Ferrostatin-1 improves prognosis and regulates gut microbiota of steatotic liver transplantation recipients in rats.

Aims: To investigate the effects of Ferrostatin-1 (Fer-1) on improving the prognosis of liver transplant recipients with steatotic liver grafts and regulating gut microbiota in rats. Methods: We obtained steatotic liver grafts and established a liver transplantation model. Recipients were divided into sham, liver transplantation and Fer-1 treatment groups, which were assessed 1 and 7 days after surgery (n = 6). Results & conclusion: Fer-1 promotes recovery of the histological structure and function of steatotic liver grafts and the intestinal tract, and improves inflammatory responses of recipients following liver transplantation. Fer-1 reduces gut microbiota pathogenicity, and lowers iron absorption and improves fat metabolism of recipients, thereby protecting steatotic liver grafts.

Development of a machine learning-based model to predict hepatic inflammation in chronic hepatitis B patients with concurrent hepatic steatosis: a cohort study.

Background: With increasingly prevalent coexistence of chronic hepatitis B (CHB) and hepatic steatosis (HS), simple, non-invasive diagnostic methods to accurately assess the severity of hepatic inflammation are needed. We aimed to build a machine learning (ML) based model to detect hepatic inflammation in patients with CHB and concurrent HS. Methods: We conducted a multicenter, retrospective cohort study in China. Treatment-naive CHB patients with biopsy-proven HS between April 2004 and September 2022 were included. The optimal features for model development were selected by SHapley Additive explanations, and an ML algorithm with the best accuracy to diagnose moderate to severe hepatic inflammation (Scheuer's system ≥ G3) was determined and assessed by decision curve analysis (DCA) and calibration curve. This study is registered with ClinicalTrials.gov (NCT05766449). Findings: From a pool of 1,787 treatment-naive patients with CHB and HS across eleven hospitals, 689 patients from nine of these hospitals were chosen for the development of the diagnostic model. The remaining two hospitals contributed to two independent external validation cohorts, comprising 509 patients in validation cohort 1 and 589 in validation cohort 2. Eleven features regarding inflammation, hepatic and metabolic functions were identified. The gradient boosting classifier (GBC) model showed the best performance in predicting moderate to severe hepatic inflammation, with an area under the receiver operating characteristic curve (AUROC) of 0.86 (95% CI 0.83-0.88) in the training cohort, and 0.89 (95% CI 0.86-0.92), 0.76 (95% CI 0.73-0.80) in the first and second external validation cohorts, respectively. A publicly accessible web tool was generated for the model. Interpretation: Using simple parameters, the GBC model predicted hepatic inflammation in CHB patients with concurrent HS. It holds promise for guiding clinical management and improving patient outcomes. Funding: This research was supported by the National Natural Science Foundation of China (No. 82170609, 81970545), Natural Science Foundation of Shandong Province (Major Project) (No. ZR2020KH006), Natural Science Foundation of Jiangsu Province (No.BK20231118), Tianjin Key Medical Discipline (Specialty), Construction Project, TJYXZDXK-059B, Tianjin Health Science and Technology Project key discipline special, TJWJ2022XK034, and Research project of Chinese traditional medicine and Chinese traditional medicine combined with Western medicine of Tianjin municipal health and Family Planning Commission (2021022).

Heme Oxygenase-1-Modified BMMSCs Activate AMPK-Nrf2-FTH1 to Reduce Severe Steatotic Liver Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is an important cause of graft dysfunction post-liver transplantation, where donor liver with severe steatosis is more sensitive to IRI. Liver IRI involves ferroptosis and can be alleviated by heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs). AIMS: To explore the role and mechanism of HO-1/BMMSCs in severe steatotic liver IRI. METHODS: A severe steatotic liver IRI rat model and a hypoxia/reoxygenation (H/R) of severe steatosis hepatocyte model were established. Liver and hepatocyte damage was evaluated via liver histopathology and cell activity. Ferroptosis was evaluated through ferroptosis indexes. Nuclear factor erythroid 2-related factor 2 (Nrf2) was knocked down in severe steatotic hepatocytes. The role of Nrf2 and AMPK in HO-1/BMMSC inhibition of ferroptosis was examined using the AMP-activated protein kinase (AMPK) pathway inhibitor Compound C. RESULTS: The HO-1/BMMSCs alleviated severe steatotic liver IRI and ferroptosis. HO-1/BMMSCs promoted ferritin heavy chain 1(FTH1), Nrf2, and phosphorylated (p)-AMPK expression in the H/R severe steatotic hepatocytes. Nrf2 knockdown decreased FTH1 expression levels but did not significantly affect p-AMPK expression levels. The protective effect of HO-1/BMMSCs against H/R injury in severe steatotic hepatocytes and the inhibitory effect on ferroptosis were reduced. Compound C decreased p-AMPK, Nrf2, and FTH1 expression levels, weakened the HO-1/BMMSC protective effect against severe steatotic liver IRI and H/R-injured severe steatotic hepatocytes, and reduced the inhibition of ferroptosis. CONCLUSIONS: Ferroptosis was involved in HO-1/BMMSC reduction of severe steatotic liver IRI. HO-1/BMMSCs protected against severe steatotic liver IRI by inhibiting ferroptosis through the AMPK-Nrf2-FTH1 pathway. HO-1/BMMSCs activate AMPK, which activates Nrf2, promotes its nuclear transcription, then promotes the expression of its downstream protein FTH1, thereby inhibiting ferroptosis and attenuating severe steatotic liver IRI in rats. Glu: glutamic acid; Cys: cystine; GSH: glutathione; GPX4: glutathione peroxidase 4; HO-1/BMMSCs: HO-1-modified BMMSCs; Fer-1: ferrostatin-1; DFO: deferoxamine; FTH1: ferritin heavy chain1; p-AMPK: phosphorylated AMP-activated protein kinase; Nrf2: nuclear factor erythroid 2-related factor 2; IRI: ischemia-reperfusion injury; MCD: methionine-choline deficiency.

Small extracellular vesicles from HO-1-modified bone marrow-derived mesenchymal stem cells attenuate ischemia-reperfusion injury after steatotic liver transplantation by suppressing ferroptosis via miR-214-3p.

Donor shortage is a major problem that limits liver transplantation availability. Steatotic donor liver presents a feasible strategy to solve this problem. However, severe ischemia-reperfusion injury (IRI) is an obstacle to the adoption of steatotic transplanted livers. Evidence from our prior studies indicated that bone marrow mesenchymal stem cells modified with heme oxygenase-1 (HMSCs) can attenuate non-steatotic liver IRI. However, the contribution of HMSCs in transplanted steatotic liver IRI is unclear. Here, HMSCs and their derived small extracellular vesicles (HM-sEVs) alleviated IRI in transplanted steatotic livers. After liver transplantation, there was significant enrichment of the differentially expressed genes in the glutathione metabolism and ferroptosis pathways, accompanied by ferroptosis marker upregulation. The HMSCs and HM-sEVs suppressed ferroptosis and attenuated IRI in the transplanted steatotic livers. MicroRNA (miRNA) microarray and validation experiments indicated that miR-214-3p, which was abundant in the HM-sEVs, suppressed ferroptosis by targeting cyclooxygenase 2 (COX2). In contrast, COX2 overexpression reversed this effect. Knockdown of miR-214-3p in the HM-sEVs diminished its ability to suppress ferroptosis and protect liver tissues/cells. The findings suggested that HM-sEVs suppressed ferroptosis to attenuate transplanted steatotic liver IRI via the miR-214-3p-COX2 axis.

Comparative Efficacy of Six Active Warming Systems for Intraoperative Warming in Adult Patients Undergoing Laparoscopic Surgery: A Systematic Review and Network Meta-Analysis.

Intraoperative hypothermia is very common and harmful in adult patients undergoing laparoscopic surgery. A variety of active warming systems has received close attention and has been researched by related scholars. However, the relative efficacy of these systems and which active warming system is preferred for such patients remain unclear. The aim of this study was to compare and rank six active warming systems regarding intraoperative warming efficacy in adult patients undergoing laparoscopic surgery. Following the PRISMA 2020 guidelines, relevant randomized controlled trials (RCTs) on the efficacy of different active warming systems in warming adult patients undergoing laparoscopic surgery were searched from five English databases and three Chinese databases. The quality of the studies was assessed using the Cochrane Risk of Bias tool (RoB2). The outcome was the final intraoperative core temperature. We estimated direct effects by using pairwise meta-analysis, estimated relative effects and ranking with the consistency model to conduct an NetworkMeta-Analysis (NMA). We used GRADE (Grading of Recommendations Assessment, Development, and Evaluation) to assess the certainty of the evidence. Sensitivity analysis was performed to test the robustness of the results. This study is registered with PROSPERO, with number CRD42022309057. In total, 19 RCTs involving 6 active warming systems and comprising 1364 patients were included in this NMA. The NMA once again confirmed the validity of forced-air warming (FAW) systems compared with other active warming systems, and further showed that underbody FAW was associated with more remarkable warming efficacy in different types of FAW systems. NMA was used to perform an exhaustive comparison of the warming efficacy of six active warming systems and indicated that underbody FAW was most likely to be the most effective warming system in adult patients undergoing laparoscopic surgery; however, considering the sparsity of the network, our results should be cautiously interpreted. Furthermore, a large number of high-quality RCTs comparing the warming efficacy of different competitive active warming systems are needed.

miR-29a-3p in Exosomes from Heme Oxygenase-1 Modified Bone Marrow Mesenchymal Stem Cells Alleviates Steatotic Liver Ischemia-Reperfusion Injury in Rats by Suppressing Ferroptosis via Iron Responsive Element Binding Protein 2.

Hepatic ischemia-reperfusion injury (IRI) is an inevitable result of liver surgery. Steatotic livers are extremely sensitive to IRI and have worse tolerance. Ferroptosis is considered to be one of the main factors of organ IRI. This study is aimed at exploring the role of ferroptosis in the effect of heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs) on steatotic liver IRI and its mechanism. An IRI model of a steatotic liver and a hypoxia reoxygenation (HR) model of steatotic hepatocytes (SHPs) were established. Rat BMMSCs were extracted and transfected with the Ho1 gene to establish HO-1/BMMSCs, and their exosomes were extracted by ultracentrifugation. Ireb2 was knocked down to verify its role in ferroptosis and cell injury in SHP-HR. Public database screening combined with quantitative real-time reverse transcription PCR identified microRNAs (miRNAs) targeting Ireb2 in HO-1/BMMSCs exosomes. miR-29a-3p mimic and inhibitor were used for functional verification experiments. Liver function, histopathology, terminal deoxynulceotidyl transferase nick-end-labeling staining, cell viability, mitochondrial membrane potential, and cell death were measured to evaluate liver tissue and hepatocyte injury. Ferroptosis was assessed by detecting the levels of IREB2, Fe2+, malondialdehyde, glutathione, lipid reactive oxygen species, glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2 mRNA, and mitochondrial morphology. The results revealed that HO-1/BMMSCs improved liver tissue and hepatocyte injury and suppressed ferroptosis in vivo and in vitro. The expression of IREB2 was increased in steatotic liver IRI and SHP-HR. Knocking down Ireb2 reduced the level of Fe2+ and inhibited ferroptosis. HO-1/BMMSC exosomes reduced the expression of IREB2 and inhibited ferroptosis and cell damage. Furthermore, we confirmed high levels of miR-29a-3p in HO-1/BMMSCs exosomes. Overexpression of miR-29a-3p downregulated the expression of Ireb2 and inhibited ferroptosis. Downregulation of miR-29a-3p blocked the protective effect of HO-1/BMMSC exosomes on SHP-HR cell injury. In conclusion, ferroptosis plays an important role in HO-1/BMMSC-mediated alleviation of steatotic liver IRI. HO-1/BMMSCs could suppress ferroptosis by targeting Ireb2 via the exosomal transfer of miR-29a-3p.

Intestinal Microbiota Participates in the Protective Effect of HO-1/BMMSCs on Liver Transplantation With Steatotic Liver Grafts in Rats.

The present study aimed to explore whether heme oxygenase-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) have a protective effect on liver transplantation with steatotic liver grafts in rats, and to determine the role of the intestinal microbiota in such protection. HO-1/BMMSCs were obtained by transduction of Hmox1 gene [encoding heme oxygenase (HO-1)]-encoding adenoviruses into primary rat BMMSCs. Steatotic livers were obtained by feeding rats a high-fat diet, and a model of liver transplantation with steatotic liver grafts was established. The recipients were treated with BMMSCs, HO-1/BMMSCs, or neither, via the portal vein. Two time points were used: postoperative day 1 (POD 1) and POD 7. The results showed that under the effect of HO-1/BMMSCs, the degree of steatosis in the liver grafts was significantly reduced, and the level of liver enzymes and the levels of pro-inflammatory cytokines in plasma were reduced. The effect of HO-1/BMMSCs was better than that of pure BMMSCs in the prolongation of the rats' postoperative time. In addition, HO-1/BMMSCs promoted the recovery of recipients' intestinal structure and function, especially on POD 7. The intestinal villi returned to normal, the expression of tight junction proteins was restored, and intestinal permeability was reduced on POD 7. The intestinal bacterial of the LT group showed significantly weakened energy metabolism and overgrowth. On POD 1, the abundance of Akkermansiaceae was higher. On POD 7, the abundance of Clostridiaceae increased, the level of lipopolysaccharide increased, the intestinal mucosal barrier function was destroyed, and the levels of several invasive bacteria increased. When treated with HO-1/BMMSCs, the energy metabolism of intestinal bacteria was enhanced, and on POD 1, levels bacteria that protect the intestinal mucosa, such as Desulfovibrionaceae, increased significantly. On POD 7, the changed intestinal microbiota improved lipid metabolism and increased the levels of butyrate-producing bacteria, such as Lachnospiraceae. In conclusion, HO-1/BMMSCs have protective effects on steatotic liver grafts and the intestinal barrier function of the recipients. By improving lipid metabolism and increasing the abundance of butyrate-producing bacteria, the changed intestinal microbiota has a protective effect and prolongs the recipients' survival time.

miR-124-3p delivered by exosomes from heme oxygenase-1 modified bone marrow mesenchymal stem cells inhibits ferroptosis to attenuate ischemia-reperfusion injury in steatotic grafts.

BACKGROUND: Steatotic livers tolerate ischemia-reperfusion injury (IRI) poorly, increasing the risk of organ dysfunction. Ferroptosis is considered the initiating factor of organ IRI. Heme oxygenase oxygen-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) (HO-1/BMMSCs) can reduce hepatic IRI; however, the role of ferroptosis in IRI of steatotic grafts and the effect of HO-1/BMMSCs-derived exosomes (HM-exos) on ferroptosis remain unknown. METHODS: A model of rat liver transplantation (LT) with a severe steatotic donor liver and a model of hypoxia and reoxygenation (H/R) of steatotic hepatocytes were established. Exosomes were obtained by differential centrifugation, and the differentially expressed genes (DEGs) in liver after HM-exo treatment were detected using RNA sequencing. The expression of ferroptosis markers was analyzed. microRNA (miRNA) sequencing was used to analyze the miRNA profiles in HM-exos. RESULTS: We verified the effect of a candidate miRNA on ferroptosis of H/R treated hepatocytes, and observed the effect of exosomes knockout of the candidate miRNA on hepatocytes ferroptosis. In vitro, HM-exo treatment reduced the IRI in steatotic grafts, and enrichment analysis of DEGs suggested that HM-exos were involved in the regulation of the ferroptosis pathway. In vitro, inhibition of ferroptosis by HM-exos reduced hepatocyte injury. HM-exos contained more abundant miR-124-3p, which reduced ferroptosis of H/R-treated cells by inhibiting prostate six transmembrane epithelial antigen 3 (STEAP3), while overexpression of Steap3 reversed the effect of mir-124-3p. In addition, HM-exos from cell knocked out for miR-124-3p showed a weakened inhibitory effect on ferroptosis. Similarly, HM-exo treatment increased the content of miR-124-3p in grafts, while decreasing the level of STEAP3 and reducing the degree of hepatic ferroptosis. CONCLUSION: Ferroptosis is involved in the IRI during LT with a severe steatotic donor liver. miR-124-3p in HM-exos downregulates Steap3 expression to inhibit ferroptosis, thereby attenuating graft IRI, which might be a promising strategy to treat IRI in steatotic grafts.

Bone marrow mesenchymal stem cells modified with heme oxygenase-1 alleviate rejection of donation after circulatory death liver transplantation by inhibiting dendritic cell maturation in rats.

Immature dendritic cells induce immune tolerance and mature dendritic cells induce acute rejection. We infused bone marrow mesenchymal stem cells (BMMSCs) expressing heme oxygenase 1 (HO-1) (HO-1/BMMSCs) into donation after circulatory death (DCD) livers using normothermic machine perfusion (NMP), and then performed transplantation, with the aim of determining the effects of HO 1/BMMSCs on liver DC maturation and graft rejection. A rat model of acute liver transplantation rejection was established from Lewis to BN rats, in which six experimental groups were set up: Sham operation, static cold storage, NMP, BMMSCs + NMP, HO-1/BMMSCs + NMP (HBP), and NMP + FK506 gavage. Flow cytometry was performed to detect the maturation of DCs and the activation of CD4+ T cells in the liver. In vitro, HO-1/BMMSCs were cocultured with liver DCs, and then the phenotype and ability to stimulate lymphocyte proliferation of DCs were measured. MAPK inhibitors were added to observe the effect of MAPK signaling on DC maturation. The resultsindicatedthatHO-1/BMMSCs could stably colonize the transplanted liver. In the HBP group, rejection was reduced, the maturation of DCs was inhibited, and the infiltration and activation of CD4+ T cells were reduced. In vitro, DCs cocultured with HO-1/BMMSCs showed an immature phenotype and inhibited T cell proliferation. HO-1/BMMSCs inhibited the maturation of DCs by blocking the phosphorylation of p38 and ERK1/2. This study suggested that infusion of HO-1/BMMSCs into DCD livers could reduce acute rejection significantly by inhibiting DC maturation. DC maturation regulation by HO-1/BMMSCs involves ERK1/2/MAPK and p38/MAPK signaling.

HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats.

BACKGROUND: Liver transplantation (LT) is required in many end-stage liver diseases. Donation after cardiac death (DCD) livers are often used, and treatment of acute rejection (ACR) requires the use of immunosuppressive drugs that are associated with complications. Bone marrow mesenchymal stem cells (BMMSCs) are used in treatment following LT; however, they have limitations, including low colonization in the liver. An optimized BMMSC application method is required to suppress ACR. METHODS: BMMSCs were isolated and modified with the heme oxygenase 1 (HO-1) gene. HO-1/BMMSCs were perfused into donor liver in vitro using a normothermic machine perfusion (NMP) system, followed by LT into rats. The severity of ACR was evaluated based on liver histopathology. Gene chip technology was used to detect differential gene expression, and flow cytometry to analyze changes in natural killer (NK) T cells. RESULTS: NMP induced BMMSCs to colonize the donor liver during in vitro preservation. The survival of HO-1/BMMSCs in liver grafts was significantly longer than that of unmodified BMMSCs. When the donor liver contained HO-1/BMMSCs, the local immunosuppressive effect was improved and prolonged, ACR was controlled, and survival time was significantly prolonged. The application of HO-1/BMMSCs reduced the number of NKT cells in liver grafts, increased the expression of NKT cell co-inhibitory receptors, and reduced NKT cell expression of interferon-γ. CONCLUSIONS: NK cell and CD8+ T cell activation was inhibited by application of HO-1/BMMSCs, which reduced ACR of transplanted liver. This approach could be developed to enhance the success rate of LT.

Corallincola luteus sp. nov., a marine bacterium isolated from surface sediment of Bohai Sea of China.

A novel Gram-stain negative, strictly aerobic, rod-shaped motile bacterium with a single flagellum, designated strain DASS28T, was isolated from surface sediment of Bohai Sea in China. Growth occurred in the presence of 1.0-4.0% NaCl (w/v, optimum 2.0%), at 10-37 °C (optimum 20 °C) on the Marine agar 2216E and pH 6.0-10.0 (optimum pH 8.0). The major fatty acids (> 10% of total fatty acids) were summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), C16:0 and C18:1ω7c. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid and two unidentified polar lipids. The major respiratory quinone was ubiquinone-8 (Q-8). The genomic DNA G + C content calculated from the genome sequence of strain DASS28T was 48.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain DASS28T belongs to the genus Corallincola and shows high 16S rRNA gene sequence similarity of 96.7% to Corallincola platygyrae JLT 2006T (= JCM18796T = CGMCC 1.10992T). On the basis of the polyphasic evidence, strain DASS28T is considered to represent a novel species in the genus Corallincola, for which the name Corallincola luteus sp. nov. is proposed. The type strain is DASS28T (= KCTC 52376T = MCCC 1K03208T).

Thalassococcus profundi sp. nov., a marine bacterium isolated from deep seawater of the Okinawa Trough.

A novel Gram-stain-negative, strictly aerobic, rod-shaped motile bacterium with a single flagellum, designated strain WRAS1T, was isolated from deep seawater of the Okinawa Trough. Growth occurred in the presence of 0.0-9.0 % NaCl (w/v; optimum, 3.0-4.0 %), at 4-45 °C (optimum, 28-37 °C) and pH 7.0-10.0 (optimum, pH 7.0-8.0). The major fatty acid (>10 % of total fatty acids) was summed feature 8, comprising C18:1ω6c and/or C18:1ω7c. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. The major respiratory quinone was ubiquinone-10. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain WRAS1T was in the genus Thalassococcus and showed the highest 16S rRNA gene sequence similarity of 97.5 % to Thalassococcus halodurans JCM13833T. Genome relatedness between strain WRAS1T and T. halodurans JCM13833T was computed using both average nucleotide identity and DNA-DNA hybridization with values of 74.11 % and 22.70±2.3 %, respectively. The genomic DNA G+C content calculated from the genome sequence of strain WRAS1T was 65.6 %. On the basis of polyphasic analyses, strain WRAS1T is considered to represent a novel species in the genus Thalassococcus, for which the name Thalassococcusprofundi sp. nov. is proposed. The type strain is WRAS1T (=CGMCC 1.16123T=MCCC 1K03253T =KCTC 52696T).

Alcanivorax profundi sp. nov., isolated from deep seawater of the Mariana Trench.

A Gram-stain-negative, rod-shaped, non-motile, strictly aerobic strain, designated as MTEO17T, was isolated from a 1000 m deep seawater sample of the Mariana Trench. Growth was observed at 10-45 °C (optimum, 37 °C), in the presence of 0.0-12.0 % NaCl (w/v; optimum, 3.0 %) and at pH 6.0-10.0 (optimum, pH 7.0-8.0). Phylogenetic analysis, based on the 16S rRNA gene sequence, revealed that strain MTEO17T belonged to the genus Alcanivorax and showed the highest sequence similarity of 97.9 % to Alcanivorax nanhaiticus MCCC 1A05629T. The estimated average nucleotide identity and DNA-DNA hybridization values between strain MTEO17T and A. nanhaiticus MCCC 1A05629T were 78.98 and 23.80 %, respectively. The significant dominant fatty acids were C16 : 0, summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The polar lipids comprised two phosphatidylethanolamines, one phosphatidylglycerol, one unidentified phospholipid and four unidentified polar lipids. The DNA G+C content of strain MTEO17T was 57.5 %. On the basis of the polyphasic evidence, strain MTEO17T is proposed to represent a novel species of the genus Alcanivorax, for which the name Alcanivorax profundi sp. nov. is proposed. The type strain is MTEO17T (=KCTC 52694T=MCCC 1K03252T).

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization.

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570 mg/L vs. 119 mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850 mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100 mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.