Plasmodium species and Toxoplasma gondii threatens disease control worldwide, implying that new agents/therapeutic targets are urgently needed. Nuclear localization signal (NLS)-dependent transport into the nucleus, mediated by members of the importin (IMP) superfamily of nuclear transporters, has shown potential as a target for intervention to limit viral infection. Here, we show for the first time that IMPα from P. falciparum and T. gondii have promise as targets for small molecule inhibitors. We use high-throughput screening to identify agents able to inhibit P. falciparum IMPα binding to a P. falciparum NLS, identifying a number of compounds that inhibit binding in the µM-nM range, through direct binding to P. falciparum IMPα, as shown in thermostability assays. Of these, BAY 11-7085 is shown to be a specific inhibitor of P. falciparum IMPα-NLS recognition. Importantly, a number of the inhibitors limited growth by both P. falciparum and T. gondii. The results strengthen the hypothesis that apicomplexan IMPα proteins have potential as therapeutic targets to aid in identifying novel agents for two important, yet neglected, parasitic diseases.The global burden of malaria and toxoplasmosis has been limited by the use of efficacious anti-parasitic agents, however, emerging resistance in  Plasmodium spp., Toxoplasma gondii, Cyclospora spp., and Cryptosporidium spp., can cause severe disease in humans [Plasmodium spp. in 2020 [T. gondii chronically infects about one-third of the human population worldwide with the majority of infections being asymptomatic, however, infection of immunocompromised individuals (such as those suffering from autoimmune diseases syndrome—AIDS) can result in severe toxoplasmosis, whilst in the case of pregnant women, hydrocephaly/microcephaly can occur in new-born babies [Organisms from the phylum apicomplexa, such as n humans ,2. The W in 2020 , with >6n babies ,5,6. Emen babies ,8, with Signal-dependent transport into and out of the eukaryotic cell nucleus, mediated by members of the importin (IMP) superfamily of proteins, is central to processes such as cell differentiation, transformation, development, and infection and immunity ,11,12,13IMPs have been used in high-throughput screens (HTS) to identify small molecules that target IMPα/β-dependent nuclear import of viral proteins central to infection for Human Immunodeficiency Virus, dengue, and Venezuelan Equine Encephalitis Virus ,22,23,24P. falciparum and T. gondii divide rapidly within the human host implies a need for efficient nuclear transport systems [P. falciparum and T. gondii have a single genomic copy of IMPα [P. falciparum IMPα (PfIMPα) is known to bind to a “classical NLS” identified in P. falciparum trimethyl guanosine synthase 1 (TGS1) that methylates the terminal phosphate groups of spliceosomal RNAs [P. falciparum and T. gondii.The fact that  systems , raising of IMPα  that is  of IMPα ,34. P. fmal RNAs ,36, yet mal RNAs , which amal RNAs ,16. ApicT. gondii IMPα (TgIMPα) and the well-characterized Simian Virus 40 T-antigen (SV40 T-ag) NLS, with the compound Bay 11-7085 showing high selectivity for PfIMPα. Thermostability assays were used to confirm the direct binding of Bay 11-7085 and other compounds to PfIMPα, and their ability to inhibit growth by both P. falciparum and T. gondii was also confirmed. The results establish the principle that apicomplexan IMPα is a viable target for drug discovery to combat malaria as well as toxoplasmosis.Here we describe, for the first time, a high-throughput screen (HTS) to identify small molecule inhibitors of PfIMPα: TGS1-NLS interaction using AlphaScreen technology ,22,23,38All restriction enzymes were obtained from Thermo Fisher Scientific , and gene amplification was performed using the KAPA HiFiTM PCR kit  unless otherwise indicated. Primers were procured from Integrated DNA Technologies . The integrity of all plasmid constructs was confirmed by DNA sequencing.CCATGGAGCGCAAGTTGGCCGATC 3′, Reverse: 5′ TCCCAAGCTTCTGGCCGAAGTTGAAGCCTC 3′; restriction sites underlined).To generate a TgIMPα C-terminal His-tagged bacterial expression construct, the TgIMPα coding sequence was PCR-amplified from cDNA and inserted between the NcoI and HindIII restriction sites of plasmid vector pET28a. Primers used to amplify the TgIMPα gene were . P. falciparum (PlasmoDB ID: PF3D7_0812400) and T. gondii IMPα (ToxoDB ID: TGGT1_252290) with C-terminal hexa-Histidine tags were subcloned as PCR products into the NcoI and Nhe I sites of the pET28a-C GST vector. The primers used for PCR amplification were as follows: , with PfIMPα amplified from the previously described hexa-Histidine-tagged expression construction [To generate a C-terminal glutathione S-transferase (GST) fusion protein expression construct in plasmid vector pET28a in place of the C-terminal hexa-Histidine tag, the GST coding sequence was PCR amplified from plasmid pGEX-6p1 and inserted into the NheI and HindIII restriction sites of pET28a. The primers used to amplify the GST gene sequence were , ΔIBBMmIMPα, and β1 (MmIMPβ) GST fusion proteins were expressed and purified essentially as previously [2+-affinity chromatography as described [PfIMPα, TgIMPα, eviously ,22,23. Hescribed . His-tagescribed . Biotinyescribed . AlphaScescribed ,29,30,38HTS in the AlphaScreen system was performed robotically as previously ,22,23 toBriefly, 5 μL of each compound in DMSO was added to quadruplicate wells of a 384-well plate along with appropriate controls including DMSO alone and ivermectin, as previously , using aPlates were read for an Alphascreen signal on an Envision plate reader , with signals from the negative control wells (no bait control) on each plate subtracted. Compounds showing more than 70% inhibition (DMSO negative control = 100%) were tested to exclude false-positives by counter-screening in the system using 5 nM hexa-His-Biotin in place of hexa-His-tagged and biotinylated proteins ,22,23.m is the temperature at which 50% of the protein is unfolded.The effect of inhibitory compounds on IMPα thermostability was tested, as previously, using the fluorescent dye SYPRO Orange  and the Qiagen Rotor-Gene Q6 plex instrument programmed in the melt curve mode ,30. IncrP. falciparum 3D7 strain in human red blood cells  were thawed and cultured using RPMI  1640 medium supplemented with 0.5% Albumax , 50 mg/L hypoxanthine , 2 g/L D-glucose , 2 g/L sodium bicarbonate , and 56 mg/L of gentamicin  at 37 °C in 5% CO2 in a humidified incubator according to the standard procedures [Red blood cells (RBCs) were derived from blood from volunteers  by density gradient centrifugation. Aliquots of cryopreserved ocedures ; suspensP. falciparum life cycle. The test compounds auranofin, Bay 11-7085, CAPE  and Zoxazolamine-MMV003270  were made up at a stock concentration of 10 mM in DMSO and diluted in RPMI as required for growth inhibition studies or IC50 analysis, with dihydroartemisinin  used as a control.Before growth inhibition assays, cultures were synchronized using 5% D-sorbitol  predominantly to obtain the ring stage of the 2. 72 h later, the plates were subjected to freeze–thaw to achieve hemolysis, and samples diluted 200-fold in H2O in transferring to another plate. A day prior to analysis, 96-well ELISA plates  were coated overnight at 4 °C with 100 µL of 1 μg/mL IgM capture antibody MPFM-55A  specific for P. falciparum HRP2. After blocking for 2 h at room temperature with 2% BSA in PBS, diluted samples were added to the wells, followed by incubation for 2 h, then the plates were washed three times with 0.05% Tween 20 in PBS. Next, 100 µL of the secondary antibody MPFG-55P  conjugated with horseradish peroxidase (0.25 μg/mL in PBS with 2% BSA) was then added to each well, and the plates incubated for 1 h at room temperature, prior to being washed three times, and 100 µL 3,3′, 5,5′ tetramethylbenzidine substrate  being added. After 10 min incubation in the dark, 50 µL of 1 M H2SO4 was added and the absorbance was read at 450 nm on a Multiscan™ FC Microplate Spectrophotometer .Growth assays in the absence and presence of inhibitors were performed using the Histidine–Rich Protein 2 (HRP2) sandwich horseradish peroxidase-linked immunosorbent assay to measure HRP2 protein levels as an indicator of growth ,41. BrieT. gondii RH strain expressing the luciferase reporter (RH-Fluc) was derived to assess the effect of the inhibitors on growth. Briefly, plasmid pCTG-EGFP [T. gondii TubulinA promoter, was electroporated into the T. gondii RH strain [T. gondii line expressing the luciferase reporter was selected for resistance to 20 µM chloramphenicol (encoded by the plasmid pCTG), and dilution cloning [7 RLU from 107 parasites) used for growth assays. T. gondii tachyzoites were maintained and cultured at 37 °C in 5% CO2 in a humidified incubator in primary human foreskin fibroblasts  [TM, Waltham, MA, USA) supplemented with 3.7 g/L sodium bicarbonate and 2.38 g/L HEPES, 10% Cosmic Calf serum  and 20 mg/L gentamicin [CTG-EGFP  was modiH strain ,44 usingH strain ,46. The  cloning  was perfF, ATCC) ,48; HFF ntamicin ,48.Growth inhibition was assessed by luminescence measurements as described ,50. BrieThe MTT assay was used to measure the cytotoxicity of the small molecules against the HFF cells as described . Briefly50) and d is the slope at the steepest part of the curve (the Hill slope).Four parameter dose response curves were fitted using non-linear regression analysis in GraphPad Prism 9.2.0  using the formula: y = a + ((b − a)/(1 + 10^(Log(c) − x) × d)), where a is the minimum asymptote, b is the maximum asymptote, c is the half-maximal inhibitory concentration value  to the TGS1 NLS, comparable to that for MmΔIBBIMPα binding to the well-characterized simian virus SV40 large tumor antigen (T-ag) . The results are summarized in To determine the extent to which the hit compounds may be selective for PfIMPα, we compared their ability to inhibit NLS binding by PfIMPα, mammalian non-autoinhibited MmIMPα (MmΔIBBIMPα) as well as the MmIMPα/β heterodimer, and the apicomplexan At 10 μM, 11 of the compounds showed stronger inhibition of the PfIMPα:NLS interaction than of the MmΔIBBIMPα:NLS or TgIMPα:NLS interactions , indicatBay 11-7085 appeared to be highly selective for PfIMPα (75% inhibition) with 0–14% inhibition of the other IMPαs and the MmIMPα/β heterodimer . MMV030750 analysis for the three IMPα:NLS interactions  compared to that for the PfIMPα-TGS-NLS interaction (2 µM)  of all three IMPα:NLS interactions  and MMV0033270  showed a similar inhibitory profile, although they were not as potent  ,Table 3,ractions , consistA thermostability assay (TSA) was used as previously ,30 to exP. falciparum and T. gondii IMPα in a physiological context, we screened the compounds against both parasites at a single concentration (10 µM). For P. falciparum, we employed the standard HRP2-based (histidine-rich protein 2) ELISA, with the clinically prescribed drug dihydroartemisinin (DHA) as a positive control against P. falciparum at 10 µM [To begin to assess formally the importance of NLS recognition by at 10 µM ,41,53. IT. gondii, we used tachyzoites constitutively expressing luciferase . Through cycles of counter screening, selectivity testing, IC50 analysis, and antiparasitic testing, it proved possible to identify several small molecules that have the ability to both inhibit PfIMPα:NLS binding, and limit the growth of P. falciparum and/or T. gondii, as we show here for the first time. Importantly, this study validates IMPα-dependent nuclear protein import as a target for therapeutic intervention in the case of apicomplexan parasites, consistent with the fact that both parasitic organisms have a single, essential IMPα gene [Emerging drug resistance on the part of MPα gene ,34.50 value), compared to both TgIMPα and MmIMPα in IMPα:NLS binding assays. Further, TSA analysis showed that binding of this compound has differential effects on PfIMPα compared to the other IMPαs; firstly, binding of Bay 11-7085 to PfIMPα increased its thermostability, in contrast to both TgIMPα and MmIMPα where binding decreases thermostability  [T. gondii tachyzoites [T. gondii. In fact, as shown here for the first time, Bay 11-7085 can kill T. gondii tachyzoites at low μM concentration, with a selectivity index of three; Bay 11-7085 and its structural analogues thus represent a viable starting point for drug development to combat T. gondii, and potentially also P. falciparum.Surprisingly, although Bay 11-7085 appeared to be a selective inhibitor of PfIMPα binding to  culture A; in con (NF-κB) ; althoughyzoites . This is50 values in the nM range for PfIMPα:NLS binding. Auranofin exhibits reactivity for the thiol group of proteins [P. falciparum [T. gondii [P. falciparum and T. gondii, indicate its potential for future investigation as an antiparasitic for malaria and toxoplasmosis.In terms of the less selective hit compounds emerging from our screen, auranofin (MMV688978) was the most potent, showing ICproteins  and is alciparum , as well. gondii , and we . gondii , its appIn summary, this is the first HTS to target PfIMPα binding to NLS-containing proteins. The fact that several hit compounds possess antiparasitic activity with low host cell cytotoxicity, validates apicomplexan IMPα as a therapeutic target, and opens the way both for larger screens using strategies similar to those described here, and for future investigation into the candidate hit molecules identified here in terms of the molecular details of the host-pathogen interaction. The latter is a priority for future work in this laboratory.


Scientific Reports 10.1038/s41598-021-88590-0, published online 10 May 2021Correction to: The original version of this Article contained an error in the Introduction, where7, {, #2306}{, 1986 #2260}{, #2306}{, #2306}, 550,000 in the USA8 and 235,000 in Canada9 at any one time.”“Figures indicate that 307,000 people are homeless in the UKnow reads:7, 550,000 in the USA8 and 235,000 in Canada9 at any one time.”“Figures indicate that 307,000 people are homeless in the UKThe original Article has been corrected.


To explore the efficacy and safety of rituximab (RTX) in the treatment of autoimmune nephropathy manifested as refractory nephrotic syndrome (RNS).A single-center prospective cohort study was conducted on RNS patients treated with RTX between March 2017 and December 2019. The subjects were divided into the primary nephropathy (PN) group and the secondary nephropathy (SN) group. Based on the estimated glomerular filtration rate (eGFR) before RTX treatment, the SN group was then divided into the SN-1 group (eGFR ≥ 30 ml/min) and the SN-2 group (eGFR < 30 ml/min). Biochemical parameters and clinical data were recorded during follow-up.P = 0.022). Kaplan–Meier analysis showed a significant difference of the renal survival among the three subgroups (P < 0.001). Multivariate Cox regression analysis showed that eGFR value before treatment was an independent predictor  for renal survival. In terms of adverse events, infection accounted for 56.6%. The incidence of severe infection was 10%, 25% and 50% in PN group, SN-1 group and SN-2 group, respectively.Fifty-four patients were followed up for at least 6 months. The overall remission rates were 65%, 66.7%, 27.3% in the PN, SN-1, and SN-2 groups, respectively (2. However, it has little effect on prognosis in patients with secondary RNS with eGFR < 30 ml/min/1.73m2, but with a high risk of severe infection.RTX may be a promising option in RNS patients with eGFR ≥ 30 ml/min/1.73m Rituximab (RTX), a chimeric anti-CD20 monoclonal antibody, has been initially approved for the treatment of non-Hodgkin's lymphoma . SubsequIn terms of primary glomerular diseases, the efficacy and safety of RTX in the treatment of IMN have been proved. Dahan et al.  found a Refractory nephrotic syndrome (RNS) is characterized by insensitivity to or dependence on glucocorticoids and immunosuppressants, and repeated recurrence, which is a difficult clinical problem. Meanwhile, among patients with secondary RNS, many suffer from protracted and recurrent stage 3–4 chronic kidney disease (CKD), but there is no study reporting the efficacy of RTX in this kind of patients. In view of the beneficial effects of RTX in the treatment of patients with primary or secondary RNS, we designed this single-center prospective cohort study to observe the efficacy and safety of RTX in autoimmune kidney diseases manifested as RNS.A total of 60 patients with nephrotic syndrome (NS) who visited the Department of Nephrology of Peking University International Hospital between March 2017 and December 2019 and were planned to receive RTX were selected. The inclusion criteria were as follows: (1) with clinical evidence of RNS, that is, NS characterized by glucocorticoids resistance, glucocorticoids dependence, or repeated recurrence; (2) with a definite renal pathological diagnosis; (3) age ≥ 18 years. The exclusion criteria were as follows: (1) with following secondary causes of NC: viral hepatitis, tumors, infections, medications, diabetes; (2) with reduced kidney volume (long diameter of either side < 9.0 cm) confirmed by B-mode ultrasound; (3) with complications including tumors, hematopathy, chronic wasting disease, pregnancy, and absence of limbs; (4) with previous application of RTX. This study was performed with approval from the ethics committee of our hospital and with informed consent from each patient.According to the diagnosis of primary diseases, the subjects were divided into the primary nephropathy group (PN group) and secondary nephropathy group (SN group). The SN group was then divided into two subgroups based on the estimated glomerular filtration rate (eGFR) level before RTX treatment, namely SN-1 group (eGFR ≥ 30 ml/min) and SN-2 group (eGFR < 30 ml/min). Both SN and PN are autoimmune diseases.A total of three TMA patients with complement-mediated hemolytic uremic syndrome were included in our study, 1 in the SN-1 group and 2 in the SN-2 group. These three patients had anti-complement factor H autoantibodies. In addition to intravascular hemolysis, all three patients had acute kidney injury combined with hypertension and nephrotic-range proteinuria, which met the diagnostic criteria for NC. They responded to plasma exchange therapy but still relapsed repeatedly, and a combination of steroid and immunosuppressants  was not effective. Therefore, they were then treated with RTX. In the SN-2 group, both cases had complications of pulmonary infection; one patient had reduced urine protein (Upro) but with progressive decline of renal function, who entered the stage of regular dialysis, while the other had stable renal function. In the SN-1 group, the patient had stable condition after treatment and urine protein turned negative.2 body surface area (BSA) weekly for 4 weeks. Maintaining treatment: an additional single dose of 375 mg/m2 BSA of RTX was administrated weekly for 1 week if the peripheral CD19+B lymphocyte count was more than 5 cells/μl during the follow-up visit. All patients were followed-up for more than 6 months.All patients treated for the first time were admitted for observation. Their previous treatment regimens  and infectious complications occurred 6 months before admission were recorded, and clinical data were collected. After admission, their condition was reassessed. Following exclusion of existing infection, RTX treatment was given. The treatment protocol was as follows. Induction treatment: RTX was intravenously injected at a single dose of 375 mg/mThe changes of parameters were examined, including 24-h Upro, serum creatine (SCr), albumin (ALB), peripheral white blood cell (WBC), red blood cell (RBC), platelet (PLT), total count of lymphocytes, CD4+lymphocyte count and CD19+lymphocyte count. CKD-EPI equation was employed to calculate eGFR [Efficacy evaluation based on remission state of nephropathy: (1) complete remission: Upro < 0.3 g/d, ALB > 30 g/l and renal function was normal; (2) partial remission: Upro was 0.3–3.5 g/d and decreased by more than 50% compared with that before treatment, ALB ≥ 30 g/L, and renal function was stable; (3) ineffective: Upro was decreased by less than 50% compared with that before treatment, ALB < 30 g/l, or renal function deteriorated. (4) recurrence: Upro > 3.5 g/d or > 50% of the baseline value in patients with complete and partial remission.Safety evaluation: the primary endpoints were death, maintenance hemodialysis, or end-stage renal disease (ESRD) (eGFR < 15 ml/min lasted for more than 3 months), and the secondary endpoint was severe infection . All adverse events were recorded during the follow-up period.χ2 test. Kaplan–Meier survival analysis was performed for endpoint events, and multivariate Cox regression  was used to analyze the risk factors affecting endpoint events. P < 0.05 suggests a statistical significance.SPSS 20.0  was used for statistical analysis. Results were expressed as frequencies and percentages for categorical variables, mean ± standard deviation for variables with continuous normal distribution, and median (interquartile range) for variables with continuous non-normal distribution. Intergroup comparison was conducted through one-way analysis of variance (ANOVA) for samples with a normal distribution and through rank sum test for samples with a skewed distribution. Categorical data, frequency of complete remission, and frequency of recurrence were analyzed using the Among the 60 patients, 54 patients were followed-up for more than 6 months (average 15.91 ± 9.78 months). In addition, two patients had infusion reactions to the first application of RTX and thus stopped medication three patients were lost to follow-up, and one patient died at the third month of treatment , the renal pathological types include IMN, MCD, and FSGS. In the SN-1 (n = 12) and SN-2 (n = 22) groups, the primary diseases leading to NC include systemic lupus erythematosus (SLE), AAV, and complement-mediated HUS (C-HUS) . The B-cell reconstitution occurred within 6 months in 5 cases (19.2%) in the PN group, 1 (7.1%) case in the SN-1 group, and 0 case in the SN-2 group.B cell depletion and reconstitution: the peripheral blood B-cell count of all patients decreased to less than 5 cells/ul within 1 month after treatment. B-cell depletion was maintained for 7.9 ± 3.0 months in the PN group, 9.0 ± 2.6 months in the SN-1 group, and 9.6 ± 3.5 months in the SN-2 group, with no statistical difference among the three groups , an increase in ALB  and no significant change in eGFR levels . The change range of each index from baseline was similar among the three groups .Remission: RTX treatment was effective in 27 (50%) of 54 patients, including 7 (12.96%) cases with complete remission and 20 (37.04%) cases with partial remission. In the PN, SN-1, SN-2 groups, the complete remission rates were 25%, 8.3%, 4.5%, respectively, and partial remission rates were 40%, 58.3%, and 22.7%, respectively, and the overall effective rates were 65%, 66.7%, and 27.3%, respectively  cases in the PN group relapsed, including two IMN patients, two MCD patients. Two IMN patients relapsed 6 months after partial remission, without B-cell constitution but with a decrease of anti-PlA2R antibody at relapse. In these two patients, the glucocorticoids maintenance dose at relapse was prednisone 10 mg QD, and partial remission was achieved again 2 months after administration of a low-dose of tacrolimus. Two MCD patients relapsed 6 months and 14 months after complete remission, respectively. The glucocorticoids dose for them was prednisone 10 mg QD and 15 mg QD, respectively, at relapse. In these two patients, after peripheral blood B-cell reconstitution was detected, the second round of RTX with a single dose of 375 mg / mχ2 = 25.846, P < 0.001). The results of Kaplan–Meier survival analysis . At the end of follow-up, 28% of patients in the SN-2 group had no endpoints, which was much lower than 95.2% in the PN group  and 100% in the SN-1 group . And there was no difference between the PN group and the SN-1 group . The time of reaching the endpoints in the SN-2 group was 3.61 ± 2.91 months, while only one patient with FSGS in the PN group received regular dialysis 12 months after RTX treatment.During the entire follow-up period, a total of two patients died, with a mortality rate of 3.33%, both of whom were in the SN-2 group. One of the two patients, with primary disease of anti-GBM combined with ANCA-associated AAV, scleroderma and heart valve disease, died of cardiac pump failure 6 months after RTX treatment. The other patient, with primary disease of AAV combined with pulmonary hemorrhage, died of pulmonary infection 3 months after RTX treatment. Sixteen patients (29.6%) reached ESRD or maintenance dialysis, including one case (5%) in the PN group, zero cases in the SN-1 group and 15 cases (68.2%) in the SN-2 group . A before-and-after study revealed no significant difference in number of patients with severe infection within 6 months before and after RTX treatment  patients had 47 adverse events in addition to endpoints. Specifically, 34 patients (56.6%) had infection, eight patients (13.3%) had WBC reductions, and five patients (8.3%) had infusion reactions. Among the complications of infection, lung and respiratory tract were the most common infection sites, and bacteria were the most common pathogens. Two cases of CMV viremia  in the PN group and 4 in the SN-2 group received antiviral therapy. Fungal infections were only seen in the SN groups, with three cases of candida albicans pneumonia in the SN-2 group and one case of Pneumocystis carinii pneumonia in each of the SN-1 group and the SN-2 group. Multiple infections  occurred in four cases in the SN-2 group and one case in the SN-1 group. Further statistical analysis was performed for severe infections that requiring hospitalization or a course of treatment for more than 2 weeks). The results revealed that the incidence of severe infections within 6 months of RTX treatment was highest at 50% in the SN-2 group, middle at 25% in the SN-1 group, and lowest at 10% in the PN group, with statistically significant differences among the three groups , that is, every 1 ml/min increase in eGFR prior to RTX treatment would decrease the risk of primary endpoints by 9.0 percent  or complete (n = 2) remission and maintaining stable renal function after RTX treatment, whereas patients who did not respond to the treatment experienced a progressive decrease in eGFR [In a prospective study by Xin Wang et al., all 36 IMN patients manifested as RNS, 15 41.7%) of them achieving partial  . InfectiIn addition, in this study, the SN-2 group had a significantly higher rate of severe infections after RTX treatment than the other two groups. Claire Trivin’s study have shown that eGFR reduction is associated with a high risk of infection . This isThis study is a single-center observational study with a small sample size, thus causing the following limitations. First, the PN group has a relatively high basic eGFR level and lacks a subgroup with eGFR < 30 ml/min. Therefore, our study cannot reflect the efficacy of RTX for PN patients with a low basic eGFR. Second, in this study, the PN group is mainly IMN, while the SN group is mostly LN, indicating an uneven distribution of disease composition and poor representativeness. Collectively, the results observed in this study cannot be blindly applied in other RNS populations.2 (including PN and SN) can effectively reduce urine protein, increase plasma ALB levels, and has no effect on eGFR. But the application of RTX in secondary RNS patients with eGFR < 30 ml/min/m2 has little benefit and a high incidence of adverse events, especially severe infectious complications.The application of RTX in RNS patients with eGFR ≥ 30 ml/min/m


Saccharina japonica (Sja); however, the underlying control mechanisms have not been studied. Our analysis of genomic and transcriptomic data from Sja shows that mannitol metabolism is a cyclic pathway composed of four distinct steps. A mannitol-1-phosphate dehydrogenase (M1PDH2) and two mannitol-1-phosphatases (M1Pase1 and MIPase2) work together or in combination to exhibit full enzymatic properties. Based on comprehensive transcriptomic data from different tissues, generations, and sexes as well as under different stress conditions, coupled with droplet digital PCR (ddPCR) and proteomic confirmation, we suggest that SjaM1Pase1 plays a major role in mannitol biosynthesis and that the basic mannitol anabolism and the carbohydrate pool dynamics are responsible for carbon storage and anti-stress mechanism. Our proteomic data indicate that mannitol metabolism remains constant during diurnal cycle in Sja. In addition, we discover that mannitol-metabolism-associated (MMA) genes show differential expression between the multicellular filamentous (gametophyte) and large parenchymal thallus (sporophyte) generations and respond differentially to environmental stresses, such as hyposaline and hyperthermia conditions. Our results indicate that the ecophysiological significance of such differentially expressed genes may be attributable to the evolution of heteromorphic generations  and environmental adaptation of Laminariales.As a carbon-storage compound and osmoprotectant in brown algae, mannitol is synthesized and then accumulated at high levels in  Ectocarpus but not in kelps Mannitol is one of the most common sugar alcohols in nature and is produced by a variety of living organisms, including bacteria, fungi, terrestrial plants, and algae Saccharina japonica (Sja) is one of the most important brown macroalgae in the order Laminariales because of its considerably high biomass and economic significance Sja species since early 1950s Sja, such as mannitol, have been widely used as food supplements, medicines, and chemical materials for chemical industry and scientific research Sja life cycle comprises three stages, including the single-cell (meiospore), multicellular filamentous , and large parenchymal individual  stages Sja makes it different from its close relatives in the genus Ectocarpus that lack the parenchymal stage Ectocarpus) and heteromorphic haploid-diploid algae (Saccharina) are not yet well understood. Moreover, Laminariales, such as Sja, are predominant in the marine ecosystems of cold and temperate coastal zones with harsh and extreme climates Ectocarpus siliculosus genome has paved the way for studying the molecular basis of mannitol biosynthesis in algae Dixoniella griseaCaloglossa continuaCaloglossa leprieuriiSpatoglossum pacificum, Dictyota dichotoma, and Laminaria digitataPlatymonas subcordiformisE. siliculosus, recombinant EsM1PDH1cat  and EsM1Pase2 have been characterized M1PDHs and two M1Pases in E. siliculosus  was influenced by the diurnal cycle, and EsM1Pase1 was highly down-regulated under hyposaline stress Sja .The availability of the M1PDH and M1Pase genes and their encoded proteins, which are known to act in the Sja mannitol biosynthesis, based on transcriptomic and proteomic data generated from different Sja tissues of various generations (including the sporophyte and gametophyte generations) or under different abiotic stress conditions. Our results extend the understanding of mannitol metabolic pathways and their regulatory mechanisms in the context of ecophysiological and evolutionary significance of Laminariales. In addition, Sja MMA genes may be used to engineer microbes for mannitol production, and also to engineer plants for increasing their tolerance to abiotic stresses and for mannitol biosynthesis for subsequent extraction.In this study, we systematically characterized the Sja samples, including two samples separately from male and female gametophytes, six from different parts of thalli , and four from Sja incubated under different abiotic stresses  . The molecular weight of most identified proteins ranged from 20 kDa to 70 kDa  . Approxiditions) . After rkDa 71%, A. The sekDa 71%, B. About kDa 71%, C.Figure Sja data, we annotated all MMA genes in 19 brown algal species belonging to Laminariales, Ectocarpales, Desmarestiales, Dictyotales, Fucales, and Ishigeales (M1PDH1 and M1PDH2 according to the naming convention of E. siliculosus M1PDHs) were found in most species; however, the third M1PDH gene  was only detected in three species belonging to Ectocarpales  gene was found in 15 brown algal species (HK) genes, HK1 and HK2, were found in all 19 species, with interspecific identities of 60.98% to 67.43%  ing site B.SjaM1PDH1, SjaM1PDH2, SjaM1Pase1, and SjaM1Pase2 in Escherichia coli to characterize their enzymatic activities. Although several attempts  were made to overexpress SjaM1PDH1, the efforts had failed. Then, the specificity of SjaM1PDH2 was determined by assaying its activity in the presence of different potential substrates, including F6P, G6P, and G1P for detecting reduction activity and M1P, F6P, G6P, and G1P for detecting oxidation activity. First, SjaM1PDH2 had only reduction activity in the mannitol synthesis pathway but not oxidation activity , indicating lower substrate-binding capacity than that of EsM1PDH1cat (mK = 0.02 mM) was about 41-fold higher than that of SjaM1Pase1 (mK = 0.83 mM), and it was the highest (by 21–315 folds) among the binding capacities of M1Pases from brown and red algae. Interestingly, both SjaM1Pases showed much higher catalytic efficiencies and catalytic rates than EsM1Pase2 .The recombinant SjaM1PDH2 and SjaM1Pases exhibited typical Michaelis-Menten kinetics when assayed in increasing concentrations of their substrates, and the apparent rk plots C. The Km1PDH1cat . The M1PsM1Pase2 ; SjaM1PaWe then assayed the salt influence on the recombinant SjaM1PDH2 and SjaM1Pases using NaCl D. The reSja MMA gene expression study relies on both transcriptomic and proteomic data (db.cngb.org/onekp/). All of the 19 Phaeophyceae species appear to express two M1Pase genes and two HK genes  were all expressed constitutively. As shown in The regulation of SjaM1PDH2 and two SjaM1Pases), which control the balance between mannitol and F6P and dynamically maintain the “carbohydrate pool” in vivo. Mannitol metabolism is closely associated with the metabolism of alginate, fucoidan, laminarin, and trehalose via the intermediate product F6P. The first gene that transforms F6P to mannitol is M1PDH (M1PDH1 and M1PDH2), and the first gene that transforms F6P to alginate and fucoidan is mannose phosphate isomerase MPI (MPI1 and MPI2) SjaM1PDH genes as well as two SjaMPI genes was compared among different tissues  were expressed at significantly higher levels in sporophytes than in gametophytes, showing 2.8-, 7.3-, 3.1-, 5.6-, and 4.7-fold changes, respectively  were down-regulated (1.4–3.0 folds) in sporophytes  that has approximately 89 gene family members. As a single-product pathway, the mannitol metabolism does not contain complex synthesis- and modification-associated genes, such as those encoding glycosyltransferases, sulfurtransferases, and mannuronate C5-epimerases, as observed in alginate and fucoidan metabolism that contains dozens to more than 100 genes Mannitol is one of the fundamental carbon-storage molecules and osmotic regulators in brown algae, and mannitol metabolism is one of the main traits that make brown algae unique as compared to other eukaryotic algae M1PDH unigenes in E. siliculosus, but only two unigenes are present in the Sja genome. Our transcriptomic data of 19 Phaeophyceae species enable the identification of the two M1PDH unigenes expressed in most brown algae samples  and secondary endosymbiotic  algae, which have only one copy of this gene HAD-M1Pase genes , and MMA genes in Laminariales may be regulated in a complex integrated system. For example, the expression levels of the first genes in the mannitol and alginate/fucoidan metabolic pathways differ substantially among tissues  has been confirmed to be active in E. siliculosus[38]. No enzymatic studies have been conducted for MIPase1; only the nucleotide sequence of MIPase1 from E. siliculosus has been reported. In this study, SjaM1Pase1 and SjaM1Pase2 are both confirmed to have M1Pase activity and assumed to be involved in mannitol biosynthesis in brown algae  than the latter , such as phosphoglucose reduction. This functional differentiation between homologous genes is similar to the MPI genes in alginate/fucoidan metabolism Gene duplication in evolution is an important general mechanism for maintaining normal biochemical metabolism under variable conditions, and it is probably in action here for the photosynthetic carbon storage in egulated . These fegulated , and we SjaM1PDH2 and SjaM1Pase2) exhibit significantly higher expression in sporophytes  than in gametophytes (filamentous generation) (P > 0.05), respectively (unpublished data).Laminariales have a heteromorphic haploid-diploid life cycle, with a macroscopic thallus sporophyte and a microscopic gametophyte among different generations eration) A. These eration) B. This rM1Pase2 in E. siliculosusSja and most other brown algae, counteracting salinity stress and acting as an antioxidant and heat protectant for protein stabilization Saccharina species are capable of transporting carbons from source (mature blade areas) to sink, producing a surplus of photoassimilates transported to intercalary carbon-requiring meristems The expression levels of MMA genes in response to environmental stresses show opposite trends between gametophyte and sporophyte generations. Under hyposaline stress, the expression of all MMA genes in gametophytes is up-regulated, consistent with the results observed for SjaM1PDH1 expression shows 3.75- and 1.87-fold increases under hyposaline and hyperthermia stresses, respectively, and the ddPCR analysis accordingly shows 3.97- and 1.30-fold increases, respectively . Most of our gene expression results are confirmed. For example, in RNA-seq analysis, ectively . NotablyM1Pase gene, from the protozoan parasite Eimeria tenellaE. tenella M1Pase (mK = 0.07 mM) is lower than that of SjaM1Pase2 (mK = 0.02 mM), and the catalytic efficiency of the E. tenella M1Pase (kcat = 430 s−1) is much lower than that of SjaM1Pase1 (kcat = 6453.48 s−1), indicating that the M1Pase genes from brown algae may be suitable candidates for engineered microbes for mannitol production. Furthermore, introduction of algal genes into transgenic plants may confer great advantage in terms of salt tolerance Mannitol is a commercially valuable compound widely used in food, pharmaceutical, medical, and chemical industries Sja haploid gametophytes  were available in our laboratory cultures , which were grown in a modified seawater medium supplemented with nutrients (4 mg/L NaNO3-N and 0.4 mg/L KH2PO4-P) at 10 °C under 30 μM photons m−2 s−1 irradiance. Fresh samples of Sja sporophytes were collected from eastern China  and kept in the laboratory under the same culture conditions as gametophytes for a few days to acclimate, and then dissected to obtain different tissues . To study the effects of abiotic factors, the female gametophytes (5 g each sample) and blade bases of sporophytes (5 g each sample) were cultured at different temperatures  and salinities  for 6 h; the female gametophytes (5 g each sample) were cultured under a 12-h/12-h photoperiod .Preserved Sja genome sequence downloaded from Genome Warehouse , the transcriptome database downloaded from 1000 plant trancriptomes , and the transcriptome and proteome databases obtained in our study, the unigenes related to mannitol metabolism pathway were verified using the BLASTX algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were performed with ClustalX http://www.ebi.ac.uk/Tools/msa/clustalo/).Based on the analysis of the SjaM1PDH1, SjaM1PDH2, and SjaM1Pase1 were cloned into pET32a, and SjaM1Pase2 was cloned into pGEX-6p-1. The inserts were cloned into the plasmids between the EcoRI and NotI sites. After ligation, the generated recombinant plasmids were transformed into E. coli BL21 (DE3) cells, and the integrity of the sequences was verified based on Sanger sequencing.Genes were codon-optimized and synthesized  to construct recombinant plasmids. 600) reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM to induce overexpression of the target genes to produce recombinant proteins, and the bacterial cultures were incubated for 16 h at 20 °C. Then, the cells were harvested by centrifugation at 12,000 g for 3 min at 4 °C, suspended in 20 ml of 50 mM Tris-HCl buffer (pH 8.0), and lysed by sonication  to release the recombinant proteins. His-binding resin and GST-binding resin were used to purify the recombinant proteins according to the manufacturer’s instructions (www.yuekebio.com). The proteins were stored at −80 °C.The recombinant strains were cultivated in 1 L LB liquid medium. When the optical density at 600 nm  and to a set of gene model annotations including MMA genes using HISAT2 Total RNA was extracted by using an improved CTAB method t-test.ddPCR analysis was conducted according to previously described methods Every five samples, including three treatments, a control, and an internal standard , were labeled as a replicate, and three replicates were taken for each measurement. Proteins were extracted using the phenol/SDS method described by Nagai K et al. Saccharina japonica transcriptome (GSA: PRJCA000815). The search parameters were used as described by Chen L et al. t-test for P value.The raw data were analyzed using Proteome Discoverer 2.1 software (ThermoFisher Scietific) with mascot 2.3 against the protein database translated from the http://bigd.big.ac.cn/gsa. The raw protein data and metadata are deposited in  the ProteomeXchange Consortium via the iProX partner repository (ProteomeXchange: PXD009642), and are publicly accessible at http://proteomecentral.proteomexchange.org. The cDNA sequences of SjaM1PDH and SjaM1Pase genes are deposited in GenBank: SjaM1PDH1 (GenBank: MF706368), SjaM1PDH2 (GenBank: MF706369), SjaM1Pase1 (GenBank: MF440344), and SjaM1Pase1 (GenBank: MF465902).The raw sequence data are deposited in the Genome Sequence Archive Shan Chi: Methodology, Formal analysis, Visualization, Writing - original draft, Writing - review & editing. Guoliang Wang: Methodology, Formal analysis, Data curation, Visualization, Software, Writing - original draft, Writing - review & editing. Tao Liu: Conceptualization, Resources, Supervision, Funding acquisition. Xumin Wang: Conceptualization, Resources, Supervision. Cui Liu: Investigation. Yuemei Jin: Investigation. Hongxin Yin: Investigation. Xin Xu: Investigation. Jun Yu: Conceptualization, Resources, Writing - review & editing, Supervision. All authors read and approved the final manuscript.The authors have declared no competing interests.


Sepsis is a serious syndrome that is caused by immune responses dysfunction and leads to high mortality. The abilities of heat shock protein 90α (HSP90α) in assessing the diagnosis and prognosis in patients with sepsis remain ill-defined to date. We conducted a study to reveal the possible clinical applications of HSP90α as biomarker for the diagnosis and prognosis in patients with sepsis.In total, 150 patients of sepsis, 110 patients without sepsis admitted to ICU and 110 healthy subjects were involved in this study. The serum HSP90α contents, sequential organ failure assessment (SOFA) scores, procalcitonin (PCT), and short-term survival status of the participants were measured and compared. Logistic and linear regression models adjusting for potential confounders were used to examine the association of HSP90α with sepsis survival. Moreover, serum IL-1β, IL-18, MIP-3α, and ENA-78 were also determined. Finally, Spearman correlation analysis was employed to reveal a possible mechanism that HSP90α contributed to the short-term deaths.P < 0.001), and even increased in patients who died within 28 days (P < 0.001). Logistic and linear regression models identified HSP90α was an independent risk factors for sepsis mortality. Receiver operating characteristic (ROC) analysis displayed that HSP90α had a considerable predictive performance for sepsis outcome, with an area under curve (AUC) value up to 0.79. Survival analysis demonstrated that the mortality of sepsis individuals at 28 days was positively associated with HSP90α levels, especially the levels of HSP90α were greater than 120 ng/mL (P < 0.001). Moreover, among sepsis patients, those who died had notably elevated cytokines, IL-1β, IL-18, and chemokines, MIP-3α, ENA-78, relative to survivors. Further correlation analysis demonstrated that there was a nominally positive correlation between HSP90α and IL-1β, IL-18, and MIP-3α.Serum HSP90α levels in sepsis patients were higher than those in ICU controls and healthy controls (HSP90α is of favorable clinical significance in sepsis diagnosis and prognosis, laying a foundation for future clinical applications. Sepsis syndrome represents a major global health issue in today’s medicine . AnnuallSince the high mortality of sepsis attribute to the untypical clinical manifestation, early diagnosis of sepsis may be of clinical significance and can save numerous lives . In thisHeat shock proteins (HSPs) are highly conserved polypeptides and essential for diverse cellular processes, such as recognition, signaling transduction, protein maturation, and cell differentiation . In partWith these issues in mind, we evaluated whether the presence of serum HSP90α on ICU admission is a potential predictor for the early-onset of sepsis and a key indicator for survival stratification. Furthermore, we explored a possible underlying mechanism that HSP90α affect the clinical outcome of sepsis patients by eliciting the secretion of pro-inflammatory cytokines and chemokines.n = 94), including 64 males and 30 females, with an age of (57.62 ± 15.68) years; Death group, including 37 males and 19 females, with an age of (58.38 ± 15.10) years. Patients who met any of the following criteria were excluded: (1) younger than 18 years or older than 80 years; (2) with acute cardiovascular and cerebrovascular events, malignancies of various systems or severe hematologic diseases; (3) females in pregnancy or lactation period; (4) with an unclear medical history which affects the accuracy of SOFA scoring; (5) infected by HIV, with autoimmune diseases, treated by immunosuppressant drugs or cytotoxic drugs; (6) incoordinate to sign Informed Consent. All subjects we induced in this study were informed of the research content and signed informed consent, and the First Affiliated Hospital of Dali University granted Ethical approval to carry out the study within its facilities  (no. 20190612)  between September 2019 and September 2020 were enrolled, including 101 males, 49 females, with an average age of (57.95 ± 15.39) years. All subjects were enrolled in agreement with the diagnostic standard described in Sepsis 3.0. A total of 110 patients treated in the ICU without sepsis or signs of infection served as controls, including 71 males, 39 females, with an average age of (56.12 ± 10.52) years. Healthy individuals for control were from the physical examination center of our hospital, including 79 males and 31 females, with an average age of (58.38 ± 15.10) years. According to clinical outcome, the 150 sepsis patients were divided into two groups: Survival group (0190612) .The hospital information system (HIS) was consulted to refer to and collect clinical pathological data of patients based on case-control approach. Data recorded were as follows: (1) general information, such as age and gender; (2) clinical manifestations, including infection route, complications, hospital stays, vital signs ; (2) laboratory indexes, including blood routine, total bilirubin , creatinine , blood urea nitrogen , lactic acid , C-reactive protein , procalcitonin , and SOFA score, which were obtained from the examinations on peripheral venous blood samples within 48 h of admission. However, the data of CRP and PCT levels, SOFA cores, ICU stay, as well as 28-day mortality of health controls were not collected, because the levels of CRP and PCT of health individuals were not tested, and no health subject was admitted to the ICU.Venous blood samples (5 mL) were collected from all subjects with an empty stomach within 48 h of admission, and then preserved in a −80 °C refrigerator following serum separation. ELISA method was applied to determine serum HSP90α level. In short, sample buffer was firstly taken to dilute the standard, quality control and serum samples, after which the diluted samples were transferred to a 96-well micro-plate. Horseradish peroxidase-conjugated antibodies were then added, and tetramethyl benzidine was used to observe the reaction after 1 h of incubation with a temperature of 37 °C. Finally, a microplate reader was used to read the optical density (OD) value of each well at 450 nm in wavelength, and corresponding standard curve was plotted to further calculate the content of HSP90α in each group.Cytokines and chemokines quantification were completed using multi-analyte flow assay LEGENDplex™ Human Cytokine Panel 2 kit  and LEGENDplex™ Human Proinflammatory Chemokine Panel . FACS analysis was carried out with a FACS Calibur flow cytometer .χ2 or Fisher’s exact test was adopted for between group comparison. To determine the diagnosis power of HSP90α, PCT and SOFA score for sepsis, receiver-operating characteristic (ROC) curves were constructed and the area under the curve (AUC) was calculated with its 95% confidence interval (CI). Spearman correlation analysis was conducted to elucidate the association of HSP90α with SOFA score, PCT. Then univariate logistic regression was employed to investigate the effect of various parameters on survival in patients with sepsis by adjusted odds ratios (ORs). We also constructed five different models. Model 1 included only HSP90α. PCT was added to Model 2. Model 3 was Model 2 with further adjustment for SOFA score. Model 4 was Model 3 with further adjustment for creatinine. Finally, more potential indicators and confounders, including urea nitrogen was added to Model 4. The mortality in 28 days was defined as the endpoint, ROC analyses and area under the ROC curve (AUC) values were used to evaluate creatinine (Crea), blood urea nitrogen (BUN), SOFA score, PCT, and HSP90α prognostic power for 28-day mortality. Kaplan–Meier analysis was implemented for the survival of sepsis patients of various groups. Finally, Spearman correlation analysis was employed to evaluate the correlation between HSP90α with IL-1β, IL-18, ENA-78, and MIP-3α. P < 0.05 refers to between-group difference of statistical significance.All statistical analyses were performed with SPSS25.0 for windows , figures were generated by GraphPad Prism 7.0  and R package pheatmap (v1.0.12). All measurement data were expressed as mean ± standard deviation (SD). One-way analysis of variance for date of normal distribution, for data of non-normal distribution, Mann–Whitney U test was implemented for between-group comparison. For numeration data, P < 0.001). Compared with health controls and ICU controls, serum HSP90α concentrations at admission were significantly increased in the patients with sepsis (P < 0.001).The demographic and laboratory profiles of the studied subjects are shown in P < 0.001) with 88.67% sensitivity and 56.52% specificity in discriminating the patients with sepsis, which was weaker than SOFA score  and superior than PCT   . These rP > 0.05). In addition, there were no significant differences in WBC, neutrophils, lymphocytes, C-reactive protein, total bilirubin, PaO2, PaCO2, and lactate . However, non-survivors had significantly higher levels of procalcitonin , higher SOFA score (P < 0.001), higher Crea levels (P = 0.006), higher BUN levels (=0.009), and higher serum HSP90α levels (P < 0.001) than survivors. Moreover, the spearman correlation analysis of all patients with sepsis yielded a positive correlation of serum HSP90α levels at ICU admission with SOFA score  was significantly linked to sepsis deaths. The addition of other cofounders, PCT , SOFA score , Crea  and BUN  only slightly enhanced this effect.Furthermore, five different multivariable models were constructed to explore the relationship between HSP90α and sepsis mortality . ConsequP < 0.001), with a 98.2% of sensitivity and a 63.8% specificity, and the optimal cut-off value is 120 ng/mL. The AUC of HSP90α is bigger than that of SOFA score , PCT , Crea , and BUN .All sepsis patients were followed up for 42 days, and the mortality in 28 days was defined as the endpoint. To investigate the ability of HSP90α in predicting 28-day mortality in sepsis patients, the ROC curve  was applP < 0.001). However, higher SOFA score (≥5) had limited ability to predict 28-day mortality in sepsis (P = 0.152). Collectively, these findings indicate that HSP90α has the valuable ability to predict the prognosis of the patients with sepsis.Moreover, a Kaplan–Meier survival analysis was used to estimate the effects of the HSP90α cut-off value (120 ng/mL) on survival rates in sepsis patients , with a i.e. IL-1β, IL-18, ENA-78, and MIP-3α) associated with sepsis death were further tested. Notably, the expression levels of these four cytokines in the sera of the non-survival group were significantly higher than those in the survival group , IL-18 , and MIP-3α , except for ENA-78 (P = 0.059)  . These fTo date, no reliable biomarkers could provide sufficient power to predict early-onset and clinical outcomes in patients with sepsis. In present study, we have found that HSP90α may be a potential predictive biomarker for the diagnosis and prognosis of septic patients. Our studies have provided three lines of evidence supporting these findings: (i) serum HSP90α content was significantly higher in patients with sepsis than that of in healthy controls; (ii) compared with surviving septic patients, the elevated serum HSP90α levels were observed in the non-surviving ones; (iii) A higher level of serum HSP90α on admission could identify the 28-day mortality of patients with sepsis. In addition, we also found that provoking the secretion of pro-inflammatory cytokines and chemokines might be causes of HSP90α affecting the prognosis of sepsis patients.Among the septic patients, the SOFA score serving as a major tool has been employed to indicate disease severity and predict mortality . Howeveri.e., CRP, PCT, SSA, lactate, and HBP, but the unsatisfied sensitivity and specificity limit their applications in clinical practice (As mentioned previously, the SOFA score was a key predictive factor for ICU deaths . In recepractice . In presAs is well known, an inflammatory cytokine storm is a crucial pathogenic factor in patients with sepsis at the early stage. The elevated levels of IL-1β, IL-6, TNF-α, IL-18, and IL-37 in sepsis have previously been reported . In otheNevertheless, limitations of our study are noteworthy in the interpretation of results. First, this retrospective study might have caused selective bias resulting in overestimated the diagnostic and prognostic value of HSP90α. Second, the serum levels of HSP90α was not monitored at sequential time points after sepsis developed. Third, the follow-up period was short, and long-term survival was not analyzed. Fourth, the fact that present study included a single center hindered our ability to draw conclusions. Fifth, a sample size estimation was not done. Quite evidently, additional studies are needed to verify these results.In current study, we provided evidence that serum HSP90α was a potential biomarker for evaluating the power of diagnosis and short-term prognosis of patients with sepsis, which could assist clinician identification of sepsis at early stage and lay the groundwork for the development of novel therapeutic target for patients with sepsis.10.7717/peerj.12997/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12997/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12997/supp-3Supplemental Information 3Click here for additional data file.


Considering melanoma is the deadliest malignancy among dermatoma and presently lacks effective therapies, there is an urgent need to investigate the potential mechanisms underlying melanoma metastasis and determine prospective therapeutic targets for precise treatment of melanoma.Hub genes in melanoma metastasis were identified by analyzing RNA‐seq data  obtained from TCGA database. Then the identified hub genes were validated in human tissues with qRT‐PCR, followed by survival analysis. Competing endogenous RNAs of the hub genes were defined to clarify potential molecular mechanism of melanoma progression. Then central gene‐related signaling pathways were analyzed, followed by immune cell abundance analysis in tumor microenvironment with CYTERSORTx.A tetrad of IL2RA, IL2RG, IFNG, and IL7R genes were determined as hub genes and verified by qRT‐PCR, which were significantly associated with unfavorable prognosis in melanoma. LINC02446, LINC01857, and LINC02384 may act as competing endogenous lncRNAs of IL2RA and IL7R through absorbing their shared miR.891a.5p and miR.203b.3p. JAK—STAT signaling pathway identified as the most relevant pathway in melanoma metastasis, as well as a wealthy of genes including TNFRSF 13B, TNFRSF17, TNFRSF9, TNFRSF8, TNFRSF13C, TNFRSF11B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7, may induce tumor autoimmune suppression through enhancing regulatory T‐cell abundance and performance in the tumor microenvironment. And regulatory T‐cell proportion was indeed critically elevated in metastatic melanoma relative to primary melanoma, as well as in highly expressed IL2RA, IL2RG, IL7R, and IFNG group than their respective counterparts.Elevated IL2RA, IL2RG, IL7R, and IFNG expression may play a central role in promoting melanoma metastasis through up regulation of intratumoral regulatory T—cell proportion mainly by activation of JAK—STAT signaling pathway. LINC02446, LINC01857, and LINC02384 may stimulate melanoma progression by reducing tumor‐protecting miR.891a.5p and miR.203b.3p. A number of identified molecules including TNFRSF13B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7 can serve as future therapeutic targets in melanoma treatment. Elevated IL2RA, IL2RG, IL7R, and IFNG expression may play a central role in promoting melanoma metastasis via increase in intratumoral regulatory T cell proportion, mainly by activation of the JAK–STAT signaling pathway. LINC02446, LINC01857, and LINC02384 may stimulate melanoma progression by reducing tumor‐protecting miR.891a.5p and miR.203b.3p. A number of identified molecules, including TNFRSF13B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7, can serve as future therapeutic targets in the treatment of melanoma. Furthermore, we utilized the CYBERSORTx,22.1Three normal skin tissues and three melanoma tissues were derived from three patients with melanoma who were all operated in China‐Japan Union Hospital of Jilin University. All skin samples were stored at −80°C before total RNA was extracted.2.2A total of 472 melanoma samples with raw RNA‐seq reads  and corresponding clinical information were garnered from TCGA database, including 105 primary melanoma patients and 367 metastatic patients. Single‐cell RNA‐seq data of immune cells were obtained from CIBERSORTx datasets.2.3Total RNA was extracted from human tissues using TRIzol reagent (Invitrogen) following the manufacturer's protocols. cDNA was synthesized using the the GoScript Reverse Transcription System Kit . Real‐time PCR primers, including human β‐Actin , IFNG , IL2RA , IL2RG , and IL7R , were designed with Primer‐BlAST and checked by Oligo (Version 6.44). Quantitative Real‐time PCR analysis was performed using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) ROX plus (Takara) on a ABI 7500 Real‐Time PCR System . Relative gene expression in each sample was calculated with the ‐ΔΔCt method.2.4All differentially expressed (DE) genes including mRNA, lncRNA, and microRNA are computed with raw data by the "edgeR" R package, which provides a procedure to normalize the raw data, filter the very low expression genes and calculate the DE genes between different groups.2.5Functional protein—protein interaction (PPI) network was established based on functional enrichment analysis by STRING, an online tool.2.6Gene set enrichment analysis (GSEA) for all genes expressed in melanoma were performed using GSEA software (version 3.0) and reexamined by DAVID Bioinformatics Resource 6.8.2.7The overlapping genes between the most relevant gene cluster identified by MCODE analysis and the most significant pathway in GSEA analysis results are determined as hub genes in melanoma metastasis.2.8p value < 0.05 are considered to have a significant association, and similarly miRNA‐lncRNA pairs with r < −0.3 and p value < 0.05, mRNA‐lncRNA pairs with |r|>0.6 and p value < 0.05 are considered to have a significant relationship.To further investigate the molecular mechanisms of hub genes, we established a competing endogenous RNA (ceRNA) network. The ceRNA theory is that any RNA transcript that contains MREs can sequester miRNAs from other targets sharing the same MREs, thereby regulating their expression.Screen the above results for significantly paired lncRNA‐mRNA, both of which is significantly correlated to the same miRNA, to obtain a miRNA‐lncRNA‐mRNA network, that is ceRNA, among which lncRNAs can adsorb miRNAs via a "sponge effect," suggesting that miRNAs are competitively bound by both their corresponding lncRNAs and mRNAs.2.9p > 0.05 to increase the accuracy of the estimated results.Proportion of immune cell type in melanoma was calculated with CYBERSORTx, an online tool developed by a scientific team from Stanford University, which enables precise estimation of cell abundance with RNA‐seq data and corresponding single cell RNA‐seq data. The operating parameters used in the present study were as follows: B‐mode, disable quantile normalization, and permutation for significance analysis 100. We filtered out the samples with 2.10t‐test. Survival analysis was performed using log‐rank test, and plotted using Kaplan—Meier survival curves. p < 0.05 was considered statistically significant.Statistical analysis was mainly performed using R Studio (version 3.6.1). Continuous values were analyzed using 33.1p < 0.05, |log2FC|>1) in metastatic melanoma compared with primary melanoma  on RNA‐seq data from TCGA via the R package "edgeR." There were divergent gene expression patterns between two groups, and a total of 1366 DE mRNAs, including 998 upregulated genes and 368 downmodulated genes, were identified  cells were considered as a pivotal immune cell type in advanced melanoma  of IL2RA and IL7R, were oncogenes, while miR‐203b‐3p and miR‐891a‐5p, as microRNA sponges of IL2RA and IL7R, respectively, act as potential tumor suppressor molecules. Collectively, these findings provide insights to the underlying mechanisms of melanoma metastasis and help to establish promising molecular targeted therapy.We demonstrated that the upregulated critical molecules, pathways and cells in metastatic melanoma were all immune‐related, supportive of the dominant role of immune function in the process of melanoma metastasis. Overexpressed IL2RA, IL2RG, IFNG, and IL7R were identified as the hub genes associated with melanoma metastasis, and may promote melanoma progression through activation of JAK—STAT signaling pathway. The related genes, including TNFRSF13B, TNFRSF17, TNFRSF8, CCR7, NRP1, ENTPD1, LAG3, NRP1, TOX, and FOXP3, were also significantly upregulated and remarkably correlated to undesired prognosis in metastatic melanoma. These factors contributed to the augment in the abundance and function of Treg cells. In addition, restricted proportion and function of naive T cells by abnormally elevated IL7R can subsequently lead to a reduced activator T cells of functional immune response,Our study revealed that IL2RA、IL2RG, IFNG, and IL7R were hub genes in melanoma metastasis and involved in JAK—STAT signaling pathway that was identified as an central signaling pathway in metastatic melanoma, which was rarely illustrated in previous studies.We also found that LINC01857, LINC02446, and LINC02384, as protective factors of IL2R and IL7R, were potential oncogenic molecules, while miR‐203b‐3p and miR‐891a‐5p were suppressive of IL2RA and IL7R respectively. At present, there are still no reports about LINC01857 in malignant melanoma, and three reports indicating that LINC01857 was a potential oncogene in glioma,reg cells in metastatic melanoma as compared with that in primary melanoma, were the pivotal intermediate components by which IL2RA, IL2RG, IL7R, and IFNG exert their immunosuppressive effect. Collective literature indicated a poor prognosis for cancers with an abundance of intratumoral FOXP3+ Treg cells,+ T‐cell infiltrates.+ T cells. Tumor‐resident Treg cells were reported to promote tumor expansion and progression by impairing antitumor immune response, subsequently stimulating tumoral angiogenesis reg subpopulations are heterogeneous, and different Treg subpopulations may have opposing effects on tumor progression.reg cells in the melanoma tumor microenvironment are driven by CD8+ T cells,reg cells could have augmented CD8+ T cells simultaneously, thus having an improved survival.Another relevant finding in this study was that overexpressed Treg cells with immune escape of melanoma are required to establish future precision medicine. Briefly, the present study is only a minor step on the long way to conquer melanoma, and is by no means an end.Our study has several limitations. For the main body of the observation were obtained through bioinformatics means, further biological assays and clinical researches are warranted to support the results. Meanwhile, further details on association of T5reg‐cell proportion by activation of JAK—STAT signaling pathway, and determined four hub gene‐associated ceRNAs, as well as functionally metastasis‐relevant molecules including TNFRSF13B, TNFRSF17, CCR7, NRP1, NRP1, TOX, FOXP3, LAG3, NRP1, and so on. Our findings should highlight immunosuppressive mechanisms underlying melanoma metastasis and provide insights into resistance rationale of immunotherapy on melanoma, paving the way to prospective targeted therapy of advanced melanoma.In summary, we demonstrated that elevated IL2RA,IL2RG, IL7R, and IFNG expression may facilitate melanoma metastasis through upregulation of tumor‐resident TThe authors report no conflict of interest in this work.Chuan Zhang, Xianling Cong are responsible for the literature review and writing the discussion and introduction of the paper. Dan Dang, Lele Cong, Hongyan Sun are responsible for the bioinformatics analysis, material and methods and results sections of the manuscript.The study was approved by the Ethics Committee of China‐Japan Union Hospital of Jilin University. Written informed consent was obtained from patients.Table S1Click here for additional data file.


Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome. Sepsis, a systemic host’s response to pathogens, is a multifaceted infection-induced syndrome of physiologic, pathologic, and biochemical abnormalities . More thIn response to pathogen infection, the activation of pattern recognition receptors (PRRs) , includiSince the 1980s, several small molecules and biologics have been tested in clinical trials against sepsis. These treatments targeted established mediators and mechanisms driving septic physiology and included antibodies against lipopolysaccharide (LPS), corticosteroids, TLR4 and IL-1β antagonists, anti-TNF agents, and drugs targeting the coagulation cascade . HoweverHere, we performed the screening of several small molecules targeting chromatin-associated enzymes, including kinases, to assess their efficacy to inhibit TNFa production in vitro. Based on the in vitro results, we selected SCH772984, an inhibitor of ERK1/2, for in vivo studies. SCH772984 significantly improved survival in a sepsis mouse model induced by a lethal dose of LPS and a sepsis model induced by cecal ligation and puncture (CLP). RNA-seq transcriptomic signatures of SCH772984 treatment point to the attenuation of pathways associated with sepsis, providing, at least in part, molecular insights about this agent’s beneficial effects.To determine which epigenetic drugs could have anti-sepsis properties, we undertook a supervised screening of several drugs targeting representatives of different classes of epigenetic regulators that have already been tested in at least early phases of clinical trials . We firs50) values. The most potent inhibitors were OTX015, AZD6244, and SCH772984, with IC50 values of 0.31 µM, 0.33 µM, and 0.44 µM, respectively, at 24 h post-LPS challenge (50 of 1.54 µM). As the inhibitors of BET proteins [JQ1 inhibitor was included as a positive control as it has already been established as a potent inhibitor of macrophages’ inflammatory responses . The effhallenge A. The inproteins  and MEK1proteins  have alrp-value < 0.05) with SCH772984 treatment at 0 h, 1 h, 4 h, and 24 h LPS time points, respectively  using the Reactome database. The functional analysis yielded 0/0, 2/0, 7/23, and 0/15 significantly  enriched pathways for down-/up-regulated DEGs in 0 h, 1 h, 4 h, and 24 h, respectively  and LPS-challenged RAW 264.7 cells  treated with SCH772984. The pair-wise analyses revealed 35, 343, 1661, and 4123 differentially expressed genes (DEGs)  at 1 h, 4 h, and 24 h time-points, respectively  inhibited the Ccl2/MCP-1 plasma level at the 12 h time point. In contrast, the plasma levels of IL-2, IP-10 (CXCL10), MIP-1α, and IL-4, IL-27 were significantly elevated at 12 h and 6 h time points post-CLP, respectively, in the CLP+SCH772984 group. Finally, MIP-1β levels were significantly higher upon SCH772984 administration at the 6 h and 12 h time points  with repetitive SCH772984 dosing every 6 h significantly improved mice survival A. SCH772e points C. The lee points . Additioe points . Togethep-value < 0.05) in the heart, kidney, lung, and livers samples at 6 h, and 1503, 6998, 6419, and 6310 DEGs at 12 h, respectively  using the Reactome database. We focused on the pathways that were recurrently altered in more than two types of samples, with a differential gene ratio higher than 10%, which yielded 38 pathways that fall into the Immune system, Hemostasis, ECM organization, Metabolism, Signal Transduction, and Developmental Biology Reactome pathways parental categories  Alteration of the immune system pathways in the kidney and liver. (ii) Overrepresentation of downregulated transcripts involved in hemostasis and platelet activation, signaling, and aggregation pathways. In this regard, coagulopathy is a hallmark of sepsis, manifesting liver dysfunction , and pla50 values of 4 and 1 nM, respectively [SCH772984 is an ATP-competitive inhibitor that binds to the unphosphorylated, inactive form of ERK1/2, and potently and selectively inhibits ERK1 and ERK2 activity, with ICectively . Severalectively ,46,47,48ectively  and dabrectively . V600 mutation, in a CLP mouse model of sepsis [Repurposing the large arsenal of existing cancer drugs is an attractive proposition to expand the clinical pipelines for sepsis treatment. A study by Rialdi et al. showed that the topoisomerase I inhibitors, approved for ovarian, lung, and colon cancer treatment, can significantly prolong survival in an animal model of sepsis by attenuating an acute inflammatory reaction in a mechanism that blocks the RNA polymerase II transcriptional activity at a set of genes that require chromatin remodeling and are activated immediately by immune cells during pathological inflammation . Anotherf sepsis . Althougf sepsis . More ref sepsis . Zeng etf sepsis ,19,20, wf sepsis . In summmIMCD-3 (CRL-2123), AML12, (CRL-2254), MLg (CCL-206), and RAW 264.7 (TIB-71) mouse cell lines were purchased from the ATCC  and were maintained under the recommended conditions. For AlphaLISA, 10000 cells/well were seeded on a 384-well plate  using the MultiFlo FX Microplate Dispenser. The next day, cells were treated with LPS  at a final concentration of 10 µg/mL, the plates were then incubated at 37 °C for 4 and 24 h, and the TNFa concentration was measured using AlphaLISA  on the Spark reader  and the AlphaScreen module as per the manufacturer’s protocol. The CellTiter-Glo Luminescent Cell Viability Assay  microplate assay was used to measure cell viability. Before the LPS challenge, cells were pre-treated for 1 h with the following drugs purchased from Selleckchem: AZD6244, SCH772984, MS-275, EX527, EPZ-5676, EPZ-6438, ORY-1001, RVX-208, OTX015, and (+)-JQ1 targeting the chromatin-associated proteins MEK 1/2, ERK 1/2, HDAC 1/2/3, SIRT1, DOT1L, EZH2, LSD1, BRD 2/3/4, BRD2/3, and BRD4 , respectively, at the concentrations of 5, 1, 0.2, 0.04, 0.008, 0.0018, and 0.00032 µM. Drugs were dispensed using the Microlab STAR unit , while the transfer and media dilutions for the AlphaScreen readout were performed with the CyBio SELMA liquid handler . Measurements were performed in 12-plicate.5 RAW 264.7 cells were seeded in the presence of 10% Fetal Bovine Serum (FBS) on a 6-well plate a day before the treatment. The next day, cells were pretreated with 500 nM of SCH772984 for 1 h and then challenged with 10 µg/mL of LPS for 1, 4, and 24 h in the presence of 500 nM of SCH772984. Cells were collected at indicated time points and pellets were frozen at −80 °C for later RNA isolation or immediately used for the ATAC-Seq protocol.Here, 5 × 103 tissue fragments were cut off in a dry ice-chilled workplace. Fragments were grinded with a disposable pestle  in 200 µL of TRI Reagent and left to dissolve further for 2 min in RT. To remove debris, samples were centrifuged at 14,000× g for 30 s. The supernatant containing RNA was further processed using the Direct-zolTM RNA MiniPrep Plus kit  following manufacturer instructions. RNA was stored at −80 °C.Total RNA was isolated from RAW 264.7 cells and tissues using TRIzol reagents . Then, 3 mm® Gene Expression Assays and the SensiFAST™ Probe Hi-ROX Kit  on the Applied Biosystems 7900HT Fast Real-Time PCR System, as described in [The RT reaction was performed using the High-Capacity cDNA Reverse Transcription Kit  with 500 ng of total RNA according to the manufacturer’s protocol. qPCRs of cDNA were carried out using TaqManribed in . The folRNAseq cDNA libraries were prepared with the TruSeq Stranded Total RNA Library Prep Kit with the Ribo-Zero Human/Mouse/Rat Sample Prep Kit  at the CeGaT GmbH  and sequenced there with the Illumina NovaSeq 6000 Platform using 100 bps paired-end reads. Raw sequences were trimmed according to quality using Trimmomatic (version 0.39)  using deThe overrepresentation of Reactome pathways in genes that were differentially expressed was performed with R package ClusterProfiler version 3.6, separately for up- and downregulated genes . A heatm5 RAW 264.7 cells were washed using 50 μL of ice-cold 1x PBS and centrifuged at 500× g for 7 min at 4 °C. To obtain nuclear pellets, cells were lysed using 50 μL of cold lysis buffer  and immediately spun at 500× g for 10 min at 4 °C. To avoid losing cells/nuclei, we carefully pipetted away the supernatant from the pellet. The nuclei pellets were resuspended in 50 μL of ice-cold transposition reaction mix: 25 μL of 2x TD buffer ; 2.5 μL of TDE1 ; and 22.5 μL of nuclease-free water. The transposition reaction was carried out for 30 min at 37 °C with 300 rpm mixing. Directly following transposition, the samples were purified using the DNA Clean & Concentrator kit  according to the manufacturer’s protocol. Libraries were constructed using: 10 μL of Transposed DNA; 10 μL of nuclease-free water; 25 μL of NEBNext® Ultra™ II Q5® Master Mix , and 2.5 μL of 25 μM of custom Nextera PCR primer 1 (i5) and primer 2 (i7) , using the following PCR conditions: 72 °C for 5 min, 98 °C for 30 s—1 cycle, followed by thermocycling at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min—5 cycles. To reduce the GC and size bias, we monitored the PCR reaction using qPCR to stop amplification before saturation. To this end, after the 5 cycles, a volume of 5 μL of the PCR reaction was mixed with: 4.1 μL of nuclease-free water; 0.25 μL of 25 μM of custom Nextera PCR primer 1 (i5) and primer 2 (i7); 0.1 μL of 100x SYBR Green I; and 5 μL of NEBNext® Ultra™ II Q5® Master Mix. The qPCR reaction was run under the following conditions: 98 °C for 30 s—1 cycle, followed by thermocycling at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min—20 cycles. To calculate the additional number of cycles needed for the remaining 45 μL reaction, we analyzed the plotted linear Rn versus cycle and determined the cycle number that corresponds to 1/3 of the maximum fluorescent intensity. Libraries were amplified for a total of 16 cycles and purified using the DNA Clean & Concentrator kit  according to the manufacturer’s protocol. Library quality and concentration were determined by the Agilent High Sensitivity DNA Kit on a Bioanalyzer 2100 . ATAC-Seq libraries were sequenced with the Illumina NovaSeq 6000 Platform with 75 bps paired-end reads at CeGaT GmbH . Two biological replicates were analyzed per treatment. ATAC-Seq reads were processed using Trimmomatic (version 0.39) using default parameters and then aligned to the mouse reference genome mm10 using bowtie2 [The ATAC-Seq was performed according to the Buenrostro et al. protocol  with ming, 4 °C, and 10 min. Protein concentration was measured with a BCA Protein Assay Kit . Then, 20 µg of total protein per sample was resolved on 10% SDS-PAGE and transferred to the PVDF membrane . Membranes were blocked in 5% Bovine Serum Albumin (BSA) in Tris-Buffered Saline, with Tween 20 (TBST) followed by incubation with specific primary and secondary antibodies. For signal detection, the Clarity Western ECL Substrate  and ChemiDoc imaging system  were used. Antibodies used: pErk1/2 (Thr202/Tyr204)  and Actb . The level of ERK1/2 phosphorylation was quantified by densitometric analysis using ImageJ software. For each blot, the background-subtracted density of the protein band was divided by the density of the loading control. The mean and standard deviations for the biological replicates were determined.Cells and tissues were lysed in RIPA buffer  supplemented with a cocktail of protease and phosphatase inhibitors . Tissues were sonicated in a Bioruptor Plus  using a 30 s on–off cycles protocol for 10 min at high intensity followed by centrifugation at 12,000× C57BL/6W (own breeding) mice males, 16–20 weeks of age, were maintained under the specific pathogen-free animal facility at the Maria Skłodowska-Curie National Research Institute of Oncology according to the institutional guidelines.Weight-matched 16–20 weeks-old male mice were injected with 20 mg/kg of LPS into the contralateral side of the abdomen.The CLP procedure to induce high-grade sepsis was performed as previously described . BrieflyMice were maintained at room temperature in cages, 4 individuals each, with access to fresh water and food throughout the experiment. Mice were monitored by two investigators every 2 h after the induction of sepsis. The severity of sepsis was assessed by five variables and the 0–4 scoring system, including physical appearance, body weight, measurable clinical symptoms, unprovoked behavior, and response to external stimuli . Mice wei.p. 2 h following LPS (20 mg/kg) dosing in the survival experiment, and then every 6 h later, the SCH772984 was administrated. For the 6 h and 12 h time points and survival, CLP experiments’ mice were injected i.p. with 10 mg/kg of SCH772984 or formulation 2 h following the CLP procedure and every 6 h with another dose of SCH772984 or formulation. In summary, mice at 6 h and 12 h time points received a single and double dose of SCH772984, respectively. At 6 h and 12 h post-CLP, anesthesia was induced with isoflurane and blood was collected via cardiac puncture. The chest was opened and the heart and lungs collected rapidly into cryovials, which were immediately snap-frozen in liquid nitrogen. The kidney and liver were collected similarly immediately thereafter. The SCH772984 at 10 mg/kg or formulation (3% DMSO + 10% polyethylene glycol) was injected Cytokine & Chemokine 26-Plex Mouse ProcartaPlex™   was used to screen cytokines in 50 μL of plasma according to the manufacturer’s instructions.Our study identified the ERK1/2 inhibitor, the SCH772984 compound, as a promising drug improving survival in a preclinical model of sepsis. Transcriptomic signatures of SCH772984 compound action in vivo highlighted its influence on immune response, the platelet-related signaling, ECM, and RA signaling pathways. Recently, to improve the pharmacokinetics of SCH772984, the medical chemistry optimization yielded the MK-8353 compound  that was