In this work, we conducted a comprehensive investigation using in vivo, in vitro, and ex vivo approaches, involving animal and cellular models, to examine the pharmacokinetic properties and absorption mechanisms of TDS. The results of in vivo studies revealed that the half-life (t1/2) of TDS was 1.380 ± 0.46 h and 1.224 ± 0.39 h following intragastric (i.g.) and intravenous (i.v.) administration of 20 mg/kg TDS, respectively. This indicates that TDS is rapidly eliminated in rats. The area under the curve (AUC) of TDS after i.g. and i.v. administration was 114.7 ± 40 ng/mL*h and 48,400 ± 19,110 ng/mL*h, respectively, and the absolute bioavailability of TDS was found to be low (0.24%). Furthermore, TDS was extensively metabolized in rats, with the AUC of the major active metabolite [1-[2-pyrimidyl]-piperazine] being approximately 16.38-fold higher than that of TDS after i.g. administration. The results from the in vitro Caco-2 cell model and ex vivo everted gut sac experiment demonstrated that TDS exhibited good permeability, and its transport was influenced by concentration, temperature, and pH. Passive diffusion was identified as the main absorption mechanism. In conclusion, TDS is classified as a Biopharmaceutics Classification System (BCS) class I drug, characterized by high solubility and permeability. The low absolute bioavailability of TDS may be attributed to its rapid metabolism. The pharmacokinetic data and absorption characteristics obtained in this study provide fundamental information for the further development and utilization of TDS.Tandospirone citrate (TDS) is commonly used for the treatment of patients with generalized anxiety disorder in clinical practice, and several studies are developing new indications for TDS. However, the  Understanding the pharmacokinetic properties and absorption mechanisms of TDS is crucial for optimizing its therapeutic use and exploring new indications.Tandospirone citrate (TDS), a partial agonist of the 5-HT1A receptor, is widely used in China and Japan for the treatment of patients with anxiety disorders caused by various neuroses, as well as anxiety states associated with physical diseases. TDS was included in China’s National Essential Drugs List in 2018, highlighting its importance in clinical practice. Its therapeutic benefits extend beyond anxiety and encompass other central nervous system (CNS) disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia . As our in vivo. It guides the selection of administration routes and optimization of drug dosage forms, ultimately improving drug efficacy. In addition, understanding the absorption characteristics of a drug is essential for various aspects of drug development and application, including drug delivery route selection, formulation design, prescription screening, process optimization, and clinical use. Therefore, the aim of this study is to investigate the absolute bioavailability and absorption mechanism of TDS, providing data support for its further development and utilization. The major active metabolite of TDS is 1-[2-pyrimidyl]-piperazine (1-PP)  . Initialsorption , we emplsorption . Therefoin vivo, in vitro, and ex vivo studies, as well as animal and cellular models. The results will provide valuable insights for selecting the appropriate administration route, developing dosage forms, combining TDS with other drugs, and making informed decisions regarding its clinical application, especially when exploring new indications for TDS.In summary, this study comprehensively investigated the pharmacokinetic properties and absorption characteristics of TDS using various approaches, including TDS was provided by Sichuan Keruide Kaihua Pharmaceutical Co. Ltd. 1-PP was supplied by J&K. Dapoxetine (DAP) was purchased from Taizhou Haichen Pharmaceutical Co., Ltd. Atenolol (ATE) was purchased from Dalian Meilun Biotechnology Co., Ltd. Propranolol hydrochloride (PRO) was provided by National Institutes for Food and Drug Control. Benzoic acid (BA) and rifampicin (RFP) were purchased from Adamas. Hank’s balanced salt solution (HBSS) was purchased from Beyotime Biotechnology. MTT was purchased from J&K Scientific. Urethane was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.2.Caco-2 cells were obtained from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, and stored at West China School of Pharmacy, Sichuan University. The cells were cultured in Dulbecco’s modified Eagle medium and maintained in an incubator at 37°C with 5% COSprague–Dawley (SD) rats  were purchased from Chengdu Dossy Experimental Animals Co., Ltd. The animals were quarantined in the animal facility at the West China School of Pharmacy, Sichuan University in Chengdu, China.By adjusting the chromatographic conditions and mass spectrometry parameters, HPLC-MS/MS with high sensitivity and accuracy was established for the simultaneous determination of TDS and its main active metabolite 1-PP. We selected DAP as the internal standard (IS) due to its availability and cost-effectiveness. The HPLC-MS/MS analysis was performed using a CAPCELL PAK C18 column  with a mobile phase for the binary gradient consisting of acetonitrile-water . The flow rate was set at 0.4 mL/min, the column temperature was maintained at 40°C, and the injection volume was 10 μL. The gradient elution program of the mobile phase is provided in On the day of analysis, acetonitrile was used in a 4:1 ratio with the sample to either precipitate plasma proteins or dilute the sample. After centrifugation at 12,000 rpm for 10 min, the organic layer was separated, filtered, and transferred into a vial for injection into the HPLC-MS/MS system.i.g. represents the area under the drug–time curve after i.g. administration and AUCi.v. represents the area under the drug–time curve after i.v. administration. Di.g. represents the dose administered in the i.g. group and Di.v. represents the dose administered in the i.v. group.Twelve male SD rats were randomly divided into two groups: the i.g. group and the i.v. group. TDS was prepared in saline (0.9% NaCl aqueous solution), and both groups were administered TDS at a dose of 20 mg/kg. Blank blood samples were collected at 0 min before administration, and blood samples were collected at 2, 5, 8, 12, 20, 30, and 45 min, at 1, 2, 4, 7, and 10 h after administration. During the sampling process, rats were allowed to drink freely and food was provided 2 h after drug administration. The collected blood samples were placed in heparinized centrifuge tubes and centrifuged at 4,000 rpm for 10 min, and the plasma was separated and stored at −70°C. The plasma drug concentration was determined using HPLC-MS/MS, and the blood drug concentration–time data were fitted using DAS 3.0 pharmacokinetic software. Subsequently, the pharmacokinetic parameters of the i.g. group and the i.v. group were obtained. The absolute bioavailability was calculated using equation (Eq.) 1. AUC4 cells per well. After 24 h of culture, the cells were fully attached. The medium was then removed, and the drug-containing medium was added. Control wells without the drug-containing medium and blank wells without cells were also prepared. The cells were incubated for 12 h at 37°C in a 5% CO2 cell incubator. After incubation, the medium was discarded, and the cells were carefully washed three times with PBS. Subsequently, 200 μL of 0.5% MTT solution was added to each well in the dark, and the culture was continued for 4 h. The culture medium was then gently aspirated, and 100 μL of DMSO was added to each well. The cells were placed on a low-speed shaker for 10 min, and the absorbance value at 490 nm was measured using a microplate reader.When Caco-2 cells reached 80% confluence, they were seeded in 96-well cell culture plates at a density of 1×100 represents the OD value of the zeroed wells, and AControl represents the OD value of the control wells.The absorbance value at 490 nm for each well was recorded as A, and the cell inhibition rate was calculated according to Eq. ® inserts with a membrane area of 0.33 cm2 and a pore size of 0.4 μm at a density of 105 cells/cm2 per well .Caco-2 cells in the logarithmic growth phase were seeded onto 24-well polyester clear Transwellper well . Apical RT is the measured resistance value, R0 is the blank resistance value, and A is the polycarbonate film area.The trans-epithelial electrical resistance (TEER) was calculated according to Eq. 2 were selected for the transport experiment,  (cm·s−1) were calculated according to Eqs. Ci represents the concentration at the sampling time point in the receiving chamber, while C0 represents the initial concentration in the donor chamber.The uptake (Qi) and the apparent permeability coefficient . The abdominal cavity was opened along the midline of the abdomen, approximately 3 cm, and a section of the small intestine (approximately 10 cm) was quickly clipped. The animals were then euthanized by cervical dislocation. The intestinal tube was rinsed with iced normal saline until no content was flowing out. One end of the intestinal tube was ligated with a surgical thread, and then the tube was inverted using a glass rod. After cleaning with the K-R buffer (pH 7.4), the other end was ligated with a plastic tube to form an intestinal sac. In total, 1 mL of the K-R buffer at 37°C was injected into the intestinal sac, which was then placed in the blank K-R buffer and equilibrated for 5 min. Subsequently, the sac was transferred to a drug-containing K-R buffer. The experiment was conducted at a temperature of 37°C, and a gas mixture of 95% O2 and 5% CO2 was passed into the test solution. Samples of 100 μL were collected from the intestinal sac at 0, 30, 45, 60, 90, and 120 min and replenished with the same volume of the blank K-R buffer.According to A). The cumulative absorption amount (Q) (Eq. Papp)  and circumference (c) of the intestinal segment were measured to calculate the surface area ( (Q) Eq.  and the app) Eq.  were calt-test or one-way analysis of variance (ANOVA). A significance level of p < 0.05 was considered statistically significant. The following notation was used for indicating levels of significance: *p < 0.05, **p < 0.01, and ***p < 0.001.All experimental data were expressed as mean ± standard deviation. The data were processed using Prism 8.0 statistical software, and statistical analysis was performed by There are limited methods for simultaneous detection of TDS and its major active metabolite 1-PP. Although the existing HPLC-MS/MS method exhibits good sensitivity and selectivity, its practical application is somewhat restricted due to the requirement of expensive TDS-d8 and 1-PP-d8 as internal standards . In thisRepresentative chromatograms are presented in The pharmacokinetic data of TDS play a crucial role in the further development and utilization of this drug. As the major active metabolite of TDS, simultaneous measurement of plasma concentrations of TDS and 1-PP was conducted in this study. The plasma concentration–time curves of TDS and 1-PP in rats following i.g. and i.v. administration were fitted and depicted in tmax was 0.161 ± 0.09 h after i.g. administration. t1/2 of TDS was determined to be 1.380 ± 0.46 h and 1.224 ± 0.39 h after i.g. and i.v. administration, respectively. These indicated rapid absorption and elimination of TDS. The AUC(0-∞) values of TDS after i.g. and i.v. administration were 114.7 ± 41 ng/mL*h and 48,397 ± 19,107 ng/mL*h, respectively. The absolute bioavailability of TDS was calculated to be 0.24%. In addition, the AUC(0-∞) of 1-PP was approximately 16.38-fold higher than that of TDS after i.g. administration, indicating extensive metabolism of TDS in rats. According to in vivo, primarily mediated by CYP3A4, with 1-PP being its primary active metabolite. Due to its significant hepatic first-pass effect, its absolute bioavailability is very low. In addition, TDS was rapidly eliminated in vivo due to first-pass elimination in the liver, resulting in a short half-life. On the other hand, 1-PP had a longer half-life, and no significant first-pass effect was observed. Therefore, close attention should be paid to the pharmacological effects of 1-PP when exploring new indications for TDS.The results for TDS revealed that −1 TDS, 0∼200 μmol·L−1 ATE, 0∼200 μmol·L-1 PRO, 0∼20 mmol·L−1 BA, and 0∼100 μmol·L−1 RFP for 12 h. The results in The growth inhibition of drugs on the cells needs to be clear before carrying out the cell transport assay. Cells were treated with 0∼100 μmol·L2, the cells formed tight junctions  of ATE (50 μmol·L−1) was (4.1 ± 0.11) ×10–7 cm·s−1 and that of PRO (5 μmol·L−1) was (2.1 ± 3.44) ×10−5 cm·s−1  for TDS in the concentration range of 10–50 μmol·L−1, whereas Papp (AP-BL) at 5 μmol·L−1 was significantly higher than the other three concentrations. All of Papp (BL-AP) were (20–30) ×10−6 cm·s−1, with no significant differences. Papp (AP-BL) of 5 μmol·L−1 TDS was significantly higher than that of ATE and higher than that of PRO. Taken together, it is speculated that TDS has good permeability in Caco-2 cells and is similar to PRO. TDS had the best permeability at low concentrations, indicating that the transport of TDS was mainly passive diffusion, with uptake transporters also involved, but the effect of transporters was masked by passive diffusion at higher concentrations.To investigate whether the transport of TDS was concentration-dependent, combined with MTT results, 5 μmol·L−1) of TDS was selected to investigate the effect of temperature on the bidirectional transport. Papp (AP-BL) and Papp (BL-AP) were significantly decreased, with a more dramatic decrease observed in Papp (AP-BL). These results indicated that the transport of TDS in Caco-2 cells was temperature-sensitive and an energy-dependent process, suggesting the involvement of transporters in the transport from AP to BL.A low concentration (5 μmol·L−1 TDS in pH 5.5 HBSS. As shown in Papp (AP-BL), whereas Papp (BL-AP) showed no significant change. These results suggest that the transport of TDS in Caco-2 cells may be influenced by pH.The effect of pH on the bidirectional transport of TDS was investigated by preparing 5 μmol·LBased on the results obtained at different concentrations and temperatures, it can be speculated that uptake transporters may be involved in the transport of TDS. The uptake transporters on the villous side of human intestinal epithelial cells mainly include OATP, MCT1, PEPT1, and ASBT. Among them, OATP is involved in the transport of various drugs, including anticancer drugs, antibiotics, lipid-lowering drugs, glucose-lowering drugs, and toxins and poisons . In addi−1 BA and 50 μmol·L−1 RFP for the bidirectional transport experiments. Papp (AP-BL) and Papp (BL-AP) in the BA group were not significantly different from those in the control group, indicating that the transport of TDS in Caco-2 cells may not be affected by the MCT1 transporter. There was no significant difference in Papp (BL-AP) between the RFP group and the control group, whereas Papp (AP-BL) was significantly reduced, suggesting that the transport of TDS from AP to BL in Caco-2 cells may be affected by OATP transporters. However, Papp (AP-BL) of TDS in the RFP group was still larger than 10 × 10-6 cm s−1, indicating that the effect of OATP transporters on the transmembrane transport of TDS was limited, and the main transport way for TDS was passive diffusion.BA  and RFP ex vivo everted gut sac method is a classical approach for studying small intestinal absorption by quantitatively characterizing the permeability of drugs that pass through the intestinal mucosa. Therefore, we applied the everted gut sac method to investigate the intestinal absorption of TDS. The permeability rate curves of TDS at low, medium, and high concentrations were plotted by calculating the cumulative absorption, Q (R2 > 0.95), which allowed for the calculation of Papp. Papp of TDS at the medium concentration was significantly higher than that at the low and high concentrations, whereas there was no significant difference between the low and high concentrations. These results suggested the possible involvement of transporters in the transport of TDS. However, when MCT1 and OATP inhibitors were added, the results did not significantly differ from Papp in the control group, indicating that MCT1 and OATP transporters may not be involved in the absorption of TDS.The ption, Q . The curFrom cellular experiments, it was determined that OATP is involved in the transport of TDS, whereas from everted gut sac experiments, it was found that OATP is not involved in the absorption of TDS. According to the in vivo process and absorption characteristics of TDS. The in vivo model examined the pharmacokinetic properties of TDS in rats and determined an oral bioavailability of approximately 0.24%. The results of the in vitro Caco-2 cell model and ex vivo everted gut sac method indicated that TDS exhibited good permeability in the intestine, with passive diffusion being the main mode of drug absorption. Overall, TDS was classified as a Biopharmaceutics Classification System (BCS) class I drug with high solubility and high permeability, but its oral bioavailability was low due to rapid and extensive metabolism in the body. Understanding the characteristics of TDS, such as high permeability and low oral bioavailability, will serve as a reference for future development, including the selection of administration routes, formulation development, combination drug use, and rational clinical application.This work systematically investigated the


Our comprehensive characterization and control experiments indicated that the improved performance can be attributed to the combined effect of the dual MOF and the presence of phosphorous, influencing the battery-type supercapacitor behavior of GO. Additionally, we fabricated an asymmetric hybrid supercapacitor (AHSC) using Ni/Co-Hemin/PrGO/Nickel foam (NF) and activated carbon (AC)/NF. This AHSC demonstrated a specific capacitance of 281 C g−1 at 1.0 A g−1, an operating voltage of 1.80 V, an impressive energy density of 70.3 Wh kg−1 at a high power density of 0.9 kW kg−1. Notably, three AHSC devices connected in series successfully powered a clock for approximately 42 min. These findings highlight the potential application of Hemin-based MOFs in advanced supercapacitor systems.In this study, we conducted direct synthesis of a dual metal–organic framework (Ni/Co-Hemin MOF) on phosphorous-doped reduced graphene oxide (PrGO) to serve as an active material in high-performance asymmetrical supercapacitors. The nanocomposite was utilized as an active material in supercapacitors, exhibiting a noteworthy specific capacitance of 963 C g To survive the earth, renewable energy is essential to reducing greenhouse gas emissions and air pollution3. Hence, new energy generation technologies like solar4, wind5, and fuel cells6 require devices to store energy. Li-ion batteries and supercapacitors are the two main electrical energy storage systems. They developed over the years for portable devices as well as smart grid deployments7. Supercapacitors can store a large amount of charge compared to conventional capacitors, deliver energy quickly, have fast charging ability, have a long lifetime, offer superior low-temperature performance, are eco-friendly, and have low costs. Moreover, unlike batteries, they do not explode even if it is overcharged11.Energy is considered the most critical scientific subject in the twenty-first century12 illustrates supercapacitors' importance in their high specific power density. Besides, the capacitance in supercapacitors is influenced by equivalent series resistance and electrode and electrolyte materials and affects the operating voltage14. Therefore, for the best performance of a supercapacitor, it needs to have high capacitance, high operating voltage, and low resistance15. From these, electrode material, among all parameters, plays an essential role in developing the supercapacitor performance15. In other words, Hybrid supercapacitor devices are crucial for the advancement of electrochemical energy storage systems that can offer high energy storage capacity at a low operational cost16.On the other hand, Ragone plot19. Structures of this type offer the following advantages: high internal surface area, high porosity, structural and chemical tunability, and good stability. Besides, MOFs can be controlled in terms of porosity due to pore uniformity and atomic-level structure, dimension, geometry, functionality, and flexibility in network topology21. But, most pristine MOFs are poor conductors23. To overcome this deficiency, one of the most common strategies is to combine MOFs with carbon materials (reduced graphene oxide and carbon nanotubes) or conductive polymers (polypyrrole and polyaniline)24. Additionally, the incorporation of reduced graphene oxide (rGO) in composites can serve as an effective means of impeding the aggregation and restacking of graphene, both during the fabrication process and in practical usage25. The exceptional mechanical and chemical durability exhibited by rGO makes it an excellent scaffold material for the active component, which can effectively mitigate structural degradation and thereby enhance the cyclic stability of the system26.One of these electrode materials that appears effective is MOFs, porous hybrid materials consisting of metal ions or metal clusters coordinated to organic linkers29. However, the performance of GO in supercapacitors is limited due to nanosheet stacking and agglomeration. This problem exists because of the van der Waals forces and π–π interactions, therefore, it dramatically decreases the specific capacitance30. To put it more simply, macroscopic properties are affected by the electrode material microstructure31. Some researchers are used different methods, such as synthesis GO with porous 3D architectures in the form of hydrogels, organic gels, and foams to provide a larger surface area and better electron transport channels32. The other method is modifying GO, including chemical doping with different heteroatoms , which provide capacitive behavior and reduce the aggregation of GO sheets34. Among these heteroatoms, phosphorus great much attention for doping GO because the electronegativity of P (2.19) is lower than C (2.55), and the atomic radius size of P (195 pm) is more than C (170 pm), which causes polarization and distortion in the graphene lattice followed by the specific capacitance, surface area, and electrochemical performance were improved36. In other words, when phosphorus is doped in GO, P electrons contribute to the graphene π-system to increase charge carriers30.This research synthesized dual MOFs/ PrGO as an active material. In other words, carbon-based materials such as GO have been extensively studied and utilized in industrial production for capacitors and batteries. The advantages of GO for use in supercapacitors are abundance, non-toxicity, facile manufacturing, low cost, high chemical stability, good electronic and mechanical properties, high specific surface area, and a vast working temperature range38 and energy production, such as hydrogen and oxygen evolution reactions40. Besides, porphyrin-base structures such as Hemin have several advantages for utilizing active materials in supercapacitors. These structures have the following benefits: non-toxicity, low cost, and highly exposed active site in the active substance due to its highly conjugated structure (M–N4)42. Therefore, we tried synthesizing dual Ni/Co-Hemin MOF (NCH) and compositing with PrGO through the one-pot co-synthesis method to increase their cycle life, specific surface area, and pseudo-capacitance behavior. This method causes the uniform distribution of Ni-MOF and Co-MOF particles, and PrGO acts as a conductive bridge. Afterward, various spectroscopic and microscopic methods studied the structure of the obtained nanocomposite. The electrochemical behavior of this nanocomposite was first investigated in a three-electrode system for supercapacitor applications. The results of this study revealed that NCH/PrGO nanocomposite is a promising active material as an AHSC with wide operating voltage and acceptable value of energy density versus several related materials  and 43.8° (101) and the disappearance of the peak at 10.8° indicated that GO was reduced successfully30. Additionally, the broadening peaks of PrGO are due to a few layers of stacked graphene sheets forming the material's framework due to the poor stacking direction of graphene sheets32. As can be seen, in the NCH pattern, the main diffraction peaks at 5.8°, 9.1°, 19.9°, 33.7°, 36°, 39.1°, 52.8°, 59.7°, 61.3°, and 63.4° suggesting the crystalline structure of this MOF. Moreover, by comparing the XRD patterns of NCH with CH and NH, the diffraction peaks of both MOFs that appear in dual MOF can be concluded. According to the XRD pattern obtained from NCH/PrGO, its index peaks are equal to the patterns of dual MOF and PrGO, so their presence of them in the final nanocomposite is confirmed. Many attempts were done to obtain single crystal, but due to the very low solubility of this structure in most solvents, attempts were unsuccessful.Accordingly, X-ray diffraction (XRD) was used to characterize the structure of GO, PrGO, NH, CH, NCH, and NCH/PrGO Fig. A,B. In t−1 are attributed to the O–H, C–H, and C=N/C=C of the protoporphyrin (IX) ring system44. The band's intensity at 1703 cm−1 was attributed to the C=O stretching vibration that decreased in the NCH spectrum, compared with the Hemin spectrum. Furthermore, 1465, 1392, and 1004 cm−1 absorption bands are corresponded to the B3u vibration of porphyrin, –CH3 of Hemin, and =C–H deformation vibration of the olefin. All results confirm the successful coordination of the metals and –COOH groups46. Besides, spectrum f reveals the NCH/PrGO is well composite. The soret (~ 400 nm) and Q (~ 630 nm) bands of Hemin appeared in the UV–Vis spectrum of NCH  spectra of Hemin, NCH, and NCH/PrGO are illustrated in Fig. 47. The O 1s peaks at 529.8, 531.7, 532.9, 533.2, and 534.9 eV are associated with the P–OH, C=O/P=O, C–O/C–O–P, P–O–P, and water/carboxylic groups respectively48. The high-resolution P 2p XPS spectrum showed two peaks at 133.2 (C–P bond) and 134.3 eV (P–O bond)48. Moreover, the P–O peak intensity is higher than the P–C peak intensity revealing the GOs and phytic acids oxygen atoms led to the partial oxidation of P–C bonding in the thermal treatment30. Therefore, the results indicated that P had been successfully incorporated into the graphene lattices. It was stated in the introduction that reversible redox reactions in the P–O and C–P groups increase the overall energy density and, consequently, improve the electrochemical performance. The peaks at 700.1 (Fe pre-peak), 708.3 (Fe 2p3/2), 713 (Fe–O), and 734.1 (Fe 2p1/2) eV in a high-resolution scan of the Fe 2p region are related to Fe3+ in Hemin49. The main peaks appeared in the high-resolution spectrum of the Co 2P attributed to Co 2p3/2 and Co 2p1/2, which indicates the existence50 of Co2+ and Co3+. Likewise, the Ni 2p3/2 and Ni 2p1/2 spectra demonstrated four peaks that contribute to Ni2+  and Ni3+ in the nanocomposite45.To characterize the chemical state of the bonded element and the phase composition of NCH/PrGO, X-ray photoelectron spectroscopy (XPS) measurements were carried out. Figure 51. The morphology of CH is illustrated in Fig. − diffusion is reduced, and the nanocomposite has sufficient space to diminish deformation and improve the cycle life during charge and discharge.Microscopy images were used for the investigation of the morphology. Figure The high-resolution TEM (HR-TEM) image of NCH MOF is shown in Fig. According to the results obtained from the reported analysis, it can be concluded that the NCH MOF is successfully synthesized, and the NCH/PrGO nanocomposite is formed.The electrochemical performance of the different electrodes was investigated by cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS) in 1.0 M KOH solution in a three-electrode system.−1) at a scan rate of 20 mV s−1 in a potential window from 0.00 to 0.80 V vs. Hg/HgO. With increasing the amount of PrGO up to 0.6 g L−1, the area of the CV curve was increased; after this amount, the area dramatically decreased. In addition, this claim was proven by the Brunauer–Emmett–Teller (BET) data. As a result, the specific surface area of CH, NH, NCH, and NCH/PrGO was measured to be 40.21, 68.41, 71.44, and 92.54 m2 g−1, respectively. According to these observations, increasing the amount of P improves the capacitive behavior of the nanomaterial because of the lattice distortions of GO. Still, over-doping with P may damage graphene's original structure and reduce its capacitance and conductivity30. Moreover, as shown in Fig. 0.6 revealed the highest capacitance (963 C g−1), and this electrode was used for subsequent investigation. On the other hand, comparing their FE-SEM images gives us similar results with electrochemical data. In the FE-SEM image of NCH/PrGO0.2, due to the low amount of PrGO, we cannot see the PrGO sheets, and in the FE-SEM image of NCH/PrGO0.8, PrGO sheets cover the porosity of dual MOF structures  are obtained by zeroing the first-order derivative of the cost function. According to the cost function estimated at 0.6, the maximum capacity is obtained, which is very consistent with the value obtained from practical experiments.−1 in the potential window from 0.0 to 0.80 V vs. Hg/HgO. The NCH/PrGO/NF CV showed the highest current density, revealing excellent faradic behavior and a larger specific capacity. The results showed that a dual MOF announced superior performance than a monometallic MOF due to the synergic effects of the metals. The peaks in the NCH/PrGO/NF CV curve are related to the overlaid peaks of two pairs of the redox peaks of cobalt and nickel. Moreover, GO plays an important role in enhancing the electrochemical properties because of increasing the surface area and conductivity. P, as a dopant, increased the charge density 53 and caused structural distortions. Unlike the conventional methods, such as chemical vapor deposition, which produces hazardous gaseous for doping GO, the technique used in the present study for doping GO is safe. Additionally, phytic acid is a natural and green P source compared with other sources.Figure − ions. The shape of the CVs shows the quasi-reversible reaction. Additionally, the cathodic peak potential was shifted to a negative potential, while the anodic peak potential was moved to a positive value  at the interface between the electrode surface/electrolyte) of NCH/PrGO/NF displays a lower Rct (1.05 Ω) than NCH/NF (1.16 Ω), and NCH/rGO/NF (2.05 Ω). Moreover, In the low-frequency region, the Warburg resistance (Rw) varies as a function of the ion diffusion resistance of the electrolyte inside the electrode materials; the steep slope of the NCH/PrGO/NF curve shows the lower diffusive resistance and the pure capacitor behavior. Therefore, this electrode revealed fast electron transfer kinetics, lower internal resistance, and good conductivity.EIS is a perfect method to better study the charge-transfer behavior, interfacial properties, and capacitive properties of the electrodes. The Nyquist plot of the NCH/NF, NCH/rGO/NF, and NCH/PrGO/NF electrode from 100 kHz to 100 mHz frequency range, with an equivalent circuit (inset) plot for fitting the EIS data of NCH/PrGO/NF electrode at open circuit potential (OCP) is shown in Fig. −1) among other electrodes due to more active sites and high conductivity  and GCD (at 1.0 A g−1) curves of NCH/PrGO/NF//AC/NF AHSC device in different potential windows are illustrated in Fig. A significant shortcoming of supercapacitors is their low energy density and limited by the voltage window. The asymmetric hybrid supercapacitor (AHSC) was fabricated to improve the voltage window; this configuration consists of two different kinds of electrodes. The NCH/PrGO/NF//AC/NF was assembled as an AHSC device to evaluate energy and power density performance. The NCH/PrGO/NF and AC/NF were applied as positive and negative electrodes, respectively. Figure −1)  Fig. D. Conseq−1 are shown in Fig. −1 at the potential of 1.75 V for 10 h and repeated ten times. For every 10 h, 5 GCD test was performed, and capacity retention of 93.1% was maintained after 120 h 2·6H2O and 0.32 mmol Co(NO3)2·6H2O were added to 20 mL of each suspension and ultrasonic for 1 h. After that, 0.068 phytic acids and 0.15 mmol cetrimonium bromide (CTAB) were added to the solution and stirred for 30 min (solution A). Besides, 20 mL (18 mL distilled water + 2.0 mL ammonium hydroxide solution) of 0.50 mM Hemin solution was prepared (solution B). Afterward, solution B was gradually added to solution A and stirred for 15 min. The resulting solution was transferred into a 70 mL Teflon-lined stainless-steel autoclave, kept at 150 °C for 8 h, cooled naturally to room temperature, and washed with distilled water several times. Finally, the dark slate grey sediments  were collected and dried at 60 °C overnight.First, 0.2, 0.4, 0.6, and 0.8 mg mLApparatus and methods are described in the −1 at 1.0 A g−1 (68.3%) retained after increasing the current density 20 times), and good cycling life. The advantages of the composition of MOF with carbon-based material such as GO that is doped with heteroatom are (1) high conductivity, (2) non-toxicity, (3) high porosity, (4) high internal and specific surface area, (5) facile synthesis, (6) good stability, (7) chemical and structural tunability, (8) superior mechanical and electronic properties, (9) low cost, (10) enhance charge carriers, (11) increase pseudo-capacitance property, (12) synergistic effect of bimetallic and (13) prevent agglomeration. Moreover, Hemin is a promising ligand for synthesizing MOF that is used in supercapacitor applications for the first time and has some advantages, such as low cost, non-toxic, and highly conjugated structure. Furthermore, the AHSC was assembled on Ni/Co-Hemin MOF/PrGO/NF (as the positive electrode) and AC (as the negative electrode), resulting in a specific capacity of 281 C g−1 with a high energy density of 70.3 Wh kg−1 and power density of 0.9 kW kg−1. The excellent electrochemical performance confirms Ni/Co-Hemin MOF/PrGO's potential as an active material for AHSCs for future energy storage systems used in electronic devices and vehicles. Finally, this work aims to show the perspective of MOF-based Hemin  for energy storage systems.To summarize, novel Ni/Co-Hemin MOF/PrGO nanocomposite was synthesized as an active material for a high-performance hybrid supercapacitor. Afterward, this nanocomposite was characterized by different spectroscopic  and microscopic (FE-SEM and TEM) techniques. This nanocomposite demonstrates excellent electrochemical properties, including high specific capacity 963 C gSupplementary Information.Supplementary Video 1.


Opiates produce analgesia via G-protein signaling, and adverse effects, such as respiratory depression and decreased bowel motility, by β-arrestin pathway. Oliceridine, a G protein-biased MOR agonist, only presents modest safety advantages as compared to other opiates in clinical trials, possibly due to its limited bias. Our previous study shown that LPM3480392, a full MOR biased agonist, is selective for the Gi pathway over the β-arrestin-2. In the present article, we evaluated the subacute toxicity of LPM3480392 in rats. The rats were administered with control article or LPM3480392 0.6, 1.2 or 2.4 mg/kg/day for 4 consecutive weeks followed by a 4-week recovery phase. Intravenous infusion was conducted at tail vein at 0.2, 0.4 or 0.8 mg/kg/day with a dosing volume of 10 mL/kg and 5 min/rat/dose, three times a day with an interval of approximately 4 h. The concomitant toxicokinetics study was conducted. Two unscheduled rats at 2.4 mg/kg/day died with no clear cause. For the scheduled necropsy, the major effects were associated with the MOR agonist-related pharmacodynamic properties of LPM3480392  in three LPM3480392 groups. In addition, LPM3480392 at 2.4 mg/kg/day also induced deep respiration and histopathology changes in testis and epididymis in sporadic individual rats. However, different from other opiates, LPM3480392 presents weak/no immunosuppression and the decreased adrenal gland weight, which may be due to LPM3480392’ full MOR bias. At the end of recovery phase, all findings were recovered to some extent or completely. In the toxicokinetics study, the dose-dependent elevation of drug exposure was observed, which partly explained the toxicity of high dose. In summary, LPM3480392 has exhibited good safety characteristics in this subacute toxicity study in rats. Acute postoperative pain is experienced in more than 80% of all surgical patients, with the moderate to severe intensity in at least 75% of these patients . OpiatesAs a G protein-coupled receptor (GPCR), MOR has been revealed as the main receptor for both the analgesic and adverse effects of morphine . Signali® Trevena, PA, United States) was approved by the US Food and Drug Administration (FDA) for the treatment of moderate to severe pain on 8 Aug 2020  without β-arrestin-2 recruitment activity , good brain penetration, a favorable pharmacokinetic profile and produced potent antinociceptive effect with reduced respiratory suppression as compared to TRV130.In the previous study, we rationally designed and synthesized a series of potent highly biased MOR agonists through the modification and structure-activity relationship study of TRV130 . Among tPreviously, we have completed a series of toxicity studies in order to support the clinical trials in agreement with the 21 CFR Part 58 of US FDA, Good Laboratory Practice (GLP) regulations. Until now, LPM3480392 has completed phase I clinical trials  and is currently under phase II clinical trial (CTR20222358) as an analgesic for the treatment of moderate to severe pain. In this article, we present the subacute toxicity study for LPM3480392 in rats.LPM3480392 maleate injection (6.45 mg/5 mL) with a chemical purity of >97.9% was provided by the State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies  .ad libitum manufactured by Beijing Keao Xieli Feed Co., Ltd (Production License Number: Jingsi license (2018) 06,073). The animals were housed under 12 h light/dark cycles at 21.24°C–25.55°C in 45.23%–72.01% relative humidity environment. All animals had free access to water and food during the experimental periods. This subacute toxicity study has been conducted in WestChina-Frontier PharmaTech Co., Ltd. (WCFP). Animal experiments complied with the relevant regulations in Institutional Animal Care and Use Committee (IACUC) in WCFP.The Sprague Dawley (SD) rats of SPF grade  were from Beijing Vital River Laboratory Animal Co., Ltd. (Production license No.: SCXK (Jing) 016–0011), with the body weight of 132.8–174.6 g and the ages of 6–7 weeks. The rats were supplied with rat/mouse breeding diet The rats were assigned to 4 groups, including control group, and three LPM3480392 injection groups, with 19 rats/sex in each group. The rats were administered with control article (0.9% sodium chloride injection) or LPM3480392 injection 0.6, 1.2 or 2.4 mg/kg/day for 4 consecutive weeks followed by a 4-week recovery phase. Intravenous infusion was conducted at tail vein at 0.2, 0.4 or 0.8 mg/kg with a dosing volume of 10 mL/kg and 5 min/rat/dose using micro-injection pump, three times a day with an interval of approximately 4 h. The first dosing day was defined as Day 1, and the day after the last dosing was defined as Recovery day 1. At 1 day after the last dosing, 10 rats per sex in each group  were anesthetized with pentobarbital sodium  followed by abdominal aorta exsanguination for a gross necropsy based on AVMA Guidelines for the Euthanasia of Animals: 2020 Edition . At the end of recovery phase, the 5 animals per sex in each group  were sacrificed. For the toxicokinetics study, 4 rats/sex/group were used in three LPM3480392 injection groups.LPM3480392 injection was diluted with 0.9% NaCl injection to the required concentration under the aseptic condition. The dose formulations were prepared at least once every 8 days. The prepared dose formulations were used within 192 h when stored at 15°C–25°C. Dose formulations in control group and LPM3480392 groups were analyzed for the first and last dosing. The sample (0.100 mL) was collected from the middle layer of control formulation, or LPM3480392 injection dose formulations at each concentration. The absence of LPM3480392 should be confirmed in control formulations. The relative error (RE%) between detected result and nominal value should be within ±10% for LPM3480392 injection dose formulations at different concentrations.For all surviving animals, a morning and an afternoon observation every day were conducted for the moribundity and death. Daily observation was carried out once within 1 h after each dosing in dosing phase, and at least once daily in recovery phase, including mental status, food and water consumption, hair, discharge, mortality and other toxic reactions. Detailed observation was performed once every week, including appearance, movement, mental status, gland secretions, skin and mucosa color, respiration, genitals, mortality and other toxic reactions.Body weight was recorded twice a week during the dosing phase and then once a week during the recovery phase. Terminal body weight was recorded before each necropsy for calculating the organ weight ratios.Food consumption of all surviving animals was recorded twice a week during the dosing phase and then once a week in last 2 weeks of recovery phase.The examination was conducted on rats in control and 2.4 mg/kg/day groups at the end of dosing phase (Day 27) and at the end of recovery phase (Recovery day 27). The mydriasis  was conducted using Mydrin-P (compound tropicamide eye drops) before examination. Binocular indirect ophthalmoscope was used to examine conjunctiva, cornea, iris, lens, anterior chamber, posterior chamber and fundus.The blood samples were collected via abdominal aorta after anaesthetization with pentobarbital sodium  on the scheduled necropsy days of dosing phase and recovery phase. Hematological examination was performed using ADVIA 2120i hematology analyzer (Germany) and CS-5100 automatic coagulaiton analyzer (Japan) . The cliUrine samples were directly collected from the metabolic cage at the end of dosing phase (Day 26) and at the end of recovery phase (Recovery Day 28), respectively. Urinalysis parameters in Complete gross necropsies were conducted on all surviving animals at the end of treatment (the day after the last dosing) and recovery periods. The following selected organs were weighed and evaluated in terms of organ/body or organ/brain weight ratios: brain, kidneys, adrenals, testes, ovaries, uterus, epididymides, thymus, spleen, heart and liver. Paired organs were weighed together. Organ-to-body weight ratio = Organ weight(g)/Body weight(g) × 100%, and Organ-to-brain weight ratio = Organ weight(g)/Brain weight(g) × 100%.The tissues in the The animals found dead were recorded for the observation or death time, and weighed followed by a complete postmortem examination to explore the cause of death. All required organs and tissue as listed in the st dosing on Days 1 and 28; at 2 min and 4 h postdose of the 2nd dosing on Days 1 and 28; at 2 min, 1 h and 4 h postdose the 3rd dosing on Days 1 and 28. Animals in control group were sampled at predose and 2 min postdose of the 1st dosing on Days 1 and 28. Approximately 0.36 mL of blood was collected from the jugular vein of surviving TK animals at each time point. Whole blood samples were anticoagulated with EDTA-K2 and centrifuged at 2°C–8°C and 1800 × g for 10 min. The harvested plasma was stored below −66°C before the analysis. LC-MS/MS methods (liquid chromatograph/mass spectrometer) were used to detect concentrations of LPM3480392 at each time point. TK parameters  were calculated by Phoenix WinNonlin (Pharsight) 8.2. The detection instruments include ultra-performance liquid chromatography  and triple quadrupole tandem mass spectrometer .Blood samples from animals in LPM3480392 groups were collected at predose and 2 min, 30 min, 1 h and 4 h postdose of the 1et al. Qualitative data  of urinalysis (including urinary specific gravity at the end of dosing phase) were presented as observed counts (frequency). When sample size was less than three, raw data of that group were directly presented without statistical comparison.Quantitative parameters were described as means ± standard deviation (X ± SD). The parameters included body weight, food consumption, hematology, clinical chemistry, urinary specific gravity (only at the end of recovery phase), organ weight and ratios, p > 0.05), they were evaluated using one-way analysis of variance (ANOVA); in the case of heterogeneity of variances (p ≤ 0.05), Kruskal-Wallis (K-W) H test was used. And if ANOVA was with significant difference (p ≤ 0.05), Dunnett’s t-test (Dunnett) was used for pairwise comparisons; if ANOVA was not significant (p > 0.05), the statistical analysis was completed. When K-W H test was significant (p ≤ 0.05), Mann-Whitney (M-W) U test was used for pairwise comparisons; if K-W H test was not significant (p > 0.05), the statistical analysis was completed. Ordinal multi-category data were analysed by K-W H test. In the case of finding significant difference (p ≤ 0.05), M-W U test was used for pairwise comparison. Binomial category data were analysed by Fisher’s exact probabilities test (EXACT). If there was significant difference (p ≤ 0.05), Fisher EXACT was used for pairwise comparisons. Analyses were performed according to sex at two-tailed probability level (α = 0.05). PRISTIMA 7.2.0 was used for statistical analysis of body weight, hematology, clinical chemistry, organ weight and ratios. Stata/IC 15.0 for Windows was adopted for the statistical analysis of the rest data.Statistical analysis was performed between each LPM3480392 group and the control group. Levene’s test was used to analyse the variance homogeneity of quantitative data. In the case of homogeneity of variance  at 2.4 mg/kg/day were found dead on Days 3 and 15, respectively. They showed decreased activity and/or increased muscle tone, sternal recumbent posture after dosing, with no drug-related abnormalities in gross necropsy or histopathology.The rats in 0.6, 1.2 and 2.4 mg/kg/day groups displayed mild to severe increased activity, starting from Days 7, 4 and 1 during the dosing phase, respectively. And the animals in 2.4 mg/kg/day showed decreased activity  only a few time points during the dosing phase. Muscle tone was also increased in 1.2 mg/kg/day group  during the early stage of dosing phase in the first 5 days, and 2.4 mg/kg/day group  during the mostly complete dosing phase, following by the gradually decreased incidence with time.The rats in 2.4 mg/kg/day group occasionally showed hunchback , piloerection  and deep respiration or rale . During the middle and late stage of dosing phase, sporadic individual rats in 1.2 and 2.4 mg/kg/day groups had fester at the application site of tail or broken tail, with no drug-related abnormalities in histopathology examination, which was maybe caused by mechanical stimulation of long-term operation.LPM3480392-treated rats showed good general health, normal spontaneous activity, with no other toxic reaction during the recovery phase.During the dosing phase, body weight gains were dose-dependently decreased in males from Day 16 to Day 27 at 0.6 mg/kg/day, and from Day 6 to Day 27 at 1.2 and 2.4 mg/kg/day. Interestingly, during the recovery phase, at the four time points, body weight gains were deeply reduced in the three LPM3480392 groups .However, in females, body weight gains were increased at every time point during the dosing phase, while the increased degree of body weight gains seem to be less during the recovery phase .During the dosing phase, at every time points, food consumption in males was dose-dependently reduced. Especially, from Day 10 to Day 17, food consumption was deeply decreased compared with other time points. And, during the recovery phase, decreased food consumption obtained the partial recovery .However, in females, during the dosing phase or the recovery phase, the food consumption was decreased at some time points while increased at the other time points, which did not present obvious pattern .At the end of dosing phase and recovery phase, both genders in 2.4 mg/kg/day group were observed with clear dioptric media and optic disk. The diameter of retinal artery was even. Veins were without apparent dilation. No abnormalities were noted in retina, optic disk, conjunctiva, cornea, iris, lens, anterior chamber or posterior chamber.At the end of dosing phase, for the males, 2.4 mg/kg/day of LPM3480392 induced the significantly increased RET/RET% and the significantly decreased EOS/EOS% and MONO, and 1.2 mg/kg/day significantly increased RET/RET% and decreased ESO. At the end of recovery phase, 2.4 mg/kg/day significantly increased RET% .At the end of dosing phase, for the females, the significantly decreased MONO% or MONO in 0.6, 1.2 and 2.4 mg/kg/day groups, the significantly decreased RBC in 1.2 and 2.4 mg/kg/day groups, the increased MCV in 1.2 mg/kg/day group and the decreased PLT in 2.4 mg/kg/day group, were observed. At the end of recovery phase, the significantly increased LYM%, and the significantly decreased NEU/NEU%, and MONO% were noted in all LPM3480392 groups .p > 0.05).With the exception of the above changes, no significant differences were observed in other parameters in males or females treated with LPM3480392 at the end of dosing phase and recovery phase as compared with the control .With the exception of the above, no significant differences were observed in other clinical chemistry parameters for males or females treated with LPM3480392 at the end of dosing phase and recovery phase as compared with the control (p ≤ 0.05) at the end of dosing phase   .p > 0.05).With the exception of the above, there were no significant differences in urinary parameters for the males or females treated with LPM3480392 at the end of dosing phase and recovery phase as compared with the control .With the exception of above changes, no significant differences were noted in the absolute weight and ratios of other organs of LPM3480392-treated males or females at the end of dosing phase and recovery phase as compared with the control , the marked decreased sperm in the lumen and the minimal to moderate cell debris in the lumen of the epididymis (1/9) at the end of dosing phase. The above changes were not identified at the end of recovery phase .With the exception of the above, no microscopic changes were observed in brain, heart, liver, spleen, lungs, kidney, adrenal gland, thymus, lymph node, and gastrointestinal tract of LPM3480392 -treated rats at the end of dosing phase and recovery phase.0–12h of LPM3480392 in plasma had no apparent gender difference, and was increased generally in a dose-proportional manner on Day 1 and Day 28. No accumulation of LPM3480392 was noted after the 28-day repeated dosing.The concomitant toxicokinetics study was conducted for LPM3480392. Over the dose range from 0.6–2.4 mg/kg/day, the mean AUC0–12h were 315, 663 and 1250 h ng/mL for rats  in each group, with the between-group ratio of 1:2.1:4.0. On Day 28, mean AUC0–12h were 349, 680 and 1490 h ng/mL for rats  in each group, with the between-group ratio of 1:1.9:4.3. Compared with Day 28, mean AUC0–12h ratios of LPM3480392 in plasma were 1.1, 1.0 and 1.2 for rats  in each group on Day 1, respectively, . The rats were intravenously injected with LPM3480392 injection from 0.3 to 1.8 mg/kg/day for 30 consecutive days. Physical dependence was induced by LPM3480392 injection in rats after the spontaneous withdrawal. Increased scores of the withdrawal behaviours such as wet dog shaking, writhing, chew, teeth chattering and eyelid ptosis were noted in LPM3480392 injection groups after the drug withdrawal. These withdrawal behaviours had a gradually recovery trend, which had reserved to normal on Recovery day 6 for rats administered with LPM3480392 injection. After the withdrawal, body weight of male rats in LPM3480392 injection group significantly decreased and then recovered to normal within the withdrawal recovery phase. There were no apparent abnormalities in body weights of female rats in LPM3480392 injection groups during dosing and recovery phases. The effects of LPM3480392 on body weight in males or females in the spontaneous withdrawal test are similar to those in the subacute toxicity study in rats.0–12h and Cmax of LPM3480392 were increased generally in a dose-proportional manner on Day 1 and Day 28. No accumulation of LPM3480392 was noted after the 28-day repeated dosing. After every LPM3480392 treatment, the drug was eliminated fast in the body . All the changes were considered not toxicologically significant because of their small magnitude of changes, evidence of reversibility, and absence of microscopic correlation.The effects of decreased food intake on a series of toxicological parameters in rats have been widely understood, including haematology, clinical chemistry, urinalysis, organ weight, and histopathology . The redIn urinalysis, the decreased grade distribution of NIT and KET in the males of 1.2 and/or 2.4 mg/kg/day groups, and the increased grade distribution of pH in the females of 2.4 mg/kg/day group were observed at the end of dosing phase; and PH changes in 0.6 mg/kg/day group were also noted at the end of recovery phase. The above changes had no mostly toxicologically significant, as no abnormalities were noted in serum renal function related parameters or histopathology of kidney and urinary system.It is generally accepted that µ-opioid agonists such as morphine and fentanyl activate the hypothalamus-pituitary-adrenal (HPA) axis in rodents , and chrAt the end of recovery phase, the decreased absolute weight and organ-to-brain weight ratio of epididymides were observed in 2.4 mg/kg/day group. Sporadic individual males at 2.4 mg/kg/day were observed with the mild tubular degeneration of the testis, the marked decreased sperm in the lumen and the minimal to moderate cell debris in the lumen of the epididymis. The changes of testis and epididymis were maybe target organ toxicity for LPM3480392. However, the above microscopic changes of testis and epididymis were recovered at the end of recovery phase.0–12h was increased generally in a dose-proportional manner. No accumulation of LPM3480392 was noted after the 28-day repeated dosing. The highest drug exposure was observed at 2.4 mg/kg/day, which partly explained the toxicity, including the unscheduled death, the clinical signs of increased activity, increased muscle tone, sternal recumbent posture, deep respiration; microscopic changes of tubular degeneration of the testis, and decreased sperm in the lumen and cell debris in the lumen of the epididymis.The toxicokinetics study shown that typical linear kinetics characteristics were presented on Day 1 and Day 28 after LPM3480392 administration, and the mean AUCIn this study, there are several limitations: (1) We missed the observation time points for the opiates withdrawal such as the decreased body weight and food consumption, and wet-dog shaking during the first days of recovery phase. (2) Morphine can induce mydriasis in rats . HoweverIn summary, LPM3480392 presents weak/no immunosuppression and the decreased adrenal gland weight, which is different from other opiates. The main reason may be that LPM3480392 is a full MOR biased agonist. Further experiments need to be warranted in order to study the effects of LPM3480392 on immune system and HPA axis.


We aimed to probe the safety and effectiveness of flexible ureteroscopic lithotripsy (FURL) with a suctioning ureteral access sheath (S-UAS) for removing upper urinary calculi under local anesthesia (LA).The clinical data of 56 patients with upper urinary calculi treated by FURL with an S-UAS under LA during the period between September 2019 and November 2022 were analyzed retrospectively. For LA, intramuscular pethidine (1.0–2.0 mg/kg) and phenergan (25 mg) were administered 30 min prior to surgery, and oxybuprocaine hydrochloride gel was administered through the urethra at the start of the surgery. The S-UAS and flexible ureteroscope were used for FURL. Demographic characteristics, stone-related parameters, and clinical outcomes were analyzed.A total of 66 procedures were performed successfully on 46 patients (Group A), who underwent unilateral surgeries, and on 10 patients (Group B) who underwent same-session bilateral surgeries. All 56 patients were operated upon without altering the anesthesia strategy, and none required additional analgesia. The mean stone sizes of the Group A and Group B patients were 20.24 ± 5.45 mm and 29.40 ± 3.89 mm, respectively. The mean operative times of the two groups were 53.04 ± 13.35 min and 90.00 ± 15.81 min, respectively. In Group A, the stone-free rates (SFRs) were 76.1% (35/46) and 85.1% (40/46) at postoperative day 1 and day 30, respectively. In Group B, the SFRs were 80.0% (16/20) and 85.0% (17/20), respectively. Four (8.7%) patients in Group A suffered complications such as fever, stent pain, urosepsis, and steinstrasse. In Group B, one (10%) patient suffered from fever.FURL, combined with an S-UAS under LA, is a feasible option and provides satisfactory clinical outcomes for appropriately selected patients. HoweverFURL is regularly performed under general or regional anesthesia  but rare2.2.1.The medical records of patients with upper urinary calculi who underwent FURL with an S-UAS under LA at the First Affiliated Hospital of Gannan Medical University during the period between September 2019 and November 2022 were retrospectively reviewed, and these patients were included in the study. The exclusion criteria were as follows: (a) lower urinary tract calculi; (b) middle or distal ureteral stones; (c) preoperative ureteral structure or calculous pyonephrosis; (d) combined with upper urinary carcinoma. For two patients, the method of treatment was changed to PCNL under LA because of a narrow ureter. Finally, a total of 56 patients were included in our study. All patients were diagnosed by preoperative urinary non-contrast computed tomography (NCCT). For patients with normal renal function, intravenous urography (IVU) was recommended. The stone size was defined as the largest diameter measured by NCCT, and for multiple stones or bilateral upper urinary stones, the size was the sum of the largest diameter of each stone. Urinalysis and urine culture were routinely examined and the stones were treated with appropriate antibiotics preoperatively. Preoperative demographic characteristics such as gender, age, American Society of Anesthesiologists (ASA) score, body mass index (BMI), surgical side, ipsilateral surgical history, midstream urine culture result, stone parameters, hydronephrosis, and preoperative ureteral stent placement were obtained according to medical records.Ethical approval for the study protocol was obtained from the Ethics Committee of the First Affiliated Hospital of Gannan Medical University (proof number: 2023032706), and the study was performed in accordance with the Declaration of Helsinki (as revised in 2013). Written informed consent was obtained from all participants.2.2.All patients were explicated with all alternative therapeutic strategies and anesthetic methods. Written informed consent was granted before the operation. For patients who selected FURL under LA, intramuscular pethidine (1.0–2.0 mg/kg) and phenergan (25 mg) were administered 30 min prior to surgery. After the patients were placed in the lithotomy position, the oxybuprocaine hydrochloride gel (10 ml gel containing 30 mg oxybuprocaine) was injected into the urethra for mucosal anesthesia and lubrication. A ureteroscopy inspection was performed by using a semirigid 6/7.5 Fr ureteroscope, and then a guide wire was inserted in the ureter. If a proximal ureter stone was detected, the stone was pushed retrogradely to the renal pelvis. Under the guidance of the wire, an 11/13 F or 12/14 F S-UAS , combined with a vacuum aspiration device, was inserted depending on the condition of the ureter , and theThe operative time, hemoglobin loss, visual analog scale (VAS) score, stone-free rate (SFR), and complication rate (CR) were analyzed. Kidney-ureter-bladder (KUB) graphy and/or urinary NCCT were performed at 1 day and 1 month after surgery, and a stone-free status was defined as “no remaining stone.” For patients who underwent bilateral FURL, the operation time was defined as “the total surgical time of two sides.” The double-J stent was routinely removed a month following surgery. For patients with ureteral stenosis or residual stones, a second procedure was performed 1 month after surgery.3.A total of 66 flexible ureteroscopic procedures were performed on 56 patients with upper urinary calculi, who included 46 patients (Group A) who underwent unilateral surgeries and 10 patients (Group B) who underwent same-session bilateral surgeries. In Group A, 22 patients underwent surgery on the left side and 24 patients on the right side. All procedures were successfully performed under local anesthesia without shifting to general or regional anesthesia, and none of them required additional analgesia during the performance of the surgeries.Females constituted the majority of the two groupings. A total of nine patients in Group A and two patients in Group B were evaluated as high anesthesia risk (ASA III-V) patients. Comorbidities of the patients were hypertension, diabetes mellitus, coronary atherosclerotic heart disease, chronic obstructive pulmonary disease, renal insufficiency, and cerebral infarction. Two patients in Group A and one patient in Group B took daily aspirin, but there was no need to stop aspirin before FURL. The mean stone size was 20.24 ± 5.45 mm in Group A patients and 29.40 ± 3.89 mm in Group B patients. Preoperative ureteral stents on the surgical side were seen in 36 patients in Group A and 8 patients in Group B. The demographic characteristics and stone-related parameters are given in A total of 44 patients were prestented, including four patients with high general anesthesia risk who underwent regular replacement of a double-J stent because of calculous hydronephrosis, three patients who received a stent for sepsis; the remaining 37 patients were prestented for ureter dilation. In Group A, all prestented patients and two patients without preoperative stent were inserted with 12/14 F S-UAS, and the remaining patients were inserted with 11/13 F S-UAS. All 10 patients in Group B were treated with 12/14 F S-UAS. The mean operative time was 53.04 ± 13.35 min in Group A patients and 90.00 ± 15.81 min in Group B patients, and the mean hemoglobin loss was −4.78 ± 9.22 g/L and −4.10 ± 9.09 g/L, respectively. The intraoperative mean visual analog scale scores and scores at 6 and 24 h after surgery in Group A patients were 3.83 ± 0.53, 2.23 ± 0.64, and 1.22 ± 0.79, respectively. In Group B patients, the intraoperative mean visual analog scale scores and scores at 6 and 24 h after surgery were 3.10 ± 0.74, 2.20 ± 0.63, and 1.20 ± 0.63, respectively. The mean postoperative hospitalization time was 3.43 ± 1.70 days and 3.20 ± 1.40 days, and 35.7% (20/56) of patients had a postoperative hospitalization time of more than 3 days, predominantly because of the intrinsic features of our medical strategy and insurance policy and the tertiary hospital referral system and not because of surgery.In our study, all patients received postoperative KUB. For patients with suspicious fragments based on KUB, a CT was done, and 30.3% (17/56) of patients were checked with NCCT. The SFRs of Group A patients at postoperative day 1 and day 30 were 76.1% and 85.1%, respectively. In Group B patients, the SFRs of the surgical ipsilateral side at postoperative day 1 and day 30 were 80.0% and 85.0%, respectively.Four (8.7%) patients in Group A suffered from the following complications: 1 (2.2%) patient suffered from fever (Clavien grade I), 1 (2.2%) patient who had stent pain (Clavien grade I) was treated with a steroidal anti-inflammatory agent, 1 (2.2%) patient who had urosepsis only needed additional antibiotics (Clavien grade II), and 1 (2.2%) patient with steinstrasse was treated with ureteroscopic lithotripsy under LA. In Group B, 1 (10%) patient suffered from fever (Clavien grade I). No ureter injuries were observed in the two groups. More details of the clinical outcomes are given in 4.Because of the miniaturization of novel FURS and the development of lithotripsy devices, FURL is being increasingly performed nowadays because of its superior minimally invasive characteristics and satisfactory success rates. FURL is usually performed under general or regional anesthesia by anesthesiologists and urologists  but is rWe first reported FURL, combined with S-UAS, for urinary calculi under LA. Unlike the LA method described in a previous study conducted by Pai et al. , which uFor patients who underwent LA, preoperative ureteral stenting was advised, and these patients constituted 78.3% in Group A and 80.0% in Group B. The benefits of prestenting were continuous relief of hydronephrosis and alleviation of obstructive pain caused by edematous mucosa . Stent pIn our study, we evaluated the intraoperative and postoperative VAS scores of patients who underwent FURL under LA. All procedures were successfully completed without additional analgesia, and the mean intraoperative VAS scores were 3.83 ± 0.53 in Group A and 3.10 ± 0.74 in Group B. A previous study indicated that high pelvis pressure was associated with intraoperative pain , 17. AccExcept for one patient who needed an oral steroidal anti-inflammatory agent after surgery because of stent pain, all patients tolerated postoperative pain. Multiple studies have focused on factors related to postoperative pain after FURL , 19–22. The SFRs at postoperative day 30 were 85.1% (40/46) in Group A patients and 85.0% 17/20) in Group B patients, which were consistent with the outcomes of the study conducted by Pai et al. /20 in Gr, our SFRThe overall CR in our study was 8.7% in the unilateral group, which was comparable with outcomes reported in a previous study , but it Same-session bilateral FURL is a favorable therapy with a satisfactory SFR and an acceptable CR; however, it may prolong operation time and cause renal damage . Ten patOur study had several limitations. A major limitation was that it was a retrospective study with a limited sample size, and therefore, potential patient selection bias could not be ruled out. Second, a control group was lacking in the study. Third, we recorded the intraoperative VAS score, but the VAS score of different surgical procedures were not recorded, these procedures included ureteroscopy inspection, UAS insertion, ureteroscopic manipulation, and lithotripsy procedures. Therefore, a professionally designed study with a large sample size is recommended for the future.5.FURL combined with an S-UAS under LA is a feasible option and provides satisfactory clinical outcomes for appropriately selected patients. A professionally designed study with a large sample size is recommended for the future.


Streptococcus pneumoniae is one of the main causes of community-acquired infections in the lung alveoli in children and the elderly. Alveolar macrophages (AM) patrol alveoli in homeostasis and under infectious conditions. However, the molecular adaptations of AM upon infections with Streptococcus pneumoniae are incompletely resolved.Streptococcus pneumoniae.We used a comparative transcriptomic and proteomic approach to provide novel insights into the cellular mechanism that changes the molecular signature of AM during lung infections. Using a tandem mass spectrometry approach to murine cell-sorted AM, we revealed significant proteomic changes upon lung infection with AM showed a strong neutrophil-associated proteomic signature, such as expression of CD11b, MPO, neutrophil gelatinases, and elastases, which was associated with phagocytosis of recruited neutrophils. Transcriptomic analysis indicated intrinsic expression of CD11b by AM. Moreover, comparative transcriptomic and proteomic profiling identified CD11b as the central molecular hub in AM, which influenced neutrophil recruitment, activation, and migration.Streptococcus pneumoniae and reveals profound alterations critical for effective antimicrobial immunity.In conclusion, our study provides novel insights into the intrinsic molecular adaptations of AM upon lung infection with   Streptococcus pneumoniae (S. pneumoniae)  . Childreumoniae) . Alveolaumoniae) , 4. Uponumoniae) , 6. Actiumoniae) . Importanfection , 9. Subsnfection , but theS. pneumoniae  is a cell surface protein that is present in various immune cells. AM express only low levels of this integrin in homeostasis , but inceumoniae . Regardieumoniae , suggestad libitum. C57BL/6 mice (WT), 8–12 weeks of age, from Charles River, were used. The animal experiments were approved by the local animal review board of the government , 81-02.04.2017.A470. Experimental mice were euthanized with cervical dislocation under isoflurane anesthesia for the isolation of organs.Mice were bred and housed under specific pathogen-free conditions in the central animal facility at the University Hospital Essen and were allowed to eat and drink S. pneumoniae SV1, ATCC 33400) was used. S. pneumoniae were grown overnight on Columbia blood agar plates  at 37°C, single colonies were resuspended and cultured in 10 ml Brain–Heart Infusion Broth  to mid-logarithmic phase , and 800 µl of culture was frozen at −80°C with 200 µl of 86% glycerol. For infection, bacteria were cultured to mid-logarithmic phase, centrifuged at 1,500×g for 10 min at 4°C, and resuspended in 550 µl PBS; 50 µl per animal corresponding to 1 × 108 CFU S. pneumoniae was used for infection.For bacterial lung infection, a pneumococcal serotype 1 strain , a 22-G cannula with a blunted end was inserted into the mouse trachea. The needle was removed, and 50 µl of the liquid was pipetted into the cannula. MiniVent 845  was connected to the cannula to evenly spread the liquid throughout the lungs and ventilate the lungs.For bronchoalveolar lavage (BAL) extraction, the fur was wetted with ethanol and dissected to expose the rib cage. The diaphragm and lower part of the rib cage were removed to visualize the lung. The trachea was exposed by the removal of salivary glands and adventitia and cannulated with a 22-G needle . Ice-cold PBS containing 2 mM EDTA was injected with a 1-ml syringe. The first lung lavage was performed with 0.7 ml and three subsequent washes with 0.5 ml of buffer. Cell-free supernatant from the first lavage was used for the assessment of soluble molecules by ELISA, and cell pellets were combined from all four washes and used to analyze the cellular compartment by flow cytometry. The determination of BAL fluid CXCL1 was carried out using the Multiplex assay from Bio-Rad Laboratories GmbH, Kapellenstr. 12, 85622 Feldkirchen, Germany (171G5018M) according to the manufacturer’s protocol. Total protein content in the BAL was determined using Pierce™ BCA Protein Assay Kit (#23225) following the manufacturer’s protocol.g, and processed for flow cytometry.To obtain a single-cell suspension of murine lungs, the fur was wetted with ethanol and dissected to expose the rib cage. The diaphragm was removed, and the whole lung was cut out and separated from the heart. Lungs were disaggregated with 3 ml of PBS supplemented with 0.5 mg/ml collagenase D and 0.2 mg/ml DNAse I and placed in a six-well plate. Each lobe was injected with the digestion medium using a 26-G needle to facilitate the diffusion of the medium through the tissue. After 5 min of incubation at RT, the lung was disrupted with fine-toothed forceps and transferred into 5 ml polystyrene tubes, which were incubated in a water bath for 30 min. The tissue was resuspended every 10 min to mechanically boost the digestion process. The resulting single-cell suspension was filtered (100 µm), pelleted for 5 min at 4°C and 400×c = 1.5 mg/ml, PZN-01700625, CSL Behring GmbH, Emil-von-Behring-Straße 76, 35041 Marburg, Germany) and 20 μl was added onto cell pellets, followed by a 20-min incubation at 4°C in the dark. Either 0.05 µg or 0.1 µg of each antibody were used per sample. Unbound antibodies were washed away, and the cell pellets were resuspended in PBS with 2 mM EDTA and 2% FCS (FACS buffer) for subsequent measurement on the BD LSR Fortessa II. Ten thousand Calibrite™ APC beads  were added to each sample to extrapolate the total cell number in the lung. BD Aria III was used for sorting AM for transcriptome and proteome analyses. Briefly, AM were identified as CD45+, CD11c+, and Siglec-F+ cells, whereas neutrophils were delineated based on CD45, CD11b, and Ly6G expression.The single-cell suspension  was liberated from erythrocytes using RCB buffer . Antibodies were mixed in a solution of human immunoglobulins . Briefly, the cell pellet was resuspended in 100 µl of 4% PFA and incubated at 4°C for 10 min. Subsequently, cells were washed with 2 ml FACS buffer, followed by a wash with 2 ml of BD Perm/Wash . A 15-min incubation step with 1 ml of perm/wash was carried out to transiently permeabilize the cells. After that, anti-mouse MPO  was added at 0.1 µg per sample and incubated for 30 min at 4°C in the dark. Next, Streptavidin Alexa Fluor 647  was added at 1 µg per sample and incubated for 30 min at 4°C in the dark. The final washing step was carried out with 1 ml perm/wash, and the samples were redissolved in FACS buffer for the acquisition.5 cells from the lung single-cell digests were resuspended in 100 µl of complete RPMI 1640 and incubated with 1 µM CellROX Deep red dye for 30 min at 37°C. Excess dye was washed away with 1 ml PBS, and cells were subsequently stained for extracellular markers.AM reactive oxygen species (ROS) production was determined by flow cytometry using the CellROX kit from molecular probes  according to the manufacturer’s protocol. Briefly, 3 × 105) was lysed in 50 mM ammonium bicarbonate buffer containing 0.1% RapiGest Surfactant. The samples were reduced with 20 mM dithiothreitol for 30 min at 60°C and subsequently alkylated with 15 mM iodoacetamide for 30 min at room temperature, protected from light. Proteins were digested using 22.5 ng of trypsin per sample overnight at 37°C. The digestion was stopped by adding 0.5% trifluoroacetic acid, and the precipitated RapiGest was removed by centrifugation. Peptides were dried in vacuo, and 350 ng of peptides in 15 µl of 0.1% trifluoroacetic acid was subjected to an Orbitrap Elite mass spectrometer coupled to an Ultimate 3000 RSLCnano system. The peptides were preconcentrated for 7 min on a trap column  and subsequently separated on an analytical column  by a gradient from 5% to 40% solvent B over 98 min . Full-scan MS spectra were acquired by data-dependent acquisition (DDA) in the Orbitrap analyzer. Tandem spectra were measured in the linear ion trap following peptide fragmentation by collision-induced dissociation.A detailed description of sample preparation, protein detection, and analysis can be found in ref . In brieMus musculus using the Mascot search engine (v.2.5). The mass tolerance was set to 5 ppm for precursor ions and 0.4 Da for fragment ions. One tryptic miscleavage was considered as well as chemical modifications of methionine (oxidation) and cysteine (propionamide). The percolator function, implemented in the proteome discoverer, was used to estimate peptide confidence, and only peptides that passed a false discovery rate (FDR) of<1% (FDR-adjusted p< 0.01) were considered for analysis. Ion intensity-based label-free quantification was performed using Progenesis QI for proteomics. To account for retention time shifts, LC-MS runs were aligned to one run automatically chosen by the software. A master list of features considering retention time and m/z was generated considering peptide ions with a minimum three isotopic peaks and charges state of +2, +3, and +4. The peptide identifications (peptide spectrum matches) from Proteome Discoverer were imported into the software and matched to the respective features. The protein abundances were calculated considering the normalized ion intensities of all nonconflicting peptides of a protein. Normalized protein intensities were further analyzed using bioinformatics and statistical analysis.Peptides were identified using the Proteome Discoverer Software (v.1.4.1.14). The mass spectra were searched against the UniProtKB/Swiss-Prot database  restricted to 5) were sorted using BD FACS Aria III in 100 µl of RLT lysis buffer , and RNA was extracted using RNeasy Micro Kit  according to the manufacturer’s protocol. Using the Affymetrix IVT Pico protocol, 10 ng of total RNA was prepared and then analyzed on the Clariom S mouse array . Hybridization, washing, and staining of the arrays were done on the GC Scanner 3000. RNA signal summarization and analysis of differentially expressed genes were performed using Partek GS v6.6 software.AM , and Cytoscape  were used for functional enrichment analysis. The normalized enrichment score (NES) was calculated according to a previously published algorithm (U test (two groups) or Kruskal–Wallis test (more than groups). A two-way analysis of variance was applied for the comparison of multiple parameters .Overrepresentation , gene set enrichment  and circlize (0.4.14).Processed transcriptome and proteome data are available as supplementary tables. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD035269. The transcriptome data have been deposited via the NCBI partner repository with the GEO submission (GSE213547) and the NCBI tracking system #23319940.S. pneumoniae, and the number of CD45+, Siglec-F+, and CD11c+ AM was determined until day 3 postinfection  and scavenger receptors, such as TLR2, TLR4, and MARCO (S. pneumoniae by cytosolic PRRs leads to the induction of type-I interferon (IFN)  , which won (IFN)  and PRRson (IFN)  signalinS. pneumoniae infections. We reveal transcriptomic and proteomic adaptations of AM during lung infection with S. pneumoniae, regulation of the integrin CD11b, and identification of profound molecular alterations in the AM compartment that are critical for effective antimicrobial immunity and regulation of inflammation.In summary, our detailed molecular and functional analysis indicated critical AM-specific adaptions in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was approved by LANUV, Recklinghausen, Germany. The study was conducted in accordance with the local legislation and institutional requirements.The experiments were performed by DK, DVT, JB, KZ, NK, NS, RC, and ST. Mass spectrometry and transcriptomic measurements, data analysis, visualization, and computational analysis were performed by BB, BS, DS, JJ, LK-H, ML, OS, PS, and TB. Critical reagents and discussion were provided by DT, FW, and VJ. The study was written by KZ, ST, OS, and DRE and supervised by DRE. All authors have read and commented on the article. All authors contributed to the article and approved the submitted version.


Impaired iron transport (IIT) is a form of iron deficiency (ID) defined as transferrin saturation (TSAT) < 20% irrespective of serum ferritin levels. It is frequently observed in heart failure (HF) where it negatively affects prognosis irrespective of anaemia.In this retrospective study we searched for a surrogate biomarker of IIT.We tested the predictive power of red distribution width (RDW), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) to detect IIT in 797 non-anaemic HF patients.p = 0.0092) in the true negative vs. false negative group. Therefore, we divided the study population according to eGFR value: 109 patients with eGFR ≥ 90 ml/min/1.73 m2, 318 patients with eGFR 60–89 ml/min/1.73 m2, 308 patients with eGFR 30–59 ml/min/1.73 m2 and 62 patients with eGFR < 30 ml/min/1.73 m2. In the first group, positive and negative predictive values were 48 and 81% respectively, 51 and 85% in the second group, 48 and 73% in the third group and 43 and 67% in the fourth group.At ROC analysis, RDW provided the best AUC (0.6928). An RDW cut-off value of 14.2% identified patients with IIT, with positive and negative predictive values of 48 and 80%, respectively. Comparison between the true and false negative groups showed that estimated glomerular filtration rate (eGFR) was significantly higher (2.RDW may be seen as a reliable marker to exclude IIT in non-anaemic HF patients with eGFR ≥60 ml/min/1.73 m These are the reasons why an impaired iron metabolism can lead to systemic consequences (Iron Deficiency (ID) is frequently reported affecting more than 2 billion people worldwide . It is tequences .As reported in several guidelines , 8 the dCurrent European Society of Cardiology (ESC) Guidelines recommend that HF patients be periodically screened for ID ; nonethe2.We retrospectively collected data of 1,496 patients hospitalized for HF between January 2016 and November 2021 in the HF Unit of Centro Cardiologico Monzino .The study inclusion criteria were: HF hospitalization, presence of ID screening and absence of anaemia. Patients were defined as anaemic when haemoglobin levels were below 13 g/dl in males and 12 g/dl in females according to WHO  definition of anaemia , iron status  and renal function .At the time of hospital admission, all patients underwent complete cardiac ultrasound evaluation including left ventricular ejection fraction (LVEF) by biplane method .The study was approved by the local ethics committee (CCM04_21 PA).2.1.t-test or Mann-Whitney U-test, while categorical variables were compared by Chi-square test or Fisher's exact test, as appropriate.Continuous variables are presented as mean and standard deviation (SD) if normally distributed, otherwise as median and interquartile range (IQR). Categorical variables are reported as frequencies and percentages. Continuous variables between groups were compared by Through estimation of the area under the curve (AUC) of the ROC curve, the predictive ability of RDW, MCV and MCHC to detect patients with possible IIT was assessed. These parameters were arbitrarily chosen because part of the routine hematopoietic laboratory analysis and all were presumed to be linked to iron status .After finding the best parameter, it was investigated whether any further predictive ability was reached by a combination of the above reported variables.A cross-validation procedure was employed to calculate the best cut-off of selected parameter(s) to identify IIT patients. The study population was randomly split in half 200 times, the best cut-off by Euclidean distance was estimated in the first arm (training set), and its sensitivity and specificity were subsequently tested in the second half (testing set). The mean value of each cut-off was considered.Thereafter we compared the true negative and false negative groups to identify the demographic (age and sex) and laboratory  features.2, 30–60 ml/min/1.73 m2, 60–90 ml/min/1.73 m2 and ≥90 ml/min/1.73 m2. In each group sensitivity, specificity, positive predictive value and negative predictive value were calculated.Since we found out that RDW's relationship with TSAT levels significantly changed with the eGFR value, a further analysis was conducted by dividing the study population according to eGFR: <30 ml/min/1.73 mp-value < 0.05 was considered statistically significant. Statistical analyses were performed with SAS software, version 9.4 .A 3.Seven-hundred-ninety-seven non-anaemic HF patients were identified and evaluated in the present report. Two-hundred-sixty-four cases, 33.1% of the study population, had TSAT values < 20% and therefore were classified as IIT patients.Demographic and clinical characteristics of the entire population and grouping patients according to the presence/absence of IIT are displayed in We tested RDW, MCV and MCHC as possible IIT predictors: RDW performed the best with an AUC of 0.6891 . In the p = 0.0092) in the true negative (72 ± 22 ml/min/1.73 m2) vs. false negative group (65 ± 23 ml/min/1.73 m2). Borderline significant differences in the two groups resulted also for gender (p = 0.0495)  and false negative (85 subjects) patients. Among the parameters analysed, eGFR by MDRD formula was significantly higher ( 0.0495) .2, 308 (38.6%) patients had eGFR ranging between 30 and 60 ml/min/1.73 m2, 318 (39.9%) patients had eGFR between 60 and 90 ml/min/1.73 m2 and 109 (13.7%) patients had eGFR ≥90 ml/min/1.73 m2. On average, RDW decreases as renal function improves. Specifically, RDW levels were 15.8 ± 2.7% in the group with eGFR <30 ml/min/1.73 m2, 14.6 ± 3.8% with eGFR between 30 and 59 ml/min/1.73 m2, 14.3 ± 4.9% with eGFR between 60 and 89 ml/min/1.73 m2 and 13.8 ± 1.3% with eGFR ≥90 ml/min/1.73 m2. IIT was identified in the eGFR group <30 ml/min/1.73 m2 with a sensitivity of 76%, specificity of 32%, PPV of 43% and NPV of 67%, in eGFR ≥30 and <60 ml/min/1.73 m2 group, they were 73%, 48%, 48% and 73% respectively, in the eGFR ≥60 and <90 ml/min/1.73 m2 group, they were 64%, 77%, 51% and 85% respectively, in the eGFR ≥90 ml/min/1.73 m2 group, they were 48%, 81%, 48% and 81% respectively (Thereafter we grouped our patients according to eGFR. Sixty-two (7.8%) patients had eGFR <30 ml/min/1.73 mectively . Accordi4.2. Similarly, a very high negative predictive value was observed in HF patients with normal or moderately reduced eGFR, meaning that in these patients the presence of ITT can be reasonably excluded in patients with normal RDW. On the other hand, the negative predictive value of the proposed cut-off moderately decreases with eGFR ranging between 60 and 30 ml/min/1.73 m2, and even more under 30 ml/min/1.73 m2.In this study we found that IIT is present in around 1/3 of non-anaemic HF patients and IIT presence can be assumed in case of elevated anisocytosis as suggested by a high RDW value. Specifically, a value of 14.2% is the most precise RDW cut-off to suggest IIT in non-anaemic HF patients. The highest specificity of RDW as an indicator of IIT was found in non-anaemic HF patients with preserved renal function with an eGFR ≥90 ml/min/1.73 m2 and high VE/VCO2 slope, being both established tools for defining prognosis in HF patients . In addition, IIT is the form of ID which is most linked with a worse prognosis in HF patients, as previously shown by a few reports . Moreovepatients , 16. Accpatients . Non-anaRDW is calculated as the standard deviation of MCV ÷ MCV × 100 and is the most common indicator of anisocytosis, i.e., a high variability of red blood cell diameter generally due to ineffective erythropoiesis. An elevated RDW value is frequently associated with chronic illness, as in HF, and associated to disease progression , 19. It Our findings extend those of Van Craenenbroeck et al. who showed that RDW may be associated with ID in HF patients with a reduction of RDW after ID treatment with ferric carboxymaltose . Indeed,This study presents confirmatory and novel data about ID in HF and specifically as regards IIT, the form of ID which is related to the worst exercise performance and HF prognosis . Of noteThe present study has a few limitations which should be acknowledged. First, the retrospective nature of the study. Second, the population of non-anaemic HF patients we studied is relatively small and larger population studies are needed. Third, HF patients were recruited regardless of the ejection fraction value, the HF etiology or severity therefore we do not know whether our findings could apply differently in specific HF phenotypes such as, for instance, patients with low, middle range and preserved ejection fraction. Similarly, we do not know whether our findings could apply in anaemic HF patients or in HF patients with different comorbidities such as hepatic, renal, neurological diseases. Fourth, we analyzed IIT and not all forms of ID. Accordingly, we do not know whether RDW is associated with ID in specific settings such as in ID patients with TSAT > 20%. Fifth, we used cut-off values for grouping patients according to RDW and renal function. Albeit clinically useful, it must be underlined that cut-off values of any parameters are not reliable to evaluate measurements that are very close to the cut-off itself. Sixth, eGFR was obtained by MDRD formula. We recognized that other formulas such as CKD-EPI may be effectively used. However, MDRD is frequently used in HF. Furthermore, no patients have acute kidney injury, which is the condition where MDRD fails more frequently to properly predict eGFR. Finally, we have not evaluated whether an effective IIT treatment influences RDW, albeit some data show a reduction of RDW in ID patients after treatment (In conclusion, albeit several HF guidelines recommend ID detection, this is not routinely performed in clinical practice. The present study suggests that RDW, a cheap and frequently available parameter, is a reliable predictor of IIT in HF patients. The presence of an elevated RDW value reinforces the need for ID evaluation in non-anaemic HF patients, while a normal RDW, particularly in subjects with preserved kidney function, is rarely associated with IIT being the latter the strongest predictive element of the present analysis.


We aimed to explore the relationship between serum phosphate concentration and 90-day mortality in critically ill children receiving continuous renal replacement therapy (CRRT).Data from the medical records of children aged <13 years who received CRRT at the Pediatric Intensive Care Unit of Hunan Children's Hospital, China from January 2015 to June 2020 were retrospectively collected. Children were grouped into four categories according to the baseline phosphate concentration before CRRT and mean serum phosphate concentration during CRRT: <0.81 mmol/L (hypophosphatemia), 0.81–1.19 mmol/L, 1.2–2.4 mmol/L , and >2.4 mmol/L (hyperphosphatemia), with the normal phosphate group serving as the comparator group. The correlation of the serum phosphate concentration before and during CRRT with the 90-day mortality after CRRT initiation was analyzed using logistic regression.P = 0.008). The mean serum phosphate concentration during CRRT was 1.2 mmol/L . The 90-day mortality rate was increased in children with a mean serum phosphate concentration >2.4 mmol/L during CRRT .A total of 177 children were included in our study. The mean serum phosphate concentration before CRRT was 1.46 mmol/L . The 90-day mortality rate was increased in children with a serum phosphate concentration >2.4 mmol/L before CRRT (adjusted odds ratio [aOR] 3.74, 95% confidence interval [CI] 1.42–9.86, Hyperphosphatemia before and during CRRT predicts a higher 90-day mortality rate. CurrentPhosphate concentration is typically abnormal in critically ill patients. There have been an increasing number of studies on the relationship between phosphate abnormalities and clinical outcomes –9. A recPhosphate, a small inorganic solute, is removed through diffusion or convection during continuous renal replacement therapy (CRRT). Patients receiving CRRT have an increased risk of developing hypophosphatemia, and this has been associated with poor prognosis , 14. Pat2.2.1.Data from the medical records of children who underwent CRRT in the Department of Critical Care Medicine at Hunan Children's Hospital from January 2015 to June 2020 were retrospectively collected. The normal serum phosphate concentration of children fluctuates slightly with age, whereas the reference value for children aged >13 years fluctuates greatly. Therefore, to facilitate group statistics, children >13 years of age were excluded from this study , 16. TheThis study was conducted with approval from the Ethics Committee of Hunan Children's Hospital . This was a retrospective observational study; data were acquired from the electronic case records database; this research did not involve aspects related to patient privacy; and there was no commercial interest involved. Thus, the ethics committee waived the requirement for informed patient consent.2.2.The equipment used for CRRT at our center was the multiFiltrate system  or PRISMAflex System , including the ancillary tubing and filters. For vascular access, a double-lumen central venous catheter was inserted in the femoral or internal jugular vein. The treatment mode was continuous veno-venous hemodialysis or hemodiafiltration, heparin or sodium citrate was the anticoagulation agent, and phosphate-free replacement and dialysis fluids were used. Electrolytes were routinely monitored, every 6–8 h, and vital signs, hemodynamics, and coagulation were closely monitored throughout the CRRT treatment process. CRRT treatment parameters were titrated, as needed.2.3.Clinical and biochemical parameters that were recorded included sex, age (months), weight, Pediatric Critical Illness Score , whetherThe main predictive factors in this study were as follows: (1) serum phosphate concentration before CRRT initiation and (2) mean serum phosphate concentration during CRRT. The serum phosphate concentration before CRRT initiation was defined as the test result closest and within 24 h prior to CRRT initiation. The primary outcome was the 90-day mortality rate after CRRT initiation.The range of normal serum phosphate concentration in children under 13 years of age fluctuated slightly with age, and the range from the lower limit of reference value 1.2 mmol/L (3.7 mg/dl) to the upper limit 2.4 mmol/L (7.4 mg/dl) was considered the range of normal serum phosphate concentration in these children , 19. Curvia slow intravenous infusion, with a maximum of 10 ml/session, and the decision to reintroduce intravenous phosphate was made according to the serum phosphate concentration.When the serum phosphate concentration was <0.81 mmol/L, 0.5–1 ml/kg/session sodium glycerophosphate was empirically administered Participants were divided into four groups according to their serum phosphate concentration: <0.81 mmol/L, 0.81–1.19 mmol/L, 1.2–2.4 mmol/L, and >2.4 mmol/L, with the 1.2–2.4 mmol/L group—the normal range for serum phosphate in children—set as the comparator group.2.4.M], and the Kruskal–Wallis H test was used for comparisons between groups.Data were analyzed using IBM SPSS Statistics, version 25.0 . Data with a normal distribution are expressed as mean ± standard deviation  of the relationship between the serum phosphate concentration and the 90-day mortality rate. Confounding factors were controlled by adjusting for clinically relevant variables, including sex, age (in months), weight, Pediatric Critical Illness Score, whether or not mechanical ventilation was used, whether or not vasopressors were used, serum creatinine, albumin, sodium, potassium, and calcium concentrations, blood pH, and blood platelet count. Covariables were selected by force entry. Four-node restricted cubic splines were constructed to study the nonlinear relationship of the serum phosphate concentration before (baseline) and during CRRT with the 90-day mortality rate.P < 0.05. Regression analyses and model constructions were performed using R-studio, version 4.2.1 ; glm was used for the logistic regression, and rms was used for the restricted cubic spline curves.Differences were considered statistically significant at 3.3.1.n = 81) required mechanical ventilation and 13.6% (n = 24) required vasopressors . The 90-day mortality rates of children with serum phosphate concentrations <0.81, 0.81–1.19, 1.2–2.4, and >2.4 mmol/L before CRRT were 50%, 35.7%, 26.6%, and 55.3%, respectively. Multivariate logistic regression analysis showed a higher 90-day mortality rate in children with a serum phosphate concentration >2.4 mmol/L before CRRT than in children with a serum phosphate concentration of 1.2–2.4 mmol/L [adjusted OR 3.74, confidence interval (CI) 1.42–9.86, ity rate . Figure 3.3.P = 0.011). Hypophosphatemia during CRRT was not significantly correlated with mortality . The 90-day mortality rates of children with a mean serum phosphate concentration <0.81, 0.81–1.19, 1.2–2.4, and >2.4 mmol/L during CRRT were 34.5%, 32.2%, 33.3%, and 63.6%, respectively. Multivariate logistic regression analysis showed a higher 90-day mortality rate in children with a mean serum phosphate concentration >2.4 mmol/L during CRRT than in children with a serum phosphate concentration of 1.2–2.4 mmol/L  mortality rates. In an earlier study, Thongprayoon et al. . This finding was consistent with those of several recent studies conducted among adults which have reported that hyperphosphatemia during CRRT predicts a higher risk of mortality (P < 0.001) and 1.50-fold  increased risk of 28-day and 90-day mortality, respectively  levels, and this directly induces the rognosis –28. Hypeity rate , 30.P > 0.05). However, urine volume was not included in the multivariate analysis to take into account the effect of pRIFLE score on results, and this is indeed a limitation. In addition, according to standard practice, the ratio of the population to variables should be 10:1. However, in the present study, we did not select covariables through single-factor analysis, and all clinically relevant variables were included: this is a potential limitation of this study.This study has some limitations. First, it is a single-center retrospective observational study, and there may be a potential for bias as well as confounding factors that could have affected our interpretation of the results. Second, the data were captured from an electronic medical records database and the system neither archived information on the causes of phosphate disturbances at baseline and during CRRT, nor denoted the causes of mortality. Therefore, the causal relationship between serum phosphate concentration and mortality remains unclear, and it is not possible to ascertain whether patient mortality was due to hyperphosphatemia or other underlying causes. Third, the efficacy of CRRT in phosphate removal varies with the effluent dose . We did Phosphate is a potential biomarker of disease severity in critically ill patients receiving CRRT and is associated with a high mortality rate. Early identification and treatment of hyperphosphatemia is therefore important in patients receiving CRRT in the ICU. In future, we plan to conduct a randomized controlled trial that incorporates the phosphate concentration into the risk stratification and explores whether reducing the phosphate concentration, such as by CRRT, may reduce mortality risk in patients with hyperphosphatemia.