The glymphatic system, a fluid-clearance pathway involved in brain waste clearance, is known to be impaired in neurological disorders, including Alzheimer’s disease (AD). For this reason, it is important to understand the specific mechanisms and factors controlling glymphatic function. This pathway enables the flow of cerebrospinal fluid (CSF) into the brain and subsequently the brain interstitium, supported by aquaporins (AQPs). Continuous CSF transport through the brain parenchyma is critical for the effective transport and drainage of waste solutes, such as toxic proteins, through the glymphatic system. However, a balance between CSF production and secretion from the choroid plexus, through AQP regulation, is also needed. Thus, any condition that affects CSF homeostasis will also interfere with effective waste removal through the clearance glymphatic pathway and the subsequent processes of neurodegeneration. In this review, we highlight the role of AQPs in the choroid plexus in the modulation of CSF homeostasis and, consequently, the glymphatic clearance pathway, with a special focus on AD. Protein aggregates are a common feature of neurodegenerative diseases, including Alzheimer’s disease (AD) . This imAlong perivascular spaces, CSF transport partly involves the metabolic waste disposal pathway in the brain known as the “glymphatic system” . The glyIn the glymphatic pathway, a convective CSF influx is compensated by the perivenous efflux of the interstitial fluid (ISF), clearing noxious proteins and peptides, including amyloid-beta (Aβ) . CSF movIn the last decade, several studies reported that soluble Aβ protein and tau oligomers are removed from the brain through the glymphatic system ,10,11,12To maintain the extracellular environment of the brain, the blood–brain barrier (BBB) and the blood–CSF barrier (BCSFB) separate the blood from the brain parenchyma and the CSF, respectively . The BCSThe choroid plexus epithelial cells are sealed by tight junctions at the luminal membrane conferring the barrier property to the BCSFB. Tight junctions are composed of proteins associated with the cell membrane of the choroid plexus epithelial cells ,18,19. OThe major function of the choroid plexus is to form the BCSFB and produce the CSF. For this purpose, the plasma that flows through the fenestrated capillaries is rapidly transported by hydrostatic pressure into the choroid plexus interstitium. Once filtered by the membranous diaphragms, the ultrafiltrate is transported through the choroid plexus epithelial cells to the ventricle, where it can be used in CSF production .Contrary to other secretory epithelia, in the choroid plexus epithelium, most ion transporters are located in the apical membrane, for example, Na+-K+ ATPase channels, K+-Cl-Na+HCO3 cotransporters, or some AQP water channels. Only a few transporters, such as K+Cl- cotransporters, are found on the basolateral membrane . In addiSeveral significant alterations have been indicated in the aging choroid plexus. A loss of 11% in the volume of human choroid plexus epithelial cells has been associated with aging. In addition to collagen fibers and dystrophic calcifications within the stroma, aged choroid plexus epithelial cells show increased pathological protein deposits called Biondi ring tangles ,38. ReduMorphological changes in the choroid plexus have been described in AD, including the atrophy of choroid plexus epithelial cells with the accumulation of lipofuscin vacuoles, stromal fibrosis, and the thickening of blood vessel walls and the basement membrane of the choroid plexus ,42,43,44Aβ presence in the choroid plexus may induce innate immune reactions, increasing the concentration of IgG that leads to capillary damage and interstitial fibrosis . It has It is well known that the primary role of the BCSFB is to prevent the free passage of molecules between the blood and the CSF, and the main function of the choroid plexus is to the production and regulation of the CSF ,14,27,59Inflammation is an important defensive response to infection or injury, and the choroid plexus is known as an entry site for pathogens, a checkpoint for peripheral immune cells into the CNS, and a regulator of cytokines and other signaling molecules in the CSF ,66,67. SAdditionally, the choroid plexus is involved in the control of circadian rhythmicity ,32,33. TIn 2012, Nedergaard and colleagues defined the glymphatic system as a network of perivascular spaces through which the CSF circulates in the brain parenchyma . The glyIt is well documented that the CSF and the cerebral ISF drain into deep cervical lymph nodes, but the CSF also drains into the perineural space. In fact, perineural pathways along the optic and olfactory nerves are considered relevant lymphatic routes ,74. HoweMost neurodegenerative diseases are characterized by the improper accumulation of cellular waste products, among which misfolded proteins are the most difficult to clear from the brain, and their aggregates lead to diseases such as AD . The glyAlthough the best-described function of the glymphatic system is solute clearance, studies are underway on other possible functions such as facilitating the transport of glucose , apolipoGlymphatic activity decreases with advanced age. Studies in mice show a reduction in the efficiency of clearance and loss of perivascular APQ4 polarization in the astrocytes of aging mice compared with young mice . AQP4 isIt has been established that the glymphatic system is more effective during sleep and is largely inactive during wakefulness ,82,83. DThe glymphatic system is one of the numerous mechanisms that contribute to the removal of soluble Aβ from the brain . AD patiAQP4, located on the perivascular astrocytic end-feet, enables the exchange of CSF and ISF and facilitates CSF influx into the brain parenchyma and its efflux back to the perivascular space . Owing tAQPs are a heterogeneous group of channel-forming proteins found in all kingdoms of life . AQPs arAQPs are tetramers and comprise monomers with identically hydrophilic membrane proteins. Each monomer includes six transmembrane alpha helix segments and two short helix segments, joined by five connecting loops. The carboxyl and terminal amino domains are located on the intracellular side. This distribution allows AQPs to operate as a hydrophilic integral membrane protein . The cenThe distribution of AQPs includes different organs and tissues, which have been associated with a variety of important physiological functions including brain water homeostasis, transepithelial fluid transport, osmoregulation, angiogenesis, wound healing, cell signaling, migration, and proliferation ,108. In In the brain, AQPs mediate the movement of water between different fluid compartments, including the CSF, the ISF, and the blood ,110. As The expression of AQPs has not been extensively studied in the choroid plexus; however, there is evidence that AQP1, AQP4, AQP5, AQP7, and AQP11 are expressed in the epithelial cells of the choroid plexus.AQP1 was observed on the choroid plexus epithelial cells of mice, where it is located in the apical membrane at the ventricular-facing surface of the choroid plexus epithelium ,140,141.In the CNS, the most frequent aquaporin is AQP4. It is usually expressed in ependymal-glial-limiting membranes, including the ependymal cells and subependymal glia that border the brain ventricles, in the subpial astrocytes of the cortex, the end-feet of the perivascular astrocytes that surround the blood vessels forming the BBB, and the choroid plexus. The presence of AQP4 in the choroid plexus of rats was reported in some previous studies, in which they showed weak and diffuse AQP4 signals ,126. HowAQP4-positive cells were found in the choroid plexus of human donors, where the AQP4 signal was mostly observed in the basolateral membrane domain . In thisIn a previous study by Iliff et al., it was found that AQP4, expressed in astrocytic end-feet in contact with the brain vasculature, facilitates CSF transport into the brain parenchyma .Although the dependence of the glymphatic system on AQP4 has been demonstrated and reported in some studies , the phyAQP5 has been also described in choroid plexus epithelial cells . MoreoveThe aquaglyceroporin AQP7 mRNA and protein levels were largely found in the choroid plexus, increasing during perinatal development of the brain, and suggesting that AQP7 could be an important structural and functional element in the choroid plexus during brain development in mice . EvidencAQP7 was first denoted as “aquaporin adipose” because it was initially described in human adipose tissue . Next, oIn rat choroid plexus a weak AQP11 expression has been reported . More reAlthough the choroid plexus is involved in the production and regulation of one of the central components of the glymphatic system, the CSF, not enough attention has been paid to research on the glymphatic system. A balance between CSF production and drainage is required for operative waste removal through the clearance glymphatic pathway. In this environment, AQPs play a major role in CSF homeostasis. As we propose in The choroid plexus comprise an intricate network of complexly organized cells that, in addition to functioning as a barrier between the blood and CSF, have now been confirmed to play a major role in neurological processes, including AD. Currently, more understanding about the connection between the choroid plexus and glymphatic system is appearing, and in this review, we summarized some key elements to contribute highlight the role of AQPs in the choroid plexus modulating CSF homeostasis and, consequently, the clearance glymphatic pathway.


Blood lipids are strong risk factors for the progression of atherosclerotic plaques. However, data on gender-specific associations are limited.To examine gender-specific associations of coronary plaque with blood lipids among a large sample of Saudi patients without CAD.Retrospective cross-sectional study was conducted among adult patients referred to  computed tomography (CT) for standard indications at the Prince Sultan Cardiac Centre  between July 2007 and December 2017. Those with pre-existing CAD were excluded. Plaques were determined based on quantification of coronary calcium and Coronary CT angiography.p < 0.001). Approximately 78.9% of all plaques were calcified. Blood lipids (mmol/L) were 4.75 ± 1.14 for total cholesterol, 2.90 ± 0.96 for LDL cholesterol, 1.20 ± 0.36 for HDL cholesterol, and 1.64 ± 1.09 for triglycerides. Males had significantly higher triglycerides and lower HDL cholesterol compared with females. In adjusted models in males and all patients, soft and/or calcified plaques were significantly associated with lower HDL cholesterol and higher triglycerides. In females, the only significant association was between soft plaques and higher triglycerides.A total 2421 patients  were included. The prevalence of any plaque was 36.6% with higher burden in males than females (41.3% versus 28.9%, Middle-aged patients without clinical CAD in Saudi Arabia have a high burden of plaques, specially calcified ones. The findings may impact the use of lipid lowering mediations, by underscoring the importance of assessing the risk of CAD in patients without clinical CAD even in case of lack of coronary calcification. Atherosclerosis is a chronic inflammatory disease characterized by build‑up of fatty plaques in the walls of arteries, eventually leading to their narrowing . RuptureHyperlipidemia particularly high levels of low-density lipoprotein (LDL) cholesterol is one of the leading risk factors of CAD and stroke , 9. OverThe prevalence of hyperlipidemia in Saudi Arabia has been estimated between 20 and 50% in the general population –15. HoweThe current study was conducted at Prince Sultan Cardiac Center (PSCC). The PSCC is 200-bed specialized cardiac center located in Riyadh that provides a major portion of the diagnostic and therapeutic cardiac services in Saudi Arabia. The PSCC has several departments including adult and pediatric cardiology, adult and pediatric cardiac surgery, cardiac anaesthesia, and advanced imaging. The current study was done at the advanced imaging unit under adult cardiology.It was a retrospective cohort study conducted between July 2007 and December 2017. The study design obtained all required ethical approvals from the ethical committee of PSCC.The study targeted adult patients (age > 18 years) referred to  computed tomographic (CT) for standard indications. Those with pre-existing CAD were excluded from the study. Pre-existing CAD was defined as myocardial infarction, angioplasty, stent placement, and coronary artery bypass grafting. Additionally, CT done for aortic assessment, for pericardial assessment and low-quality CT with artifacts were excluded from the study. Finally, those who were missing blood lipid (N = 441) or plaque (n = 20) testing results were excluded, leaving 2421 for analysis. The number of patients is sufficient to detect a gender-specific difference in total cholesterol of 0.15 mmol/L with more than 90% power.Patients were scanned during a single breath-hold using a 64  CT scanner (Philips Brilliance). A retrospective gating protocol with thickness of 0.5 to 2.5, FOV 220, and the average radiation dose is 6–9 mSv. The scanning protocol was designed to minimize the radiation dose based on BMI. Indications of coronary CT included chest pain in patients with intermediate risk of CAD, impaired left ventricular function in asymptomatic patient, before non-coronary cardiac surgery in patients with intermediate risk of CAD, to rule out coronary anomaly, and in case of arrhythmia with atypical chest pain.Plaques were determined based on quantification of coronary calcium and coronary CT angiography. Classification of plaques as calcified or soft  was based on the presence or lack of coronary calcification (respectively). The later was expressed using Agatston score, which reflects the total area of calcium deposits and the density of the calcium in the entire coronary tree. Therefore, those with coronary stenosis on coronary CT angiography and had above zero coronary calcium score were classified as calcified plaque Fig. A while tStratification of the risk of CAD among patients was done using the number of risk factors and the systematic coronary risk evaluation (SCORE) of the European Society of Cardiology (ESC). Risk factors for CAD were defined as history of hypertension, diabetes, dyslipidemia, smoking, family history of premature CAD (before the age of 65 years), and obesity (BMI > 30). The SCORE estimating the 10-year risk of developing fatal cardiovascular disease in populations with high cardiovascular disease risk was calculated according to standard methodology .Study data collection sheet was initiated for patients who underwent coronary CT and meeting the study eligibility criteria. Clinical information including medical history, traditional risk factors, and cardiac comorbidity were then abstracted from the electronic patient chart system.P-value < 0.05 was considered as significant. SPSS software  was used for all statistical analyses.Demographic characteristics, risk stratification, and blood lipids were compared by the plaque and gender groups. Chi-square or Fisher exact tests (as appropriate) were used to detect differences in categorical variables. Analysis of variance (ANOVA) or Kruskal–Wallis test (as appropriate) were used to detect plaque differences in continuous variables while t-test or Mann–Whitney test (as appropriate) were used to detect gender differences in continuous variables. In case of plaque groups, pairwise differences were calculated using Bonferroni adjustment method for multiple comparisons. Generalized linear regression models was used to detect differences in log-transformed blood lipid levels between plaque and gender groups after adjusting for other CAD risk factors. Log data were transformed back to normal before presentation in Table p < 0.001). Approximately 78.9% of all plaques were calcified. The prevalence of calcified plaques was 28.9% and was higher in males than females . The prevalence of soft plaques was 7.7% and was higher in males than females .A total 2421 patients  were included in the current analysis. As shown in Fig. Table Table Table Table The current study reported gender-specific prevalence of plaques, levels of blood lipids, and their associations among patients without history of CAD in Saudi Arabia. Approximately 37% of the patients in the current study had plaques, with 79% of them had calcified plaques. The current finding confirms the high burden subclinical atherosclerosis among middle-aged patients without clinical CAD . AdditioThe levels of blood lipids in the current study were slightly different from global levels . For exaSimilar to previous studies, coronary plaques in the current study were associated with traditional risk factors of CAD including the SCORE , 27, 28.The current study is considered the first study in Saudi Arabia and the Gulf region to examine gender-specific associations of plaques and blood lipids. The study examined a large number of patients seen over 10 years in a large referral center. This allowed gender specific presentation of data. Both univariate and multivariate analysis was used to detect independent associations. Nevertheless, few limitations are acknowledged. The cross-sectional design can determine association but not causation. Being a single center experience without follow up data about clinical CAD may limit the generalizability of the findings. However, it should be fine for a similar population without evidence of clinical CAD. As the patients were first seen at the CT, there was lack of information about the use of and adherence with lipid lowering medications. Patient rather than segmental plaque assessment may have overestimated calcified plaques. However, we believe that these limitations have minor impact on the study finding (if any).In conclusion, middle-aged patients without clinical CAD in Saudi Arabia have a high burden of plaques, specially calcified ones. Plaque-gender interactions were detected in HDL cholesterol and triglycerides. Males who had higher burden of plaques had higher triglycerides and lower HDL cholesterol compared with females. Plaques were associated with lower HDL cholesterol and higher triglycerides, with stronger associations in males than females in both univariate and multivariate analysis. The current findings may impact the use of lipid lowering mediations, by underscoring the importance of assessing the risk of CAD in patients without clinical CAD even in case of lack of coronary calcification.


Donor-characteristics and donor characteristics-based decision algorithms are being progressively used in the decision process whether or not to accept an available donor kidney graft for transplantation. While this may improve outcomes, the performance characteristics of the algorithms remains moderate. To estimate the impact of donor factors of grafts accepted for transplantation on transplant outcomes, and to test whether implementation of donor-characteristics-based algorithms in clinical decision-making is justified, we applied an instrumental variable analysis to outcomes for kidney donor pairs transplanted in different individuals.This analysis used (dis)congruent outcomes of kidney donor pairs as an instrument and was based on national transplantation registry data for all donor kidney pairs transplanted in separate individuals in the Netherlands , and the United Kingdom . Incident early graft loss (EGL) was used as the primary discriminatory factor. It was reasoned that a scenario with a dominant impact of donor variables on transplantation outcomes would result in high concordance of EGL in both recipients, whilst dominance of asymmetrical outcomes could indicate a more complex scenario, involving an interaction of donor, procedural and recipient factors.Incidences of congruent EGL  were slightly lower than the arithmetical (stochastic) incidences, suggesting that once a graft has been accepted for transplantation, donor factors minimally contribute to incident EGL. A long-term impact of donor factors was explored by comparing outcomes for functional grafts from donor pairs with asymmetrical vs. symmetrical outcomes. Recipient survival was similar for both groups, but a slightly compromised graft survival was observed for grafts with asymmetrical outcomes in the UK cohort: (10-years Hazard Ratio for graft loss: 1·18 [1·03-1·35] p<0·018); and 5 years eGFR .Our results suggest that donor factors for kidney grafts deemed acceptable for transplantation impact minimally on transplantation outcomes. A strong reliance on donor factors and/or donor-characteristics-based decision algorithms could result in unjustified rejection of grafts. Future efforts to optimize transplant outcomes should focus on a better understanding of the recipient factors underlying transplant outcomes.None. Donor-characteristics, and donor characteristics-based algorithms are often used in the decision-process whether or not to accept an offered donor kidney for transplantation. Yet, epidemiological evaluations suggests that donor factors and decision-algorithms (risk indices) only modestly predict outcomes and therefore a reliance only on the donor factors and/or decision algorithms may result in discard of potentially suitable grafts.Using national registry data for the Netherlands and the UK, an instrumental variable analysis based on an outcome comparison of donor kidney pairs, that were transplanted in two different recipients, was applied. Results from this evaluation suggest that once kidneys are considered eligible for transplantation, donor factors minimally impact outcomes.A strong focus on donor factors in the decision-making process whether or not to accept an offered graft for transplantation might result in unjustified rejection or even discard of viable donor organs.Alt-text: Unlabelled box,,7In an era of pressing donor shortages, decisions when to accept or decline a donor graft for transplantation, and questions how to optimize allocation of available donor organs remain major challenges to the field.The modest performance of donor risk indices may reflect incomplete attribution of relevant donor aspects, failure to fully capture all procedural characteristics in the donation and procurement process and/or a possible recipient-donor organ interaction. Moreover, since most risk indices relate to 5-year post-transplant outcomes, they are affected by differences in the quality and organization of post-transplant care.In an effort to estimate the contribution of donor factors (of grafts accepted for transplantation) to transplantation outcomes in kidney transplantation, we here report an instrumental variable analysisIt was reasoned, that a scenario with a dominant impact of donor variables on transplantation outcomes would result in high concordance of EGL in both recipients, whilst dominance of asymmetrical outcomes could indicate a more complex scenario, involving an interaction of donor, procedural and in particular recipient factors.The study is based on data from two national registries in The Netherlands and the United Kingdom (UK): the NOTR (Netherlands Organ Transplant Registry) and NTxD . The study is based on fully anonymized registry data, as such no formal medical ethical consent is required according to Dutch law. The scientific review boards of the NOTR and NTxD approved the study. The original first data-set retrieved from the NOTR, was analyzed and evaluated. To study the robustness of the findings, subsequently, a second similar data-set was generated in NTxD with help of the Clinical Trials and Studies Unit of NHS Blood and Transplant (NHSBT) in the UK for comparison with the results from the NOTR and to allow evaluation of long-term (i.e. day 90 and beyond) outcomes. Data used in the study is accessible through NOTR and NTxD.The study protocol was approved by the respective governing committees of both NOTR that is hosted by the Dutch Transplant Foundation (NTS) and of UK NTxD that is hosted by the NHSBT. The reported clinical and research activities are consistent with the Principles of the Declaration of Istanbul as outlined in the 'Declaration of Istanbul on Organ Trafficking and Transplant Tourism. Patients and public were not involved in the study.The NOTR and NTxD are both mandatory nationwide registries that include granular donor and recipient demographics, procedural and outcome data of all types of organ transplantation carried out in both The Netherlands and the UK, and include data provided by the organ procurement organization as well as all participating transplant centers. In the first year after transplantation, registry follow-up is at month 3, thereafter on a yearly basis. Quality checks are performed by on-site polls, business rules in application and cross checks with the national dialysis registry. Data on multiple variables as kidney donor and recipient characteristics as well as transplant outcomes for all procedures performed in The Netherlands from 1st January 1990 through 1st January 2018, and subsequently in the UK from 1st January 2000 through 1st January 2018 were retrieved. There were no missing data for type of donor, graft survival or 90-day recipient survival in both data sets. Sixteen respectively 27% of the 1 and 5 years eGFRs values used for the secondary outcome analysis were missing. Missing values were handled as -missing at random- and list-wise deleted.This study only includes data for procedures in which both donor kidneys were transplanted in two different recipients and for which outcome information for both kidneys was available. Combined organ procedures including paired kidney transplantations, procedures involving recipients younger than 18 years old, and procedures with grafts donated after uncontrolled circulatory death  were excluded from the analysis.EGL was defined as graft loss within 90 days after transplantation. Delayed Graft Function (DGF) was defined as the need for dialysis in the first postoperative week(s). The Modification of Diet in Renal Disease (MDRD) equation was used to estimate the glomerular filtration rate (eGFR). The non-scaled, donor-only version of the Kidney Donor Risk Index (KDRI) was calculated as described by Rao et al.Main (primary) outcomes for the study were: (I) the observed vs. predicted concordance of EGL for donor kidney pairs, and (II) long-term graft outcomes  of functional kidneys -from donor pairs from which the contralateral graft experienced EGL- vs. symmetrically functional pairs (no EGL). Main exposure variables are summarized in Tables IBM SPSS Statistics 23.0  was used for statistical analysis. Comparisons between the groups were performed using the independent t-test for normal-distributed data, the Mann-Whitney rank test for non-parametric data, and the chi-square test for categorical data.Simple univariate correlations were performed to map correlations between donor, procurement and recipient characteristics and outcomes. Recipient and graft survival were depicted with Kaplan-Meier curves, and survival differences explored through log-rank tests. Cox proportional hazards models were applied to estimate differences in the risk of recipient mortality or in graft loss. All survival models were adjusted  to calculate the Hazard Ratio (HR) for variables statistically relevant  in the univariate analysis. Results are represented as HR with the corresponding 95% confidence intervals (CI). (Backward) binary logistic regression analyses, and Analysis of Covariance (ANCOVA) were used to examine the association between donor, procedural and recipient characteristics, and functional outcomes . Factors included in the binary logistic regression analyses and covariates included in the ANCOVA are summarized in the table legends.2 for the simple correlations or Nagelkerke's r2 for the regression analyses.The amount of variation explained by the variable or set of variables was estimated by rMissing data was considered as missing at random. There was less than 2% missing data for the primary outcome graft survival and key characteristics such as donor and recipient age and sex, donor type and cold ischemia time.The primary analysis was based on death-censored EGL, i.e. excluding patients who died within 90 days after transplantation with a functioning graft. The following three sensitivity analyses were performed: (1) group allocation based on a more extended definition of EGL that included all graft losses occurring in the first 90 days following transplantation (i.e. including graft loss caused by recipient death within the first 90 days) as the primary outcome measure, and (2) and (3) group allocation based on more restricted (more donor-factors focused) definitions i.e.: (2) only EGL attributed to primary non function, thrombosis or vascular operative problems, or (3) exclusively EGL as the consequence of primary non function.There were no specific funding sources involved in the study. Access to the data set was handled by CK and LM. The decision to submit for publication was unanimously made by all authors.The impact of donor factors (of grafts accepted for transplantation) to transplantation outcomes in kidney transplantation was first explored by evaluating the concordance of EGL for transplanted donor pairs. It was reasoned that a high concordance would be consistent with prominent role of donor factors on outcome, and a low concordance would be consistent with a more modest role. A first exploration was performed for all paired transplantations performed in the 1990-2018 interval in the Netherlands. Out of the 10307 transplantations, paired outcome data was available for 5,816 kidneys A. For 4,death censored rate in the Dutch cohort of 5,396 kidneys was 7·6%, with an incidence of double-sided  EGL of 1·2% , national registry data (NTxD) for the UK were used for validation and further exploration. The distribution of different donor types such as donation after brain death (DBD), extended criteria donation (ECD) and controlled donation after circulatory death (DCD) is comparable in The Netherlands and the UK. However, as the UK does not participate in an international organ exchange program, interference caused by missing data related to cross-border exchange is not present. It was decided to limit the evaluation to the 2000-2018 interval in the UK in order to reduce the impact of time-related changes in incident EGL and outcomes.death censored for the UK cohort was 6.2%, and the incidence of double-sided  EGL was 0·7%  of the variation in EGL  of statistical prediction models that include all major donor characteristics suggest that the contribution of donor factors to EGL might be less than commonly assumed.The slightly lower than expected by change alone incidences of paired EGLs, and modest performance characteristics (rcensored for death with functioning graft), and symmetrically functional grafts (donor pairs of which neither graft experienced EGL). The Kaplan Meier curves for the Dutch and the UK cohorts  with a reduced 1 and 5-years eGFR for functioning grafts from asymmetrical outcome pairs p< ·001, , a diffecensored for death with functioning graft as the selection criterium for the asymmetrical outcome group in the functional outcome analysis, three sensitivity analyses were performed on the UK registry data .Conclusions of these analyses are potentially interfered by the complexity of the mechanisms underlying EGL. EGL may involve recipient factors, similarly graft quality (donor factors) that may contribute to recipient's 90-days mortality. To test the robustness of the conclusions based on an analysis using EGLall EGLs: 1·17 , p< ·13 resp ·25, supplemental Figures 2B respectively 2C).A first sensitivity analysis included all EGL's . The second analysis only included EGLs caused by primary non-function, vascular thrombosis or operative problems (i.e. largely excluding immunologic aspects), and a third analysis  exclusively included contralateral EGL caused by primary non-function. Conclusions from the first and second sensitivity analyses  showed compromised graft survival in the asymmetrical outcome group . This difference that persisted following correction for the year of transplantation, donor and recipient age, donor type and a history of diabetes in the donor (HRConclusions with respect to graft function (eGFR) were minimally influenced by the sensitivity analyses, or by the statistical strategy chosen .It could be argued that these conclusions are interfered by differences between the donor populations in the USA, British Columbia and The Netherlands, as e.g. in the latter donors are older, there are more DCDs, and the prevalence of hepatitis C is minimal. Therefore, it is important to point out that the reported performance characteristics (c-statistics) for the optimized KDRI (c-statistics: 0.652),Therefore, we decided for an alternative, indirect approach, and performed this instrumental variable analysis based on the outcomes of donor kidney pairs with grafts transplanted in different recipients. We reasoned that a prominent role of donor-associated factors would result in a high concordance of transplant outcomes, whereas a low concordance would be consistent with a more diffuse model. Although it could be argued that EGL is of limited clinical relevance given that is it both infrequent and notoriously difficult to predict, we chose EGL as it constitutes the most unequivocal outcome measure for this conceptual study. Longer-term outcomes such as graft survival and/or eGFR are particularly prone to interference by confounders, such as recurrent disease, differences in surveillance, and (adherence to) immune suppressive therapy. On the same token, clinically applied definitions for the short-term outcome measure -delayed graft function- are diverse, and its diagnosis is interfered by timing of the dialysis preceding transplantation, fluid overload and/or hyperpotassaemia/hyperphosphataemia, as well as by contrasting impacts in DBD and DCD donor grafts.With respect to an outcome comparison, we thought two aspects to be relevant. First, the degree of symmetry in incident EGL for donor pairs. It was reasoned that a high concordance (“symmetry”) of EGL would be consistent with a prominent role for donor factors in this outcome. The second aspect considered relevant was a comparison of transplantation outcomes for procedures with asymmetrical outcomes (i.e. grafts from a donor pair from which the other graft was lost due to EGL), with the outcomes for symmetrically successful transplantations (i.e. both grafts from a donor pair were successfully transplanted in two recipients). It was reasoned that prominent involvement of donor factors would result in segregation of transplantation outcomes with superior outcomes in symmetrically functional grafts, whereas a more discrete impact of donor factors would translate in more congruent outcomes.Registry data for both The Netherlands and the UK indicated a slightly lower incidence of symmetrical EGL than would be predicted on basis of chance alone, and it was concluded that for the vast majority of donor pairs (>85%) EGL was asymmetrical. Consequently, these data support a more granular diffuse etiology for EGL, with a limited impact of donor factors for grafts that were both accepted for transplantation.A limited contribution of donor factors is also reflected by the moderate differences in adverse donor factors for kidney pairs with symmetrical EGL, hence no specific donor factor was found to be dominantly associated with co-incident EGL. Similarly mapping of procedural and recipient characteristics associated with EGL in the asymmetrical outcome group indicated a relative (granular) enrichment of adverse factors for procedures in the EGL arm, but no specific procedural or recipient factor was found to prominently associate with co-incident EGL. Therefore, once the decision to accept a donor kidney pair for transplantation has been taken and the grafts have been transplanted, the impact of donor factors on early graft failure appears to be very limited.The high incidence of asymmetrical EGLs allowed for a second, alternative strategy to test the hypothesis that, once grafts are accepted for donation, donor factors have a limited impact on transplant outcomes. In this second strategy we compared the long-term outcomes of functional grafts from donor pairs with asymmetrical outcomes (i.e. pairs from which one graft was lost because of EGL) with outcomes of symmetrically successful transplantations.This analysis showed similar graft survival for the Dutch cohort, and a slightly compromised graft survival for grafts in the asymmetrical outcome group in the UK data. Because incident EGL also involves recipient-related factors, and because donor-factors may contribute to death-with-a-functioning graft, we performed a number of sensitivity analyses to evaluate the robustness of our findings. Although the conclusions of the sensitivity analyses are to some extend inferred by reductions in sample size, conclusions from the sensitivity analysis fully aligned with the primary analysis, and even suggested that the primary analysis overestimated the potential impact of donor factors.Functional data (1- and 5-years eGFR) for the UK cohort also allowed for a further, functional analysis. This analysis indicated a 5-10% lower eGFR in the asymmetrical outcome group. This lower eGFR did not impact recipient survival, but may (in part) contribute to the poorer graft survival in the asymmetrical outcome group  Consequently, results from this second evaluation follow the conclusions for short-term outcomes, and indicate a limited role of donor factors on long-term outcomes of transplanted donor kidney pairs.,It is thus concluded that, once donor graft have been accepted for transplantation, donor characteristics will minimally impact transplant outcomes. The strong focus on donor characteristics or donor risk indices in the decision process whether or not to accept a transplantable kidney graft could result in an unjustified increase of discarded viable donor kidneysOur study has some limitations as this is a registry-based study on outcome evaluation of donated, and transplanted kidney pairs. This strategy obviously results in a degree of bias and confounding since the analysis is restricted to the registered variables, information for kidney pairs not accepted for transplantation is missing, and as it excludes kidneys from a single donor from whom one kidney was declined for transplantation. Moreover, although the NOTR and NTxD registries are mandatory registry for all transplant centers and several quality checks are performed, missing data and registration errors remain an issue. For example, in the UK registry a quarter of the EGL cases the underlying cause was not coded. This may result in an incomplete inclusion in the sensitivity analysis applying a more restricted definition of EGL. A second limitation is that it is exclusively based on transplanted donor kidney pairs. It is well conceivable that this results in a bias towards more favorable donor characteristics.,Also, the vast majority of the patients in this evaluation are Caucasian. Given the potential impact of race on transplant outcomes,Finally, conclusions in this study are potentially influenced by confounders such as time-effects with improved outcomes over time, and by medical decision-making such as the introduction of the old-for-old transplantation program in the Netherlands and the longevity matching in the UK.In conclusion, this study based on the data of two national registries suggests that, once the decision to accept kidneys from a deceased donor for transplantation has been taken, outcomes for most transplants reflect a complex interplay of donor-, procedural- and, in particular recipient factors. Efforts to optimize transplant outcomes should focus on a better understanding of the recipient factors underlying transplant outcomes.AFS: conceptualization, data interpretation, drafting manuscript. MK: Data acquisition, reviewing. LM: Data acquisition, reviewing. MR: Data acquisition, reviewing. RJ: Data acquisition, reviewing. MK: Data analysis, reviewing . FJB: Data acquisition, reviewing . JvdW: Data acquisition, reviewing. ADvZ: Data acquisition, reviewing. MHLC: Data acquisition, reviewing. MCB: Data acquisition, reviewing. ASN: Data acquisition, reviewing. SPB: Data acquisition, reviewing. EB: Data interpretation, reviewing. APJdV: Data acquisition, reviewing. ES: Data analysis, data interpretation, reviewing. RJP: Data acquisition, data interpretation, reviewing. JHNL: Data analysis, data interpretation, drafting manuscript.This study is based on data made available by the Netherlands Organ.Transplant Registry (NOTR) and the UK National Transplant Database (NTxD). Analyses can be available upon reasonable request to the corresponding author.None.The authors have nothing to disclose.


Retrospective studies demonstrate similar efficacy between oral (PO) and intravenous (IV) antibiotics at discharge for gram negative bacteremia (GNB), with reduced duration of hospitalization, hospital costs, and duration of therapy with PO. Understanding current physician discharge practices can help guide future antimicrobial stewardship initiatives.Escherichia coli, Klebsiella species, or Proteus mirabilis and a PO antibiotic available at discharge were included. The primary outcome was the percentage of patients discharged on PO antibiotics. Secondary outcomes included duration of hospitalization, total length of therapy, and 30-day readmission secondary to clinical failure or therapeutic complication in the IV and PO at discharge groups.A multicenter, retrospective cohort study was conducted on adult inpatients admitted between July 1, 2020 and June 30, 2021 with urinary source GNB. Patients with both blood and urine cultures positive for Of the 157 included patients, 128 (82%) were discharged on PO antibiotics. Patients discharged on IV and PO had similar median quick Pitt bacteremia scores . Mean duration of hospitalization for patients discharged on IV vs. PO therapy was 5  days vs. 4  days, p=0.001, respectively. Mean total length of antibiotic therapy was 21.6  days as compared to 13.5  days, p=0.017, for the IV vs. PO arms, respectively. Thirty–day readmission rates were 11/29 (38%) and 23/128 (18%), p=0.035, in the IV and PO arms, respectively. Notably, 21/29 (72%) of the IV arm had an organism from blood culture that was susceptible to trimethoprim-sulfamethoxazole (TMP-SMX).Most patients with urinary source GNB were discharged on PO antibiotics. Patients discharged on IV antibiotics were more likely to be readmitted within 30 days. Future efforts to decrease IV antibiotic use may focus on increasing TMP-SMX use at discharge.Matthew Song, PharmD, BCIDP, Merck: Stocks/Bonds|ObsEva: Stocks/Bonds.


Troponin elevation is common in ischemic stroke (IS) patients. The pathomechanisms involved are incompletely understood and comprise coronary and non-coronary causes, e.g. autonomic dysfunction. We investigated determinants of troponin elevation in acute IS patients including markers of autonomic dysfunction, assessed by heart rate variability (HRV) time domain variables.Data were collected within the Stroke Induced Cardiac FAILure (SICFAIL) cohort study. IS patients admitted to the Department of Neurology, Würzburg University Hospital, underwent baseline investigation including cardiac history, physical examination, echocardiography, and blood sampling. Four HRV time domain variables were calculated in patients undergoing electrocardiographic Holter monitoring. Multivariable logistic regression with corresponding odds ratios (OR) and 95% confidence intervals (CI) was used to investigate the determinants of high-sensitive troponin T (hs-TnT) levels ≥14 ng/L.2 0.71; 95% CI 0.61–0.84), systolic dysfunction , diastolic dysfunction , atrial fibrillation , and increasing levels of C-reactive protein . We did not identify an independent association of troponin elevation with the investigated HRV variables.We report results from 543 IS patients recruited between 01/2014–02/2017. Of those, 203 (37%) had hs-TnT ≥14 ng/L, which was independently associated with older age , male sex , decreasing estimated glomerular filtration rate (OR per 10 mL/min/1.73 mCardiac dysfunction and elevated C-reactive protein, but not a reduced HRV as surrogate of autonomic dysfunction, were associated with increased hs-TnT levels in IS patients independent of established cardiovascular risk factors.https://www.drks.de/drks_web/; Unique identifier: DRKS00011615.Registration-URL: The online version contains supplementary material available at 10.1186/s12883-022-03017-1. Cardiac troponin levels are sensitive and specific markers of myocardial injury  and are Troponin elevation may be caused by a thrombotic acute coronary syndrome preceding or concomitant to acute IS. Postulated mechanisms of troponin elevation due to non-acute coronary causes comprise heart failure , 9, impaComparability of prevalence and determinants of troponin elevation in IS patients between existing studies , 8–12 isMarkers of autonomic function  are not routinely measured in IS patients and may be influenced by environmental stress . Hence, Therefore, we examined determinants of troponin elevation >99th percentile using a high-sensitivity assay within prospectively collected data undergoing detailed cardiac phenotyping at baseline including HRV time domain variables.Patients were recruited within the prospective Stroke Induced Cardiac FAILure in mice and men (SICFAIL) study, a hospital-based, investigator-initiated cohort intended to investigate the prevalence and natural course of cardiac dysfunction after IS . The methodology of the SICFAIL study has been previously described . In briePatients or next of kin knowledgeable about the patient’s history were interviewed about socio-demographic factors, previous medical history , [Cardiac structure and function were assessed using transthoracic echocardiography by certified echocardiography technicians  of the Cilable),  tricuspiilable), . Becauseilable), , diastolilable), . Clinicailable), , as previlable), .High-sensitive troponin T (hs-TnT), high-sensitive C-reactive protein (hs-CRP), and creatinine were measured from fasting blood samples drawn at median 3 days (quartiles 2–4) after symptoms onset and stored at − 80 °C in the Interdisciplinary Bank of Biomaterials and Data Würzburg (ibdw)  (for furPatients underwent ECG Holter monitoring as part of the routine diagnostic workup using a 3-channel (12-lead) Holter ECG . Initial inspection of QRS complexes for ectopy and artifact labelling was performed using commercially available software (Philips Zymed Holter 2010 Plus/1810 Series). Annotated beat-to-beat files underwent a second stage of manual editing of R-R intervals using dedicated software . The following time domain variables were calculated in accordance with published recommendations:  (1) stanOnly records fulfilling the following prespecified criteria were included in this analysis: normal sinus rhythm with ≥18 hours of 5-minute segments with ≥80% normal R-R intervals and < 20% ectopic beats, recorded in all three channels and including the entire night (midnight to 6 AM). Patients with pacemaker, long segments of atrial fibrillation, and atrioventricular or bundle branch block were excluded  with 95% confidence intervals (CI). The variables of interest were selected a priori based on background clinical knowledge: age, sex, systolic dysfunction, diastolic dysfunction in absence of systolic dysfunction, atrial fibrillation, eGFR, history of coronary artery disease (CAD), severity (assessed by the NIHSS), insular stroke, and hs-CRP. Collinearity was investigated using the variance inflation factor. Concentrations of hs-CRP were logarithmically transformed due to skewed distribution. In a sensitivity analysis, we investigated: (1) the consistency of the association of the previously selected variables with troponin elevation after adjusting for atherogenic cardiovascular risk factors ; and (2) the association of single HRV time domain variables with hs-TnT ≥14 ng/L in patients with available HRV data. Due to the limited amount of events, we used a parsimonious model for the second sensitivity analysis and adjusted for age (model 1), or age and eGFR (model 2), based on previous data identifying age and eGFR as the single most relevant variables associated with hs-TNT ≥14 ng/L in non-coronary patients [We tested differences between groups using the χpatients . HRV varn = 41, withdrawal of consent n = 13) and patients without available hs-TnT levels (n = 153), 543 patients were included in the present analysis  but did not differ otherwise from included patients , and increasing hs-CRP levels  , and 203 patients (37%) had levels ≥14 ng/L. Troponin elevation was independently associated with older age , male sex , systolic dysfunction , diastolic dysfunction in absence of systolic dysfunction , atrial fibrillation , decreasing eGFR . We did not find an independent association between the investigated HRV time domain variables and increased hs-TnT. To the best of our knowledge, this is the first study investigating the association of reduced HRV with troponin elevation among IS patients.n = 19,501, age range 18–98 years) reported an URL for hs-TnT as high as 47.1 and 38.6 ng/L for men and women ≥70 years, even after exclusion of individuals with known cardiovascular disease [Rates of elevated hs-TnT in our study are in line with previous publications in IS patients applying comparable kits, ranging between 30 and 60% , 8, 28.  disease . While i disease , 31. Thi disease .We found a strong correlation of eGFR with hs-TnT ≥14 ng/L, in line with previous reports , 12. TroWe found an association of both systolic and diastolic dysfunction with increased hs-TnT. Comparability with previous studies is limited, since most studies lacked structural and functional cardiac investigation , 8, 11, Nearly all patients with atrial fibrillation have detectable troponin levels and increasing values are associated with increasing risk of stroke . Thus, IOnly one study has previously reported an association of an inflammation marker  with troponin elevation , althougAutonomic dysfunction with sympathetic overweight is another proposed mechanism to explain troponin elevation in IS patients . We are Overall, our results suggest that troponin elevation in IS patients is predominantly associated with some traditional cardiovascular risk factors and symptomatic or asymptomatic cardiac disease. Thus, the prognostic value of troponin in IS patients probably reflects an increased baseline cardiovascular risk and may explain the inconsistent association of troponin with poor outcome found in a recent systematic review, which was more often non-significant when adjusted for other relevant cardiac prognostic factors (such as cardiac comorbidities or biomarkers) . Our datThe major strength of our study is the assessment of the determinants of hs-TnT elevation within a large, prospective study of IS patients undergoing detailed and standardized cardiac examination. Our study has, however, limitations. First, this analysis was not prespecified and we analyzed the determinants of hs-TnT levels only at baseline. Thus, we might have missed transient troponin elevation during the hyperacute phase. However, previous results using the same hs-TnT assay do not suggest a clear dynamic during the first four days after IS . We cannAlmost a third of patients with acute IS exhibited elevated troponin levels. Our data suggest that older age, male sex, clinical or subclinical cardiac disease, impaired renal function, and elevated hs-CRP are associated with increased troponin, while it did not support an association with reduced HRV. The clinical interpretation of elevated troponin levels must account for a broad spectrum of comorbidities and clinical characteristics. The pathophysiological relevance of autonomic dysfunction –especially in older, comorbid IS patients and in patients with severe stroke– and other markers of inflammation beyond hs-CRP in troponin elevation in IS patients as well the potential use of troponin to predict cardiovascular events after acute IS must be addressed in further studies.Additional file 1: Appendix I. Methods. Appendix Table I. Comparison of patients with and without available troponin measurement . Appendix Table II. Comparison of patients with and without available heart rate variability analysis . Appendix Table III. Determinants of elevated troponin levels in patients with ischemic stroke in multivariable logistic regression analysis after adjustment for cardiovascular risk factors. Appendix Table IV. Determinants of elevated troponin levels in patients with ischemic stroke in multivariable logistic regression analysis after adjustment for time point of blood sampling. Appendix Table V. Association of time domain variables of the heart rate variability with troponin elevation


Receiving an opioid prescription during childhood increases the risk of hazardous prescription opioid (PO) use during emerging adulthood. Instruction on how to safely use POs plays an essential role in pediatric patients’ capacity to utilize as well as to discontinue POs appropriately. This study aimed to evaluate pediatric PO label instructions provided to a large sample of pediatric outpatients. Data were extracted from the electronic healthcare records system identifying pediatric patients who received a PO between 2016 and 2019 from pediatric outpatient medical clinics were affiliated with a northwestern United States medical center and children’s hospital. Pediatric patients  between 0–17 years old who received a PO during outpatient care were included. Patients with chronic health conditions  or who received their PO from an inpatient medical setting were excluded. Patient demographics, medication instructions, associated diagnoses, and other prescription information  were examined using automated text classification. Many label instructions did not include any indication/reason for use (20.8%). Virtually none of the POs (>99%) included instructions for how to reduce/wean off POs, contact information for questions about the POs, and/or instructions around how to dispose of the POs. Efforts are needed to ensure that pediatric PO instructions contain essential elements to improve comprehension of when and how to use POs for pediatric patients. Prescription opioids (POs) given to children during the course of routine outpatient treatment for pain can increase risk for hazardous PO use during young adulthood . ChildhoMany adolescents are first introduced to POs through legitimate prescriptions to treat pain by medical providers ,4. No foMedication instructions play an essential role in parents′ management of pediatric medications and represent a key modifiable target within the domain of “opioid stewardship.” Specifically, when instructions are vague, they can be misinterpreted, leading to confusion and incorrect use of pediatric PO medications ,14,15. AAdult studies show that high levels of instruction complexity, specifically related to dosing, have been associated with greater confusion or error in medication use; medication labels with multistep instructions have been reported to be more difficult to use as prescribed . Prior sMore data are needed to understand the nature of pediatric PO instructions for young patients. Complicating these instructions, the vast majority of pediatric PO instructions specify that medication is to be taken “as needed”, but many fail to include how, when, or why to use and how, when, or why to discontinue use ,23. AddiData were collected using the Research Data Warehouse (RDW), a service provided by the Oregon Clinical and Translational Research Institute (OCTRI) at a northwest medical school. The RDW provided a repository of data from the electronic medical records of pediatric patients who were eligible for the study. Prescription information for children ages 0 to 17 years who received a pediatric PO between 1 January 2016 and 31 December 2019 in the context of routine pediatric outpatient or ambulatory care met inclusion criteria and was extracted from the RDW. Information related to patient demographics, type of PO, dose, quantity dispensed, instructions provided on the prescription label, and associated diagnoses were extracted. In order to capture pediatric POs given in outpatient settings for generally healthy children, patients with chronic diseases , patients with blood disorders or cancer/neoplasms, patients with congenital disorders, those who received their pediatric PO through an inpatient unit, and routes of administration other than oral were excluded. This investigation was approved by the participating Institutional Review Board. Pediatric PO instruction features posited to impact instruction clarity  were identified and included . An initAn iterative process was used to refine classification by identifying frequently repeated pediatric PO phrases not automatically classified in the preceding round and including them in the algorithm for the subsequent round; an additional 5% of pediatric POs were manually classified to verify the accuracy of automatic classification. Elements that were not automatically classified in the preceding process were reviewed by two study team members. Analyses were performed in RStudio (Version 1.3.1093). Pediatric PO instructions specifying a route of pediatric PO administration other than oral were excluded from analysis as these are less representative of the low complexity pediatric ambulatory care population examined in this study. Frequency tables were developed for pediatric PO instruction features posited to impact instruction clarity , as well as elements that occurred frequently during the iterative review of pediatric PO instructions by study team members. Frequency of each pediatric PO instruction element was compared between surgical and non-surgical prescribing departments and between patients 13 and older and those younger than 13 for those pediatric PO-instruction elements with significant variation across pediatric PO prescriptions (>5% in each category). Chi-square tests were used for these analyses. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated.Surgical departments included those specialties recognized by the American College of Surgeons: cardiothoracic surgery, colon and rectal surgery, general surgery, gynecology and obstetrics, gynecologic oncology, neurological surgery, ophthalmic surgery, oral and maxillofacial surgery, orthopedic surgery, otorhinolaryngology, pediatric surgery, plastic and maxillofacial surgery, urology, and vascular surgery. All other departments were classified as non-surgical. Non-surgical departments included outpatient general and specialty clinics.A total of N = 11,213 pediatric PO patients ages 0–17 were identified. In line with this metropolitan region, this sample was predominately non-Hispanic white (78%), English-speaking (91.1%), with a mean age of 11.1 (SD = 5.5), and N = 2032 (18.1%) Hispanic. In addition, N = 6563 (58.5%) pediatric PO patients were male and N = 4650 (41.4%) were female see .p < 0.001) and less likely to contain potentially confusing language  than pediatric PO instructions from a non-surgical department were (p < 0.001) and to specify the pain severity for which pediatric PO use was intended  compared with pediatric PO instructions from a non-surgical department.Characteristics of pediatric PO instructions are listed in ent were . Pediatrp < 0.001) and more likely to contain pediatric PO instructions to limit use  than pediatric PO instructions written for patients under 13 years old (p < 0.001) than pediatric PO instructions written for patients under age 13. There was no significant difference in the frequency with which pediatric PO instructions specified the pain severity for which the pediatric PO use was intended between these age groups . Pediatric PO instructions written for patients who were at least 13 years old were significantly more likely to specify a discrete amount of pediatric PO to take at one time , and 99.4% included a route of administration. An amount of pediatric PO to take per day was observed in 99.4% of instructions, and among them, 75.8% contained a specified discrete amount  rather than a range . Lastly, an indication  was specified in 78.9% of the instructions, and among these, 89.7% included a severity level . Indications were considered highly important for safe pediatric PO use by supporting pediatric patients’ and their families’ understanding of the steps necessary to take the pediatric PO in the manner that the provider intended .n = 142), only 68 (0.5%) included specific instructions as to how to wean. Only 3.5% included a maximum total amount of pediatric PO to take, and 3.2% included the maximum duration the pediatric PO should be taken. Increasing the rate at which prescribers include these elements might be beneficial for patient and parent understanding of how and when to safely discontinue. Thus, these data indicate that major areas of improvement include using more specific instructions on the amount of pediatric PO to take, providing clearer directions on when or in what situations/circumstances to use, and how to wean off of or discontinue. Areas for pediatric PO improvement were also identified. For example, 21.1% pediatric PO labels did not include an indication, and 14.9% included a range  rather than a specific amount to take . Further, there were no clear directions on when to use and how and when to discontinue use. The vast majority (98.1%) instructed children with pain to take “as needed” rather than including specific instructions on when pediatric POs should be taken or in what situations/circumstances the pediatric patient and/or parent should consider the pediatric PO as being medically “needed”. Most (98.9%) pediatric PO instructions did not contain directions regarding when to discontinue or wean off of the pediatric PO, and among the minority of instructions that mentioned weaning , requiring a higher reading level to comprehend , or for previously unspecified but clearly confusing text . Zheng et al.  reportedIt can be especially challenging for patients to interpret and follow the pediatric PO instructions when they contain abbreviations or medical jargon, and this may even be more pronounced for less formally-educated families and/or for families for whom English is not their first language. The average U.S. adult reads at an eighth-grade level . Given UAdditionally, contact information for families’ follow-up questions were provided in fewer than 0.001% of the pediatric PO instructions; this is highly problematic given the need for additional information/clarification from the prescribing provider. While families may have this information in other locations , making this information easily accessible may be helpful for increasing patient–provider communication around pediatric PO use and discontinuation. Similarly, fewer than 0.001% of pediatric PO labels included instructions on how to dispose of leftover pediatric POs. In fact, recent findings indicate that nearly half of adolescents who use POs in hazardous ways receive them from friends and relatives , suggestResults of analyses indicate that patients 13 years of age and older were more likely to receive more precise pediatric PO instructions and less likely to receive potentially confusing instructions than patients younger than age 13. Prior research indicates that 25% of individuals who were prescribed opioids at 13 years or younger may be at enhanced risk for transitioning into OUD [Our study has numerous strengths, including a careful empirical evaluation. At the same time, results should be interpreted in light of the following limitations. Because data were extracted from medical records of pediatric patients who received a pediatric PO, information about other variables of interest could not be evaluated. For example, future research may examine whether provider demographics and other patient characteristics are associated with the quality of written pediatric PO instructions provided, given that these characteristics can exacerbate problems in patient–provider interactions, quality of care, treatment adherence, and continuity of care . FurtherAddressing the ambiguity, technicality, and variability of pediatric PO label instructions is among one of the most actionable avenues to improve comprehension around safe pediatric PO use by young people and their families. Increased efforts to develop structured systems or tools to help standardize label directions, such as embedding a comprehensive set of direction components into the electronic health record, could improve the quality of these directions and potentially increase appropriate pediatric PO use by patients . In addi


Background: Myocardial mechano-energetic efficiency (MEE) is the capability of the left ventricle (LV) to convert the chemical energy obtained from the cardiac oxidative metabolism into mechanical work. The aim of present study was to establish normal non-invasive MEE and MEEi reference values. Methods: In total, 1168 healthy subjects underwent physical examinations, clinical assessment, and standardized transthoracic echocardiographic (TTE) examination. MEE was obtained by TTE as the ratio between stroke volume (SV) and heart rate (HR): MEE = SV/HR [HR expressed in seconds (HR/60)]. Because MEE is highly related to left ventricular mass (LVM), MEE was then divided by LVM with the purpose of obtaining an estimate of energetic expenditure per unit of myocardial mass . Results: The mean values of MEE and MEEi in the overall population were 61.09 ± 18.19 mL/s; 0.45 ± 0.14, respectively. In a multivariable analysis, gender, body surface area (BSA), diastolic blood pressure, left atrial volume indexed to BSA, E/e’ and tricuspid annular plane systolic excursion (TAPSE) were the independent variables associated with MEE, while age, gender, BSA and TAPSE were the independent variables associated with MEEi. Conclusions: The knowledge of age- and gender-based MEE and MEEi normal values may improve the global assessment of LV cardiac mechanics and serve as a reference to identify phenotypes at high risk of cardiovascular events. Myocardial mechano-energetic efficiency (MEE) is defined as the capability of the left ventricle (LV) to convert the chemical energy obtained from the cardiac oxidative metabolism into mechanical work . It has The study population consisted of 1168 healthy subjects (volunteers or subjects undergoing work ability assessment (mean age 43.4 ± 14.0); 123 (45.7%) men) referred to the echocardiographic laboratories of the Cardiology Division, “Cava de’ Tirreni-Amalfi Coast”, Heart Department, University Hospital of Salerno, Italy, and the Department of Cardiology and Emergency Medicine of San Antonio Hospital, San Daniele del Friuli, Udine, Italy ,10. The 3 − LVID3] + 0.6 g. The left ventricular outflow tract (LVOT) diameter was measured in the zoom mode from the parasternal long axis view using standard electronic calipers in mid-systole, between the hinge points of the aortic valve leaflets from inner edge to inner edge. The LV stroke volume (SV) was calculated as the product of LVOT area and LVOT velocity time integral (VTI), obtained by a pulsed wave Doppler: SV = π × (LVOT/2)2 × LVOT VTI [A TTE with continuous ECG recording was performed with commercially available equipment on all subjects , according to the American Society of Echocardiography/European Association of Cardiovascular Imaging Guidelines, as previously described ,12,13. ALVOT VTI .MEE can be defined as the ratio between the external systolic work and the total amount of energy produced by cardiomyocytes, estimated by the rate pressure product, which is an indirect measure of MVO2 ,4,5. LV As MEE is highly related to LVM, MEE was divided by LVM to obtain an estimate of energetic expenditure per unit of myocardial mass  .p-value less than 0.05 was considered significant. Holm’s correction was used for multiple hypothesis correction, if necessary. Spearman’s rank correlation was carried out for continuous variables to assess univariate associations. The variables were selected according to their clinical relevance. Multivariable linear regression analysis, including all variables that showed a significant correlation from the univariate analysis, was constructed to assess the independent associations of these variables with MEE and MEEi. Inter-observer agreement was tested with two independent observers remeasuring echocardiographic parameters in 20 randomly selected cases. Intra-observer variability was considered in 20 randomly selected cases by repeating the measurements on 2 occasions. The inter- and intra-observer variability were examined using both paired t tests and intraclass correlation coefficients (ICCs). An ICC >0.9 indicated excellent agreement. A statistical analysis was performed using R software  [Continuous variables are expressed as means and standard deviations (SD). The data were tested for normality through the Shapiro–Wilk test. The Wilcoxon rank-sum test or t-test was used, as required, for comparisons of continuous variables between groups. Categorical variables are expressed as percentages and were compared using the chi- square test or the Fisher’s exact test. To compare more than two groups, the Kruskal–Wallis test was used. A two-tailed Austria) .The demographic data of the study population are reported in The mean values of MEE and MEEi in the overall population were 61.09 ± 18.19 mL/s and 0.45 ± 0.14, respectively. The upper and lower limits (CI 95%) of normal MEE and MEEi were 62.13–60.05 and 0.46–0.44, respectively .p-value < 0.001)   A. In con< 0.001) B. The up< 0.001) .p = 0.092) was found; conversely, a significant negative correlation between MEEi and age  was found   and between group 1 and group 3 (>60 years)   . On the  0.0011) .p-value < 0.001) and positively with BSA (r = 0.32), BMI (r = 0.11), left atrial volume indexed to BSA (LAVI) (r = 0.22) and TAPSE (r = 0.24) . A Significant but weak and negative correlation between MEE and DBP , E/e’  was found. No significant correlation between MEE and SBP, PP, mean blood pressure (MBP), left ventricular ejection fraction (LVEF), tricuspid regurgitation velocity (TRV) was seen in this sample of normal adults   A.p-values < 0.05)  .p = < 0.001), BSA (r = −0.2), BMI (r = −0.27), SBP (r = −0.25), DBP (r = −0.24), PP (r = −0.12), MBP (r = −0.28), E/e’ (r = −0.12) and TAPSE (r = 0.12) . The MEEi values significantly correlated with gender . No significant correlation between MEEi and LAVI, LVEF and TRV was found   and with age  B.p-values < 0.05)  .The overall inter- and intra-observer agreement rates were similar, showing non-significant variability. The interobserver variability analysis revealed an ICCs of 0.96 . The intraobserver agreement rates were 0.97 .MEE provides useful information about LV cardiac mechanics, representing the capability of the LV to transform the chemical energy obtained from oxidative metabolism into mechanical work . It appeSeveral investigators reported the MEE and MEEi values of subjects with CV risk factors, including arterial hypertension, obesity, diabetes, hyperlipemia and former or current smoking habits .p value ≤ 0.78). As a note, hypertensive patients with low LV mechanical efficiency  had much higher HR, systolic BPs, and pulse pressure than those exhibiting normal LV mechanical efficiency . Low MEE was also associated with inappropriately high LV mass (p < 0.0001).De Simone et al. reported the MEE values of 255 subjects  without CV risk factors, as a normotensive control group of 306 hypertensive patients  free of CV disease [Furthermore, among 12,353 hypertensive patients , De Simone et al. showed that reduced MEE was associated with altered metabolic profile, LVH, and concentric LV geometry and independently predicted hard CV events, reducing the statistical impact of LVH [p for trend < 0.05) [The fat-associated CV dysfunction (FATCOR) study explored the association of MEEi with LV systolic circumferential and longitudinal myocardial function in 480 subjects with increased body mass index (BMI), without known CV disease . Patients with lower MEEi values were more frequently men with obesity, hypertension, dyslipidemia, and a higher insulin resistance index  predicted incident heart failure, after adjustment for LVH, prolonged relaxation and associated CV risk factors, including hypertension, obesity, diabetes, and smoking habits [Interestingly, Losi et al., among 1912 unselected participants of a population-based cohort of American Indians with normal baseline EF, demonstrated that the lowest MEEi quartile  comprehensively assessed the full range of MEE and MEEi values in a large cohort of 1168 healthy individuals stratified by age and gender; (b) demonstrated that the MEE values were higher in males than in females, whereas the MEEi values were significantly higher in females; (c) showed that the MEEi values were reduced in older age, while MEE was not significantly correlated with age; (d) revealed gender, BSA, DBP, LAVI, E/e’ and TAPSE as independent variables associated with MEE, and age, gender, BSA and TAPSE as independent variables associated with MEEi.In the present study, the normal MEE values measured by the Doppler method were significantly lower than the values reported by De Simone et al.  in an older population . In the latter study, SV was calculated as the difference between 2 D volumes (SV = LV end-diastolic volume—LV end-systolic volume), using the biplane method of disc summation (modified Simpson’s rule). This difference may also be consistent with the hypothesis that SV and CO are significantly lower if calculated by 2D rather than by Doppler and 3D methods  . Thus, dp value). These differences may be attributable to gender-related differences in biometrics characteristics. In this regard, a significant and independent association between the MEE and MEEi values and BSA was found.There are well-known gender differences in LV structure and function, including larger volumes, greater LVM, and higher CO and SV in men compared with women ,19,20. SFurthermore, our findings suggest a physiological impact of the aging process on MEE and MEEi (more evident), highlighting the need to adapt the normal reference values to age. This is consistent with previous results demonstrating that, in both genders, CO and SV tended to decrease with age . Of noteOn the other hand, the significant and independent association of MEE and MEEi values with TAPSE may be indicative of the ventricular interdependence concept  .First, the study was limited to Caucasian healthy subjects. Thus, in the present study, the clinical relevance of MEE and MEEi for different races and pathologic states was not investigated. Secondly, additional echocardiographic techniques such as strain imaging and 3D echocardiography (3DE) were not performed.We reported the physiologic ranges of MEE and MEEi measured by TTE in a large population of healthy subjects. MEE was significantly correlated with gender  and BSA. Instead, MEEi was significantly decreased in older age and correlated significantly with gender  and BSA. The knowledge of age-, BSA- and gender-based normal MEE and MEEi values may serve as reference to identify phenotypes at high risk of CV events.


The optimal groove design of a MEMS piezoresistive pressure sensor for ultra-low pressure measurement is proposed in this work. Two designs of the local groove and one design of the annular groove are investigated. The sensitivity and linearity of the sensor are investigated due to the variations of two dimensionless geometric parameters of these grooves. The finite element method is used to determine the stress and deflection of the diaphragm in order to find the sensor performances. The sensor performances can be enhanced by creating the annular or local groove on the diaphragm with the optimal dimensionless groove depth and length. In contrast, the performances are diminished when the local groove is created on the beam at the piezoresistor. The sensitivity can be increased by increasing the dimensionless groove length and depth. However, to maintain low nonlinearity error, the annular and local grooves should be created on the top of the diaphragm. With the optimal designs of annular and local grooves, the net volume of the annular groove is four times greater than that of the local groove. Finally, the functional forms of the stress and deflection of the diaphragm are constructed for both annular and local groove cases. Microelectromechanical system (MEMS) pressure sensors occupy the largest market share in the world market of MEMS devices ,2,3. TheThe MEMS piezoresistive pressure sensor can detect pressure by realizing the effect of piezoresistance on the piezoresistors, where the relative change in resistance essed as (1)ΔRR0=rding to , as(2)Δo (2005) . In the The MEMS piezoresistive pressure sensor for ultra-low pressure measurement in applications, such as biomedical devices and HVAC systems, requires high sensitivity D=Ej3r (1991)  increase. 2010)  proposed. (2010) . Many re. (2010)  reported. (2010)  reported. (2010) , but the. (2010)  proposed. (2010)  proposed. (2010)  proposed. (2010)  investig10  propo. (2010)  proposed. (2010) ,37,38,39Because the MEMS piezoresistive pressure sensor for ultra-low pressure measurements with optimized geometric parameters of boss, cross-beam, peninsular structures and petal edge was proposed by Thawornsathit et al. (2022) , as showTo design the groove, there are three important geometric parameters, i.e., the groove width e theory , the gove (1979)  proposedThis dimensionless diaphragm deflection is adopted in this work. The stress components e (1979) . TherefoBecause According to several previous works ,37,38,39n et al. 18b [20],The second design is also the local groove The third design is the annular groove To determine the stress and deflection of the diaphragm over a range of the applied pressures of 1–5 kPa, the finite element method is performed by using the commercial software ANSYS Mechanical version 18.1. Because of the symmetry in the middle of the sensor, only a quarter of the sensor is created for the finite element model, as shown in The performances of the sensor with the local groove Therefore, the sensor with the local groove The stress difference distributions of the sensor with the annular groove It clearly shows that the increment of The performances of the sensor with the annular groove The performances of the sensors with three optimal groove designs, i.e., The nonlinearity error at each measurement point , after substituting According to The functional forms of the stress differences of the sensor with the annular groove According to To validate the functional forms of The functional forms of the averaged stress differences in terms of Three selected groove designs, i.e., Three groove designs for the MEMS piezoresistive pressure sensor were investigated in the present work. There were two designs for the local groove type (


Trans-Tephrostachin (TT) in zebrafish brain. Neurospecific PNIPAM NGs are functionalized with polysorbate 80 (PS80) to overcome the Blood Brain Barrier (BBB). The TT loaded with NG (NG + TT) was confirmed in UV-spectroscopy and transmission electron microscopy (TEM) with 90% efficiency of controlled release at 37 °C. The neurospecificity of NG was confirmed in 144 hours post fertilization (hpf) larvae with PS80 surface-treated rhodamine-B (Rh–B) conjugated NG and visualized in the zebrafish CNS. Oral gavaging of TT loaded NG with PS80 surface treatment (NG + TT + PS80) was confirmed to cross the BBB in adult zebrafish at 37 °C. TT release was detected by RP-HPLC. LC50 was determined as 250 μg/ml for NG, 172 μg/ml for NG + TT, and 0.9 μg/ml for TT at 96 hpf and confirmed the lesser toxicity in TT bound NG. Delays in growth and malformations were observed at concentrations above the 96 hpf-LC50. The behavior outcomes were varied with phase - and concentration-dependent hypo- or hyperactivity. The altered expression of genes associated with Alzheimer’s disease (AD) was found at 96 hpf of its LC50 concentration. The expression of appa was significantly increased for TT and supporting the TT to bind NG without altering the AD genes. Thus the study suggests the biocompatible potential of PNIPAM and its neurospecific delivery to the brain.Biocompatible Poly(N-isopropylacrylamide) (PNIPAM) nanogels (NGs) were developed at 40–65 nm to deliver  PNIPAM Nanogel; Polysorbate 80; Trans-Tephrostachin; Neurospecific; Zebrafish; Toxicology. Clinical trials of CNS drugs are challenging due to the impermeability of the blood-brain barrier (BBB) . It actsPolymer nanoparticles with 1–1000 nm have been grown rapidly for clinical medicine  and exteDanio rerio) is a complex vertebrate organism with higher homology to humans. Major neurotransmitters, hormones, receptors, and apolipoproteins of zebrafish are similar to humans  total was the total amount of TT entrapped in the nanoparticles.The cumulative release of TT from NG in 0.1 M Phosphate buffered saline (PBS) (pH 7.4) was measured by dialysis bag technique. The solution was centrifuged at 3000 rpm for 10 min to separate the released TT from the NGs and dissolved in 1 ml of CHCl2.6The mouse primary fibroblast cells were purchased from National Center for Cell Science, Pune, India. The cells were grown in 96-well cell culture plates until 80% confluent. The culture media was supplemented by a fresh medium containing diluted NGs in 0.1% DMSO in full medium and the cells were exposed to the NG, NG + TT, or TT for 24 h. The MTT assay was carried out. MTT -2,5- diphenyltetrazolium bromide, Sigma, 250 μg/mL final concentration) was added to the cells for 2 h and measured at 540nm (Ensight Multimode reader PerkinElmer).2.7Fibroblast cells were grown until 80% confluency in 24-well culture plates. The culture medium was replaced with a medium containing NG + Rh–B diluted at 14%  and the cells were exposed to the NGs. Further the cells were washed with PBS and Rh–B fluorescence was observed.2.8Adult zebrafish 5D strain was maintained in a recirculating stand-alone system  at 28 °C on a 10:14 h dark: light cycle with a pH range of 7.1–7.3 and conductivity of 470–550 μS. Fishes were bred to attain embryos for experiments in breeding tanks . All pro2.9Nanogels were injected in the aortic region by microinjections to analyze the neurospecificity of the NG at 144 hpf zebrafish. The coating with PS80 permitted the crossing of NG through the BBB. The toxicity of PS80 was previously established in zebrafish and optimized in this study . 5 nl of2.10Five Adult zebrafish were taken in each batch for the administration of TT, NG + TT, NG + TT + PS80, or negative control (water only). Venflon syringe of 24G was used to administer 5 μl volume of the drug to adult zebrafish by inserting the plastic needle 1 cm below the gills to reach the end of the esophagus . These f2.11Fish Embryo Toxicity (FET) was carried out as per the OECD guidelines . Embryos were distributed in a 24 well plate containing 3 mL of the culture water with several dilutions of NG , NG + TT , and TT  respectively and LC50 were calculated at 96 hpf. The quantitative data were expressed as mean ± SD using GraphPad Prism version 9.11 .2.12Danio vision Observation Chamber following exposure to NG , NG + TT , or TT  for 4 to 120 hpf. Each treatment group within a biological replica consisted of twenty-four larvae (subsamples). Following a 10 min dark acclimation period, the Noldus white light routine was used to test visual motor response by exposing larvae to 10 min intervals by alternating light and dark periods for 50 min in a previous study , Science and Engineering Research Board [SB/S9/Z-03/2017-XXV (2018–2019)].Arjun Pitchai was supported by Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


Colony stimulating factor 1 receptor (CSF-1R) is a single channel III transmembrane receptor tyrosine kinase (RTK) and plays an important role in immune regulation and the development of various cancer types. The expression of CSF-1R in colon adenocarcinoma (COAD) and its prognostic value remain incompletely understood. Therefore, we aim to explore the prognostic value of CSF-1R in COAD and its relationship with tumor immunity.high and CSF-1Rlow tumor-associated macrophages (TAMs) were investigated. Gene enrichment analysis was used to explore the signal pathways related to CSF-1R. In addition, the relationship between CSF-1R in tumor microenvironment (TME) and tumor immunity was also studied.CSF-1R expression in a COAD cohort containing 103 patients was examined using immunohistochemistry (IHC). The relationship between CSF-1R expression and clinicopathological parameters and prognosis was evaluated. Dual immunofluorescence staining was conducted to determine the localization of CSF-1R in COAD tissues. Univariate and multivariate Cox regression analysis were performed to evaluate independent prognostic factors. Transcriptomic profiles of CSF-1RIHC analysis showed that CSF-1R was overexpressed in COAD, and higher expression was associated with shorter overall survival (OS). Immunofluorescence staining showed that CSF-1R was co-localized with macrophage marker CD68. Univariate and multivariate Cox regression analysis showed that CSF-1R was an independent prognostic factor for COAD. The results of gene enrichment analysis showed that CSF-1R was involved in tumor immune response and regulation of TME. In addition, CSF-1R was significantly correlated with TME, immune cell infiltration, TMB, MSI, Neoantigen, and immune checkpoint molecules.CSF-1R can serve as an independent prognostic factor of COAD and promising immunotherapeutic target of COAD. According to the latest cancer statistics, Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer-related mortality in the world . It is wvia hypoxia-inducible factor-1α (HIF-1α) and NF-κB pathways, leading to a tumor-promoting TME , natural killer T (NKT) cells, Treg and CTLs, are central components of immunosuppressive TME . TAMs arironment , 22. HowColony-stimulating factor 1 receptor (CSF-1R) is a single channel III transmembrane receptor tyrosine kinase (RTK) and acts as a cell-surface receptor for colony-stimulating factor 1 (CSF-1) and interleukin 34 (IL34). CSF-1R signaling plays an indispensable role in the regulation of survival, proliferation and differentiation of macrophages and monocytes . Emerginhigh TAMs were associated with pro-tumor inflammatory environment. Our study provides a theoretical basis for targeting CSF-1R as an immunotherapeutic strategy against COAD.In this study, we reported that CSF-1R expression was significantly more abundant in cancerous tissues than in adjacent cancerous tissues, and CSF-1R expression may serve as an independent prognostic predictor for worsened survival in patients with COAD. Immunofluorescence analysis revealed that CSF-1R was mainly expressed in TAMs of COAD. High levels of CSF-1RWe conducted a retrospective study and recruited 103 COAD patients who underwent radical resection surgery of COAD in Nantong University Affiliated Hospital between 2010 and 2013. The clinicopathological and follow-up data of all patients were obtained and summarized in Tissue microarrays (TMA) was prepared from COAD specimens by histological department of Nantong University Affiliated Hospital and subjected to immunohistochemistrical analysis. Briefly, TMAs were deparaffinized, hydrated, and heated to 121°C using an autoclave in sodium citrate buffer for 20 min to retrieve the antigen. Thereafter, the sections were blocked with 10% goat serum in 0.01M phosphate buffered saline (PBS) for 2h at room temperature, followed by incubation with an anti-CSF-1R rabbit polyclonal antibody  overnight at 4°C. The section was incubated with horseradish peroxidase (HRP)-conjugated sheep anti-rabbit IgG at 37°C for 30 min. The immunoreactivity was developed with DAB reagent . Finally, the slides were counterstained with hematoxylin. The immunostaining of each specimen was analyzed by two independent pathologists who were blinded to the patients’ clinic pathologic data. They evaluated the staining score of each specimen using the H score. The H score ranged from 0–300, multiplying the percentage of positive cells by the staining intensity . The best cutoff value for the staining score was selected with X-title Software. The patients were divided into two groups: CSF-1R-high (n = 57) and CSF-1R-low (n = 46).high TAMs in five different sections was examined and analyzed using the Image J software. Cells with immunostaining scores significantly higher (2.5-fold increase) than non TAMs were defined as CSF-1Rhigh TAMs.For immunofluorescence staining, paraffin sections were deparaffinized, hydrated and antigen-retrieved, as mentioned above. Next, the sections were rinsed three times with PBS, and incubated with anti-CSF-1R antibody (1:50 dilution) and CD68 antibody  overnight at 4°C. After washing using PBS for 3 times, and the slides were incubated with the following secondary antibodies for 1h at 37°C: Alexa Fluor488 goat anti-rabbit IgG ; Alexa Fluor555 donkey anti-mouse IgG . Finally, the sections were mounted with Antifade Mounting solution containing 10μg/ml DAPI. Representative visual fields were acquired using a Leica DM5000 B microscope . Next, the fluorescence intensity of the CSF-1Rhttp://gdc.cancer.gov). The TCGA data were all analyzed by the R software (version 4.1.0) and survival package. The expression data of CSF-1R in various CRC cells were obtained from the DepMap portal  database.The COAD expression data and corresponding clinical data were obtained from The Cancer Genome Atlas (TCGA) official website . Then put it on the ice and ship it to the laboratory. The tumor tissue was digested with human Tumor Dissociation Kit (Miltenyi biotec), and then the gentleMACS C test tube containing enzyme mixture and tissue was inverted and connected to gentleMACS Dissociator  (Miltenysingle cell suspension was prepared as described previously (low TAMs (CD45+ CD14+ CSF-1Rlow) and CSF-1Rhigh TAMs (CD45+ CD14+ CSF-1Rhigh) were obtained from single-cell mixed suspension by flow cell sorting. Total RNA was isolated from the sorted samples using Qiagen RNeasy kit and subjected to cDNA library preparation using Smart-seq2 (For fluorescence activated cell sorting (FACS), eviously  from freart-seq2  scheme. art-seq2 .https://www.bioconductor.org/packages/release/bioc/html/limma.html) to analyze differentially expressed genes (DEGs) between CSF-1Rhigh TAMs and CSF-1Rlow TAMs and used ggplot2 and pheatmap packages to draw volcano map and heat map respectively. We selected | log2 (FC) | >1 and p <0.05 as the criteria for differential gene expression. Then, according to the changes of gene expression, DEGs were divided into two groups: up-regulated genes and down-regulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed by DAVID (https://david.ncifcrf.gov/tools.jsp).We used R software (version 4.0.2) and Limma package (by DAVID  tool (hthttp://sangerbox.com/) and Pearson method to evaluate the correlation between CSF-1R expression and Tumor Mutation Burden (TMB), Microsatellite Instability (MSI) and Neoantigen. And Pearson’s correlation test was also used to evaluate the correlation between CSF-1R and immune checkpoint molecules.We used Sanger Box data analysis platform  to determine differentially expression genes (DEGs) between these two populations. According to the changes of gene expression, we identified 1298 up-regulated genes and 371 down-regulated genes. In order to explore the biological functions of these DEGs, we used DAVID tools to carry out GO and KEGG enrichment analysis. GO analysis showed that the up-regulated DEGs were mainly enriched in genes involved in inflammatory response, immune response and the regulation of TME and the down-regulated DEGs were strongly linked to respiratory chain and mitochondrial electron transport . PPI network showed that 10 genes  were functionally related to CSF-1R  family, plays an important role in the development of solid tumors . While Cy tumors , 27. Oure manner . Moreovee manner . What isTo date, the value of targeting CSF-1R as an immunotherapeutic strategy in COAD remains unclear. It’s worth noting that CSF-1R expression was significantly correlated with TMB, MSI and Neoantigen in COAD. Furthermore, we found that CSF-1R was significantly correlated with tumor-related immunosuppressive molecules . PDCD1, also termed as PD1, is a member of the CD28-B7 family. The expression of PDCD1 on cancer cells is considered to be a key mechanism leading to tumor immune evasion . CTLA4, high and CSF-1Rlow TAMs, underscoring complexity of the origination of the two TAM populations. Further studies are urgently needed to clarify the mechanism underpinning the regulation of CSF-1R expression in TAMs.Last, we admit that our research has several limitations. First, our studies were carried out using limited sample sizes, which may, to some extent, compromise the rigorousness of our conclusion. Secondly, the clinical information of TCGA database is limited, and the clinical information of individual samples is lost, which may affect our results. Finally, the molecular mechanisms underlying CSF-1R expression remain obscure. Indeed, our RNA-seq data indicated that CSF-1R may exhibit strong expression difference (fold change up to 100) between CSF-1Rhigh TAMs represent a vital cell population in driving tumor immune tolerance. In addition, CSF-1Rhigh TAMs is involved in multiple immune response pathways and the recruitment of immune cells, such as Treg. These findings provide a theoretical basis for targeting CSF-1R as an immunotherapeutic strategy against COAD.In summary, our study illustrates the prognostic value of CSF-1R in COAD, and CSF-1R can be regarded as an independent risk factor for the prognosis of COAD. Our study clarified that CSF-1R plays a critical role in COAD immune environment. CSF-1R is not mainly expressed in tumor cells and has very limited effects in directly regulating tumor malignancy in COAD. Instead, CSF-1R is strongly distributed in TAMs and CSF-1Rhttps://www.ncbi.nlm.nih.gov/geo/, GSE193814.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Ethics Committee of Nantong University Affiliated Hospital. The patients/participants provided their written informed consent to participate in this study.XW and BH designed and performed the experiments. XW drafted the manuscript. FQ and JZ collected the clinical specimens and analyzed the data. FQ revised the manuscript. All authors agree to be accountable for the content of the work. All authors contributed to the article and approved the submitted version.This study was supported by National Natural Science Foundation of China (81502057), and the Natural Science Foundation of the Jiangsu Higher Education (21KJB320016).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.