Background: Ischemia injury affects the recovery of liver allograft function. We propose a novel technique aimed at avoiding a second ischemic injury: transplanting an extended criteria donor (ECD) liver directly under normothermic machine perfusion (NMP) without recooling. We studied two cases to evaluate the efficacy and safety of this technique.Methods: The perioperative characteristics and postoperative outcomes of two recipients of ECD livers were analyzed. Both transplantations were performed with continuous normothermic machine perfusion without recooling.Result: In case 1, the cause of donor death was anoxia, and the donor liver had hypernatremia before procurement. The recipient was diagnosed with decompensated cirrhosis. His model for end-stage liver disease (MELD) score was 38. In case 2, the donor liver was from a donor after cardiac death (DCD), and the donor had elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The recipient was diagnosed with acute hepatic failure. His MELD score was 35. Both donor livers were maintained under NMP and then transplanted without recooling. The peak ALT and AST levels after surgery were 452 and 770 U/L in case 1 and 100 and 592 U/L in case 2. Neither early allograft dysfunction (EAD) nor primary graft non-function (PNF) was present in these two cases.Conclusion: In conclusion, our results demonstrate that continuous NMP without recooling is efficacious and safe for LT with extended criteria donor livers. Further investigations of this technique will be performed to confirm these promising results. Liver transplantation (LT) is a successful treatment option for end-stage liver disease (ESLD) of various etiologies . The 1-yAll the procedures were performed in accordance with the ethical standards of the responsible committee on human institutional and national experimentation and with the Helsinki Declaration of 1964 and its later revisions.Two patients who had undergone OLT with continuous NMP without recooling were included in the study. The recipient pre-transplantation data and the perioperative data, including the perfusion parameters and postoperative complications, were assessed and analyzed.via an interposition vascular graft (the donor iliac artery), and the gastroduodenal artery (GDA), respectively, then connected to a liver assist device for perfusion. The perfusate is described in Details about this technique have been described in previously published articles. Briefly, the donor liver was procured under a standard perfusion technique, and the organ was preserved in a University of Wisconsin (UW) solution prior to perfusion. Afterwards, the donor liver was reprocessed in NMP to 37°C following the flush of lactated Ringer's solution. After ligation of the cystic duct, a tube was placed in it to drain bile. The cannula was placed separately into the infrahepatic inferior vena cava (IHIVC), portal vein (PV) After dissection of the diseased liver, the donor liver was transplanted with an undergoing NMP circuit. We first anastomosed the donor suprahepatic inferior vena cava (SHIVC) and the recipient counterpart in an end-to-end fashion. Subsequently, PV and HA were anastomosed with the donor's counterpart in an end-to-end fashion. During the anastomosis, the blood supply was not stopped. Afterwards, the clamps on the recipient's PV and HA were removed to restore the blood supply to the donor liver soon after NMP was stopped, after removal of the HA and PV cannula . The IHIAfter the transplantation, all patients were transferred to the intensive care unit with the endotracheal tube. After the tubes were removed, the patients were transferred to the general ward for further care. Biomedical values including regular blood test, transaminase, and bilirubin were tested and documented every day in the first week. Routine Doppler ultrasound of the liver graft blood flow and biliary tract was performed once every 2 days for 7 days. The immunosuppressive regimen was similar for all patients and included tacrolimus and mycophenolate mofetil. Basiliximab (20 mg) was administered intraoperatively before closing the abdomen and the 4th day postoperation by intravenous pump for induction immunotherapy. The blood levels of tacrolimus were 8–12 ng/ml in the first month, followed by 4–8 ng/ml thereafter.Data of these two cases are presented in The recipient patient in case 1 was diagnosed with hepatitis B virus-associated (HBV) decompensated cirrhosis. He had a history of esophageal and gastric variceal bleeding and abdominal bleeding. His serum AST, ALT, and Tbil levels were 11 U/L, 39 U/L, and 152.8 μmol/L, respectively. His MELD score was 38. The recipient in case 2 was diagnosed with acute hepatic failure and hepatic encephalopathy. He had a history of cerebral infarction, hypertension, and HBV infection. His AST, ALT, and Tbil levels were 50 U/L, 35 U/L, and 440.8 μmol/L, respectively. His MELD score was 38.NMP was performed for a total of 7 h for each donor liver . During Postoperative data are presented in In conventional LT, cryopreservation can reduce the physiological metabolism of cells but cannot slow down the damage to cell integrity . IRI repECD livers are thought to be of lower-than-average quality and to be associated with poorer postoperative outcomes . Maring The limitations of our study are as follows. First, the preservation  and the implantation are complex and need to be further modified. In addition, its effectiveness needs to be proven in a RCT.In conclusion, our results demonstrate that continuous NMP without recooling is efficacious and safe for LT with extended criteria donor livers.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Institutional Ethics Committee for Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.WJ and MC: conceptualization. ZG and XHe: methodology. ZC, XHo, TW, and SH: writing–original draft. YM, CH, YG, and QZ: data collection. MC: writing—review and editing. WJ: supervision. XHe: project administration. WJ, MC, and XHe: funding acquisition. All authors contributed to the article and approved the submitted version.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.


Acacetin  is the major bioactive component of the traditional Chinese medicine “Snow lotus”. As a natural flavonoid compound, it has been shown to have good pharmacological effects such as anti-inflammatory, anticancer, and anti-obesity. Among them, its prominent role in cardiovascular diseases (CVD) has received extensive attention from scholars in recent years. In this review, the protective effects of acacetin on a variety of cardiovascular diseases, as well as the existing problems and prospects, are discussed and summarized. This review also highlights the great potential of acacetin, a natural-derived Chinese medicine, as a cardiovascular agent candidate. Cardiovascular disease (CVD), including coronary heart disease, hypertension, myocardial infarction, and heart failure, is not only the main cause of death worldwide but also a major public health problem , 2. AlthTraditional Chinese medicine (TCM) has a history of more than 2,000 years. It is not only popular in China but also highly recognized in many countries in the world . In receRobinia pseudoacacia, Chrysanthemum indicum, and other species [Acacetin , one of the flavones of flavonoids, is a monomethoxy flavonoid, which exists in a variety of plants in free form or glycoside form, including  species , 10  is one of the most common cardiac arrhythmias in clinical practice, and its incidence increases with age . ComplicIKur (ultra-rapidly activated delayed rectifier potassium current), Ito (transient outward potassium current), IKACh (acetylcholine activated potassium current), and SKca , while it had no effect on the Na+ current, L-type Ca2+ current, or inward-rectifier K+ current in guinea pig cardiac myocytes [Ikur is specifically expressed in the human atrium but not in the ventricle, which is an ideal target for atrial-selective antiarrhythmic drugs [IKACh inhibitors prevent AF induced by vagal stimulation [Recently, Li et al. have demonstrated that acacetin can preferentially inhibit myocytes , 20. Amoic drugs , 22. In mulation , 24. Intmulation . The stuIto and Ikur were further investigated [Ito) and hKv1.5 (coding Ikur) channels. Acacetin, as an antagonist of the hKv4.3 channel, interacts with T366A and T367A located in the P-loop helix and V392A, I395A, and V399A located in the S6-segment [The molecular determinants of acacetin's ability to block stigated , 27. The-segment . In addiIn addition, the combination of acacetin and sodium channel blockers has also been shown to be a promising strategy for the treatment of AF .J wave syndromes, mainly including Brugada syndrome (BrS) and early repolarization syndrome (ERS), are characterized by an abnormal transition point between the terminal part of the QRS complex and the ST segment, that is, the J point in the electrocardiogram. It is well known that J wave syndromes are associated with serious ventricular tachycardia (VT), ventricular fibrillation (VF), and other life-threatening events [J wave syndrome. But the drugs for the diseases are extremely limited. Quinidine, an Ito inhibitor, is one of the few drugs that are effective in treating J wave syndromes [to density, action potential notch, and J wave area [g events , 30. Impyndromes . More reave area .α, thus playing a protective role on the myocardium after myocardial infarction (MI) [In 2014, Yang et al. for the first time demonstrated that acacetin in vitro can reduce the hypoxia/reoxygenation injury to the cardiac myocardium and has a protective effect on the myocardium by reducing lipid peroxidation and enhancing the antioxidant capacity . In thision (MI) . Furtherion (MI) . Interesion (MI) .Myocardial infarction is one of the most prominent cardiovascular diseases, contributing significantly to death. Cardiac remodeling is an important process after myocardial infarction, which is closely related to the prognosis of the disease. Recent experiments have shown that acacetin can inhibit myocardial remodeling such as myocardial hypertrophy and fibrosis, reduce myocardial infarct size, and enhance left ventricular function after myocardial infarction . The proAtherosclerosis used to be regarded as a chronic inflammatory disease related to dyslipidaemia, but more and more evidence shows that oxidative stress is also involved . ConsideDoxorubicin is an anthracycline antibiotic, which is widely used as an anticancer drug in clinics . HoweverAgeing is considered an independent risk factor for cardiovascular disease, and cardiac senescence is closely related to the occurrence and development of cardiomyopathy in the elderly . Since pAlthough acacetin is a safe and promising candidate for the treatment of CVD, there are still many problems to be resolved in the future. One of the most difficult problems is the poor solubility and rapid metabolism of acacetin in vivo, which will be a major obstacle to the clinical development and utilization of acacetin in the future . In 2016On the other hand, although the pathophysiological conditions of various CVDs are simulated in animals, there are few applications of uniform animal experimental models that are recognized around the world. The in-depth research of acacetin in this area in the future will be an important milestone in promoting preclinical experiments.In addition, the clinical pharmacological information of acacetin, which is considered to be an important part of drugs, such as absorption, metabolism, and toxicity, is not sufficient at present , 54. TheIn general, this review emphasizes the great potential of acacetin as a traditional Chinese medicine on cardiovascular disease and summarizes the remarkable effects and related possible mechanisms of acacetin against atrial fibrillation, ischemia-reperfusion injury, atherosclerosis, doxorubicin cardiomyopathy, and cardiac senescence. Further studies are warranted to resolve the current problems of acacetin, such as poor solubility and rapid metabolism in vivo, which may help prevent and treat cardiovascular diseases in the future.


Intraventricular hemorrhage (IVH) and post-hemorrhagic hydrocephalus (PHH) have a complex pathophysiology involving inflammatory response, ventricular zone and cell–cell junction disruption, and choroid-plexus (ChP) hypersecretion. Increased cerebrospinal fluid (CSF) cytokines, extracellular matrix proteins, and blood metabolites have been noted in IVH/PHH, but osmolality and electrolyte disturbances have not been evaluated in human infants with these conditions. We hypothesized that CSF total protein, osmolality, electrolytes, and immune cells increase in PHH.CSF samples were obtained from lumbar punctures of control infants and infants with IVH prior to the development of PHH and any neurosurgical intervention. Osmolality, total protein, and electrolytes were measured in 52 infants  IVH, 13 high grade (HG) IVH, and 11 PHH). Serum electrolyte concentrations, and CSF and serum cell counts within 1-day of clinical sampling were obtained from clinical charts. Frontal occipital horn ratio (FOR) was measured for estimating the degree of ventriculomegaly. Dunn or Tukey’s post-test ANOVA analysis were used for pair-wise comparisons.CSF osmolality, sodium, potassium, and chloride were elevated in PHH compared to control (p = 0.012 − < 0.0001), LGIVH (p = 0.023 − < 0.0001), and HGIVH (p = 0.015 − 0.0003), while magnesium and calcium levels were higher compared to control (p = 0.031) and LGIVH (p = 0.041). CSF total protein was higher in both HGIVH and PHH compared to control (p = 0.0009 and 0.0006 respectively) and LGIVH (p = 0.034 and 0.028 respectively). These differences were not reflected in serum electrolyte concentrations nor calculated osmolality across the groups. However, quantitatively, CSF sodium and chloride contributed 86% of CSF osmolality change between control and PHH; and CSF osmolality positively correlated with CSF sodium , potassium , chloride , but not total protein across the entire patient cohort. CSF total cells (p = 0.012), total nucleated cells (p = 0.0005), and percent monocyte (p = 0.016) were elevated in PHH compared to control. Serum white blood cell count increased in PHH compared to control (p = 0.042) but there were no differences in serum cell differential across groups. CSF total nucleated cells also positively correlated with CSF osmolality, sodium, potassium, and total protein (p = 0.025 − 0.0008) in the whole cohort.CSF osmolality increased in PHH, largely driven by electrolyte changes rather than protein levels. However, serum electrolytes levels were unchanged across groups. CSF osmolality and electrolyte changes were correlated with CSF total nucleated cells which were also increased in PHH, further suggesting PHH is a neuro-inflammatory condition.The online version contains supplementary material available at 10.1186/s12987-021-00295-8. High grade intraventricular hemorrhage (HGIVH), defined as IVH with blood filling more than 50% of the ventricular volume (Papile grades III–IV) , affectsPHH is a complex condition that involves neuro-inflammation , alteratWe hypothesized that PHH in human preterm infants is associated with alterations in the biochemical profile of CSF, including CSF electrolytes, total protein, and osmolality. To examine this hypothesis, we measured each of CSF osmolality, protein levels, and electrolyte concentrations in lumbar CSF and serum obtained from human infants with no known neurological injury, IVH (grades I/II and III/IV), and PHH. PHH was associated with increased CSF protein, osmolality, and specific electrolytes likely to impact osmotic and oncotic pressure. These findings provide novel insights into the putative pathophysiology of ventriculomegaly in PHH. To our knowledge, this is the first study quantifying the biochemical profile of CSF in human infants with PHH.We retrospectively analyzed human CSF prospectively collected at Washington University/St. Louis Children’s Hospital between November 2009 and December 2017. Included were infants born at post-menstrual age (PMA) < 34 weeks with birth weight < 2000 g who underwent lumbar puncture (LP) in the St. Louis Children’s Hospital (SLCH) Neonatal Intensive Care Unit (NICU) as a part of routine care. Neonates with no known neurologic disease who underwent a LP for sepsis evaluation where the evaluation and all cultures were negative were included as controls. Papile grading scale  was usedAll CSF samples were acquired via standard LP and serum samples were acquired via routine clinical venipuncture PHH patients underwent temporizing neurosurgical treatment with a ventricular reservoir followed by permanent treatment with a CSF shunt, or, in some cases, endoscopic third ventriculostomy with choroid plexus cauterization. LP in IVH and PHH group was performed after the occurrence of IVH but prior to PHH diagnosis and any neurosurgical intervention.All protocols and procedures were approved by the Washington University Human Research Protection Office and Institutional Review Board with a waiver of written informed consent (HRPO# 201203126).The general health status of the neonates was assessed with the clinical risk index for babies minus temperature (CRIB II – T)  and a moCSF samples were collected by the NICU clinical team at SLCH using standard, sterile procedures for LP. CSF samples were sent to the SLCH clinical laboratory for clinically indicated microbiological culture (5-day sutveillance) and laboratory evaluation. Excess CSF or “saved” CSF samples were centrifuged at 1000 g for six minutes and the supernatant aliquoted, frozen -20 °C for 3 months before transfer to the Washington University Neonatal CSF Repository for freezing and storage at − 80 °C until the time of experimental analysis, as described previously , 50. CSFOsmolality was measured using the VAPRO vapor pressure osmometer (Wescor 5520), as described previously for analysis of CSF and other biofluids –53. The CSF electrolyte  measurements were performed in the Washington University core clinical laboratory for clinical studies after instrument validation using artificial CSF  were performed and, if the data were normally distributed, ANOVA with Tukey post-test analyses were conducted. Otherwise, Kruskal–Wallis ANOVA with Dunn’s post-test analyses were performed for non-parametric data. Pearson or Spearman correlation test were also performed based on normality test result.A total of 52 patients were included in this study: 18 Control, 10 LGIVH, 13 HGIVH, and 11 PHH infants. There was a higher percentage of males in all groups , total nucleated cells (p = 0.0005), and percent monocytes (p = 0.016) were higher in PHH ; 270.5 (30.0); 262.0 (73.0); and 323.5 (60.7) mmol/L, (median (Interquartile range [IQR])) respectively  concentration was increased in PHH (median 170 mmol/L with IQR of 58) compared to all other groups (median (IQR): control 141.0 [+ concentration in PHH compared to control. Similarly, CSF potassium (K+) concentration was elevated in PHH (median 4.4 mmol/L with IQR of 1.0) compared to control (median 3.1 mmol/L with IQR of 0.6), LGIVH (median 3.1 mmol/L with IQR of 0.7), and HGIVH (median 3.2 mmol/L with IQR of 0.4). Median and IQR for CSF chloride (Cl−) concentration in Control, LGIVH, HGIVH, and PHH were 119.0 (16.0); 121 (5.8); 123.0 (23.0); and 138.0 [− concentration was elevated in PHH compared to control by 21 mmol/L. Notably, serum values for Na + , K + , and Cl- were similar across all groups.CSF sodium  nd 138.0  mmol/L r2+) concentration was elevated in PHH (3.7 ± 0.8 mg/dL) compared to control  and LGIVH  but not HGIVH  (Table 2+) was significantly higher in PHH (4.3 ± 0.3 mg/dL) compared to control , LGIVH , but not HGIVH . This is a 1.0 mg/dL or 0.07 mmol/L absolute increase in mean Mg2+ concentration and 1.5 mmol/L increase in Ca2+ in PHH compared to control. Carbon dioxide (CO2) concentration, a reflection of bicarbonate ion concentration and pH, showed variable differences among groups with differences observed between PHH (19.8 ± 1.0 mmol/L) and HGIVH (19.5 ± 2.3 mmol/L) compared to LGIVH  but not control (17.3 ± 4.3 mmol/L). Similarly, there is no change in serum calcium or bicarbonate across all groups.CSF magnesium , followed by chloride (20.9 mmol/L) Table . Total pTo further evaluate the possible causes of osmolality and electrolyte changes and its relationship to ventricular volume, we calculated the correlations shown in Additional file Osmolality and thus electrolyte concentration have been implicated in the development of ventriculomegaly , 54, 55.Mean control CSF osmolality (280.8 mmol/L), Na (142.3 mmol/L), K (3.1 mmol/L), Cl (122.4 mmol/L), CO2 (17.3 mmol/L), were within the range of previously published studies . Serum eOsmolality is one of the driving forces for water movement across a cell layer , 58. TheThe elevation in PHH CSF osmolality is interesting, as one would expect fluid movement to achieve equilibrium at or near normal CSF osmolality. Indeed, we found that PHH CSF osmolality was uniformly elevated relative to serum. This is likely due to limitation of water transport and thus ventricular dilatation by the ventricular wall compliance, hydrostatic pressure, and ICP . In addiWater transport into the ventricular space could be originating from the ChP capillaries via AQP1 channels (if the BCSFB is intact) on the ChP apical membrane , 73 or fSimilar to previous studies , 37, 76,PHH, though described clinically by signs and symptoms of ventriculomegaly and elevated ICP, is likely preceded by complex cytopathologies such as ChP CSF hypersecretion , 59, 77,+ and HCO3− ion transport, implicating BCSFB disruption rather than CO2 diffusion. The sodium-hydrogen exchanger (NHE1), a channel that delivers sodium into the CSF while moving hydrogen ion out, might also be involved in CSF bicarbonate changes. If hyperactive in PHH, this could be contributing to the increased Na and pH in the CSF of infants with PHH. Further studies to examine the activity of both channels in PHH should be conducted.CO2, as a surrogate for bicarbonate which is the major driver of CSF pH , was notMg is also significantly increased in PHH compared to control, but it is unclear at this point what might be leading to this change. One possibility is the inflammatory response as Mg is positively correlated with nucleated cells. Total nucleated cells were also positively correlated with Na, K, osmolality, and total proteins indicating that inflammation might also be involved in the elevation of other CSF electrolytes and total protein. Many animals, in vitro, and human studies have shown increased cytokines and chemokines in PHH which might contribute to the total protein concentration , 18. HowThe disruption of the BCSFB is another pathology that might contribute to the electrolyte, total protein, and osmolality changes , 87, 88.Finally, maintaining a normal CSF biochemical profile is essential for normal neurodevelopment and neuronal activity especially at the neonatal age period . CSF conA few limitations in this study must be acknowledged. IVH/PHH is more prevalent in males with about 60% of cases affecting males –95 but, In this study, we report the novel finding of increased CSF concentration of Na, K, Cl, Ca, Mg, and osmolality in infants with PHH. Changes in CSF osmolality are most likely driven by changes in sodium and electrolyte concentrations rather than protein concentrations. These changes in the CSF were independent of the serum as we found no changes in serum electrolytes across the groups. However, we observed positive correlations between CSF total nucleated cells and CSF osmolality, Na, and K. Total cells and nucleated cells in the CSF were increased in PHH. Serum WBC count was also increased in PHH but no differences in the WBC differential. These findings provide important insights into the pathophysiology of IVH and PHH.Additional file 1: Table S1. CSF and serum electrolyte concentrations in control individuals as well as CSF/Serum electrolyte ratio in control and PHH. Table S2. Correlations between electrolytes and osmolality, and CSF cell counts. Table S3. Subgroup analysis by sex of cerebrospinal fluid total protein, osmolality, and electrolytes in PHH. Sample size limitations permitted analysis only of the male cohort; there were insufficient samples for analysis by female sex. To address this limitation, regression analysis examining sex as an explanatory variable was also performed (please refer to Table S4). Values recorded as mean ± standard deviation. In the “P value’ column, ANOVA summary p-value was recorded on the top row while significant pairwise post-test P value were recorded on the bottom row. All pairwise comparisons were analyzed based on Dunn’s or Tukey’s post-test ANOVA depending on the distribution of the data . n, represents sample size. Table S4. F, t, and p values from regression analyses evaluating sex as an explanatory variable. Analysis of all CSF parameters resulted in non-significant p-values indicating sex is not an explanatory variable for differences in the CSF parameters. F=F-value; t=t-value; and p=p-value. Figure S1. Ultrasonographic image representation of Control, IVH and PHH.


Gaining information about the habitat environment and biological interactions is important for conserving gold-spotted pond frogs, which are faced with a threat of local population extinction in Korea due to artificial habitat changes. Based on stable isotope ratios, we estimated the ecological niche space (ENS) of gold-spotted pond frogs in an ecological wetland park and a rice paddy differing in habitat patch connectivity and analyzed the possibility of their ENS overlapping that of competitive and predatory frogs. Gold-spotted pond frogs showed a broader ENS in the ecological wetland park, wherein predation was relatively easy, than in the rice paddy. However, the ENS of the gold-spotted pond frogs was highly probably overlapped with that of other competing frog species that shared some of the food sources. Nevertheless, since the stable isotope analysis showed that gold-spotted pond frogs fed on more diverse prey than their competitors, it would remain relatively easy to procure alternative food sources, which are less affected by the competition in an environment with abundant food. Therefore, for stable settling of gold-spotted pond frogs into habitats and preserving their population, establishing habitat environments with highly diverse food sources is crucial, following consideration of their feeding behavior.Pelophylax chosenicus) is an endangered amphibian species in South Korea. In order to obtain ecological information regarding the gold-spotted pond frog’s habitat environment and biological interactions, we applied stable isotope analysis to quantify the ecological niche space (ENS) of frogs including black-spotted pond frogs (P. nigromaculatus) and bullfrogs (Lithobates catesbeianus) within the food web of two different habitats—an ecological wetland park and a rice paddy. The gold-spotted pond frog population exhibited a broader ENS in the ecological wetland park than in the rice paddy. According to the carbon stable isotope ratios, gold-spotted pond frogs mainly fed on insects, regardless of habitat type. However, the results comparing the range of both carbon and nitrogen stable isotopes showed that gold-spotted pond frogs living in the rice paddy showed limited feeding behavior, while those living in the ecological wetland park fed on various food sources located in more varied trophic positions. Although the ENS of the gold-spotted pond frog was generally less likely to be overlapped by that of other frog species, it was predicted to overlap with a high probability of 87.3% in the ecological wetland park. Nevertheless, gold-spotted pond frogs in the ecological wetland park were not significantly affected by the prey competition with competitive species by feeding on other prey for which other species’ preference was low. Since these results show that a habitats’ food diversity has an effect on securing the ENS of gold-spotted pond frogs and prey competition, we recommend that the establishment of a food environment that considers the feeding behavior of gold-spotted pond frogs is important for the sustainable preservation of gold-spotted pond frogs and their settlement in alternative habitats.The gold-spotted pond frog ( The niciversity ,3,4,5. Siversity ,7.Current understanding of niche width estimation is based on the heterogeneity of the feeding behavior of target organisms and ecological indices  . To cons13 C) provides insights into the food sources of a consumer, as the fixed value does not change significantly from photosynthesis through feeding, digestion, and assimilation, with a difference lower than 1‰ between prey and predator. In contrast, nitrogen (N) stable isotope ratios (δ15 N) are typically enriched in consumers relative to the concentrations in the diets, and the concentrations in predators can be up to 3.4‰ higher than those in prey [Stable isotopes have been used as alternative tools for analyzing food sources and overcoming the limitations of existing methods. These isotopes can accurately evaluate trophic structure based on the fractionation effect exhibited by each isotope during food assimilation into the tissues of consumers in the upper trophic levels ,8. The c in prey . A bi-pl in prey ,11. One 13 C–δ15 N stable isotope bi-plots are useful for identifying the isotopic niches of organisms within a food web and for the quantitative interpretation of community structure based on niche characteristics . However, stable isotope bi-plots have some limitations for assessing the impacts of individual species on trophic diversity and redundancy within communities. Layman et al. [13 C–δ15 N bi-plot and five metrics derived from it, δ15 Nrange (NR), δ13 C range, mean distance to centroid (CD), mean nearest neighbor distance (NND), and standard deviation of the NND (SDNND), reveals the responses of a population to environmental changes in the form of numerical values, which can be used not only to compare populations within a community, but also to analyze spatiotemporal changes in the overall community structure. However, community-wide metrics are estimates that do not consider uncertainties that may occur during sampling and population mean calculations, whereas TA, which forms the basis of community-wide metrics, is sensitive to sample size, presenting challenges when comparing populations (or communities) with different sample sizes. To address this problem, multivariate ellipse-based metrics, based on Bayesian inference techniques, that incorporate standard error in the estimates are used. Here, as standard ellipse area (SEA) can be derived by considering the sample size within a community , communities with different sample sizes can be directly compared [δcompared . Such stcompared ,12,13,14Pelophylax chosenicus)  have decreased in number in South Korea because of habitat destruction and the introduction of bullfrogs, an invasive species [All amphibians feed heavily on invertebrates; thus, their impact on this food source can be prodigious. In many habitats, amphibians are a major ‘conveyor belt’ that provides the transfer of invertebrate energy sources to predatory animals higher up the food chain. Amphibians have an important role in ecosystem energy flow and nutrient cycling in many ecosystems . Gold-sp species . The gol species . Gold-sp species ,18. In a species .Recently, programs to establish alternative habitats for conserving gold-spotted pond frog populations have been pursued ,21,22. AIn this study, we used stable isotope ratios to measure the niche widths of gold-spotted pond frogs and other frog species, with which they have competitive and predatory interactions, using frogs sampled from different habitats. The aim of the study was to investigate how different environments influence their feeding behavior and positions within the food web. Community-wide metrics were used to compare community characteristics, while niche overlap probability values were used to quantitatively assess how biological interactions with other frog species in different environments influenced gold-spotted pond frog trophic diversity. The results of this study provide basic ecological data on habitat environments and interspecific competition, which may facilitate the establishment and maintenance of gold-spotted pond frog populations in alternative habitats in conservation programs.Gold-spotted pond frogs are typically found in wetland environments. Therefore, when alternative habitats are created to protect their populations, the establishment of artificial habitats such as ecological parks is often considered . We seleNelumbo nucifera), common reed (Phragmites communis) and cattail (Typha angustifolia), and each pond is connected to a small water channel to facilitate free movement of the organisms along the waterways. Frogs in the rice paddy habitat were sampled mostly from rice paddies and waterways on the banks of Mankyeong River. Common reed (P. communis) was the dominant plant, and other hydrophytes, such as wormwood (Artemisia selengensis f. serratifolia), water chestnut (Trapa japonica), and silver grass (Miscanthus sacchariflorus) were found close by. The rice paddy habitat is vulnerable to habitat fragmentation because of concrete channels and human interference, including the use of farming equipment during cultivation seasons  to eliminate lipids to ensure that the C stable isotope ratio would accurately reflect food consumed recently [The captured frogs were ethically killed as painless as possible using MS-222 and then processed for analysis. Individuals sampled were transported to the Isotope Ecology and Environmental Science Lab, Hanyang University, under refrigerated conditions and subsequently freeze-stored (−23 °C) until stable isotope sample extraction. Appropriate amounts (1–2 g) of stable isotope samples were extracted from the muscles of each frog. Other tissues, such as bone, blood, and skin, were excluded to ensure accurate measurements. All samples were homogenized after freeze-drying (−85 °C) for at least 48 h, and then the samples divided into C and N stable isotope samples. The C stable isotope samples were treated with acid (HCl) to eliminate inorganic C . The samrecently ,31. No s13 C and δ15 N Appendix, C and N stable isotope ratios in the samples were analyzed using an elemental analyzer-isotope ratio mass spectrometer . Because the absolute quantities of C and N stable isotopes in the samples were very low, the isotope ratio was converted and expressed as a relative value compared to a reference standard. Vienna Pee-Dee Belemnite and air were used as the reference standards for C and N stable isotopes, respectively, and the abundances of the isotopes in the samples relative to in the reference standards were calculated as parts per thousand and expressed as δ13 C–δ15 N bi-plots that were constructed using the measured C and N stable isotope ratios. ENS values of frog populations between habitats were compared to determine their positions within the food webs, and differences in food sources and trophic levels of the populations were compared according to the habitat environments. ENS was calculated using the Stable Isotope Bayesian Ellipses function (SIBER) in R v3.5.1 based on the obtained stable isotope ratios [The ENS, or isotopic niche widths, of the frog populations, including the gold-spotted pond frogs in the two habitats, were analyzed based on δe ratios ,32. SIBEe ratios ,13. In aA with respect to species B was based on the overlapping area between the two species within the total niche of species B. Because interspecies-overlap has directionality, values measured in the reverse direction (niche overlap of species B with respect to species A) could also be derived. Niche overlap probability was calculated based on the degree of influence of the resource utilization capacities of the species, relative abundances within habitats, and C and N stable isotope ratios, enabling a comprehensive interpretation of the results [In order to evaluate the impacts of competitive and predatory interactions on the ENS of gold-spotted pond frog populations, the nicheROVER package in R was used to calculate the probability (%) of interspecies-niche overlap . Niche overlap of species The raw data of the C and N stable isotope ratios measured from the collected frogs specimen are summarized in 2 and 0.38‰2, respectively, indicating broader niches in the ecological wetland park habitat  and terrestrial insects  range from −28‰ to −23‰, whereas those of zooplankton and aquatic plants were reported to range from −34‰ to −29‰, and −30‰ to −26‰, respectively [Enallagma cyathigerum, Ephemerella dentata, Neocaridina denticulate, Neverita didyma, and Paracercion calamorum, Orders Odonata and Ephemeroptera) exhibited C stable isotope ratios ranging from −32‰ to −24‰ (unpublished data). When the C stable isotope ratio value ranges in the gold-spotted pond frog populations from the ecological park habitat were compared to those of the aquatic insects, the ranges were mostly similar, suggesting that these insects were the primary food sources of the gold-spotted pond frog populations in the wetland habitat.The stable isotope ratios of the gold-spotted pond frogs from the ecological wetland park habitat exhibited a wide distribution along the horizontal and vertical axes. The difference in N stable isotope ratios between trophic levels is generally 3.4‰ . ConsideIn contrast, gold-spotted pond frog populations from the rice paddy habitat exhibited narrow C and N stable isotope ratio ranges, indicating that their food sources were much more limited than that of the gold-spotted pond frogs from the wetland habitats, and that the individuals were largely feeding on similar food sources. The C stable isotope ratios ranged from −24‰ to −23‰, indicating very levels of low prey diversity. Given that there was little difference in the N stable isotope ratios among individuals these frogs may have fed on prey of similar trophic positions.Gold-spotted pond frogs are strongly omnivorous, feeding on either plant material or animals depending on feeding opportunities; therefore, their food sources vary depending on the environment . Food so2 and 0.42‰2, respectively, indicating a similar ENS regardless of habitat  and in the black-spotted pond frogs (0.42‰2) in the same habitat  of overlap with the ENS of the gold-spotted pond frogs in the ecological wetland park habitat, whereas such probability was very low (9.06%) in the rice paddy habitat. In contrast, gold-spotted pond frogs exhibited low probability of niche overlap based on the ENS of black-spotted pond frog in the ecological park habitat (4.6%) and rice paddy habitat (9.41%) A. AlthouGold-spotted pond frogs have a high preference for active food sources which have a low probability of being consumed by black-spotted pond frogs . TherefoThe above are some factors that may influence niche overlap. In addition, the influence of predators on other frog species and interspecific interactions should be considered, particularly in the case of the rice paddy habitats where bullfrog individuals were found. When ENS was compared between gold-spotted pond frogs and bullfrogs, a competitive relationship was inferred because the results did not reveal differences in trophic diversity B. The ovOur results highlight the need for environments with high trophic diversity when selecting or establishing alternative habitats for gold-spotted pond frogs. Here, trophic diversity is not limited to one ecosystem but applies to both aquatic and terrestrial ecosystems. Therefore, it is essential to create an environment in which preferred food sources can be accessed freely from the two ecosystems, with no ecosystem fragmentation. Further studies are required to investigate essential food sources for gold-spotted pond frogs that would increase their competitiveness in biological interactions when establishing alternative habitats. In addition, gold-spotted pond frogs exhibited a tendency to consume foods with relatively high N stable isotope ratio values as they grew, representing higher trophic diversity, whereas there was no correlation between growth and N stable isotope ratio values in the black-spotted pond frogs in a previous study . Such spWe used stable isotope ratios to analyze the ENS of gold-spotted pond frogs in two habitats to determine the influence of these habitats on feeding habits. The findings were applied to evaluate the influence of biological interactions based on comparisons with similar species. Measured C stable isotope ratios indicated that the sampled gold-spotted pond frog individuals fed on insects as their primary food source, regardless of the habitat, whereas N stable isotope ratios revealed slightly different distributions regarding trophic diversity depending on the habitat. Based on the habitat characteristics and stable isotope ratios, gold-spotted pond frogs in the ecological wetland park habitat with relatively high food abundance exhibited wider vertical ENS distribution, whereas those in the rice paddy habitat with somewhat limited food availability and individual mobility within the habitat exhibited narrow ENS with densely aggregated stable isotope ratio values.In conclusion, habitat environments with broad food source varieties were more favorable for securing ENS in gold-spotted pond frogs. The degree of niche overlap with competitive species was greater in the ecological park wetland habitat. Black-spotted pond frogs were sharing the ENS of gold-spotted pond frogs, and the degree of overlap with competitive species may have increased with the increasing diversity of food sources available to gold-spotted pond frogs within the ecological park habitat. Nevertheless, concurrently, the impact of niche overlap does not seem to have played a large role as gold-spotted pond frogs could also secure competition-free food sources. In contrast, in the case of the rice paddy habitat, where gold-spotted pond frogs and its competitors were estimated to exhibit limited feeding activity through narrow ENS, the competition among species may have been be intensified through continuous deterioration of the food environment, despite the probability of the calculated niche overlap being lower than that of the ecological wetland park habitat. In addition, bullfrogs in the rice paddy habitat exhibited greater competition than predation to the gold-spotted pond frogs; however, the overlap degree of not only the ENS but also of the C stable isotope ratio range indicated that food diversity was low, meaning that the bullfrogs did not strongly influence the feeding activities of the gold-spotted pond frog populations. Unfortunately, we used a relatively small number of samples for ENS analysis and could not directly investigate the available food sources in the habitat and the prey fed on by the frog species. Considering factors such as food preference analyses in addition to ecological characteristics (population density and distribution trends of frog species) within a habitat using quantitative data (stable isotope ratios in food sources) and their impacts on competition and feeding environments obtained from further study to supplement these limitations would reveal a more comprehensive picture of the biological and environmental factors present, and facilitate the establishment of more appropriate alternative habitats for conserving endangered species such as gold-spotted pond frogs.


C-X-C chemokine ligand 13 (CXCL13) is frequently elevated in cerebrospinal fluid (CSF) in a variety of inflammatory central nervous system (CNS) diseases, has been detected in meningeal B cell aggregates in brain tissues of multiple sclerosis patients, and proposedly recruits B cells into the inflamed CNS. Besides B cells also follicular helper T (Tfh) cells express the cognate receptor C-X-C chemokine receptor type 5 (CXCR5) and follow CXCL13 gradients in lymphoid tissues. These highly specialized B cell helper T cells are indispensable for B cell responses to infection and vaccination and involved in autoimmune diseases. Phenotypically and functionally related circulating CXCR5+CD4 T cells occur in blood. Their co-recruitment to the inflamed CSF is feasible but unresolved.We approached this question with a retrospective study including data of all patients between 2017 and 2019 of whom immune phenotyping data of CXCR5 expression and CSF CXCL13 concentrations were available. Discharge diagnoses and CSF laboratory parameters were retrieved from records. Patients were categorized as pyogenic/aseptic meningoencephalitis , neuroimmunological diseases , and non-inflammatory neurological diseases . ANOVA models and Spearman’s Rank-Order correlation were used for group comparisons and associations of CXCL13 levels with immune phenotyping data.In fact, intrathecal CXCL13 elevations strongly correlated with CXCR5+CD4 T cell frequencies in the total cohort , and ME  and NIMM  patients. Moreover, the ratio of CSF-to-peripheral blood (CSF/PB) frequencies of CXCR5+CD4 T cells strongly correlated with CXCL13 levels both in the total cohort  and ME subgroup , indicating selective accumulation. ME, NIMM and NIND groups differed with regard to CSF cell counts, albumin quotient, intrathecal IgG, CXCL13 elevations and CXCR5+CD4 T cells, which were higher in inflammatory subgroups.The observed link between intrathecal CXCL13 elevations and CXCR5+CD4 T cell frequencies does not prove but suggests recruitment of possible professional B cell helpers to the inflamed CSF. This highlights CSF CXCR5+CD4 T cells a key target and potential missing link to the poorly understood phenomenon of intrathecal B cell and antibody responses with relevance for infection control, chronic inflammation and CNS autoimmunity.The online version contains supplementary material available at 10.1186/s12987-021-00272-1. The chemokine C-X-C motif chemokine 13 (CXCL13) has been found elevated in cerebrospinal fluid (CSF) in a variety of inflammatory central nervous system (CNS) diseases , and proBoth are physiological functions of CXCL13 in secondary lymphoid organs where CXCL13 is crucially involved in the recruitment of B and T lymphocytes and their compartmentalization within B cell follicles of lymph nodes. (SLO) , 12. IniRecent research highlighted CXCR5+CD4 T cells in blood as phenotypically and functionally similar circulating counterparts of lymphoid Tfh cells , 16. CirCXCR5+CD4 T cells also occur in normal CSF at frequencies similar to peripheral blood . First dWe thus set out and retrospectively analyzed patient data to learn whether an association between intrathecal CXCL13 elevations and occurrence of CXCR5+CD4 T cells in the inflamed CSF could be established. In light of their potential B cell helper capacities, such evidence would highlight them as relevant targets for further research to improve our understanding of intrathecal B cell and antibody responses to neuroinfection and immune-mediated CNS diseases.The study included 57 patients admitted to the Department of Neurology of the Paracelsus Medical University (PMU) in Salzburg, Austria between August 2017 and December 2019 who had undergone lumbar puncture for diagnostic purposes and of whom immune phenotyping data of CXCR5 expression and concentrations of CSF CXCL13 were available. CSF parameters, immune phenotyping results, CXCL13 concentrations and discharge diagnosis were retrospectively collected from chart records.Based on discharge diagnosis, patients were clinically assigned into three groups: pyogenic & aseptic meningoencephalitis (ME), neuroimmunological disease (NIMM), and non-inflammatory neurological disease (NIND). Diagnoses in the ME group (n = 29) included bacterial meningoencephalitis (n = 2), epidural abscess (n = 1) , tick-borne meningoencephalitis (n = 1), Varicella-Zoster-Virus (VZV) meningoencephalitis (n = 5) Epstein-Barr virus (EBV) meningoencephalitis (n = 3), Influenza B virus meningoencephalitis (n = 1), HIV meningoencephalitis (n = 1), aseptic meningoencephalitis of unknown cause (n = 8), and lyme neuroborreliosis . Diagnoses in the neuroimmunological disease group (n = 22) included multiple sclerosis  untreated), autoimmune encephalitis (AIE) or myelitis (n = 6), neuromyelitis optica , chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids , neurosarkoidosis (n = 1), steroid responsive encephalopathy associated with autoimmune thyroiditis , and immunological encephalitis not further classifiable (n = 3). Diagnoses in the NIND group (n = 6) included polyneuropathy (n = 1), narcolepsy (n = 1), dementia (n = 1), back pain (n = 1), astrocytoma (n = 1), and epilepsy (n = 1).The responsible ethics committee of the country of Salzburg consented to the study (415-E/2218/10–2020).CSF laboratory analyses were performed at the Department of Laboratory Medicine of the PMU . CSF cells were enumerated using the Fuchs Rosenthal Counting chamber, IgG and albumin concentrations of CSF and serum samples were measured by immunonephelometry and used to calculate the CSF/serum quotients for albumin (QAlb) as measure for blood-CSF barrier integrity and for IgG (QIgG). Intrathecal IgG synthesis was quantified as the relative intrathecal IgG fraction (% IgG) using the CSF/serum quotient diagrams according to Reiber . CSF CXCData consistency was checked, and data screened for normal, log-normal and gamma distributions by using Kolmogornov Smirnov tests. Very often continuously distributed data deviated severely from normal distributions and therefore generalized linear models with Tweedie distributions were used to compare groups. Additionally, ANOVA models were used to compare normal distributed variables. The logarithm was used as link function. Chi-Squared Test  was used for group comparisons (ME vs NIMM vs NIND). Spearman’s Rank-Order correlation was used to test the relation of CXCL13 levels with routine CSF parameters and immune phenotyping results. Data of CXCL13 ELISA below the lower limit were arbitrarily set to half the limit and values above the upper limit of quantification were set to twice the limit in analogy to Markowicz et al. . All repThis study included 57 patients  with a median age of 52 years (range 20–88) categorized according to discharge diagnosis as pyogenic & aseptic ME n = 29; 51%), NIMM n = 22; 39%), and NIND . Groups did not differ with regard to age and gender distribution. Table %, NIMM nExpectedly, the two inflammatory subgroups showed higher CSF cell-counts p < 0.0001, Fig. , Fig. 1AComparing the two inflammatory subgroups showed higher CSF cell counts p < 0.0001), QAlb (p = 0.005), and CXCL13 levels (p = 0.002) in ME compared to NIMM patients, and corresponding to higher cell counts, higher absolute numbers of all five investigated lymphocyte subpopulations and subsets  but not with total CD4 + and CD8+ T cells. The strongest association was between CXCL13 concentrations and CXCR5+CD4 T cell frequencies .CXCL13 levels furthermore correlated with QAlb in the total cohort  and ME  and NIMM  subgroups, but not with intrathecal IgG synthesis Table .We next certainly were interested whether intrathecal CXCL13 also correlated with the CSF-to-peripheral blood (PB)-ratio of CXCR5 + T and B cell frequencies to support a possible function in recruitment to the CNS. Corroborating the above findings, we found an association of intrathecal CXCL13 concentrations with the CSF/PB CXCR5+CD4 T cell ratio for the total cohort  due to Morbus Wegener , AIE, NMO, and follicular B cell lymphoma. We had to exclude them from analyzing the CSF/blood ratio, because peripheral blood B cell frequencies > 50% would have resulted in misleadingly low ratios and dividing by 0% is not possible.Based on manifold evidence linking intrathecal CXCL13 elevations with CSF pleocytosis, B cells, plasma cells, and intrathecal immunoglobulin synthesis in neuroinflammation , 27, 30,In fact, our data revealed increased presence of CXCR5+CD4 T cells along with intrathecal CXCL13 elevations in a broad spectrum of inflammatory CNS diseases. Intrathecal CXCR5+CD4 T cell frequencies, absolute numbers of CXCR5+CD4 T cells, and the CSF/PB ratio of CXCR5+CD4 T cell frequencies positively associated to higher CXCL13 levels in CSF, highlighting their preferential recruitment to the inflamed CSF.Subgroup analysis showed that the positive association of CXCL13 elevations with CXCR5+CD4 T cell frequencies applied in both, ME and NIMM patients and was independent of LNB. The reason why we excluded LNB cases was to ensure that high CXCL13 levels, which are typical for LNB, did not bias results. This sub-analysis accordingly underlined a broader function of CXCL13/CXCR5 signaling in meningoencephalitis and corroborated earlier observations linking persistent CXCL13 elevations with severe disease courses in bacterial and viral CNS infections .Following the argumentation of others regarding the proposed role of intrathecal CXCL13 in the recruitment of B cells , our datThe importance can be seen all the more as we know that B cells not only migrate from blood to CSF but also expand, mature and differentiate within the CNS –35. HoweAs to the question how B cells may expand, mature and differentiate within the CNS, we propose CXCR5+CD4 T cells as missing link. These so-called “circulating counterparts” of professional B helper Tfh cells, might also be relevant intrathecally as helpers initiating and maintaining antibody-related B cell activities. Our data are no proof but support our thesis of such interactions taking place in the CNS. Latest research in MS using a single cell transcriptomics and gene set enrichment analysis approach came to a similar conclusion. Schafflick and colleagues identified a compartmentalized increase of Tfh cells in CSF, which they proposed to drive the expansion of B lineage cells .Another interesting conclusion touching intrathecal cTfh cells is that of Enose-Akahata and colleagues who characterized the CSF B cell phenotype of human T-lymphotropic virus 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients . They reLimitations of our study are the small patient number, retrospective study-design, and the resultant heterogeneous patient population regarding underlying diseases, causative pathogens, disease stage, disease course, comorbidities, and medication. On the other hand, one might rate the same limitations as strength, since the link between intrathecal CXCL13 levels and CXCR5+CD4 T cells was evident despite small group sizes, patient heterogeneities, and possibly medication-associated decreases of CSF CXCL13 concentrations in response to anti-inflammatory , 45, B cSumming up, we found a clear link between intrathecal CXCL13 elevations and CXCR5+CD4 T cells in particular in meningoencephalitis patients and CXCR5+CD4 T cell correlating with B cell frequencies in neuroimmunological disorders. The sample size is small, but the observation biologically meaningful. In light of their potential B cell helper capacities, the finding suggests intrathecal CXCL13 elevations facilitating compartmentalized proximity and possible productive T cell:B cell interactions in a broad spectrum of inflammatory CNS diseases. We thus propose investigating the intrathecal CXCL13/CXCR5-chemokine axis and CXCR5+CD4 T cell repertoire key to enhance our understanding about B cell activities and intrathecal antibody production for immune defense in CNS infection and immune pathogenic processes leading to chronic inflammation and autoimmune CNS disorders.Additional file 1.  Detailed information about the flow cytometry protocols for processing of CSF and EDTA whole blood and illustration.


The COVID-19 pandemic created significant disruptions to kidney transplantation in the United States. Particularly in the initial surge, there was a dramatic decrease in deceased donor transplants, a near complete cessation in living donor kidney transplants, and considerable mortality among kidney transplant candidates.P < 0.001; The overall number of deceased donors dropped dramatically during the initial COVID-19 surge  from a mean (SD) of 252 (±15) donors weekly presurge to a low of 156 donors the week beginning March 25, 2020 . From anMore kidneys were discarded from donors who were older and female, and donors with higher terminal creatinine and a history of diabetes, hypertension, or smoking history, but kidneys from donors with a history of drug use and Public Health Service–Increased Risk status were more frequently transplanted . More thn = 43, 20%) or reduced  the number of deceased donor kidney transplants being performed. Although some transplant centers  maintained relatively stable transplant activity (within ±25% of their presurge transplant volume), the remaining centers , at least one in each of the OPTN regions, increased their deceased donor transplant activity during the surge (P < 0.0001), from a median of 47 (interquartile range: 26–69) to a low of 33 (interquartile range: 18–55) in the week beginning April 8, 2020. Centers that decreased or had no change in their transplant volume had significantly lower KDPI during the surge , whereas centers that increased their transplant volume did not have a significant decrease in the KDPI of organs transplanted  followed by a partial recovery afterward but remains significantly below prepandemic levels  in the United States, most transplant centers either stopped (he surge . HoweverP < 0.001) but returned to presurge levels by the end of 2020 , there was a significant and sustained increase in the proportion and absolute number of pre-emptive deceased donor transplants until the last quarter of 2020 compared with pre-surge  but was significantly lower in the final quarter of 2020  compared with presurge patterns (29%).Along with the increased selectivity of kidneys transplanted during the initial surge, centers also transplanted patients with better estimated post-transplant survival scores (EPTS). The median EPTS prior to the surge was 52 (interquartile range: 23–79), and it dropped to 47 (interquartile range: 19–76) during the surge ( of 2020 d. AlthouOur data highlight the pandemic’s effects on deceased donor kidney utilization and transplantation practices in the United States in 2020. Although transplant activity decreased dramatically during the spring surge in the United States, some centers maintained or increased their deceased donor transplant activity, with at least 1 transplant center in each OPTN region increasing its transplant volume. The decrease in the KDPI of transplanted kidneys and the EPTS of recipients is evidence of increased selectivity of both organs and recipients during the pandemic, especially for centers that decreased their transplant activities, underscoring the potentially subjective nature of some allocation decisions and the ability of centers to make informed choices. Although this abrupt change of transplant center behaviors suggests wide recognition of the need for caution early in the pandemic, it is notable that the median KDPI has not yet returned to prepandemic levels and pre-emptive transplants are still trending up as of the end of the year.,,Although the discard rate was high throughout the surge period, the discard rate was lower post surge, and the annual discard rate was 21%, suggesting that improved use rates later in the year balanced out the higher numbers of weekly discards earlier in the year. The donor characteristics of the discarded and nonprocured kidneys remained consistent with previous years, but the most common reason noted for discard changed from “biopsy findings” to “no recipient located” in 2020.


All consecutive patients from whom a CSF and a serum sample had been collected between August 2013 and June 2016 were eligible for inclusion. Patients suspected of LNB were classified as definite, possible, or non-LNB according to the guidelines of the European Federation of Neurological Societies (EFNS). Due to the limited number of LNB patients in the predefined study period, additional LNB patients were included from outside this period. In total, 156 patients  were included. Seven (4.5%) were classified as definite, eight (5.1%) as possible, and 141 (90.4%) as non-LNB patients. Receiver operating characteristic (ROC) curve analysis comparing definite-LNB patients with non-LNB patients showed a cutoff value of 85.9 pg/ml for the Quantikine CXCL13 ELISA and 252.2 pg/ml for the recomBead CXCL13 assay. The corresponding sensitivity was 100%  for both, and the corresponding specificities were 98.6%  for the CXCL13 ELISA and 97.2%  for the recomBead CXCL13 assay. This study showed that CXCL13 in CSF can be of additional value for the diagnosis of LNB.Recent studies have shown elevated levels of the B-cell chemokine (C-X-C motif) ligand 13 (CXCL13) in the cerebrospinal fluid (CSF) of patients with early Lyme neuroborreliosis (LNB). In this retrospective study, we evaluated the diagnostic performance of the Quantikine CXCL13 enzyme-linked immunosorbent assay (ELISA)  and the  Borrelia burgdorferisensu lato group. The most frequent manifestation of LB is a local skin rash referred to as erythema migrans , occurs in about 2 to 13% of LB cases (3Borrelia bacterium by PCR or culture is not very sensitive (–Borrelia-specific antibodies together with the presence of pleocytosis (≥5 leucocytes/μl) in the cerebrospinal fluid (CSF)   is a tick-borne disease caused by spirochetes of the  migrans , 2. WhenB cases 36. In theB cases 3. Patientensitive –9. Insteid (CSF) . The int6 weeks) , 12. Secreatment , 13. In ffective , 15.Borrelia-specific antibodies in the CSF  ligand 13 (CXCL13) in CSF samples of LNB patients were detected by using a protein expression array  and an e the CSF . Elevate the CSF , 23. Sin species , even th species , 24–29 aes 24–25––32, 54 cs 24–25––, 28, 33,s 24–25––, 34. As recomBead CXCL13 assay , which makes use of the innovative Luminex xMAP technology.Several commercial assays for the detection of CXCL13 are available, and various studies have been performed to investigate the usefulness of CXCL13 in CSF as a marker for the diagnosis of LNB. One of the assays studied the most is the human B-lymphocyte chemoattractant/B cell-attracting chemokine 1 (BLC/BCA-1) immunoassay . The instruction manual for this assay, however, does not mention the use of CSF for the detection of CXCL13 and consequently lacks a cutoff value for CXCL13 in the CSF. Various studies have shown the potential of the detection of CXCL13 in the CSF for the diagnosis of LNB; however, a broad range of cutoff values was found, ranging from 18 pg/ml to 1,229 pg/ml , 35, 36.Borrelia-specific antibodies. If all criteria were met, then the patient was classified as a definite-LNB patient; if two criteria were met, then the patient was classified as a possible-LNB patient. In all other cases, the patient was classified as a non-LNB patient. As the samples that were collected within the framework of this study were also used for another study that evaluated various commercial LNB assays , the CSF and serum sample pairs were only included if at least 1,250 μl of CSF and 110 μl of serum were available  had been sent to the microbiology laboratory of the Diakonessenhuis Hospital, Utrecht, the Netherlands, between August 2013 and June 2016 were selected. The patients were divided into three groups based on the criteria for LNB as defined by the EFNS . These cvailable . Patientvailable , 40. ForDue to the relatively low number of LNB patients with sufficient material in the predefined study period, additional LNB patients (definite or possible LNB) were chosen from outside the study period (from February 2011 to July 2013 and from July 2016 to November 2017). The LNB patients who were eligible had taken part in two other studies of our research group , 42, andAll samples used in this study were anonymized. According to the rules of our hospital, our study was exempt from approval of the local Ethics Committee because the main goal was to validate a new assay for the diagnosis of LNB for which leftover material could be used. We did, however, obtain approval from the hospital board to conduct this study.Borrelia-specific total immunoglobulin (IgM and IgG) in serum was done by using the C6 enzyme-linked immunosorbent assay (ELISA) , and equivocal and positive C6 ELISA results were confirmed by using the recomLine IgM and IgG immunoblot test (Mikrogen Diagnostik GmbH) as described previously  as described previously (Borrelia-specific IgM (or IgG), CSF and serum of a CSF-serum pair were tested simultaneously in the same run. The IDEIA LNB assay is based on the capture ELISA principle and determines the fraction of Borrelia-specific IgM (or IgG) as part of the total amount of IgM (or IgG) in CSF and serum. If the fraction of Borrelia-specific IgM (or IgG) in the CSF exceeds the fraction in serum, then intrathecal synthesis of Borrelia-specific IgM (or IgG) is proven. Consequently, a correction for a dysfunctional blood-CSF barrier was not needed.The detection of eviously . The deteviously . To deteBorrelia species PCR was used, for which the 23S rRNA gene was used as a target. A total of 190 μl of the CSF sample was processed together with an internal control consisting of Synechococcus DNA  by using the MagnaPure LC total-nucleic acid, high-performance isolation kit  on a MagnaPure LC instrument (Roche Diagnostics). The amount of internal control used had to result in a threshold cycle value in the diagnostic range, and 10 μl was sufficient, which was established empirically. The isolated DNA was eluted in 100 μl of elution buffer (Roche Diagnostics). The real-time PCR contained 12.5 μl TaqMan universal PCR mastermix , 10 μl of eluted DNA, and 2.5 μl of a primer and probe mix. The primer and probe mix consisted of a 300 nM concentration of the forward primer (5′-GGGCGATTTAGTTAGATGTGGTAGA-3′), a 900 nM concentration of the reverse primer (5′-CAAGCTTCAGCCTGGCCATA-3′), and a 200 nM concentration of the probe (5′-6-carboxyfluorescein [FAM]-AASCCGAGTGATCTAT-3′ minor groove binder [MGB] nonfluorescent quencher). The Borrelia species PCR was performed in a 96-well plate (ThermoFisher Scientific) on an ABI 7500 system (ThermoFisher Scientific). The program consisted of 1 cycle of 2 min at 50°C, 1 cycle of 15 min at 95°C, and 45 cycles that consisted of 15 s at 95°C followed by 1 min at 60°C. A positive control  and a negative control  were included in each experiment to control for amplification and contamination.An in-house real-time y axis and the concentration on the x axis as a log/log graph in the Dynex DS2 automated ELISA instrument (Dynex Technologies). Subsequently, the OD values from the CSF samples were interpreted by using this standard curve. Concentrations below the lower detection limit of the standard curve (<7.81 pg/ml) were assigned a value of 7.81 pg/ml, and concentrations above the upper detection limit of the standard curve (>500 pg/ml) were assigned a value of 500 pg/ml.CSF samples of all patients were tested by using two CXCL13 assays according to the manufacturer’s instructions. The first assay was the Quantikine CXCL13 ELISA , hereinafter referred to as the CXCL13 ELISA, which was performed on a Dynex DS2 automated ELISA instrument  and analyzed with the DS-Matrix software (Dynex Technologies). The manufacturer of this CXCL13 ELISA recommends the assay for use in the quantitative measurement of CXCL13 in cell culture supernatants, serum, plasma, and saliva but does not mention its use for CSF . The finrecomBead CXCL13 assay (Mikrogen Diagnostik GmbH). CXCL13 concentrations (in pg/ml) were measured by using the Luminex xMAP technology. Bead analysis was done on a Bio-Plex 200 instrument  by using the Bio-Plex Manager software, version 6.1 (Bio-Rad Laboratories). The final CXCL13 concentration of a CSF sample was calculated by using a calibrator, together with the batch-dependent 4-parameter logistic (4-PL) coordinates of a standard curve by using the recomQuant evaluation software version 4.9.5 (Mikrogen Diagnostik GmbH). The measurement range is located between 9.00 and 1,000 pg/ml. CXCL13 concentrations of less than 9.00 pg/ml were assigned a value of 9.00 pg/ml, and CXCL13 concentrations of more than 1,000 pg/ml were assigned a value of 1,000 pg/ml. According to the instruction manual, CSF CXCL13 concentrations of less than 190 pg/ml are normal, between 190 and 300 pg/ml are borderline, and more than 300 pg/ml are elevated, for which an active LNB infection is suspected if symptoms match  Pearson’s chi-squared test, and quantitative data were analyzed by using the exact Wilcoxon-Mann-Whitney test. Results are shown as the (geometric) mean value with the 95% CI or as the median value with the range. For construction of the dot plots, GraphPad Prism  was used.For all statistical analyses, Rstudio  was used . The dat2 + (1 − specificity)2]. CSF CXCL13 levels above the optimal cutoff value in each assay were classified as positive. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) with 95% CIs for each assay were calculated by using the optimal cutoff value. The performances of both assays were compared by comparison of the ROC curves using the clinfun package and 106 permutations  curve was constructed for each assay separately and used to calculate the area under the curve (AUC) by using the pROC package . Therefoutations .P values of <0.050 were statistically significant; however, they were interpreted after correction for the multiple statistical analyses in this study, for which the Benjamini-Hochberg procedure (BH) was applied  or 28 (n = 3) days, following the Dutch guidelines for LB (Seven (4.5%) of the 156 patients fulfilled the three criteria for definite LNB set by the EFNS  Table 1Table 1. s for LB ; the treBorrelia species PCR result , cranial neuropathy , or peripheral neuropathy . All of the possible-LNB patients were treated for LNB according to the Dutch guidelines for LB (n = 5) or 28 (n = 1) days. One possible-LNB patient had started with intravenous ceftriaxone (2 g/day) but after 5 days switched to oral doxycycline (100 mg twice a day) for 25 days because of an allergic reaction. The remaining possible-LNB patient received oral doxycycline (100 mg twice a day) from the start for 30 days (data not shown).Eight (5.1%) of the 156 patients fulfilled two of the three EFNS criteria for definite LNB  and, thuR result . LNB-spes for LB ; the treBorrelia species PCR result (n = 1), Streptococcus meningitis (n = 1), tuberculous meningitis (n = 1), and viral meningitis (n = 2). Other diagnoses that were found at least twice among the non-LNB patients included peripheral neuropathy (n = 22), demyelinating conditions (n = 17) that included seven cases of multiple sclerosis, radiculopathy (n = 9), non-CSF infectious disease (n = 8), spinal stenosis (n = 7), (transient) facial nerve paralysis (n = 6), nonspecific tendon-myogenic pain (n = 4), cerebrovascular accident (n = 3), microvascular white matter lesions (n = 3), headache/migraine (n = 3), cancer (n = 3), residual complaints after a previous CSF infection , epilepsy (n = 2), sleep disorder (n = 2), psychogenic disorder (n = 2), and arthralgia (n = 2). Eighteen non-LNB patients had a diagnosis that was found to be unique in this study population; for 22 non-LNB patients, a diagnosis was never established (data not shown).One hundred forty-one (90.4%) of the 156 patients were classified as non-LNB patients, as they did not fulfil at least two of the three EFNS criteria for definite LNB  10). No. No10). R result . The fivrecomBead CXCL13 assay were higher than those measured by using the CXCL13 ELISA. Both assays, however, showed similar distributions between the three EFNS groups . The median CSF CXCL13 concentration among the non-LNB patients was 7.81 pg/ml , and this was significantly lower  than among both the definite-LNB and the possible-LNB patients   than among both the definite-LNB and possible-LNB patients (The CSF CXCL13 concentrations among definite-LNB patients measured by using the 00 pg/ml . Among p > 5.0%) . The medpatients .recomBead CXCL13 assay, ROC curves were created by comparison of the CXCL13 concentrations among cases  with those among controls . For both assays, the optimal cutoff value was assessed by using the point on the ROC curve for which the distance to the upper left corner, where both sensitivity and specificity are 100%, was shortest. For the CXCL13 ELISA, the optimal cutoff value was determined to be 85.9 pg/ml, with a sensitivity of 100% and a specificity of 98.6%. For the recomBead CXCL13 assay, the optimal cutoff value was determined to be 252.2 pg/ml, with a sensitivity of 100.0% and a specificity of 97.2%  was found between the diagnostic performance of the two assays  definite-LNB patients, three (37.5%) possible-LNB patients, and two (1.4%) non-LNB patients. Five (71.4%) of the seven definite-LNB patients had CSF CXCL13 concentrations above the upper detection limits in both assays. The duration of their symptoms ranged from 21 to 108 days. In contrast, the two definite-LNB patients with shorter durations of symptoms had lower CXCL13 concentrations (both within the detection limits in the two assays). One of them, who had CSF CXCL13 concentrations of 138 pg/ml in the CXCL13 ELISA and 260 pg/ml in the recomBead CXCL13 assay, had had symptoms for 3 days. The other had had symptoms for 14 days and had CSF CXCL13 concentrations of 196 pg/ml in the CXCL13 ELISA and 421 pg/ml in the recomBead assay.Twelve (7.7%) of the 156 patients included in this study had a positive CSF CXCL13 result in both assays, which included all (recomBead CXCL13 assay. This patient had a shorter duration of symptoms (22 days) than the other two patients .The three possible-LNB patients with positive CSF CXCL13 results in both assays all had pleocytosis. For two of them, the CSF CXCL13 concentrations in both assays exceeded the upper detection limits of the assays. The third possible-LNB patient had CSF CXCL13 concentrations of 358 pg/ml in the CXCL13 ELISA and 654 pg/ml in the recomBead CXCL13 assay. The patient with neurosyphilis had CSF CXCL13 concentrations of 500 pg/ml in the CXCL13 ELISA and 982 pg/ml in the recomBead CXCL13 assay. Three (2.1%) of the 141 non-LNB patients had a positive CSF CXCL13 result in the recomBead CXCL13 assay only  than the levels in the other three groups. Nine (5.8%) of the 156 patients were classified in this group, as they had a dysfunctional blood-CSF barrier and intrathecally produced total antibodies. Six of these nine patients had intrathecal total-antibody synthesis of both the IgM and the IgG class (Table S2). Five of them were diagnosed with definite LNB, and the other patient was the one diagnosed with neurosyphilis. The remaining three patients were diagnosed with possible LNB, and all of them had intrathecal total-antibody synthesis of the IgM class only. Fifteen (9.6%) of the 156 patients had a normal blood-CSF barrier and proof of intrathecal total-antibody synthesis; however, none of them had elevated CSF CXCL13 levels in either of the CXCL13 assays (Reibergram group 4)  patients had a normal blood-CSF barrier in the absence of intrathecal total-antibody synthesis and, thus, were classified in Reibergram group 1 . Only ongroup 4) . None ofrecomBead CXCL13 assay. Significantly higher CSF CXCL13 levels were found among definite-LNB patients than among non-LNB patients . Our study results are in accordance with the results published by other research groups  or possible LNB (n = 3). These results indicate a clear association between elevated CSF CXCL13 levels, a dysfunctional blood-CSF barrier, and intrathecal total-antibody synthesis among LNB patients. The three possible-LNB patients lacked intrathecal Borrelia-specific antibody synthesis, and this emphasizes the added value of using Reibergrams in the early diagnosis of LNB   . Similar13 assay . The broBorrelia-specific serum antibodies among the non-LNB patients (22.0%) ; however, the number of freeze-thaw steps was expected to be limited. We therefore do not believe this has negatively influenced the results in this study, as CXCL13 is stable when subjected to repeated freeze-thaw cycles, as shown by Hytonen et al. (for up to a maximum of five freeze-thaw cycles) (n = 84) in that year, and the Dutch population in 2010    exceeded (22.0%) , which s cycles)  and by p cycles) . We did  cycles) . Prolong cycles) , 28, 38.es) n = 8 in that 600,000) , 64. In As discussed above, this study was based on a cross-sectional design, which can be considered a strength of this study as discussed by Rupprecht et al. and Leeflang et al. , 38. AnoBorrelia-specific antibodies.In conclusion, this retrospective, cross-sectional study confirms that determining the CSF CXCL13 levels can aid in the diagnosis of LNB. None of the definite-LNB patients had a negative CSF CXCL13 result in both assays. Furthermore, we show the added value of this marker for the group of possible-LNB patients who have pleocytosis without intrathecally produced


Sparganii rhizoma, Sargentodoxa cuneata and Patrinia scabiosifolia. Studies have revealed that acacetin potentially exerts anti-inflammatory and antioxidative effects on sepsis. In this study, we investigated the potential protective effect of acacetin on sepsis and revealed the underlying mechanisms using a network pharmacology approach coupled with experimental validation and molecular docking. First, we found that acacetin significantly suppressed pathological damage and pro-inflammatory cytokine expression in mice with LPS-induced fulminant hepatic failure and acute lung injury, and in vitro experiments further confirmed that acacetin attenuated LPS-induced M1 polarization. Then, network pharmacology screening revealed EGFR, PTGS2, SRC and ESR1 as the top four overlapping targets in a PPI network, and GO and KEGG analyses revealed the top 20 enriched biological processes and signalling pathways associated with the therapeutic effects of acacetin on sepsis. Further network pharmacological analysis indicated that gap junctions may be highly involved in the protective effects of acacetin on sepsis. Finally, molecular docking verified that acacetin bound to the active sites of the four targets predicted by network pharmacology, and in vitro experiments further confirmed that acacetin significantly inhibited the upregulation of p-src induced by LPS and attenuated LPS-induced M1 polarization through gap junctions. Taken together, our results indicate that acacetin may protect against sepsis via a mechanism involving multiple targets and pathways and that gap junctions may be highly involved in this process.Sepsis is a dysregulated systemic response to infection, and no effective treatment options are available. Acacetin is a natural flavonoid found in various plants, including  Sepsis is a severe systemic inflammatory response to microbial infection, and lipopolysaccharide (LPS) is well known to induce this phenomenon . D-GalacSparganii rhizoma, Sargentodoxa cuneata and Patrinia scabiosifolia  activity , an O-methylated flavone monomer, is a bioactive constituent found in various plants, including osifolia . Acacetiosifolia . Variousactivity  and to pactivity . Moreoveactivity . These rvia bioinformatics analyses , LPS , and Gap27 (MedChemExpress) were used in the experiments.n = 5): the control group, acacetin treatment group, LPS-induced ALI model group, LPS + acacetin treatment group, LPS/GalN-induced FHF model group and LPS/GalN + acacetin treatment group. ALI was induced by the intratracheal injection of LPS (5 mg/kg)  were purchased from the Animal Experimental Centre of the Fourth Military Medical University, China. All mice were bred in a specific pathogen-free animal facility at the Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Xi’an Jiaotong University Health Science Center. All animal experiments were approved by the Institutional Animal Ethics Committee of Xi’an Jiaotong University (no. 2017–666). All animal experiments were performed in accordance with standard animal research guidelines. C57BL/6 mice were randomly assigned to one of six groups (5 mg/kg) , and FHF5 mg/kg) . An equiThe lung and liver tissues were collected and fixed in a 4% paraformaldehyde solution for 48 h. The tissues were dehydrated, embedded in paraffin, and sliced into 5 μm-thick sections. The sections were stained with haematoxylin and eosin (H & E) reagent and visualized under a light microscope.To assess the effects of acacetin on pro-inflammatory mediators, appropriate ELISA kits  were used. The levels of the pro-inflammatory mediators TNF-α, IL-6, and IL-1β in lung and liver homogenates from LPS-induced mice were measured.2. This cell line, used to screen for molecular mechanisms, was cultured in DMEM  supplemented with 10% FBS  and 1% streptomycin/penicillin. The cells were passaged when they reached 70–80% confluence.RAW264.7 macrophages were obtained from academician Rikard Holmdahl . The cells were cultured in a thermostatic incubator at 37°C and 5% CO3 cells/well. Cells in the treatment groups were administered different amounts of acacetin  dissolved in DMSO, while those in the model group were administered LPS (100 ng/ml), and those in the control and blank (no cells) groups were administered an equivalent amount of medium before growth for an additional 24 h. Then, MTT solution was added and continuously incubated at 37°C for 4 h. The medium in each well was discarded, after which 150 μl of DMSO was added, and the cells were shocked on a microvibrator for 5 min. The optical density at 490 nm was determined with a plate reader .RAW264.7 cells were cultured in a 96-well plate at 5 × 10The experimental groups were divided into a control group, an LPS model group, and treatment groups that were administered different amounts of acacetin . Groups other than the control and LPS model groups were pre-treated with acacetin for 4 h and then stimulated with LPS (100 ng/ml) for 24 h. Flow cytometry was performed on single-cell suspensions of RAW264.7 macrophages. For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm solution . The following monoclonal antibodies were used: the M1-type surface markers CD86-PE  and iNOS-PEcy7 . Samples were measured by flow cytometry , and the data were analysed using Flow Jo software .The cell grouping, stimulation, and culture were carried out as described for the flow cytometry experiment. The cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) buffer and phenyl methyl sulfonyl fluoride (PMSF), and the protein content was confirmed by the BCA method. Equivalent amounts of protein from each sample were loaded onto 10% SDS-PAGE gels and then transferred onto PVDF membranes . After being blocked with 10% skimmed milk for 2 h, the blots were probed with the following primary antibodies: CD86 , iNOS , p65 , p-p65 , Src , p-Src , Cx43  and GAPDH . The cells were gently incubated overnight at 4 °C and subsequently incubated with the corresponding secondary antibodies diluted 1:5,000 with 5% skimmed milk. The membranes were washed with TBST, incubated with ECL reagent  and developed. Quantity One software  was used to analyse the collected images.−ΔΔCt comparative method.The cell culture and stimulation were performed as described for Western blot analysis. Only one acacetin (10 μmol) treatment group was used. After stimulation, the cells were harvested with TRIzol reagent , and total RNA was extracted and reverse transcribed into cDNA using a ReverTraAce RT kit . Real-time quantitative PCR was carried out using SYBR Green Master Mix  according to the manufacturer’s protocols. The following gene primer sequences were obtained from Shanghai Gene Pharma: CD86, 5′-ACGGAGTCAATGAAGATTTCCT-3′ (forward), 5′-GATTCGGCTTCTTGTGAC-ATAC-3′ (reverse); iNOS, 5′-GTTTACCATGAGGCTGAAATCC-3′ (forward), 5′-CCTCTTGTCTTT-GACCCAGTAG-3′ (reverse); and β-actin, 5′-CACGATGGAGGGGCCGGACTCATC-3′ (forward), 5′-TAAAGACCTCTATGCCAACACAGT-3′ (reverse). mRNA abundance was quantified using the Ct values obtained by 2http://swisstargetprediction.ch/) for drug-target prediction analysis, and the keyword “sepsis” was then searched in the Gene Cards database (https://www.genecards.org/) to identify disease targets. The overlapping targets among compound and sepsis targets were used to draw a Venn diagram.The acacetin chemical formula was imported into the Swiss Target Prediction database (https://string-db.org) with the species limited to “Homo sapiens” and a confidence score >0.9. The results were further visualized by Cytoscape 3.7.0. The degree of freedom was reflected by the node size and colour. Subsequently, the top four key targets were identified as hub targets based on their degree, centrality and proximity to hub proteins.The protein-protein interaction (PPI) network was obtained by introducing these potential overlapping targets of acacetin and sepsis into the STRING database (www.ensembl.org/biomart/), and the OmicShare server (http://www.omicshare.com/) was then used for the analysis. The converted Ensembl gene names were used as input, and Gene Ontology (GO) enrichment and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were performed. The molecular function (MF), biological process (BP) and cellular component (CC) terms enriched in the targets were determined, and pathway enrichment of the top 20 pathways was visualized with the R package.The hub targets were converted into their Ensembl gene names . The original ligand active site residues were identified from the PDB coordinates and recorded. The protein structures were prepared by removing all nonreceptor atoms, including water, ions, and miscellaneous compounds, using Discover Studio Visualizer. Docking experiments were performed using CDOCKER with receptor-ligand interactions. The -CDOCKER_INTERACTION_ENERGY value was determined to evaluate bonding. The binding results were visualized as 3D and 2D diagrams using Discovery Studio Visualization version 4.5 .The 3D crystal structures of ESR1 (PDB code: 5ACC), COX-2 , SRC (PDB code: 4U5J) and EGFR (PDB code: 6JZ0) were obtained from the Protein Data Bank (PDB). The 2D structure of acacetin was downloaded from the ZINC15 database . Multiple comparisons were performed by one-way ANOVA followed by Tukey’s post hoc significant difference test. p < 0.05), and the ALI model group showed more inflammatory areas in the portal vein and more necrotic hepatocytes (p < 0.05). Compared with the FHF and ALI model groups, the acacetin group showed less severe lung and liver injury symptoms as determined by HE staining (p<0.05). The levels of IL-1β, IL-6 and TNF-α in the liver and lung tissues of the model group were significantly higher than those in the control group tissues, while those in the acacetin group were decreased compared to those in the control group (p < 0.05). These results indicated that acacetin improves liver and lung injury by decreasing the levels of IL-1β, IL-6 and TNF-α.To assess the protective effect of acacetin against sepsis, we utilized LPS-induced FHF and ALI models. Compared with the control group, the FHF model group showed congestion, oedema, inflammatory cell infiltration, and a broadened pulmonary interval . These phenomena revealed that acacetin helped to quickly reduce the CD86 and iNOS levels in M1-type macrophages.Macrophage polarization is involved in the pathogenesis of sepsis. To assess the protective effect of acacetin on sepsis and the underlying mechanism The canonical SMILES string for acacetin was input into the Swiss Target Prediction database to identify the targets of acacetin, and a total of 99 potential drug targets were found. There were 2,497 potential targets of sepsis in the GeneCards database. As shown in p <0.05).Molecular docking was carried out to investigate potential binding modes and interactions between acacetin and its key targets . For thep<0.05). Together, these results suggest a prospective role for gap junctions in attenuating the effect of acacetin on M1-type macrophages.Further network pharmacological analysis and GO and KEGG analyses indicated that gap junctions may be highly involved in the protective effects of acacetin against sepsis. We confirmed the role of gap junctions in the effects of acacetin on LPS-induced M1 polarization in RAW264.7 cells. As shown in Sparganii rhizoma, Sargentodoxa cuneata and Patrinia scabiosifolia. Our results indicated that acacetin improved liver and lung injury by decreasing the levels of IL-1β, IL-6 and TNF-α, and acacetin expedited the reduction in CD86 and iNOS expression in M1-type macrophages. Previous reports have indicated that acacetin has anti-inflammatory effects. For example, acacetin was shown to suppress the LPS-induced upregulation of iNOS and COX-2 in murine macrophages and TPA-induced tumour promotion in mice (Sepsis is a global healthcare issue and continues to be the leading cause of death from infection. In clinical practice, the use of antimicrobial agents to fight infection is key to the treatment of sepsis. Over the past few decades, natural products, such as anti-bacterial and anti-inflammatory drugs, have been used for the herbal treatment and prevention of human diseases . Acaceti in mice . Acaceti in mice  and to b in mice . These rShenFuHuang formulation in a zebrafish model, was associated with septic syndrome in patients with COVID-19 (in vitro. Our western blot results suggested that phosphorylated Src plays an important role in the anti-septic activity of acacetin. Together, our results showed that acacetin had a preferential affinity for all four targets, confirming the prediction results obtained by network pharmacology analysis.We herein further investigated the mechanisms underlying the effects of acacetin on sepsis by performing network pharmacology analysis coupled with experimental validation and molecular docking. All potential targets of acacetin and sepsis were methodically obtained, and the top four overlapping targets in the PPI network were EGFR, PTGS2, SRC and ESR1. EGFR prevents the translocation of gut-residing pathogenic and cancer-associated protein kinases and is a key target for late-onset neonatal sepsis and cancer . Our resCOVID-19 . Our resCOVID-19 . With reGO and KEGG analyses were performed to analyse the interactions among multiple targets and pathways, and the top 20 enriched BPs and signalling pathways were shown to be associated with the therapeutic effects of acacetin on sepsis. Membrane raft, oestrogen receptor activity, and human cytomegalovirus infection are related to the effects of acacetin on sepsis. In a previous study, lipid raft microdomains were shown to be essential components of phagolysosomal macrophage membranes and to play an essential role in antifungal immunity . Moreovevia a mechanism involving multiple targets and pathways and that gap junctions are highly involved in this process.Our results suggest that acacetin protects against sepsis