhC . A hypotm 14028s  and Δace8s (aceB  in TM waficantly . Intereswn in MM , S7D, whwn in MM , we obseSalmonella enterica in agricultural environments. We provided candidate genes regulated in particular ecological niches, which could be a premise for future mechanistic studies. As an example of leaf environment-related genes, aceE and aceB were studied in more detail. We postulate that the improved persistence of ΔaceB might be caused by the accumulation of fumarate in bacterial cells present in leaves, and the deficient persistence of ΔaceE was also fumarate-related. The understanding of the mechanisms used by Salmonella to utilize carbon sources could lead to prospective strategies to diminish or perhaps prevent Salmonella's long-term persistence in agricultural environments.Taken together, we described a general pattern of adaptation to carbon sources used by the human pathogen The original contributions presented in the study are included in the article/MHa, JS, SJ, and AS conceived the research project and contributed to the experimental design. Experimental study was performed by MHa with the assistance of YD. MN and MHe provided bacterial mutants and complemented strains. JW and MN-S executed GC-MS and raw data analysis. MHa performed data analysis, designed figures, and wrote the first version of the manuscript. All authors contributed to the manuscript's improvement and revision.