Using conventional broth microdilution assays,the synthetic phenolic antioxidant 2,6-di-tert-butyl-4-methylphenol(butylated hydroxytoluene,BHT)was screened for synergistic antimi-crobial activity with β-lactam antibiotics.Further validation through checkerboard assays,growth curve analysis,and time-kill curve experiments confirmed the synergistic effect of BHT and β-lac-tams against methicillin-resistant Staphylococcus aureus(MRSA)(FICI≤0.5).To elucidate the underlying mechanism of synergy,this study evaluated changes in membrane permeability and pro-ton transmembrane gradients-key factors in biofilm functionality-along with critical indicators of bacterial energy metabolism,including ATP levels and the NAD+/NADH ratio.The results dem-onstrated that BHT disrupts the proton motive force(PMF),impairs membrane potential,and perturbs bacterial energy homeostasis,thereby significantly enhancing the antibacterial efficacy ofβ-lactams.Furthermore,a murine pneumonia infection model was established to assess the in vivo therapeutic efficacy of BHT combined with amoxicillin.The combination therapy alleviated pulmo-nary tissue damage,reduced bacterial loads in target organs(liver,spleen,and lungs),and decreased systemic inflammatory responses.This study elucidates the synergistic antimicrobial action and mechanistic basis of BHT combined with amoxicillin,offering a novel combinatorial therapeutic strategy to address MRSA resistance.The findings hold significant clinical potential and research value for overcoming antibiotic resistance in MRSA infections.

Salmonellosis represents a global epidemic, and the emergence of extensively drug-resistant (XDR) Salmonella and its sustained transmission worldwide constitutes a significant public health concern. Flagellum-mediated motility serves as a crucial virulence trait of Salmonella that guides the pathogen toward the epithelial surface, enhancing gut colonization. Artemisia argyit essential oil, a traditional herb extract, demonstrates efficacy in treating inflammation-related symptoms and diseases; however, its effects on flagellum assembly and expression mechanisms in anti-Salmonella activity remain inadequately explored. This study aimed to elucidate the mechanism by which Artemisia argyit essential oil addresses Salmonella infections. Network pharmacological analysis revealed that Traditional Chinese Medicine (TCM) Artemisia argyit exhibited anti-Salmonella infection potential and inhibited flagellum-dependent motility. The application of Artemisia argyit essential oil induced notable motility defects through the downregulation of flagellar and fimbriae expression. Moreover, it significantly reduced Salmonella-infected cell damage by interfering with flagellum-mediated Salmonella colonization. In vivo studies demonstrated that Artemisia argyit essential oil administration effectively alleviated Salmonella infection symptoms by reducing bacterial loads, inhibiting interleukin-1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) production, and diminishing pathological injury. Gas chromatography-mass spectrometry (GC-MS) analysis identified forty-three compounds in Artemisia argyit essential oil, with their corresponding targets and active ingredients predicted. Investigation of an in vivo model of Salmonella infection using the active ingredient demonstrated that alpha-cedrene ameliorated Salmonella infection. These findings suggest the potential application of Artemisia argyit essential oil in controlling Salmonella, the predominant food-borne pathogen.
Artemisia/chemistry* ; Oils, Volatile/chemistry* ; Animals ; Flagella/drug effects* ; Salmonella Infections/microbiology* ; Humans ; Mice ; Anti-Bacterial Agents/pharmacology* ; Salmonella/pathogenicity*

OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.

Using conventional broth microdilution assays,the synthetic phenolic antioxidant 2,6-di-tert-butyl-4-methylphenol(butylated hydroxytoluene,BHT)was screened for synergistic antimi-crobial activity with β-lactam antibiotics.Further validation through checkerboard assays,growth curve analysis,and time-kill curve experiments confirmed the synergistic effect of BHT and β-lac-tams against methicillin-resistant Staphylococcus aureus(MRSA)(FICI≤0.5).To elucidate the underlying mechanism of synergy,this study evaluated changes in membrane permeability and pro-ton transmembrane gradients-key factors in biofilm functionality-along with critical indicators of bacterial energy metabolism,including ATP levels and the NAD+/NADH ratio.The results dem-onstrated that BHT disrupts the proton motive force(PMF),impairs membrane potential,and perturbs bacterial energy homeostasis,thereby significantly enhancing the antibacterial efficacy ofβ-lactams.Furthermore,a murine pneumonia infection model was established to assess the in vivo therapeutic efficacy of BHT combined with amoxicillin.The combination therapy alleviated pulmo-nary tissue damage,reduced bacterial loads in target organs(liver,spleen,and lungs),and decreased systemic inflammatory responses.This study elucidates the synergistic antimicrobial action and mechanistic basis of BHT combined with amoxicillin,offering a novel combinatorial therapeutic strategy to address MRSA resistance.The findings hold significant clinical potential and research value for overcoming antibiotic resistance in MRSA infections.

OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.

Objective To compare the effects of different respiratory signal acquisition methods on the delineation of moving tumor targets. Methods A cube phantom containing a sphere was placed on a motion platform to simulate respiratory movement by setting motion period, frequency, and direction. Respiratory signal was acquired by real-time position management (RPM) method and GE method independently. Target delineation was conducted using the maximum intensity projection (MIP) sequence. The difference between the reconstructed volume and the theoretical moving volume was compared under the two respiratory signal acquisition methods for cube and sphere targets. Results Under the same respiratory signal acquisition method, the same respiratory amplitude, and different respiratory frequencies, reconstructed volume changes were relatively small. For the sphere target, the deviation between the reconstructed volume and the theoretical moving volume was −1.5% to 5.7% with the RPM method and −1.3% to −13.8% with the GE method (both P < 0.05). For the cube target, the deviation between the reconstructed volume and the theoretical moving volume was 0.2% to 0.9% with the RPM method and −2.6% to 0.9% with the GE method, with no statistical significance. Conclusion For small-volume sphere targets, the target volumes obtained from MIP images by the two respiratory signal acquisition methods are both smaller than the actual moving volume. For large-volume cube targets, there is no significant difference between the reconstructed and theoretical results with any respiratory signal acquisition method. The RPM method produces smaller deviation and better image quality when reconstructing small-volume targets.

Objective:To compare the dosimetric differences between the VenusX accelerator with an orthogonal dual-layer multi-leaf collimator (MLC) and the Varian′s CLINAC IX and EDGE accelerators with a single-layer MLC for hippocampus protection in the whole-brain radiotherapy (WBRT).Methods:Forty patients with multiple brain metastases admitted to the Radiotherapy Department of the Shanghai General Hospital from June 2021 to February 2023 were selected in this study. Three whole-brain treatment plans were designed based on the above three accelerators for each patient. Under the same prescription dose, radiation field, and plan constraints, the three plans were compared in terms of the dosimetric differences in target volumes, hippocampi, and adjacent organs at risk (OARs), as well as the execution efficiency.Results:For the planning target volume (PTV), there were statistically significant differences in approximate maximum dose ( D2) between the VenusX and IX plans ( t = 4.94, P < 0.05), in approximate minimum dose ( D98) between the VenusX and EDGE plans ( t = 5.98, P < 0.05), in the target conformity indices (CIs) between VenusX plan and EDGE plans, and between the VenusX and IX plans ( t = -6.84, -14.30; P < 0.05), and dose homogeneity indices (HIs) between the VenusX and IX plans ( t = 3.48, P < 0.05). For OARs, the maximum doses ( Dmax) and average doses ( Dmean) to bilateral hippocampi of the VenusX plan were lower than those of the EDGE and IX plans ( t = 8.59-17.11, P < 0.05); the maximum doses ( Dmax) to bilateral lenses, bilateral optic nerves, and optic chiasma of the VenusX plan were lower than those of the other two plans ( t = 2.10-20.80, P < 0.05); and the differences between the maximum doses ( Dmax) to the brain stem of the VenusX and EDGE plans were statistically significant ( t = 3.86, P < 0.05). In terms of plan execution efficiency, the number of machine jumps (MU) and the treatment time of the VenusX plan were higher than those of the EDGE and IX plans, with statistically significant differences ( t = -56.48, -56.90, P < 0.05). Conclusions:The doses to target volumes of the three treatment plans all meet the prescription requirements, and the VenusX plan outperforms the EDGE and IX plans in the protection of OARs. Despite the reduced execution efficiency, the VenusX plan shortens the actual treatment time by improving the dosage rate, thus meeting the clinical requirements.

AIM: To evaluate the effect of inhibiting ubiquitin-specific protease 14 (USPl4) activity on oxidative stress induced by H2O2 of H9c2 cells.METHODS: The H9c2 cells were incubated with H2O2 at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group, H2O2 group, IU1 group (25 μmol/L or 50 μmol/L) and IU1+ H2O2 group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS: Compared with control group, the cell activity and the viability rate in H2O2 group were decreased (P<0.05), while the intracellular ROS, the protein levels of Bax/Bcl-2, P53, p-ERK1/2, p-JNK and p-P38 were increased (P<0.05).Compared with H2O2 group, the cell activity and the viability rate of the H9c2 cells in IU1+H2O2 group were increased (P<0.05), while the intracellular ROS, the protein levels of Bax/Bcl-2, P53, p-ERK1/2, p-JNK and p-P38 were decreased (P<0.05).CONCLUSION: Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.

Objective To investigate the neuroprotective effect and possible mechanism of nicorandil in mice under deep hypothermic low flow (DHLF). Methods A total of 105 3-week-old male C57/BL-6 mice were randomly divided into 7 groups: sham operation, model, nicorandil (5, 10, and 20 mg/kg), nicorandil 20 mg/kg + LY294002, and LY294002 groups (n = 15 in each group). A DHLF model was induced. At 24 h after reperfusion, the brain tissues of mice were taken out for HE and TUNEL staining. The pathological changes of cerebral cortical neurons and apoptosis were observed. Western blot was used to detect the expression levels of the total Akt, phospho-Akt (p-Akt), Bcl-2, and Bax. Results HE pathological staining showed that cortical neuronal injury was reduced, the phenomena of cel membrane depression, nuclear condensation, concentrated dye, and the blurring of the nucleus were decreased significantly in nicorandil group. The morphology of neurons was basicaly restored to normal. TUNEL staining showed that the apoptosis index in various dose groups of nicorandil was decreased significantly compared with the model group (al P < 0. 05). Western blot analysis showed that the expression levels of p-Akt and Bcl-2 proteins increased significantly in various dose groups of nicorandil compared with the model group (al P < 0. 05), and the expression level of Bax protein was decreased significantly (al P < 0. 05 ). After adding the phosphatidylinositol 3-kinase (PI3K) specific inhibitor LY294002, there was no significant difference in neurons pathological injury in the cortex compared with the model group. There was no significant difference in the apoptosis index, and the expression levels of p-Akt, Bcl-2, and Bax compared with the model group. Conclusions Nicorandil has a certain neuroprotective effect in mice under DHLF. Its mechanism may be associated with the activation of PI3K/Akt signaling pathway, and then further regulation of the downstream protein Bcl-2 and Bax expression.

Objective To evaluate the effects of limited fluid resuscitation on systemic inflammatory responses in rats with traumatic hemorrhagic shock through comparing with unlimited fluid resuscitation.Methods Sixty pathogen-free male Sprague-Dawley rats,aged 2-3 months,weighing 250-290 g,were randomly divided into 6 groups (n =10 each) using a random number table:sham operation group (group S),no fluid resuscitation group (group NF),unlimited fluid resuscitation group (group ULF),limited crystalloid fluid resuscitation group (group LR),and limited colloid fluid resuscitation groups (group LSG and group LHES).Traumatic uncontrolled hemorrhagic shock was induced by withdrawal of blood from the femoral artery at 2.5 mL/100 g over a 20-minute period,followed by tail amputation at 10 min after the end of blood withdrawal.At 10 min after the end of blood withdrawal,fluid resuscitation was performed.Lactated Ringer's solution (ULF and LR groups),4 ％ succinylated gelatin (group LSG),or 6 ％ hydroxyethyl starch 130/0.4 (group LHES) was infused intravenously.The initial infusion rate was 2 ml · kg-1 · min-1.The target MAP was maintained at 50 mm Hg in rats with limited fluid resuscitation,while at 80 mm Hg in rats with unlimited fluid resuscitation.After 60 min of fluid resuscitation,bleeding in the tail was stopped by ligation and fluid infusion was replaced with blood resuscitation.After 60 min of blood resuscitation,180 main of observation was started.At 10 min after catheterization of the femoral artery and vein (T0),10 min after the end of blood withdrawal (T1),the end of fluid resuscitation (T2),the end of blood resuscitation (T3),and the end of observation (T4),arterial blood samples were collected to measure hematocrit (Hct)and concentrations of plasma tumor necrosis factor-alpha (TNF-α),interleukin (IL)-6,and IL-10.Blood samples were collected from the femoral artery at T2 for determination of the expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) and activity of nuclear factor-kappaB (NF-κB) in monocytes.The amount of blood loss from the tail and volume of fluid infused were also recorded.Another 120 Sprague-Dawley rats were randomly divided into 6 groups (n =20 each) and resuscitation was performed according to the method previously described.The rats were observed for 72 h survival rate.Results Compared with group S,Hct was significantly decreased,the concentrations of plasma TNF-α,IL-6,and IL-10 and activity of NF-κB were increased,and the expression of TLR4,and MyD88 in monocytes was up-regulated in the other groups (P ＜ 0.05).Compared with group NF,the concentrations of plasma TNF-α and IL-6 and NF-κB activity were significantly increased,and the concentration of plasma IL-10 and Hct were decreased,and the expression of TLR4 and MyD88 in monocytes was up-regulated in ULF,LR and LSG groups,and the concentrations of plasma TNF-α and IL-6 were significantly increased,the concentration of plasma IL-10 and Hct were decreased in group LHES (P ＜ 0.05).Compared with group ULF,the concentrations of plasma TNF-α and IL-6 and NF-κB activity were significantly decreased,the concentration of plasma IL-10 and Hct were increased,the survival rate was higher,the expression of TLR4 and MyD88 in monocytes was down-regulated,and the amount of blood loss from the tail was decreased and the volume of fluid infused was reduced in LSG,LHES and LR groups (P ＜ 0.05).Compared with group LR,the concentrations of plasma TNF-α and IL-6 and NF-κB activity were significantly decreased and the expression of TLR4 and MyD88 in monocytes was down-regulated (P ＜ 0.05),and no significant change was found in the concentration of plasma IL-10 in group LHES (P ＞ 0.05),and the volume of fluid infused was reduced and the survival rate was increased (P ＜ 0.05),and no significant change was found in the amount of blood loss from the tail in LSG and LH-ES groups (P ＞ 0.05).Conclusion Compared with unlimited fluid resuscitation,limited fluid resuscitation exerts less effect on systemic inflammatory responses in rats with traumatic hemorrhagic shock,especially when resuscitation with 6％ hydroxyethyl starch 130/0.4 is performed,and inhibition of TLR4/NF-κB signaling pathway is involved in the mechanism.