Hydrophilic and Hydrophobic Compounds Antithetically Affect the Growth of Eicosapentaenoic Acid-Synthesizing Escherichia coli Recombinants

The growth of Escherichia coli DH5α recombinants producing eicosapentaenoic acid (EPA) (DH5αEPA+) and those not producing EPA (DH5αEPA–) was compared in the presence of hydrophilic or hydrophobic growth inhibitors. The minimal inhibitory concentrations of hydrophilic inhibitors such as reactive oxygen species and antibiotics were higher for DH5αEPA+ than for DH5αEPA–, and vice versa for hydrophobic inhibitors such as protonophores and radical generators. E. coli DH5α with higher levels of EPA became more resistant to ethanol. The cell surface hydrophobicity of DH5αEPA+ was higher than that of DH5αEPA–, suggesting that EPA may operate as a structural constituent in the cell membrane to affect the entry and efflux of hydrophilic and hydrophobic inhibitors

The novel function of eicosapentaenoic acid in bacterial cell membranes

第2回極域科学シンポジウム/第33回極域生物シンポジウム 11月17日（木） 統計数理研究所 3階リフレッシュフロ

Risk factors for CAR-T cell manufacturing failure among DLBCL patients: A nationwide survey in Japan

CAR-T細胞製造を成功させるためのレシピ --アフェレーシス前の下ごしらえでの工夫--. 京都大学プレスリリース. 2023-04-27.For successful chimeric antigen receptor T (CAR-T) cell therapy, CAR-T cells must be manufactured without failure caused by suboptimal expansion. In order to determine risk factors for CAR-T cell manufacturing failure, we performed a nationwide cohort study in Japan and analysed patients with diffuse large B-cell lymphoma (DLBCL) who underwent tisagenlecleucel production. We compared clinical factors between 30 cases that failed (7.4%) with those that succeeded (n = 378). Among the failures, the proportion of patients previously treated with bendamustine (43.3% vs. 14.8%; p < 0.001) was significantly higher, and their platelet counts (12.0 vs. 17.0 × 10⁴/μL; p = 0.01) and CD4/CD8 T-cell ratio (0.30 vs. 0.56; p < 0.01) in peripheral blood at apheresis were significantly lower than in the successful group. Multivariate analysis revealed that repeated bendamustine use with short washout periods prior to apheresis (odds ratio [OR], 5.52; p = 0.013 for ≥6 cycles with washout period of 3–24 months; OR, 57.09; p = 0.005 for ≥3 cycles with washout period of <3 months), low platelet counts (OR, 0.495 per 105/μL; p = 0.022) or low CD4/CD8 ratios (<one third) (OR, 3.249; p = 0.011) in peripheral blood at apheresis increased the risk of manufacturing failure. Manufacturing failure remains an obstacle to CAR-T cell therapy for DLBCL patients. Avoiding risk factors, such as repeated bendamustine administration without sufficient washout, and risk-adapted strategies may help to optimize CAR-T cell therapy for DLBCL patients

Membrane eicosapentaenoic acid is involved in the hydrophobicity of bacterial cells and affects the entry of hydrophilic and hydrophobic compounds

Eicosapentaenoic acid (EPA)-producing Shewanella marinintestina IK-1 (IK-1) and its EPA-deficient mutant IK-1Δ8 (IK-1Δ8) were grown on microtitre plates at 20 °C in a nutrient medium that contained various types of growth inhibitors. The minimal inhibitory concentrations of hydrogen peroxide and tert-butyl hydroxyl peroxide were 100 μM and 1 mM, respectively, for IK-1 and 10 and 100 μM, respectively, for IK-1Δ8. IK-1 was much more resistant than IK-1Δ8 to the four water-soluble antibiotics (ampicillin sodium, kanamycin sulphate, streptomycin sulphate, and tetracycline hydrochloride) tested. In contrast, IK-1 was less resistant than IK-1Δ8 to two hydrophobic uncouplers: carbonyl cyanide m-chloro phenylhydrazone (CCCP) and N,N′-dicyclohexylcarbodiimide (DCCD). The hydrophobicity of the IK-1 and IK-1Δ8 cells grown at 20 °C was determined using the bacterial adhesion to hydrocarbon method. EPA-containing (∼10% of total fatty acids) IK-1 cells were more hydrophobic than their counterparts with no EPA. These results suggest that the high hydrophobicity of IK-1 cells can be attributed to the presence of membrane EPA, which shields the entry of hydrophilic membrane-diffusible compounds, and that hydrophobic compounds such as CCCP and DCCD diffuse more effectively in the membranes of IK-1, where they can fulfil their inhibitory activities, than in the membranes of IK-1Δ8

Possible Biosynthetic Pathways for all cis-3,6,9,12,15,19,22,25,28-Hentriacontanonaene in Bacteria

A very long chain polyunsaturated hydrocarbon, hentriacontanonaene (C31:9), was detected in an eicosapentaenoic acid (EPA)-producing marine bacterium, which was isolated from the mid-latitude seashore of Hokkaido, Japan, and was tentatively identified as mesophilic Shewanella sp. strain osh08 from 16S rRNA gene sequencing. The geometry and position of the double bonds in this compound were determined physicochemically to be all cis at positions 3, 6, 9, 12, 15, 19, 22, 25, and 28. Although C31:9 was detected in all of the seven EPA- or/and docosahexaenoic acid-producing bacteria tested, an EPA-deficient mutant (strain IK-1Δ8) of one of these bacteria had no C31:9. Strain IK-1Δ8 had defects in the pfaD gene, one of the five pfa genes responsible for the biosynthesis of EPA. Although Escherichia coli DH5α does not produce EPA or DHA inherently, cells transformed with the pfa genes responsible for the biosynthesis of EPA and DHA produced EPA and DHA, respectively, but not C31:9. These results suggest that the Pfa protein complex is involved in the biosynthesis of C31:9 and that pfa genes must not be the only genes responsible for the formation of C31:9. In this report, we determined for the first time the molecular structure of the C31:9 and discuss the possible biosynthetic pathways of this compound