Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes.

The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.

Postischemic Housing Environment on Cerebral Metabolism and Neuron Apoptosis after Focal Cerebral Ischemia in Rats / 华中科技大学学报（医学）（英德文版）

The purpose of this study was to evaluate the roles of different housing environments in neurological function,cerebral metabolism,cerebral infarction and neuron apoptosis after focal cerebral ischemia.Twenty-eight Sprague-Dawley rats were divided into control group (CG) and cerebral ischemia group,and the latter was further divided into subgroups of different housing conditions:standard environment (SE) subgroup,individual living environment (IE) subgroup,and enriched environment (EE) subgroup.Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO).Beam walking test was used to quantify the changes of overall motor function.Cerebral infarction and cerebral metabolism were studied by in vivo magnetic resonance imaging and 1H-magnetic resonance spectra,respectively.Neuron necrosis and apoptosis were detected by hematoxylin-eosin and TUNEL staining methods,respectively.The results showed that performance on the beam-walk test was improved in EE subgroup when compared to SE subgroup and IE subgroup.Cerebral infarct volume in IE subgroup was significantly larger than that in SE subgroup (P＜0.05) and EE subgroup (P＜0.05) on day 14 after MCAO.NAA/Cr and Cho/Cr ratios were lower in MCAO groups under different housing conditions as compared to those in CG (P＜0.05).NAA/Cr ratio was lower in IE subgroup (P＜0.05) and higher in EE subgroup (P＜0.05) than that in SE subgroup.NAA/Cr ratio in EE was significantly higher than that in IE subgroup (P＜0.05).Cho/Cr ratio was decreased in MCAO groups as compared to that in CG (P＜0.05).A significant decrease in normal neurons in cerebral cortex was observed in MCAO groups as compared to CG (P＜0.05).The amount of normal neurons was less in IE subgroup (P＜0.05),and more in EE subgroup (P＜0.05) than that in SE subgroup after MCAO.The amount of normal neurons in EE subgroup was significantly more than that in IE subgroup after MCAO (P＜0.05).The ratio of TUNEL-positive neurons in EE was significantly lower than that in SE subgroup (P＜0.05) and IE subgroup (P＜0.05).Correlation analysis showed that the beam walking test was negatively correlated with NAA/Cr ratio (P＜0.05).Cerebral infarct volume was negatively correlated with both NAA/Cr ratio (P＜0.01) and Cho/Cr ratio (P＜0.01).The amount of normal cortical neurons was positively correlated with both NAA/Cr ratio (P＜0.01) and Cho/Cr ratio (P＜0.05).The TUNEL-positive neurons showed a negative correlation with both NAA/Cr ratio (P＜0.01) and Cho/Cr ratio (P＜0.01).This study goes further to show that EE may improve neurological functional deficit and cerebral metabolism,decrease cerebral infarct volume,neuron necrosis and apoptosis,while IE may aggravate brain damage after MCAO.

Postischemic Housing Environment on Cerebral Metabolism and Neuron Apoptosis after Focal Cerebral Ischemia in Rats / 华中科技大学学报（医学）（英德文版）

The purpose of this study was to evaluate the roles of different housing environments in neurological function,cerebral metabolism,cerebral infarction and neuron apoptosis after focal cerebral ischemia.Twenty-eight Sprague-Dawley rats were divided into control group (CG) and cerebral ischemia group,and the latter was further divided into subgroups of different housing conditions:standard environment (SE) subgroup,individual living environment (IE) subgroup,and enriched environment (EE) subgroup.Focal cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO).Beam walking test was used to quantify the changes of overall motor function.Cerebral infarction and cerebral metabolism were studied by in vivo magnetic resonance imaging and 1H-magnetic resonance spectra,respectively.Neuron necrosis and apoptosis were detected by hematoxylin-eosin and TUNEL staining methods,respectively.The results showed that performance on the beam-walk test was improved in EE subgroup when compared to SE subgroup and IE subgroup.Cerebral infarct volume in IE subgroup was significantly larger than that in SE subgroup (P＜0.05) and EE subgroup (P＜0.05) on day 14 after MCAO.NAA/Cr and Cho/Cr ratios were lower in MCAO groups under different housing conditions as compared to those in CG (P＜0.05).NAA/Cr ratio was lower in IE subgroup (P＜0.05) and higher in EE subgroup (P＜0.05) than that in SE subgroup.NAA/Cr ratio in EE was significantly higher than that in IE subgroup (P＜0.05).Cho/Cr ratio was decreased in MCAO groups as compared to that in CG (P＜0.05).A significant decrease in normal neurons in cerebral cortex was observed in MCAO groups as compared to CG (P＜0.05).The amount of normal neurons was less in IE subgroup (P＜0.05),and more in EE subgroup (P＜0.05) than that in SE subgroup after MCAO.The amount of normal neurons in EE subgroup was significantly more than that in IE subgroup after MCAO (P＜0.05).The ratio of TUNEL-positive neurons in EE was significantly lower than that in SE subgroup (P＜0.05) and IE subgroup (P＜0.05).Correlation analysis showed that the beam walking test was negatively correlated with NAA/Cr ratio (P＜0.05).Cerebral infarct volume was negatively correlated with both NAA/Cr ratio (P＜0.01) and Cho/Cr ratio (P＜0.01).The amount of normal cortical neurons was positively correlated with both NAA/Cr ratio (P＜0.01) and Cho/Cr ratio (P＜0.05).The TUNEL-positive neurons showed a negative correlation with both NAA/Cr ratio (P＜0.01) and Cho/Cr ratio (P＜0.01).This study goes further to show that EE may improve neurological functional deficit and cerebral metabolism,decrease cerebral infarct volume,neuron necrosis and apoptosis,while IE may aggravate brain damage after MCAO.