In this review, we discuss recipient risk assessment for allo-HCT regarding comorbidities present at baseline to predict non relapse mortality. We further reviewed the incorporation of remission status and cytogenetic risk prior to allograft transplantation to predict relapse rates for hematologic malignancies. HCT-CI and DRI are tools available to physicians to assess the risk–benefit of allo-HCT in patients referred for transplantation. Next, we discuss our algorithm for donor selection and criteria for donor selection in case matched donors are not available. Finally, we discuss our approach for stem cell mobilization, especially in donors failing G-CSF, and our approach for the use of plerixafor and data supporting its use. Allo-HCThe Center for International Blood and Marrow Transplant Research (CIBMTR) is responsible for data collection on US and international patients undergoing allogeneic hematopoietic cell transplantation (Allo-HCT) as part of the Stem Cell Therapeutic and Research Act, which was first established in 2005. The CIBMTR database remains the most reliable source for evaluating the trends and outcomes in allo-HCTs. Based on the most recent data available for the reporting period ending in 2021, 8,000 allogenic stem cell transplants were performed in the United States. The majority of allo-HCTs (43%) were performed using a matched unrelated donor (MUD), which now exceeds the number of matched related-donor transplants (22%). Demographic trends show increasing utilization of haploidentical donors (24%), which were used in approximately 2,000 allo-HCTs, slightly more than the total number of matched sibling donor (MSD) transplants (22%) performed in the last reporting period of 2021 Figure 1Among adult patients with AML receiving MUD transplants between 2009 and 2019, the 3-year probabilities (95% CI) of survival following transplantation with a disease status of CR1, CR2+ (2nd or subsequent complete remission), and telapsed disease/never in CR (including primary induction failure) were 56% (55%–57%), 54% (52%–57%), and 31% (30%–33%), respectively.Approximately 800 allo-HCTs are performed annually in the City of Hope, and AML is the leading indication for allo-HCT. Our standard MAC regimen used was FTBI with cyclophosphamide or etoposide. Using this regimen, the 6-year OS rate was 60% and the non-relapse mortality rate was 15%. Chronic GVHD rates were relatively high at 70%, resulting in a 1-year GRFS rate of 45%. For older patients not eligible for intensive conditioning, Flu/Mel-based RIC is the preferred regimen. Our standard GVHD prophylaxis uses tacrolimus and sirolimus (T/S), based on promising studies performed initially at the Dana Farber Cancer Institute 96 and further phase 2 studies at COH. In the City of Hope, we were the first to use the Flu/Mel regimen in combination with T/S prophylaxis initially in the MRD setting. The aGVHD rates (gd 2–4) were 29% and NRM at day + 100 was 6%, confirming the efficacy and safety of T/S prophylaxis with Flu/Mel in a matched related donor (MRD) setting  used in combination with systemic chemotherapy and matched donors. Using this regimen, we observed 1-year overall survival rates greater than 55% in high-risk populations , 11. FurComorbidities are any distinct or additional clinical condition that may occur during the clinical course of a patient with a primary disease . The exiThe predictive value of HCT CI scores was confirmed in an independent cohort of patients , and the tool correctly predicted 2-year NRM and overall survival in patients Table 2;http://www.hctci.org/.The HCT-CI score was subsequently validated at two large transplant centers in acute myeloid leukemia patients undergoing allogeneic transplantation in the first complete remission. On multivariate analysis, a high HCI-CI score was associated with the highest hazard ratios impacting NRM and overall survival among cohorts across both centers. This tool is now routinely used to assess comorbidities in patients before allogeneic stem cell transplantation. Online tools are now available for easy calculation of HCT-CI: To standardize patient assessment and data acquisition for calculation of HCT-CI, developers of this tool recommend a systematic three-step process that takes approximately 15 min  Figure, age indThe disease risk index (DRI) for patients undergoing allo-HCT was first developed by investigators at the Dana-Farber Cancer Institute to account for variations in transplant outcomes based on disease-related variables, such as remission status and prognostic factors for hematologic malignancies  preceding transplantation , 30. DifLow risk disease: AML with favorable cytogenetics, CLL, CML, Hodgkin’s lymphoma, and non-Hodgkin lymphoma1. Intermediate risk disease: ALL, AML or MDS with intermediate cytogenetics, myeloproliferative neoplasms, and multiple myeloma2. High-risk disease: AML or MDS with adverse cytogenetics, and extranodal T-cell lymphomas.3. 1. Low risk by stage: CR 1, CR greater than 1(for MAC), PR 1, untreated disease, CML chronic phase.2. High risk by stage: CR greater than 1(RIC), PR greater than 1, induction failure or active relapse, CML with accelerated or blast phase.In this study, investigators found that HCT-CI was also independent prognostic factor for overall survival , suggesting that both should be used for the determination of clinical outcomes in patients undergoing a transplant consultation. This finding is consistent with other studies that have shown that patients with high-risk diseases have greater NRM , 32. IncThe DRI was subsequently validated in a larger study of 13,000 patients who underwent allo-HCT between 2008 and 2010 and whose outcome data were reported to the CIBMTR. The DRI stratified patients into four groups with 2-year overall survival ranging from 64% to 24%. DRI was the strongest prognostic indicator for 2-year overall survival irrespective of age, conditioning regimen, graft source, or donor type . The DRIhttps://cibmtr.org/CIBMTR/Resources/Research-Tools-Calculators/Disease-Risk-Index-DRI-Assignment-ToolDespite its widespread adoption, further refinement based on additional information regarding risk stratification provided by molecular mutations in AML  and miniThe European Group for Blood and Marrow Transplantation (EBMT) also developed a risk-scoring model for patients undergoing allo-HCT initially for CML, which was a common indication for allo-HCT when the score was first developed. Using patient- and disease-related features such as stage of CML, age/sex of donor/recipient and histocompatibility match, and time from diagnosis to allo-HCT, a scoring system was developed that accurately predicted clinical outcomes such as OS/LFS/RFS and NRM in CML patients . This scwith DNMT3A, FLT3-ITD, TET2, and PTPN11. AML with mutated chromatin, RNA splicing genes, or both were detected in 15% of AML patients and frequently co-mutated genes were RUNX1, SRSF2, MLL, DNMT3A, ASXL1, STAG2, NRAS, TET2, and FLT3-ITD. AML with TP53 mutations was observed in 13% of the patients and was associated with a complex karyotype, monosomy 5, monosomy 7, 17p deletion, trisomy 8, and 12p deletion. Other subgroups were as follows: AML with inversion 16 or t (CBFB-MYH11), AML with allelic CEBPA mutations, AML with t(15:17) PML-RARA, AML with t RUNX1-RUNX1T1, AML with MLL fusion genes, AML with inversion 3 (GATA2) or t MECOM, AML with IDH-2 mutations, and AML with t DEK-NUP214. This genetic calcification in females has prognostic clinical applications as patients with driver mutations in the chromatin-spliceosome group were older, presented with lower WBC counts and blast counts, and had lower response rates of induction chemotherapy, high relapse rate, and poor long-term clinical outcomes similar to patients in adverse risk groups. The most recent European Leukemia Net AML guidelines published in 2022 incorporated somatic mutations to describe AML/MDS with recurrent genetic abnormalities, taking precedence over other cytogenetically defined categories  in causing hematologic malignancies.Papaemmanuil et al. enrolled 1,540 patients from three prospective trials of AML patients between the ages of 18 to 65 years who received intensive therapy with anthracycline and cytarabine . Their gtegories . In patitegories Table 3.Patients with newly diagnosed AML should undergo extensive cytogenetic, molecular, and immunophenotyping for accurate risk assessment at diagnosis at a center with expertise in performing these sophisticated tests. This initial assessment is crucial for accurate risk assessment, initiation of induction therapy based on the presence of somatic mutations, e.g., FLT3-ITD, IDH mutations or KMT2A rearranged AML or CBF AML, and in making decisions regarding post-induction consolidation strategies. Studies have shown no adverse effects of waiting for molecular testing to complete therapy .NPM1) or fusions . An emerging area of research is next-generation sequencing (NGS)-based analysis for MRD detection. The ELN has made specific recommendations regarding MPFC-based analysis for the accurate production of MRD  and CHIP mutations  should be excluded. Similarly, mutations in signaling pathways  should be excluded, as they may represent subtotal populations of cells and have low negative predictive value. Currently, there is no validated NGS panel for MRD testing; however, efforts are ongoing to standardize these tests in a prospective trial (MEASURE). Currently, PCR-based approaches for MRD testing and AML patients are limited to approximately 40% to 60% of patients harboring targetable mutations, including NPM1, RUNX1-RUNX1T1, CBFB-MYH11, PML-RARA, KMT2A-MLLT3, DEK-NUP214, BCR-ABL, and WT1. Recent data suggest that PCR-based approaches for certain mutations (NPM-1 and FLT3-ITD) have prognostic value in relapse prediction, indicating the importance of NGS-based testing as an MRD tool  measured by specialized techniques; otherwise, it is in morphologic remission, as noted by conventional techniques. Multiple studies have shown a poor prognostic impact of pre HCT MRD positivity and allo-HCT outcomes . Acute mn of MRD . A diagnMRD tool . For the regimen , additio regimen , 47. Stu regimen  and hapl regimen  may be p regimen , 51.In conclusion, the assessment of variables that have been validated to impact HCT outcomes is crucial to help both patients and physicians better understand the relative risk benefit of HCT. This will allow treating physicians to make educated decisions regarding escalation or de-escalation of conditioning regimens based on patient-related  and disease-related factors . Accurate risk assessment may also influence the choice of GVHD prophylaxis and maintenance strategies to improve the GRFS outcomes. All available tools, such as DRI, HCT-CI, and EBMT scores, are validated in large patient cohorts for all common hematologic malignancies, and the decision to use one versus the other often depends on physician choice and institutional practice.Some of the key factors that impact transplant outcomes are the underlying hematological conditions, recipient comorbidities, and suitables donor. Selection of an appropriate donor is crucial for successful transplantation.Patients’ human leukocyte antigen (HLA) typing should be performed once a diagnosis of a hematological condition is made for which stem cell transplantation is indicated. Family history should be obtained, and if patients have full siblings (same parents), they should be typed, as this is the quickest way to identify HLA-matched donors.HLA is a major histocompatibility complex (MHC) system located on the short arm of chromosome 6 that contains the most polymorphic genetic region of the entire human genome . Each paEach individual had two HLA alleles inherited by each parent. Therefore, the probability of full siblings sharing the same haplotype was approximately 25%. HLA-matched sibling donors are the best donors for patients undergoing allo-HCT. Several studies on a variety of diseases have shown that MRD is associated with the best survival. However, the HLA 8/8 allele-matched URD had similar TRM and all-cause mortality compared to MRD. However, MRD is associated with a lower risk of acute and chronic GVHD, which is a serious and common complication of allo-HCT –56.https://wmda.info/). Recent advances include precise HLA-typing, supportive care, and immunosuppressive strategies. This can be shown by the NMDP data of one-year improvement in survival  prophylaxis. This has led to improvements in GVHD prophylaxis and the use of post-transplant cyclophosphamide (PTCy) which is now commonly used. Other strategies include CD34+ cell-selective T-cell depletion as well as other graft manipulations such as CD34+ selection and add back memory CD45RA+ T, α/β T-cell depletion .Recent advances, including T-cell depletion and PTCy-based regimens, have allowed the use of partially matched related donors as viable options for allo-HCT. HHD are related donors  that share one HLA haplotype and mismatch at other HLA haplotypes by a variable number of HLA genes. Several retrospective studies have shown comparable outcomes between haploidentical transplants and MUD transplants , 60.UCB is another alternative if there is no available MRD or MUD. It offers several advantages over the MUD. There is more room for HLA mismatch, so 1–2 mismatches, relatively quicker time to graft identification and acquisition, and no risk to the donor. However, some of the limitations are limited cell dose, no option for second collection, and donor lymphocyte infusion (DLI). In terms of transplant, a higher infectious risk is due to slow immune reconstitution, count recovery, and graft failure. The number of UCB transplants has recently declined owing to the increased use of HHD . Our appIn addition to HLA typing, the following factors are important when selecting an appropriate donor. A detailed discussion of all the factors is beyond the scope of this review; however, the important features are discussed below. The reader is referred to previously published excellent reviews for donor selection , 63.All patients undergoing donor search should undergo HLA-antibody screening to detect donor-specific anti-HLA antibodies (DSA). DSA is associated with an increased risk of graft failure and decreased survival . TherefoLarge retrospective studies have shown an improvement in survival after MUD in younger donors compared to older donors. There was a 5.5% increase in the HR for overall mortality for revery 10 years increasement in donor age. Similarly, data from large retrospective studies have shown that an increase in donor age by a decade was associated with a decrease in OS, PFS, and a higher risk of acute GVHD but not chronic GVHD . Recent CMV seropositivity is determined in both the recipient and the donor. Matching the patient/donor CMV status affects transplant outcomes. Some studies have shown that the risk of CMV reactivation is more common after allo-HCT in CMV mismatches between the donor and recipient , 69. HowCompared with solid organs, allo-HCT can be safely performed despite ABO/Rh incompatibility. However, this incompatibility may result in complications, including hemolysis, slow red cell engraftment, GVHD, and PRCA , 71. TheNatural killer (NK) cells are part of the innate immune system and play a key role in the early immune response against infections and tumors. In allo-HCT settings, donor NK cells can exert a GVL effect when they express inhibitory KIR that does not engage with recipient cell MHC class I molecules . NK cell6 CD34+ cells/kg recipient body weight  is crucial for ensuring timely and sustained engraftment following hematopoietic cell transplantation (HCT). The generally accepted minimal HSC dose is 2 × 10y weight , 75. They weight , 77, buty weight . In geneneic HCT . Howeverell dose .via apheresis. In 1986, it was shown that the administration of chemotherapy resulted in a temporary increase in the number of stem cells in peripheral blood during hematopoietic recovery (Most HSCs reside in the bone marrow (BM). However, BM harvesting of HSCs is accompanied by procedural risks and lower CD34+ cell yields . Alternarecovery . Subsequrecovery . G-CSF irecovery . G-CSF iHowever, conventional mobilization regimens are associated with mobilization failures or inability to collect enough PBSCs in 5%–40% of autologous HCT candidates  and up tPlerixafor  is a newer but valuable addition to the arsenal of mobilization agents. Mechanistically, plerixafor is synergistic with G-CSF and enhances the release of HSCs into peripheral blood by disrupting the adhesion between CXCR4 expressed on CD34+ cells and its ligand CXCL12  expressed by BM stromal cells . InitialFor optimal efficacy, plerixafor is administered subcutaneously in the evening, preferably 10–14 h prior to each apheresis collection session occurring the following morning , at a doPlerixafor has been demonstrated to be effective and safe in healthy donors when administered as the sole mobilization in a single dose shortly before collection, favorable collection outcomes have been observed in multiple studies –100. How6 CD34+ cells/kg of recipient body weight. After starting plerixafor, there was a 0.75- to 7.74-fold increase in the CD34+ yield from the previous day. The median 2.94-fold increase with plerixafor was similar to that reported in previous studies evaluating the use of plerixafor in allogeneic donors (6 CD34+ cells/kg recipient weight on day one, none of those who received G-CSF-only mobilization but no plerixafor achieved the goal of ≥4.0 × 106 CD34+ cells/kg recipient weight over 2 days, but 59.2% of donors who received rescue plerixafor did. The selection of plerixafor has allowed the vast majority of donors to collect sufficient CD34+ cells to proceed with allogeneic HCT for the recipient at our center with an adequate cell dose (At our center, over an 8-year period, 4.1% or 41 of the 1,008 allogeneic donors received one dose of plerixafor in addition to G-CCSF due to poor collection yield after one day of collection, generally <60% of the desired collection target, or <2.0–2.5 × 10c donors , 102. AmIn conclusion, the primary goal of PBSC mobilization and collection is to obtain sufficient HSCs to allow for successful and durable engraftment following HCT. This can be accomplished with the use of conventional regimens of myeloid growth factors such as G-CSF in allogeneic donors, and the judicious use of adjunct agents such as plerixafor when mobilization is suboptimal. Assessment of the risk factors for inadequate mobilization and close monitoring of the mobilization and collection progress in donors can significantly improve the collection success rate while maintaining cost-effectiveness. The criteria for using plerixafor in allogeneic donors should be developed to allow individualization in specific cases. Centers should develop their own specific criteria and triggers for plerixafor use based on evidence in the literature and their own clinical data, which should also be continuously assessed and validated using institutional data and donor collection outcomes.ex vivo graft manipulation strategies, have been approved for the treatment of established cGVHD and novel strategies for prevention. The success of allo-HCT depends on appropriate donor selection, and we discuss our strategy for the selection of sibling, unrelated, and alternative donors. Nationwide, the utilization of cord blood HCTs is declining and haplo-HCTs are increasing. Donor age is now recognized as an important variable determining the success of HCT in both matched donors and haploidentical HCTs, and this is now one of the determining factors in choosing a donor for allo-HCT. Finally, we discuss strategies for HPC collection in an allogeneic setting, focusing on the role of plerixafor in patients who mobilize poorly using G-CSF alone.Recent CIBMTR data suggest that trends in allo-HCT are changing, which may change the practice in the coming years. Increasingly, older patients are being transplanted using PBSC grafts with unrelated donors. These practice changes indicate that the incidence of acute and chronic GVHD will continue to rise, increasing the clinical burden of HCT-related complications. Fortunately, new drugs, such as PTCy, use of JAK inhibitors and AS conceived, wrote and reviewed the manuscript. SY and HA contributed to writing the manuscript. All authors contributed to the article and approved the submitted version.


Heart transplantation (HTx) represents the current best surgical treatment for patients affected by end-stage heart failure. However, with the improvement of medical and interventional therapies, the population of HTx candidates is increasingly old and at high-risk for mortality and complications. Moreover, the use of “extended donor criteria” to deal with the shortage of donors could increase the risk of worse outcomes after HTx. In this setting, the strategy of donor organ preservation could significantly affect HTx results. The most widely used technique for donor organ preservation is static cold storage in ice. New techniques that are clinically being used for donor heart preservation include static controlled hypothermia and machine perfusion (MP) systems. Controlled hypothermia allows for a monitored cold storage between 4°C and 8°C. This simple technique seems to better preserve the donor heart when compared to ice, probably avoiding tissue injury due to sub-zero °C temperatures. MP platforms are divided in normothermic and hypothermic, and continuously perfuse the donor heart, reducing ischemic time, a well-known independent risk factor for mortality after HTx. Also, normothermic MP permits to evaluate marginal donor grafts, and could represent a safe and effective technique to expand the available donor pool. However, despite the increasing number of donor hearts preserved with these new approaches, whether these techniques could be considered superior to traditional CS still represents a matter of debate. The aim of this review is to summarize and critically assess the available clinical data on donor heart preservation strategies employed for HTx. However, despite improvement in the management of HTx recipients, the rate of primary graft dysfunction (PGD) continues to be relatively high, being a severe complication that still represents the leading cause of 30-day mortality after HTx –3. A recContinuous improvements of medical and interventional therapies, as well as the wide employment of mechanical circulatory support supports (MCS), have allowed an increasingly old population with multiple comorbidities to be considered as HTx candidates. On the other hand, the use of extended donor criteria to face graft shortage has increased the risk of worse outcomes after HTx. Therefore, in this setting, the strategy of donor organ preservation could play a central role in improving HTx recipient outcomes and in preventing PGD.The use of static cold storage (SCS) for donor graft preservation, aims to stabilize biological tissues by influencing metabolic pathways. Such strategy slows the cellular and extracellular biochemical processes that are responsible for organ degradation during ischemic storage, thus extending a safe storage time up to several hours. On the other hand, machine perfusion (MP) systems permit to continuously perfuse the coronary arteries, reducing ischemic time and potentially mitigate the deleterious effects of ischemia/reperfusion injury. Despite the increasing number of donor hearts preserved with MP, whether MP could be considered superior to traditional CS still represents a matter of debate.The aim of this paper is to summarize and critically assess the available clinical data on the donor heart preservation strategies currently employed for HTx.2.Employment of SCS aims to stabilize biological tissues by slowing the cellular and extracellular biochemical processes that are responsible for organ degradation during ischemic storage, thus extending the safe storage time. The temperature dependence of chemical reaction rates follows the “Arrhenius equation”, used to describe the temperature-depending metabolic changes: for every 10°C reduction of temperature below the physiological temperature the metabolic rate for living biological tissues reduces by 50%. Hence, cold storage slows but does not completely arrest cellular metabolism. Consequently, progressive ischemic injury is an inevitable consequence of prolonged SCS and the results of HTx are suboptimal when graft ischemic time is greater than 6 h.2.1.The traditional ice-cold SCS remains the most commonly used technique for donor graft preservation, being both user friendly and cost-effective. In brief, after the donor heart is retrieved, it is placed into a sterile bag filled with 1,000 ml of preservation saline solution at 4°C which is then sealed into a second bag containing 1,000 ml of cold solution, and eventually in a third bag. Then, the heart is placed in a rigid sterile container filled with cold solution which is sealed and inserted into a cooler filled with ice for transportation.Using conventional ice-cold SCS, prolonged ischemic time is known to be an independent risk factor for PGD and mortality after HTx –7. MoreoAs reported by the ISHLT Consensus Statement on donor heart and lung procurement , the ide2.2.The Paragonix SherpaPak™ cardiac transport system (PSP) is able to guarantee a constant, homogeneous and controlled temperature of the donor heart between 4°C and 8°C, thus minimizing tissue injury due to ice-cold temperature exposure.The PSP device consists of two canisters, one internal and one external . The intp = 0.12), PSP preservation significantly reduced severe PGD rate, compared to ice-cold storage  (p = 0.03) and employment of mechanical circulatory support after HTx , and thus the recipient hospital length of stay  and normothermic (NMP) machine perfusion. As reported below in this paper, NMP could also provide the opportunity to assess the metabolic and functional status of the donor graft.However, MP generally expensive and require well-trained and specialized personnel to set up the devices and manage any complications and malfunctions, which could otherwise damage the donor organ. Unfortunately, these limitations hamper a routine adoption of MP, and therefore their extensive employment is made even more difficult in centers with a low volume of activity and with scarce economic resources.3.1.The rationale of HMP preservation consists in reducing the metabolic requirements of the heart with an optimal and homogeneous cooling (below 10°C), while providing continuous metabolic support though perfusion with oxygenated, nutrient-enriched medium to limit as much as possible intracellular anerobic metabolism and consequent acidosis. Experimental studies performed on large animal models have suggested that compared to SCS, HMP could attenuate tissue injuries and provide superior myocardial function after HTx ; neverthThree single-center clinical studies have so far analyzed the effects of HMP using three different perfusion solutions: Wicomb et al. in 1984 used crystalloid cardioplegic solution in 4 patients , Hill etThe Lifecradle® Heart Preservation System is a HMP, currently under development, that uses hypothermic, oxygenated, nutrient perfusion at 5°C, in a controlled and monitored environment. The safety and efficacy of this device will be defined on the basis of clinical evidence, currently pending.3.1.1.The XVIVO Heart Perfusion System consists of a roller pump, an oxygenator, a leukocyte filter and a cooler/heater. After cardiectomy, the donor heart is connected to the XVIVO device with an aortic cannula. Then, it is submerged into the reservoir, filled with 2.5 L of perfusion solution to which 500 ml of donor and recipient immunologically-compatible irradiated blood are added. The oxygenated perfusion solution (with a hematocrit of about 15%) is pumped into the aortic root to maintain the pressure of 20 mmHg to provide a coronary blood flow between 150 and 200 ml/min in a non-beating heart state. The temperature is constantly maintained at 8°C and the pH at 7.4 value. During transportation the XVIVO device does not need continuous monitoring and power source.The initial Australian experience on 13 patients with the Xvivo NIHP for HTx showed promising results. In fact, there was no post-operative mortality and only 1 patient required veno-arterial extracorporeal membrane oxygenation (ECMO) due to secondary graft failure. The authors reported a median donor graft ischemic time of 404 min but since the period of non-ischemic perfusion was included this data could be misleading .The XVIVO innovative technology was employed for xenograft preservation during the modified pig-to-human cardiac xenotransplantation performed at the Maryland University on January 2022 .Normothermic MP systems perfuse the heart with oxygenated blood and enriched solutions, keeping it beating and at a near-physiological temperature of about 34°C. Currently the Organ Care System  represents the only NMP system commercially available for clinical use in HTx. 3.2.1.ex vivo perfusion two other solutions are infused into the circuit: the catecholamine solution, containing epinephrine to replenish the depleted catecholamine level and the maintenance solution enriched with adenosine, aiming to modulate the coronary artery resistance.The OCS consists of a portable platform and is composed of a wireless monitor/controller  and a ciThe donor blood is oxygenated and maintained at 34°C by a heater-membrane oxygenator module, and it is delivered into the aortic inflow cannula by a peristaltic pump, after both venae cavae are closed. The blood perfuses the coronary vessels, reaches the coronary sinus and eventually the pulmonary artery, where an outflow cannula collects it closing the perfusion circuit . The blo4.ex vivo perfusion has emerged as a key factor in expanding the cardiac donor pool, as it favors a safer employment of donor hearts selected using extended criteria by limiting ischemic time and allowing graft assessment. The following are some of the advantages of using NMP.During the last decade, normothermic 4.1.The continuous coronary perfusion by means of oxygenated enriched blood is the main advantage of normothermic machine perfusion. The results of the PROCEED II controlled trial demonstrated the non-inferiority of the NMP compared to the traditional ICS. Although patient and graft survival between both study arms were similar, the OCS group reported a significantly shorter graft ischemic time. Interestingly, the 4 hearts of OCS group that were discarded because of an increasing lactate concentration, after histological analysis revealed signs of infarction, contusion and severe unrecognized left ventricular hypertrophy .The OCS could be an effective tool to ensure graft quality when the expected graft ischemic time exceed the traditional “safe threshold” of 4 h, favoring long-distance organ retrieval. Two case reports presented successful HTx after preservation times of a donor heart as long as 10 h  and 16 hIn 4.2.NMP has shown interesting and promising results when utilized in extended-criteria DBD donors, and DCD. Extended-criteria were generally defined according to these parameters: >50 years of age, a history of drug abuse, cardiac resuscitation, coronary artery disease (CAD), expected graft ischemia time >4 h, left ventricular ejection fraction (LVEF) <50%, or interventricular septum thickness (IVS) >14 mm.p = 0.04). These results are also supported by histopathological and ultrastructural evidence, suggesting better myocardial preservation in NMP grafts . The 30-day post-HTx survival rate was 94.6% and the incidence of severe PGD in the first 24 h was 10.7%. Moderate to severe PGD was observed in 14.7% of patients .In our experience, out of 74 grafts preserved with NMP, a total of 9 grafts were discarded (88% utilization rate) due to a progressive increase in lactate concentration, expression in most cases of severe left ventricle hypertrophy, scarring and undiagnosed coronary artery disease. In one case, NMP real-time evaluation of lactate trend permitted to discard an apparently adequate organ which, at gross pathological examination, revealed a dissection of the right coronary artery at 4 mm from its origin . Considein situ” and if adequate recovery is observed it is retrieved and preserved with OCS or ICS  or normothermic regional perfusion (NRP). In DPP, the heart is removed after confirmation of death and expeditiously reperfused using the OCS –38. InstS or ICS , 40.The introduction of NMP in clinical practice has permitted to utilize DCD donor hearts with gratifying results –40. FurtThe method of retrieval (DPP or NRP) was not associated with different outcomes after HTx according to the results reported in the experience of Messer et al. .4.3.p = 0.03) (Heart MP could also play a protective role in high-risk recipients, particularly in those supported by durable mechanical circulatory support or who have undergone previous complex operations , 29, 31.4.4.in situ reperfusion during HTx, hearts preserved with ICS are frequently affected by myocardial injury, with damage of contractile myofilaments and organelles, including mitochondria. The ex situ perfusion with OCS is reported to be effective in reconditioning donor hearts, that are maintained metabolically active and able to heal ultrastructure changes (Sarcomere changes, such as Z-line thickening and/or non-orthodox banding were reported in donor hearts immediately after retrieval . After i changes , 42.Donor hearts, selected according to expanded-criteria, appear to be best treated by NMP, especially when severe hypotension or cardiac arrest occurs during the retrieval phase and since this technique could hamper the negative effect of cold storage on ultracellular cardiac function .5.MP systems could be useful platforms for cardiac conditioning before transplantation, since they create a “safe period” between procurement and transplantation during which the organ could potentially be manipulated. Graft immunomodulation, via infusion of viral vectors , 44 or mIn HTx, donor infusion of MSCs has been shown to prolong the survival of a semi-allogeneic HTx in a mouse model through the generation of regulatory T cells . In addiIn an effort to further suppress tissue metabolism and thus increase a safe preservation duration, sub-zero preservation techniques have been investigated in preclinical studies . IsochorVarious pharmacological agents have also been investigated in order to better preserve the graft by interfere with the ischemia-reperfusion injury mechanism. Donor simvastatin treatment might significantly improve graft function after transplantation , while vInterfering with the immune system during graft preservation could lead to a smooth immunological response of the recipient against the organ, and thus reduce the degree and number of rejections. Antioxidative agents, and inhibitors of cytokines production and activity and maturation of lymphocytes, as well as inhibitors and modulators of cellular receptors involved in signalling pathways and adhesion molecules expression are described as intriguing potential treatments .6.The field of graft preservation is subject of notable innovations. ICS was the standard technique for 50 years, but it does not allow temperature monitoring and exposes the heart to freezing damage. Increasingly, the use of ICS is being replaced with controlled hypothermic preservation using the Paragonix SherpaPak™ device, that in preliminary reports seems to offer advantages over ICS in therms of better organ preservation and clinical outcomes. Paragonix SherpaPak™ transport system has all the premises to be considered the near-future standard for donor heart cold storage, being able to allow temperature control, avoid tissue freezing, be relatively cheap and simple to use.On the other hand, MP systems can represent the opportunity to assess and recondition the donor heart and are increasingly employed worldwide in an attempt to expand the donor pool for HTx. However, despite interesting results, the role of MP in HTx remains still debatable, mainly because of higher costs and training needs than those required for CS.At present, while the clinical effectiveness of HMP has to be investigated, NMP with the OCS seems to allow safe utilization of DCD and extended-criteria donor organs, combining two major advantages: to limit the graft ischemic time and to verify cardiac function by direct visual inspection and through assessment of metabolic values, and haemodynamic parameters. Maintenance of myocardial aerobic metabolism during preservation could lead to better donor heart quality compared to traditional CS. Thus, the NMP represents an effective technique that permits to expand the donor pool, allowing acceptance of grafts which would have otherwise been refused, while maintaining satisfactory safety levels. In fact, NMP allows to identify unsuitable grafts and discard them before transplantation, reducing the risk of PGD and its life-threatening sequelae , 27, 31.Some issues related to MP systems should be more thoroughly investigated in the near future to further improve this technique, such as additional metabolic support, solution components and optimal perfusion settings. Also, the identification of other functional parameters or biomarkers, apart from lactate levels, could be of paramount importance to increase the sensitivity of MP to help clinicians in assessing suitability of perfused donor grafts.ex vivo perfusion, and to promptly intervene in case of machine malfunction or user error. In fact, since the donor heart is preserved in a beating normothermic state, the margin of safety is limited in case of complications or non-appropriate NMP management due to the risk of catastrophic and irreversible warm ischemia of the graft (The main drawback of NMP is that it requires an experienced and well-trained professional team, to manage the interaction between the donor organ and the he graft .In conclusion, the satisfactory results reported in HTx with high-risk recipients and extended-criteria donors highlight the effectiveness of NMP in complex cases, particularly in unfavorable combinations of donor, procedural and recipient characteristics. The results of ongoing multicenter clinical trials investigating on heart MP are required to confirm these expectations.


The mechanisms of cerebrospinal fluid (CSF) production by the ventricular choroid plexus (ChP) have not been fully deciphered. One prominent hypothesized mechanism is trans-epithelial water transport mediated by accumulation of solutes at the luminal ChP membrane that produces local osmotic gradients. However, this standing osmotic gradient hypothesis has not been systematically tested.To assess the plausibility of the standing gradient mechanism serving as the main driver of CSF production by the ChP, we developed a three-dimensional (3D) and a one-dimensional (1D) computational model to quantitatively describe the associated processes in the rat ChP inter-microvillar spaces and in CSF pools between macroscopic ChP folds (1D only). The computationally expensive 3D model was used to examine the applicability of the 1D model for hypothesis testing. The 1D model was employed to predict the rate of CSF produced by the standing gradient mechanism for 200,000 parameter permutations. Model parameter values for each permutation were chosen by random sampling from distributions derived from published experimental data.Both models predict that the CSF production rate by the standing osmotic gradient mechanism is below 10% of experimentally measured values that reflect the contribution of all actual production mechanisms. The 1D model indicates that increasing the size of CSF pools between ChP folds, where diffusion dominates solute transport, would increase the contribution of the standing gradient mechanism to CSF production.The models suggest that the effect of standing osmotic gradients is too small to contribute substantially to CSF production. ChP motion and movement of CSF in the ventricles, which are not accounted for in the models, would further reduce this effect, making it unlikely that standing osmotic gradients are the main drivers of CSF production.The online version contains supplementary material available at 10.1186/s12987-023-00419-2. There i+-ATPase . However+-ATPase . This me+-ATPase –11 or is+-ATPase , 12.standing gradient (SG) model [The origin of the idea of local osmotic gradients as the driving force behind CSF production can be traced back to experiments on water transport across the intestinal epithelium . Water aG) model , which wG) model .Diamond and Bossert considered the intracellular and the lateral intercellular spaces as the first and the second compartment, respectively . They asUnlike the intestinal epithelium, which is “absorptive” in nature, the ChP epithelium is “secretory,” meaning that for ChP, net water flux is from the basolateral to the luminal side, i.e., from blood to the CSF space. While in absorptive epithelia a local increase in osmolyte concentration in the lateral intercellular space may serve as a driving force for water transport, in secretory epithelia, such a concentration gradient would oppose it. With the epithelial tight junctions localized on the luminal side in the ChP, the lateral intercellular spaces are on the ‘wrong’ side to enable local osmotic gradient-driven fluid transfer . HoweverNecturus renal proximal tubule could be responsible for the observed quasi-isotonic water reabsorption. Consequently, there would be no need for an osmotic gradient in the lateral intercellular space. Schafer et al. [Since direct experimental assessment of the SG hypothesis is difficult, mathematical models have been used to aid in its evaluation. Most of this work has been done for absorptive epithelia: for example, Sackin and Boulpaep  suggester et al.  came to r et al. , 21. Howr et al. . Pedley r et al.  used a mr et al.  on waterr et al. . HoweverOne aspect common to the mentioned models is their high sensitivity to input parameters and boundary conditions such as solute–solvent transport coupling at cellular membranes. This has blunted the impact of the corresponding studies on the scientific discourse. Therefore, models covering large anatomic and physiologic parameter ranges are required to allow for a more robust assessment of the SG hypothesis. In the current study, we tested whether standing osmotic gradients at the surface of the ChP epithelium could be the predominant factor for CSF production by the ChP in rats. To this end, we developed a one-dimensional (1D) computational model to describe the conjugate fluid and solute transport within a subunit of inter-microvillar space  and between macroscopic ChP folds, under the assumption that standing osmotic gradients are the only mechanism contributing to CSF production. We then compared the calculated CSF production rate to measured rates of CSF production, which include the contributions of all actual mechanisms. We emphasize that, unlike the original SG model, we did not limit solute transport to the bottom 10% of the FU , but conWe simplified the ChP luminal membrane to a surface covered with homogeneously distributed cylindrical pins representing microvilli as shown in Fig. mentclass2pt{minimSince the microvilli are arranged in a recurring pattern, we defined a representative functional unit (FU) as the space between four adjacent microvilli Fig. b, c. MicTo derive the 1D model, we simplified the FU to a cylindrical channel with equivalent hydraulic diameter, Here, The transport of fluid and solutes in the FU are coupled. Solute transport is governed by diffusion and convection, whereas fluid transport is driven by trans-membrane solute concentration differences. The fluid velocity mentclass2pt{minimHere, quations  and 5)  \documenquations  indicatequations  represenmentclass2pt{minimThe solute flux used in Eq.  is calcued (Eqs. –8)).9\d).9\d\docHere, The 3D model considers coupled fluid and solute transport, taking into account viscous forces that were not included in the 1D model. The fluid velocity and pressure and the solute concentration distributions are governed by Eqs. –15). Th. Th15). Here, Equations  and 11)11) descrn in Eq. . We noten in Eq. ). This iFor verification of our 1D model against the original implementation of the standing gradient one , we empl+, the most relevant ion for the SG mechanism. We computed the distribution of the ChP apparent area and luminal membrane permeability based on the distribution of other related parameters   5) were To calculate the CSF production rate with the 1D model based on the parameter distributions described in Table For immunohistochemistry, anesthetized Sprague–Dawley rats (P21) were transcardially perfused with 4% paraformaldehyde, and the excised brain was immersed in the fixative at 4 °C overnight before embedding in paraffin blocks and sectioned in a microtome. Sections were deparaffinized and rehydrated in xylene and ethanol following standard protocols prior to labeling . Sections were mounted with ProLong Gold DAPI mounting medium (Dako) and imaged using a Zeiss LSM700 point laser (Argon Lasos RMC781272) scanning confocal microscope with a Zeiss Plan-Apochromat 63 × /1.4 numerical aperture oil immersion objective .To verify the implementation of the 1D SG model, we selected the same parameter values as used by Diamond and Bossert for their original standing gradient one . Since tWe assessed the applicability of the 1D SG model for testing the hypothesis that standing osmotic gradients are the main drivers of CSF production in rats by comparing its production rate predictions with those of the more intricate 3D SG model. The set of parameter values given in Table After having verified the implementation of the 1D SG model and having shown that it likely over- rather than underestimates CSF production by the SG mechanism, we employed it to calculate the CSF production rate based on the parameter distributions given in Table We aimed to test the hypothesis that standing osmotic gradients at the surface of the ChP epithelium are the main drivers of CSF production. To this end, we extended and reformulated the original standing gradient model, which was designed for absorptive epithelia, to reflect the conditions on the ChP epithelial surface. Upon verification of this new 1D SG model with the results of Diamond and Bossert , we compBoth models require the prescription of solute flux into the FU. Since solute flux into the inter-microvillar space has not been experimentally quantified, we assumed that all solutes in the ventricular space originate in inter-microvillar spaces, and that all solutes exit the ventricles driven by bulk CSF flow. Under steady-state conditions, ventricular CSF production is equal to the amount of CSF leaving the ventricles. Therefore, the cumulative solute flux from all FUs corresponds to the bulk solute concentration multiplied by the CSF production rate. This means that the actual CSF production rate is an input to the models, and the calculated production rate corresponds to the fraction that can be attributed to the SG mechanism. Since there is a wide range of measured CSF production rates in rats reported in the literature , 30–33, Neither the 1D nor the 3D model accounts for ChP motion, which could be caused by cardiovascular and respiratory action or by head and spine movement. Any such motion would work against CSF production by the SG mechanism both by disturbing standing gradients in the inter-microvillar spaces and by flushing pools of protected CSF from ChP folds. In the 1D model, the effect of protected pools is considered by the protected length, which is defined purely based on anatomic considerations. To obtain more accurate predictions of the CSF production rate, the effective protected length should be considered instead, noting that its value is location-dependent and difficult to determine. For the purposes of this study, it is sufficient to note that the values of protected length considered here are higher than the corresponding effective values. To understand the impact of changes in protected length on CSF production, we simplified Eqs. –8) usin usin8) uThe 1D model predicts a higher CSF production rate than the 3D model. This can be attributed to a difference in the area ratio of the FU solute input surface (active surface) to its total surface. In the 1D model, solutes enter the FU through the whole side surface  results in a larger protected length. Assuming an active to total surface ratio of one for the FU results in a high transmembrane water transport rate. Finally, considering the SG mechanism as the sole driving force for CSF production discounts the effects of other mechanisms, such as hydrostatic pressure gradients, that would reduce standing gradients. Consequently, the here-reported contribution of the SG mechanism to ChP CSF production likely constitutes an upper limit.We have implemented and verified a one-dimensional standing osmotic gradient model of CSF production on the ChP luminal surface that also accounts for the effect of protected pools in ChP folds. Calculations with this model, based on probabilistic parameter value distributions derived from experimental measurements, suggest that potential local osmotic gradients in the inter-microvillar spaces are too small to contribute substantially to CSF production; this even though the underlying model assumptions, which include protected pools of CSF, favor the SG mechanism. ChP motion and movement of CSF in the ventricles, both not accounted for in the model, would reduce local osmotic gradients, making it unlikely that they are the main drivers of CSF production.Additional file 1. Derivation of the functional unit tip boundary condition, estimation of choroid plexus surface area and luminal membrane permeability, Péclet number calculation, and estimation of the protected length


Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H2O2-driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H2O2 (po-H2O2) emission impact the autophagic process and to which extent peroxisomes with a disturbed H2O2 metabolism are selectively eliminated through a process called “pexophagy”. To address these issues, we generated and validated HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H2O2 can be modulated. We demonstrate that (i) po-H2O2 can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H2O2 emission act as a prime determinant of pexophagy, and (iii) high levels of po-H2O2 impair autophagic flux by oxidative inhibition of enzymes involved in LC3II formation. Unexpectedly, our analyses also revealed that the autophagy receptor optineurin can be recruited to peroxisomes, thereby triggering pexophagy. In summary, these findings lend support to the idea that, during cellular and organismal aging, peroxisomes with enhanced H2O2 release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris.Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H Autophagy is an evolutionarily conserved multistep process that delivers superfluous or damaged components of the cytoplasm, including organelles, to the lysosome for turnover . During 2O2), which has the capacity to promote disulfide bond formation within or between proteins  through the oxidation of redox-sensitive cysteine residues [Autophagy is a redox-regulated process that can be activated by oxidative stress, thereby protecting cells from the accumulation of oxidatively damaged macromolecules and organelles ,7,8. Thiresidues ,13. Thisresidues  and TFEBresidues ), (ii) aresidues , ATM [17residues ), (iii) residues , ATG4A [residues , ATG4B [residues , and ATGresidues ), (iv) cresidues ,22, SQSTresidues ,24, and residues ). Importresidues .ATG4 proteins exert a dual role in autophagy: on the one hand, they activate LC3 by cleavage of pro-LC3, thereby exposing a C-terminal glycine residue that is required for conjugation to phosphatidyl-ethanolamine (PE) at the autophagosome membrane; on the other hand, they are necessary for degreasing LC3-PE (termed LC3-II) to a cytosolic isoform (termed LC3-I). Interestingly, disulfide bond formation of the human orthologs of ATG4 has been shown to render these cysteine proteases enzymatically inactive ,20. This2O2 as part of their catalytic cycle, and catalase, an enzyme that decomposes H2O2, they have the intrinsic ability to initiate and modulate H2O2-driven signaling events, including (selective) autophagy [Peroxisomes are dynamic organelles that rapidly adapt their protein content, morphology, and number in response to cellular needs and environmental cues . In mammutophagy . To mainutophagy ,31. Howeutophagy .2O2 (po-H2O2) affects general autophagy and pexophagy. Here, it is pertinent to mention that we recently developed a unique and powerful Flp-In T-REx 293 cell model that allows the selective generation of H2O2 inside peroxisomes in a time- and dose-dependent manner (i) through controlled expression of a destabilization domain (DD)-tagged version of D-amino acid oxidase (DAO), and (ii) by regulating its activity with the supplementation of a D-amino acid  to the assay medium [2O2 impacted pexophagy and general autophagy.The aim of this study was to investigate how the production of peroxisome-derived Hy medium . In thisy medium , or po-my medium , and to agatctaccatggtgagcgtgatcgccaagcag-3’ and 5′-tccccgcggccgcttacagcttgctcttgcccagcagggagtggcgggcgatg-3’ ) and ligating the Bgl II/Not I-digested PCR product into Bgl II/Not I-restricted pEGFP-N1 . The plasmid encoding OPTN-GFP was obtained from Addgene  [Nb-PM (pMF1968) [TOP10F’ E. coli strain  was used as a cloning and plasmid amplification host. All plasmids were validated by DNA sequencing .The plasmid encoding po-mKeima (pMF2005) was generated by amplifying the corresponding cDNA via PCR ); primers: 5′-gggIL, USA) ). The plIL, USA) , c-roGFPIL, USA) , mCherryIL, USA) , or GFPNpMF1968)  have bee2 incubator in regular minimum essential medium Eagle α , 10% (v/v) fetal bovine serum , 2 mM UltraGlutamine I , and 0.2% (v/v) MycoZap ). Electroporations were performed with the Neon Transfection System (Thermo Fisher Scientific) and conducted in 10 µL tips with the following electroporation parameters: 1150 V, 20 ms pulse width, 2 pulses (for 293 cells); 1500 V, 20 ms pulse width, 1 pulse ). To generate DD-DAO Flp-In T-REx cell lines constitutively expressing po-mKeima or mRFP-GFP-LC3, the cells were transfected with pMF2005 or p4-RFP-GFP-LC3, respectively, and cultured for 2–3 weeks in rMEMα supplemented with 10 µg/mL blasticidin , 100 µg/mL hygromycin , and 200 µg/mL (for po-mKeima) or 400 µg/mL (for mRFP-GFP-LC3) G418 . Stably transformed clones of flow-sorted pools  were selected and further cultured in the same medium containing 200 μg/mL of G418. The DD-DAO Flp-In T-REx 293 and HeLa cell lines were generated as detailed elsewhere . Cells wTo selectively express DD-DAO in peroxisomes, DD-DAO Flp-In T-REx cells were cultured for 3 days in rMEMα containing 1 μg/mL doxycycline  and 500 nM Shield1  and subsequently for 1 day in the same medium lacking DOX/Shield1 . 2O2 inside peroxisomes, the DOX/Shield1-chased cells were incubated in (i) DPBS  supplemented with 10 mM 3-amino-1,2,4-triazole , (ii) modified DMEM (mDMEM) lacking glucose and glutamine  but supplemented with 5% FBS, 2 mM UltraGlutamine I, 10 mM sodium pyruvate , 10 mM 3-AT, and 50 µM dehydroepiandrosterone , or (iii) modified MEMα (mMEMα) containing 10 mM 3-AT and 200 µM DHEA, always in combination with 20 mM L- or D-alanine (L/D-Ala) as indicated. To generate HFor starvation, the cells were first washed three times with Earle’s balanced salt solution  and subsequently incubated in the same buffer for the indicated time. To stimulate autophagy, the cells were treated for 8 or 14 h with 1 µM Torin-1  or 50 nM rapamycin , respectively; to block autophagosome formation, the cells were pretreated for 24 h with 5 mM 3-methyladenine , and to prevent autophagosome-lysosome fusion, the cells were pretreated for 2 or 8 h with 200 µM chloroquine  or 200 nM bafilomycin A1 , respectively. The same volume of DMSO  was included as the vehicle control. To image lysosomes, the cells were incubated with LysoTracker Green DND-26 . 2HPO4, 9.1 mM sodium citrate (dihydrate), 10.9 mM citric acid, 1.2 mM CaCl2, 0.8 mM MgSO4; pH 4.5).To investigate the effect of acidic pH on po-mKeima, the cells were incubated in a citrate buffer  or immunoblotting (IB): rabbit anti-ATG3 , anti-ATG4B , anti-ATG7 , anti-SQSTM1 , anti-OPTN , anti-CACO2/NDP52 , anti-LC3B , anti-TUBA1A , anti-USP30 , anti-DNM1L , mouse anti-PEX14  , and ant2) was used as the staining substrate.Sample preparation for IB was carried out as previously described . A modifFluorescence microscopy was essentially carried out as described previously . The folv/v) 2-mercaptoethanol  was added and samples were heated to 100 °C for 10 min.Electrophoretic mobility shift assays (EMSAs) were essentially performed as described previously . A slighg, 1 min), resuspended in the appropriate assay buffer (as specified elsewhere), and analyzed using flow cytometry . For po-mKeima, the cells were analyzed by using 405- and 561-nm lasers in combination with a 600–620 nm emission filter; for mRFP-GFP-LC3, the cells were analyzed by using 488 and 561 nm lasers in combination with 515–545 nm and 600–620 nm emission filters, respectively. For each sample, 100,000 events were collected and analyzed using BD FACSDiva v8.0.1 software (BD Biosciences).The cells were washed with DPBS and dislodged from the plate by trypsinization. Next, the cells were pelleted . Data are expressed as the mean ± standard deviation (SD). A p-value less than 0.05 was considered statistically significant.Statistical analysis was performed by one-way analysis variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons, and Student’s unpaired two-tailed 2O2 on pexophagy, we generated DD-DAO Flp-In T-REx 293 and HeLa cell lines stably expressing a peroxisome-targeted variant of mKeima (po-mKeima). The excitation spectrum of this coral-derived monomeric red fluorescent protein (emission peak: 620 nm) is pH-dependent  [To establish robust and sensitive cell models that can be used to study the effects of po-H 586 nm) , a propeTo functionally validate po-mKeima, DD-DAO/po-mKeima Flp-In T-REx 293 cells were subjected to starvation, a stressor known to induce pexophagy . To dist2O2 emission impinges on pexophagy, we first established assay conditions yielding different net levels of po-H2O2 in the cytosol. To that end, the DOX/Shield1-chased DD-DAO/po-mKeima Flp-In T-REx cells were incubated in different assay media: (i) DPBS supplemented with 10 mM 3-amino-1,2,4-triazole , indicated with “DPBS”, and 10 mM L- or D-Ala; (ii) modified DMEM (mDMEM) lacking glucose and glutamine but supplemented with 10 mM 3-AT, 50 μM dehydroepiandrosterone , and 20 mM L- or D-Ala; or (iii) modified MEMα (mMEMα) containing 10 mM 3-AT, 200 μM DHEA, and 20 mM L- or D-Ala. Note that the L-Ala conditions served as a negative control. To document the release of po-H2O2 on the addition of D-Ala, we used a cytosolic (c-) variant of the ratiometric H2O2 biosensor roGFP2-Orp1 [2O2 levels can be assessed.To investigate to what extent po-HFP2-Orp1 . On oxid2O2 inside peroxisomes slightly acidified the organelle lumen  uted [2O2 B, and -based translocation assay  1,10-phenanthroline-induced pexophagy in Chang liver cells coincides with elevated levels of po-H2O2, and that this process can be completely counteracted by N-acetyl-L-cysteine [2O2 [2O2 act as the primary trigger for pexophagy.In an analogy with mitochondria , pexophatabolism , it is rcysteine , (ii) incysteine  as well cysteine , and  H2O2-mediated phenomenon. Regarding the second observation, OPTN is a multifunctional autophagy receptor that contributes to mitophagy, aggrephagy, and xenophagy through ubiquitin signaling [This study also revealed two unexpected but interesting side observations: (i) DAO-mediated production of po-Hme lumen C,D, and me lumen . Regardiignaling . Our stu2O2 emission and pexophagy, a topic not examined thus far, we established HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H2O2 can be controlled in a dose- and time-dependent fashion. Our results demonstrate that neither mild, moderate, nor high levels of po-H2O2 act as a primary trigger for pexophagy. However, po-H2O2 has the potential to oxidatively modify redox-sensitive SARs  and core autophagy proteins , and enhanced levels of po-H2O2 potently inhibit late-stage autophagy. Finally, this study provides compelling evidence that OPTN can act as a pexophagy receptor, a novel finding that opens new avenues for exploring and exploiting the importance of peroxisome clearance in normal physiology and human disease.To investigate the direct relationship between po-H


The levels of synaptic markers synaptosomal-associated protein 25 (SNAP-25) and neurogranin (Ng) have been shown to increase early in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD) and to have prognostic potential. However, no validation studies assessed these biomarkers' diagnostic and prognostic value in a large clinical setting cohort of rapidly progressive dementia.n = 220) or non-prion rapidly progressive dementia (np-RPD) (n = 213). We evaluated and compared the diagnostic accuracy of each CSF biomarker and biomarker combination by receiver operating characteristics curve (ROC) analyses, studied SNAP-25 and Ng CSF concentrations distribution across CJD subtypes, and estimated their association with survival using multivariable Cox regression analyses.In this retrospective study, using commercially available immunoassays, we measured the levels of SNAP-25, Ng, 14–3-3, total-tau (t-tau), neurofilament light chain (NfL), and phospho-tau181 (p-tau) in CSF samples from consecutive patients with CJD . SNAP-25 diagnostic accuracy  exceeded that of 14–3-3 , t-tau , and the t-tau/p-tau ratio . In contrast, Ng performed worse  than all other surrogate biomarkers, except for NfL . SNAP-25 maintained a relatively high diagnostic value even for atypical CJD subtypes . In Cox regression analyses, SNAP-25 levels were significantly associated with survival in CJD (hazard ratio [HR] 1.71 95% CI 1.40–2.09). Conversely, Ng was associated with survival only in the most rapidly progressive CJD subtypes (sCJD MM(V)1 and gCJD M1) (HR 1.81 95% CI 1.21–2.93).CSF SNAP-25 and Ng levels were higher in CJD than in np-RPD (SNAP-25: 582, 95% CI 240–1250 vs. 115, 95% CI 78–157 pg/ml, In the clinical setting, CSF SNAP-25 is a viable alternative to t-tau, 14–3-3, and the t-tau/p-tau ratio in discriminating the CJD subtypes from other RPDs. Additionally, SNAP-25 and, to a lesser extent, Ng predict survival in CJD, showing prognostic power in the range of CSF t-tau/14–3-3 and NfL, respectively.The online version contains supplementary material available at 10.1186/s13195-023-01300-y. PRNP) and the type (1 or 2) of misfolded PrP (PrPSc) accumulating in the brain . About 35% of sCJD cases show mixed phenotypic features due to the co-occurrence of PrPSc types 1 and 2 and are classified accordingly ) , and NfL (AUC 0.649 [0.593–0.705]) . The diagnostic accuracy of CSF Ng slightly exceeded that of NfL  but was inferior to that of SNAP-25 , t-tau , and 14–3-3 . The combined use of p-tau increased the diagnostic accuracy of both synaptic biomarkers, with the CSF SNAP-25/p-tau ratio performing slightly better than the t-tau/p-tau ratio (AUC 0.924 [0.900–0.948])   and 69% (AUC 0.697 [0.626–0.767]) in discriminating between CJD and np-RPD. The diagnostic performance of CSF SNAP-25 was better than that of t-tau (AUC 0.878 [0.845–0.901]) .In the differential diagnosis between the "atypical," slowly progressive sCJD subtypes 2C, MM2T, and VV1) and np-RPD, CSF SNAP-25 diagnostic accuracy (AUC 0.792 [0.729–0.854]) was in the range of that of t-tau, t-tau/p-tau ratio, and SNAP-25/p-tau ratio. CSF SNAP-25 and t-tau outperformed CSF Ng in this sub-analysis 1 and VV2), CSF SNAP-25 yielded an extremely high diagnostic accuracy (AUC 0.982 [0.971–0.994]). In this sub-analysis, the correction of SNAP-25 values with p-tau did not significantly impact the diagnostic performance of the biomarker (AUC 0.988 [0.979–0.996]). In the same comparison, CSF Ng diagnostic value (as that of the other biomarkers) improved as well (AUC 0.809 [0.727–0.891]), although its diagnostic power remained way lower than that of SNAP-25, t-tau, and 14–3-3.P = 0.0003; SNAP-25 vs. t-tau/p-tau, P = 0.0002; SNAP-25 vs 14–3-3, P < 0.0001; SNAP-25 vs. NfL, P < 0.0001) (Table P = 0.0003). In this analysis, p-tau-corrected biomarkers yielded a lower diagnostic accuracy than the single correspondent biomarkers   , except for NfL (AUC 0.839 [0.782–0.896]). In this analysis, all p-tau-corrected biomarkers showed better diagnostic accuracy than the corresponding single biomarkers  was outperformed by 14–3-3 (AUC 0.942 [0.916–0.967]) , even after accounting for covariates known as prognostic factors in prion disease as codon 129 genotype, age at LP and time from onset to LP. When stratifying for the clinicopathological subtype, CSF SNAP-25 levels correlated with survival in both “typical” MM(V)1 CJD  and “non-MM(V)1 CJD”  groups. In the multivariate Cox regression, CSF SNAP-25 were significantly associated with survival in both the whole prion cohort , and “typical CJD” , but not in “non-MM(V)1 CJD”.When considering the whole CJD cohort, CSF SNAP-25 was significantly associated with survival .Conversely, we found no significant associations between CSF Ng and survival when considering the whole CJD cohort and the “non-MM(V)1 CJD” subtypes. CSF Ng levels significantly correlated with survival in “typical CJD” in both univariate and multivariate Cox regression  and multivariate Cox regression analyses, yielding a prognostic performance in the range of that of SNAP-25 (in case of t-tau and 14–3-3) and Ng (in case of NfL).Within the whole CJD cohort, CSF t-tau, 14–3-3, and NfL were significantly associated with survival in both univariate , while Ng showed no significant association . The disease stage did not correlate with biomarker values across CJD subtypes, apart from SNAP-25 in MV2K . Data regarding the association between biomarkers levels and disease stages are shown in Supplementary Fig. Regarding the possible correlation between CSF biomarkers levels and disease stage within the whole CJD cohort, higher SNAP-25 was weakly associated with later disease stage , CSF SNAP-25 discriminatory power exceeded that of t-tau (and the t-tau/p-tau ratio), which is currently considered the gold standard screening test in this specific clinical scenario , 15. TheThe differential diagnosis between CJD and rp-ND, mostly rapidly progressive Alzheimer's disease (AD), is a common clinical scenario in which t-tau yields a discrete number of false positive results. CSF SNAP-25 and t-tau diagnostic power fell slightly behind 14–3-3, currently considered the most helpful assay in this clinical context , 15, 34.Our results indicate that SNAP-25 is a viable alternative diagnostic marker to 14–3-3 and t-tau for RPDs. Although it is unlikely to provide a significant clinical advantage over t-tau in discriminating between CJD and rp-ND overall, the clinical impact of SNAP-25 could be relevant when the differential diagnosis with non-neurodegenerative np-RPD is an issue. The SNAP-25/p-tau ratio provides no additional diagnostic value compared to SNAP-25 alone, except in the differential diagnosis with rp-ND.Regarding the synaptic biomarkers distributions across CJD subtypes, the mean CSF SNAP-25 levels were higher in MM(V)1 and VV2 than in MV2K, MM2C, MM2T, and VV1. Patients with gCJD M2T showed the lowest levels of the whole CJD cohort. The distribution pattern of mean CSF SNAP-25 levels among CJD subtypes was substantially similar to that previously reported for CSF t-tau, CSF 14–3-3, and CSF α-synuclein , 27. In Interestingly, CSF Ng levels distribution across CJD subtypes slightly differs from those of the other synaptic and neuroaxonal biomarkers, especially regarding the relatively low concentrations observed in VV2. Ng is a neuronal protein, mainly expressed in the cerebral cortex and hippocampus and, to a lesser extent, in the amygdala, caudate, and putamen . Based oPRNP codon 129 genotype, and time between symptoms onset and LP. Consistent with previous observations [In the present study, in line with recently proposed prognostic models , 39, we rvations , we founrvations , are likRegarding the strength of the association between biomarkers levels and survival in the whole CJD cohort, CSF SNAP-25 yielded an HR similar to CSF t-tau, CSF 14–3-3 ones (data not shown), and the one we previously reported for CSF α-synuclein . In contRegarding the possible correlation between biomarkers levels and clinical variables, we found a weak association between SNAP-25 concentrations and disease stages in the whole cohort. SNAP-25 concentrations did not vary significantly between stages in each sCJD subtype but the MV2K. These results suggest that SNAP-25 levels increase early and remain stable during the disease course in the most common subtypes 1 and VV2), as previously reported . In the We are aware that the low number of neuropathologically-verified np-RPD cases is a limitation of this study. Similarly, the classification of participants with probable CJD into a specific clinicopathological subtype could have been mistaken due to the lack of a definite diagnosis. However, we are confident that through the thorough screening of medical records, codon 129 genotyping, and the second-generation prion RT-QuIC, we have effectively tackled the risks of patient misdiagnosis and misclassification. We recognize that some of the biomarkers used for sCJD subtype determination  in the cases lacking neuropathology share parts of their pathophysiological background with SNAP-25 or Ng and that this may cause a verification bias when the biomarker levels are compared between  subtypes. As an additional limitation, the fact we used akinetic mutism in place of time to death when life-extending treatments were adopted might have introduced a bias in survival calculation, given that we did not use the same variable for all patients. Unfortunately, the information on the onset of akinetic mutism was unavailable for some patients. However, we thought that eliminating the significant effect of life-extending treatments would be more accurate than non-considering this variable, which would also introduce a bias in the calculation of disease duration among patients. We also know that small sample sizes are an additional study limitation, especially in subanalyses concerning rare sCJD subtypes (MM(V)2C, MM2T, and VV1). Finally, this study's retrospective and unicentric nature could limit the generalizability of the results. As the main strength, in this study, we assessed the diagnostic value of CSF SNAP-25 and Ng in a large cohort of patients presenting with a rapidly progressive neurological syndrome, thus reflecting the clinical scenario in which these biomarkers are expected to be employed.In conclusion, our results suggest that CSF SNAP-25 is a viable alternative to established diagnostic CSF surrogate biomarkers such as t-tau, 14–3-3, and the t-tau/p-tau ratio in discriminating CJD among RPD cases, especially from those of non-neurodegenerative etiology. When differential diagnosis with rp-ND is an issue, the biomarkers' accuracy can be optimized by calculating the SNAP-25/p-tau ratio. In contrast to SNAP-25, CSF Ng diagnostic performance is significantly worse than that of CSF t-tau and CSF 14–3-3. Moreover, our study suggests that CSF SNAP-25 and, to a lesser extent, CSF Ng can predict survival in CJD, with the former yielding a predictive performance comparable to that of CSF t-tau, 14–3-3, and α-synuclein. Finally, since CSF levels of synaptic biomarkers seem to recapitulate the prevalent regional and sub-cellular pathology of prion diseases, they could prospectively serve as valuable tools for patient stratification in clinical trials and patient-tailored medical management.Additional file 1: Supplementary material. Supplementary methods, probable sCJD and np-RPD patients’ classification. Supplementary table 1, np-RPD cohort diagnostic categories. Supplementary table 2, distribution of biomarkers levels in the subgroups of the CJD cohort. Supplementary table 3, associations of t-tau, 14-3-3, and NfL CSF levels with survival time in the whole CJD cohort and after stratification according to the disease subtype. Supplementary figure 1, t-tau, 14-3-3, and NfL prognostic performance. Supplementary figure 2, Correlation analysis between SNAP-25 or Ng levels and disease stages.


A water-soluble acacetin prodrug has been synthesized and reported by our group previously. Acetaminophen (APAP) overdose is a leading cause of acute liver injury. We found that subcutaneous injection of acacetin prodrug  decreased serum ALT, AST, and ALP, corrected the abnormal MDA and GSH in liver, and improved intrahepatic hemorrhage and destruction of liver structures in APAP (300 mg/kg)-treated mice. Molecular mechanism analysis revealed that the expressions of endoplasmic reticulum (ER) stress markers ATF6, CHOP, and p-PERK, apoptosis-related protein BAX, and cleaved caspase 3 were decreased by acacetin in a dose-dependent manner in vivo and in vitro. Moreover, via the acacetin-upregulated peroxisome-proliferator-activated receptor gamma (PPARγ) of HepG2 cells and liver, the suppressive effect of acacetin on ER stress and apoptosis was abolished by PPARγ inhibitor (GW9662) or PPARγ-siRNA. Molecular docking revealed that acacetin can bind to three active pockets of PPARγ, mainly by hydrogen bond. Our results provide novel evidence that acacetin prodrug exhibits significant protective effect against APAP-induced liver injury by targeting PPARγ, thereby suppressing ER stress and hepatocyte apoptosis. Acacetin prodrug is likely a promising new drug candidate for treating patients with acute liver injury induced by APAP. APAP overdose is the major cause of acute liver injury and death in the United States and Western countries ,2,3,4. IAcacetin is a di-hydroxy and mono-methoxy flavone reported to be present in 92 plants. The biological activities of acacetin in anti-cancer, anti-obesity, anti-diabetic, anti-inflammatory, anti-oxidant, and anti-microbial roles were well reviewed recently . ConsideIt was found that acacetin prodrug administrated by subcutaneous injection effectively lowered the elevation of serum ALT, AST, and ALP and restored the decrease of liver GSH in APAP-treated mice, and protected APAP-induced liver damage. Mechanistically, APAP-induced ER stress and downregulation of PPARγ was improved in acacetin-prodrug-treated mice. More importantly, PPARγ was proven to be essential for suppression of ER stress by acacetin because the protective effect of acacetin was abolished by PPARγ siRNA or the specific inhibitor. Meanwhile, computer molecular docking uncovered the interaction between acacetin and PPARγ, supporting the hypothesis that PPARγ is the target of acacetin. Our present study indicates the potential application of acacetin prodrug against AILI in clinic in future.p < 0.01 vs. untreated, n = 3). Here, 5 mM APAP was selected for further experiments. It was found that acacetin could reverse the suppression of APAP on cell proliferation in a dose-dependent manner, but not a time-dependent manner . APAP-induced hepatocellular apoptosis has been reported [p < 0.05). These data strongly indicate the suppressive effect of acacetin on the HepG2 cell apoptosis induced by APAP.reported . Flow cyp < 0.01). However, treatment with acacetin prodrug  by subcutaneous injection reduced the levels of serum ALT, AST, and ALP significantly (## p < 0.01). The PPARγ activator rosiglitazone , which was used as a positive control, also reduced serum ALT, AST, and ALP significantly in APAP-treated mice, as expected  is common to set up the acute liver injury animal model. After treatment for 4 h, significant increases in serum ALT, AST, and ALP activities and markers of hepatocellular damage were observed in APAP-treated mice compared with normal mice .The effects of acacetin prodrug on hepatic lipid peroxidation during APAP-induced acute liver injury were analyzed. As shown in Histological analysis of liver samples with hematoxylin and eosin (H&E) staining strongly supported the protective effect of acacetin prodrug on hepatocytes. As shown in p < 0.05, ** p < 0.01, *** p < 0.001 vs. normal, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. APAP).ER stress was reported to play an important role in APAP-induced acute liver injury in mice. To explore whether acacetin prodrug treatment alleviated ER stress caused by APAP administration, the gene and/or protein expressions of CHOP, ATF6, PDI, and p-PERK in livers were measured. In addition, the protein expressions of BAX, Caspase-3, and cleaved caspase-3 were also analyzed. p < 0.05, ** p < 0.01 vs. untreated, # p < 0.05, ## p < 0.01 vs. APAP). These results are consistent with the observations in mice. Subsequently, we found that PPARγ was downregulated by APAP both in vivo and in vitro. However, this downregulation could be significantly reversed by acacetin in vitro or acacetin prodrug in vivo .To study the mechanism of how acacetin suppresses ER stress, HepG2 cells were treated with APAP or APAP + acacetin for 15 h. First, the changes in gene expression of CHOP, ATF6, BIP, and PDI were reversed by acacetin in a dose-dependent manner in HepG2 cells treated with APAP . As expected, the APAP-induced high gene expressions of CHOP, BIP, and ATF6 were decreased by acacetin (30 μM), but these decreases were abolished by PPARγ-siRNA or GW9662; the expression of PDI, a suppressor of ER stress, was decreased by PPARγ-siRNA or GW9662 compared with acacetin . The protein expressions of ATF6, CHOP, p-PERK, PERK, BAX, Caspase-3, and cleaved caspase-3 were measured in cells treated with APAP, APAP + acacetin, APAP + acacetin + GW9662, and APAP + acacetin + PPARγ-siRNA. p < 0.05, ** p < 0.01 vs. normal, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. APAP, $ p < 0.05, $$ p < 0.01 vs. acacetin). In order to further confirm the important role of PPARγ in AILI, GW9662 was employed to treat HepG2 cells incubated with APAP and/or acacetin, and then cell viability was evaluated. It was found that GW9662 could abolish the protective effect of acacetin on HepG2 cells  map of acacetin provides a visual representation of the electronic density, chemically active sites, and the reactivity of the atoms. The different values of electrostatic potential at the MEP surface are represented by different colors: red, blue, and green represent the regions of most negative, most positive and zero electrostatic potential, respectively. Specifically, the MEP of acacetin clearly shows one major negative potential region around the oxygen atoms of the carbonyl group (represented by red color). The H atom indicates the strong attraction and the oxygen atom indicates the repulsion. The value of the electrostatic potential is largely responsible for the binding of a substrate to its receptor binding sites and thus leads to further development of the receptor-ligand interaction A upper.To further explore the energetic behavior, the 3D plots of the HOMO, HOMO + 1, LUMO and LUMO + 1 frontier orbitals of acacetin are depicted in In order to verify the functional effects of acacetin in non-alcoholic fatty liver, the interaction between acacetin and PPARγ was tested and verified randomly. First, the active pockets were speculated by DoGisteScorer B. Then, The present study investigated the therapeutic effect of a water-soluble acacetin prodrug, sodium phosphate of acacetin, on AILI. The data showed that acacetin administration in vitro significantly attenuated APAP-induced HepG2 cell apoptosis. Acacetin prodrug treatment in vivo decreased APAP-induced elevation of serum ALT, AST, and ALP levels. MDA and GSH in liver tissue were also analyzed, and the increase in MDA or hepatic GSH depletion caused by APAP were restored after acacetin prodrug treatment. It has been reported that ER stress is an important event during AILI ,20. Our Although PPARγ is predominantly expressed in adipocytes, where it plays an essential role in regulation of adipogenesis; it is also expressed in many other cells, including hepatocytes, smooth muscle cells, and macrophages. A number of studies have revealed the regulatory role of PPARγ in apoptosis and inflammation ,31. HoweER stress is an important intrinsic stress of integrated stress response (ISR), which is a conserved intra-cellular signaling network and promotes cell survival and homeostasis. Both intrinsic and extrinsic stresses are sensed by four specialized kinases: PERK, GCN2, PKR, and HRI. Subsequently, the central player of ISR elF2α is phosphorylated . At the A previous publication reported the protective effects of acacetin on D-galactosamine and LPS-induced fulminant hepatic failure in mice, in which acacetin was dissolved in 5% DMSO-saline solution and administrated by intraperitoneal injection. The dose of acacetin used in that study was as high as 100 mg/kg, and their results show that acacetin protects against liver injury via suppressing TLR4 signaling and enhancing autophagic flux . In addiThe biggest contradiction of the studies on natural flavone including acacetin is that it is very easy to obtain results from cell culture or enzyme assays; however, they may not have real biological significance because of poor bioavailability and rapid metabolism in animal experiments . TherefoOf note, CYP 2E1 is also expressed in the kidneys, so kidney injury is a common event in APAP overdose. In fact, acute kidney injury could contribute to the increased morbidity and mortality observed in AILI . The norHowever, the present study has several limitations. First, in vivo study is needed to confirm the molecular target of acacetin, for example, PPARγ knockout mice should be employed to evaluate the effect of acacetin. In addition, although computer molecular docking displays the interaction between PPARγ and acacetin, this should be further demonstrated with specific experiments. Second, it is still unclear how ER stress is regulated by PPARγ. We don’t know whether PPARγ regulates ER stress directly or indirectly. In addition, the toxicity of the compound also needs to be evaluated.In summary, the present study demonstrated the protective effects of acacetin prodrug on AILI. Our results provide evidence that acacetin prodrug targets PPARγ in modulation of ER stress in the progression of AILI. Thus, acacetin prodrug is a promising candidate for treatment of liver injury in future.Both acacetin and the water-soluble acacetin prodrug were kindly provided by Dr. Gui-Rong Li from Nanjing Amazigh Pharma Ltd. . The synthesis process and in vivo conversion of the acacetin prodrug were reported previously ,24. APAP2 at 37 °C. The culture medium was changed every two days with a conventional cell culture procedure.Human liver cancer cell line HepG2 provided by Procell Life Science & Technology Co., Ltd.  was cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin  and maintained in a cell incubator containing 5% CO4 cells per well for 8 h, then cultured in the medium containing APAP, acacetin, or RSG for different time periods. MTT  solution was then added to each well and incubated at 37 °C under dark for 4 h. Then the medium was removed and DMSO was applied to each well. The plates were read using a microplate spectrophotometer at 570 nm with 630 nm as a reference. Cell viability was normalized as a percentage of control wells.HepG2 cells were seeded in 96-well plates at a density of 1.8 × 10Six-week-old ICR male mice were purchased from the Shanghai Experimental Animal Center . The standard animal diet and water were given according to a 12 h light-dark cycle at the Experimental Animal Center of Ningbo University under the condition of controlled temperature (22 °C). The experimental procedure was approved by the Committee on the Use of Live Animals in Teaching and Research of Ningbo University (Approved No: 2019-48).After adaptive feeding for 1 week, mice were randomly divided into six groups: vehicle control , APAP (300 mg/kg), APAP (300 mg/kg) + acacetin prodrug (5 mg/kg), APAP (300 mg/kg) + acacetin prodrug (10 mg/kg), APAP (300 mg/kg) + acacetin prodrug (20 mg/kg), and APAP (300 mg/kg) + rosiglitazone (20 mg/kg); there were 5 mice in each group. APAP, acacetin prodrug, and rosiglitazone were dissolved in 0.9% saline. After the mice fasted for 16 h, acacetin prodrug and rosiglitazone were administered via subcutaneous injection and intraperitoneal injection, respectively. After 1 h, APAP was administrated by intraperitoneal injection. Another 4 h later, all mice were sacrificed and blood and livers were collected for blood biochemical and molecular biology analysis.g for 10 min at 4 °C, the supernatant was collected for analysis. Liver tissues were homogenized in PBS buffer and centrifuged at 2500× g for 10 min at 4 °C, then the supernatant was collected. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), malondialdehyde (MDA), and glutathione peroxidase (GSH) were detected according to the manuals of the kits which were purchased from Nanjing Jiancheng Institute of Bioengineering .The blood samples were kept at room temperature for 30 min. After centrifugation at 1000× Liver samples were fixed with formalin, dehydrated, embedded in paraffin, sliced, and stained with H&E. The pathological changes were observed under a light microscope and photographed with 200× magnification. Finally, the degree of liver injury and structural changes were evaluated.TM reverse transcript (RT) reagent Kit  used for quantitative real-time PCR. Quantitative RT-PCR reactions were prepared with SYBR Premix Ex TaqTM II  and primers specific for human or mouse GAPDH, CHOP, BIP, ATF6, PDI, and PPARγ , and RNA concentration was measured using a NanoDrop 2000 and normalized among the experimental sample set. cDNA was generated using the PrimeScriptnd PPARγ . The reag for 20 min at 4 °C. After transferring the supernatant to a fresh ice-cold tube, protein concentration was quantified with a BCA assay kit, then mixed with SDS sample buffer and denatured at 95 °C for 5 min; samples were resolved with 10% SDS-page gel. Gels were then transferred onto PVDF membrane papers and the membranes were blocked with 5% non-fat milk in TTBS for 1 h. Then membranes were incubated with primary antibodies overnight at 4 °C. Anti-β-actin  was purchased from proteintech, Wuhan, China. Anti-CHOP/GADD , anti-ATF6 , anti-Caspase-3 , anti-Bax , anti-PERK , and anti-PPARγ  antibodies were purchased from SantaCruz Biotechnology. Anti-p-PERK  was purchased from Cell Signaling Technology. After washing with TTBS, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h. The membranes were washed again with TTBS and then the blots were analyzed using an enhanced chemiluminescence detection system.The Western blotting was performed with the procedure described previously. Briefly, 20 mg liver tissue or HepG2 cells were lysed with modified RIPA buffer for 30 min on ice; cell lysates were then centrifuged at 12,000× 6 cells per dish for 8 h. After incubation in the medium containing APAP, acacetin, and RSG for 15 h, the cells were collected and washed with PBS. Then 1 × 105 cells were resuspended in 100 μL binding buffer containing AnnexinV-APC and PI following the kit manual . The cells were detected using a BECKMAN CytoFlex S flow cytometer. For each sample, no less than 10,000 events were acquired.HepG2 cells were seeded into 33 mm dishes at a density of 1 × 10Short interference RNA (siRNA) molecules targeting human PPARγ  had target-specific nucleotides. The siRNA molecules (150 nM) were transfected into HepG2 cells with 60–80% confluence using lipofectamine 2000 (Invitrogen) following the manual. The transfected cells were used for RNA extraction and Western blotting analysis.LUMO + EHOMO)/2, η is the value from (ELUMO − EHOMO)/2, and σ is the value from 1/η. The analysis of frontier molecular orbitals (FMO) and electrostatic potential were also finished by Gauss View.For investigating the quantum chemical properties of acacetin (Pubchem ID: 5280442), the Gaussian 09 program was used to prepare the initial structures and to perform the ab initio calculation at the Becke-3-Lee–Yang–Parr/6-31G level. The geometric optimization of valacyclovir was carried out with the 6-311G++ basis set and B3LYP functional, which were chosen for the high-quality theoretical approach. The frontier molecular orbital parameters, such as the highest occupied molecular orbital (HOMO), the lowest unoccupied molecular orbital (LUMO), HOMO-LUMO energy gaps, the chemical potential (χ), chemical hardness (η), and chemical softness (σ) were calculated based on the outputs of Gaussian 09, in which χ is the value from ). The active pockets were identified based on DoGisteScorer (https://proteins.plus/ (accessed on 6 June 2022)). The AutoDock Tools (ADT) graphical user interface was used to prepare the protein by removing water and adding polar hydrogens and the required charges. Autodock 4.2 (http://mgltools.scripps.edu/downloads (accessed on 6 June 2022)) was used to find the minimum binding energy. PyMOL 2.4.0 and Discovery Studio software were performed the dehydration of the receptor protein.To obtain a deeper understanding about the association of active ingredients with PPARγ , molecular docking was applied to evaluate the strength and mode of interactions. The crystal structure of PPARγ were obtained from the RCSB Protein Data Bank (PDB, p < 0.05 were considered to indicate statistical significance.All graphical data were expressed as means ± SEM. Unpaired Student’s t-tests were used as appropriate to evaluate the differences between two group means, and analysis of variance was use for multiple groups. Values of


This study aimed to investigate the factors influencing Procalcitonin (PCT) in the cerebrospinal fluid (CSF) of patients with high fever and suspected intracranial infection after neurosurgery and its clinical application value.Between February 2021 and August 2022, CSF and serum samples were collected via lumbar puncture from patients with high fever and suspected intracranial infection in the Intensive Care Unit(ICU) of our hospital. Multivariate logistic regression analysis was performed to analyze the factors influencing elevated PCT in CSF. The diagnostic efficacy of each index was assessed using receiver operating characteristic (ROC) curves.p < 0.01] and hemorrhagic CSF  were factors influencing CSF PCT, while trauma , epileptic seizure , age  and Glasgow Coma Scale (GCS) score  did not influence CSF PCT. The CSF PCT and serum PCT levels in the intracranial infection group and the case‒control group were 0.13  ng/ml and 0.14  ng/ml and 0.14  ng/ml and 0.23 ng/ml, respectively, with no statistically significant difference. The median values of CSF lactate in the intracranial infection group and the case‒control group were 6.45  mmol/l and 3.2  mmol/l, respectively, with a statistically significant difference between the groups.The areas under the ROC curve of CSF PCT, serum PCT,CSF lactate, CSF PCT combined with lactate were 0.59, 0.63, 0.82,and 0.83,respectively.A total of 183 CSF samples were collected, of which 148 had increased PCT levels, including 73 cases of intracranial infection and 75 cases in the case‒control group. Multivariate logistic regression analysis showed that intracranial infection [OR = 0.117, 95% CI: 0.025–0.559; Intracranial infection and hemorrhagic CSF are influencing factors for elevated CSF PCT following neurosurgery. It should be noted that the diagnostic value of intracranial infection by CSF PCT elevated alone is limited, but the combination it with other indicators can help improve diagnostic efficacy. Intracranial infection is a common complication following neurosurgery, with an incidence of 1.5–19.4% . DiagnosProcalcitonin (PCT) is an inflammatory index that increases significantly during severe infections. It is highly sensitive and not influenced by other factors. The International Sepsis Conference has designated PCT as a diagnostic indicator of sepsis, and it is widely utilized in the diagnosis and treating of bacterial infections , 4. ReceThis study was conducted in accordance with the Declaration of Helsinki (2000) of the World Medical Association. This study was performed in the ICU of People's Hospital of Ningbo University between February 2021 and August 2022 and was approved by the Ethics Committee of the Affiliated People's Hospital of Ningbo University [2021(006)].CSF and serum samples were collected from all ICU neurosurgery patients with high fever (T > 38.5 °C) who were suspected to have intracranial infection. Patients diagnosed with other nonintracranial infections , renal failure, immunosuppressant use, and confirmed intracranial infections were excluded from this study. Patients under the age of 18, pregnant women, patients with immune-mediated encephalitis, and patients with metabolic encephalopathy were also not eligible.In this study, we considered elevated CSF PCT levels to be those exceeding the normal reference value (0.05 ng/ml). All participants were divided into the intracranial infection groupor nonintracranial infection group (referred to as the case–control group in subsequent text).The diagnostic criteria for intracranial infection were as follows: (1) clinical manifestations such as postoperative fever, headache, neck stiffness, or change in consciousness; (2) glucose level of CSF < 2.5 mmol/L or a CSF/serum glucose ratio < 0.4; (3) a CSF white blood cell (WBC) count ≥ 1000 cells/µl and a polykaryocyte percentage ≥ 75%; and (4) positive CSF culture. The diagnosis of intracranial infection was made individually for patients meeting the fourth criterion. Intracranial infection was also diagnosed in patients with negative CSF culture results who met the other three diagnostic criteria.PCT detection method: CSF specimens were acquired by lumbar puncture for all patients. Protein, chlorine, glucose and lactate levels were determined using an automated chemical analyzer . PCT was detected by enzyme-linked fluorescence . The lower limit of positivity was 0.05 ng/ml. Peripheral venipuncture blood was collected under aseptic conditions no later than 30 min after CSF collection.p < 0.05), continuous data were expressed as the median, and nonparametric tests were used for comparisons between two groups, while counting data were compared by chi-square tests. Factors with a p value < 0.2 on univariate analysis were further analyzed by multivariate logistic regression for influencing CSF PCT. ROC curve analysis was used to analyze the diagnostic efficacy. P < 0.05 was considered statistically significant. The figures were drawn with GraphPad Prism, version 6.01 .Data were analyzed using SPSS 25.0 statistical software . Since the data of all parameters showed a skewed distribution . Among the collected samples, a total of148 were identified as having increased CSF PCT levels, including 73 in the intracranial infection group and 75in the case‒control group  groups.Howere, there were no significant differences in age, gender, Glasgow Coma Scale (GCS) score, epilepsy, serum and tumor Table . Multiva9] Table .Table 3Up > 0.05, Fig. p > 0.05, Fig. p < 0.05, Fig. p > 0.05, Fig. The median CSF PCT levels in the intracranial infection group and the case‒control group were 0.13 0.11, 0.25) ng/ml and 0.14  ng/ml, respectively, with no significant difference between the two groups (5 ng/ml a. However, there are conflicting results regarding whether this increase is due to the secretion of brain tissue or the elevated serum PCT levels after breaching the blood–brain barrier. The potential use of CSF PCT in diagnosing intracranial infection after neurosurgery remains a topic of debate. Additionally, it has been observed that some patients without intracranial infection also exhibit increased CSF PCT levels, suggesting that the elevation of CSF PCT may not be specific to infection.Studies have provided evidence that PCT in CSF increases in in cases of intracranial infection following neurosurgeryThis particular study discovered that not only did 73 patients with intracranial infection exhibit increased CSF PCT levels, but 75 patients without intracranial infection also showed similar elevations.These finding suggesting that the increase in CSF PCT may not be specific to infection and is instead nonspecific. Furthermore, when performing multivariate analysis, it was revealed that hemorrhagic CSF could also impact CSF PCT results. This finding aligns with a previous study by Wang et al. that reported increased PCT in the CSF of patients with subarachnoid hemorrhage . HoweverThe finding of this study may be biased due to the absence of multicenter support and the relatively small number of cases. The analysis of the specific pathogens causing intracranial infections was limited due to the small number of samples available. Furthermore, the correlation between the degree of hemorrhagic CSF and the level of PCT in CSF was not investigated in this study. Furthermore, this study did not investigate the source of CSF PCT, which is not conducive to the diagnostic value of CSF PCT for intracranial infection after neurosurgery.The presence of elevated PCT in the CSF of patients with high fever after neurosurgery is nonspecific and can be influenced by factors other than intracranial infection. Hemorrhagic CSF is an independent risk factor for increased CSF PCT. Therefore, relying solely on CSF PCT for diagnosing intracranial infections has limited diagnostic value. However, combining CSF PCT with other indicators may improve its diagnostic efficacy. It is important to note that the small sample size of this study warrants further investigation with larger samples and a multicenter approach.To gain a better understanding of the mechanism of CSF PCT changes during intracranial infections, we plan to establish a rabbit model of bacterial ventriculitis using different Gram staining types. This will allow us to explore the specific role of CSF PCT in the context of different types of bacterial infections. By conducting this study, we hope to enhance our understanding of CSF PCT as a diagnostic marker and contribute to the development of more accurate diagnostic tools for intracranial infections after neurosurgery.