Human carboxylesterase 2A (hCES2A) plays pivotal roles in prodrug activation and hydrolytic metabolism of ester-bearing chemicals. Targeted inhibition of intestinal hCES2A represents a feasible strategy to mitigate irinotecan-triggered gut toxicity (ITGT), but the orally active, selective, and efficacious hCES2A inhibitors are rarely reported. Here, a novel drug-like hCES2A inhibitor was developed via three rounds of structure-based drug design (SBDD) and structural optimization. Initially, donepezil was identified as a moderate hCES2A inhibitor from 2000 US Food and Drug Administration (FDA)-approved drugs. Following two rounds of SBDD and structural optimization, a donepezil derivative (B7) was identified as a strong reversible hCES2A inhibitor. Subsequently, nine B7 carbamates were rationally designed, synthesized and biologically assayed. Among all synthesized carbamates, C3 showed the most potent time-dependent inhibition on hCES2A (IC₅₀ = 0.56 nmol/L), excellent specificity and favorable drug-like properties. C3 could covalently modify the catalytic serine of hCES2A with high selectivity, while this agent also showed favorable safety profiles, high intestinal exposure, and impressive effects for ameliorating ITGT in both human intestinal organoids and tumor-bearing mice. Collectively, this study showcases a rational strategy for developing drug-like and serine-targeting covalent inhibitors against target serine hydrolase(s), while C3 emerges as a promising orally active drug candidate for ameliorating ITGT.

Iron metabolism is a critical factor in tumorigenesis and development. Although TP53 mutations are prevalent in glioblastoma (GBM), the mechanisms by which TP53 regulates iron metabolism remain elusive. We reveal an imbalance iron homeostasis in GBM via TCGA database analysis. TP53 mutations disrupted iron homeostasis in GBM, characterized by elevated total iron levels and reduced ferritin (FTH). The gain-of-function effect triggered by TP53 mutations upregulates itchy E3 ubiquitin-protein ligase (ITCH) protein expression in astrocytes, leading to FTH degradation and an increase in free iron levels. TP53-mut astrocytes were more tolerant to the high iron environment induced by exogenous ferric ammonium citrate (FAC), but the increase in intracellular free iron made them more sensitive to Erastin-induced ferroptosis. Interestingly, we found that Erastin combined with FAC treatment significantly increased ferroptosis. These findings provide new insights for drug development and therapeutic modalities for GBM patients with TP53 mutations from iron metabolism perspectives.
Ferroptosis/drug effects* ; Humans ; Iron/metabolism* ; Glioblastoma/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; Homeostasis/physiology* ; Ferritins/metabolism* ; Brain Neoplasms/genetics* ; Mutation ; Astrocytes/drug effects* ; Cell Line, Tumor ; Piperazines/pharmacology* ; Quaternary Ammonium Compounds/pharmacology* ; Ferric Compounds

Objective:To explore the mechanism by which Pien Tze Huang improves liver Kupffer cell damage induced by lipopolysaccharide (LPS) by regulating the expression of miR-155.Methods:LPS induced liver Kupffer cells to establish a cell injury model to simulate septic liver injury. RT-qPCR was used to detect the expression of miR-155 in damaged cells, and RT-qPCR, Western Blot, ELISA and flow cytometry were used to evaluate the inflammatory response and apoptosis of damaged cells. Then we treated LPS-induced Kupffer cells with Pien Tze Huang at different concentrations (0 mg/L, 5 mg/L, 10 mg/L and 15 mg/L), and detected the expression of miR-155 in the cells, the inflammatory response of the cells and Apoptosis rate. MiR-155 was silenced in the cell injury model, and RT-qPCR, Western Blot, ELISA and flow cytometry were used to evaluate the effect of miR-155 on inflammatory response and apoptosis of model cells. Overexpression of miR-155 in damaged cells treated with Pien Tze Huang was used to detect changes in cellular inflammatory response and apoptosis. Data are expressed in the form of mean ± standard deviation, and each group of data is analyzed using t test or one-way analysis of variance.Results:In the LPS-induced liver Kupffer cell injury model, the expression of miR-155 was significantly increased ( P<0.05), the expression levels of pro-inflammatory factors IL-6 and TNF-α were significantly increased, and the anti-inflammatory factor IL-10 was significantly increased. was inhibited ( P<0.05), and the cell apoptosis rate was significantly increased ( P<0.05). After Pien Tze Huang treatment, the expression of miR-155 in damaged liver cells was inhibited ( P<0.05), the levels of cellular inflammatory factors IL-6 and TNF-α were inhibited, and the expression of anti-inflammatory factor IL-10 was promoted ( P<0.05). Inhibit cell apoptosis ( P<0.05). Silencing miR-155 reduced the inflammatory response and apoptosis rate of cells ( P<0.05). Overexpression of miR-155 can reverse the effect of Pien Tze Huang on liver cell injury ( P<0.05). Conclusions:In the model of LPS-induced liver Kupffer cell injury, Pien Tze Huang can inhibite the inflammatory response and apoptosis of cells by inhibiting the expression of miR-155.

Objective To study the immunoadjuvant effects of chitosan oligosaccharide(COS),including the immune activation and the triggering of lysosomal escape,and to explore whether COS can be used as an adjuvant for attenuated live bacteria vector vaccines.Methods 1)Mouse macrophages RAW264.7 cells were cultured with COS at 0 mg/mL(the control group)and 0.1-4 mg/mL for 24 h and the effect on cell viability was measured by CCK8 assay.Mouse macrophages RAW264.7 were treated with COS at 0(the control group),1,2,and 4 mg/mL for 24 h.Then,the mRNA expression levels of the cytokines,including IFN-γ,IL-10,TGF-β,and TLR4,were determined by RT-qPCR assay.2)RAW264.7 cells were treated with 1 mL of PBS containing different components,including calcein at 50 μg/mL,COS at 2 mg/mL,and bafilomycin A1,an inhibitor,at 1 μmol/mL,for culturing.The cells were divided into the Calcein group,Calcein+COS group,and Calcein+COS+Bafilomycin A1 group accordingly.Laser scanning confocal microscopy was used to observe the phagocytosis and the intracellular fluorescence distribution of calcein,a fluorescent dye,in RAW264.7 cells in the presence or absence of COS intervention to determine whether COS was able to trigger lysosomal escape.3)LM?E6E7 and LI?E6E7,the attenuated Listeria vector candidate therapeutic vaccines for cervical cancer,were encapsulated with COS at the mass concentrations of 0.5 mg/mL,1 mg/mL,2 mg/mL,4 mg/mL,and 8 mg/mL.Then,the changes in zeta potential were measured to select the concentration of COS that successfully encapsulated the bacteria.Phagocytosis of the vaccine strains by RAW264.7 cells was measured before and after LM?E6E7 and LI?E6E7 were coated with COS at 2 mg/mL.Results 1)CCK8 assays showed that,compared with the findings for the control group,the intervention of RAW264.7 cells with COS at different concentrations for 24 h was not toxic to the cells and promoted cell proliferation,with the difference being statistically significant(P＜0.05).According to the RT-qPCR results,compared with those of the control group,the COS intervention up-regulated the mRNA levels of TLR4 and IFN-γ in RAW264.7 cells,while it inhibited the mRNA expression levels of TGF-β and IL-10,with the most prominent effect being observed in the 4 mg/mL COS group(P＜0.05).2)Laser scanning confocal microscopy revealed that the amount of fluorescent dye released from lysosomes into the cells was greater in the Calcein+COS group than that in the Calcein group.In other words,a greater amount of fluorescent dye was released from lysosomes into the cells under COS intervention.Furthermore,this process could be blocked by bafilomycin A1.3)The zeta potential results showed that COS could successfully encapsulate the surface of bacteria when its mass concentration reached 2 mg/mL.Before and after the vaccine strain was encapsulated by COS,the phagocytosis of LM?E6E7 by RAW264.7 cells was 5.70%and 22.00%,respectively,showing statistically significant differences(P＜0.05);the phagocytosis of LI?E6E7 by RAW264.7 cells was 1.55%and 6.12%,respectively,showing statistically significant differences(P＜0.05).Conclusion COS has the effect of activating the immune response of macrophages and triggering lysosomal escape.The candidates strains of coated live attenuated bacterial vector vaccines can promote the phagocytosis of bacteria by macrophages.Further research is warranted to develop COS into an adjuvant for bacterial vector vaccine.

There are gradual increases in the incidence rates of metabolic associated fatty liver disease （MAFLD） and type 2 diabetes mellitus （T2DM）， with close relationship and mutual interaction between the two diseases， but the specific mechanism is still unclear. Studies have shown that T2DM and MAFLD may cause aggravation of each other through insulin resistance， inflammation， some hepatocyte factors， and cellular senescence and protect each other through some hepatocyte factors. Further research on the association between T2DM and MAFLD and the mechanism of comorbidity is of great significance for the clinical prevention and treatment of the two diseases.

Objective To construct Listeria monocytogenes(LM)and Listeria ivanovii(LI)balanced lethal systems expressing cervical cancer antigens,to study their basic biological characteristics,and to provide reference data for the immunotherapy of cervical cancer.Methods Through seamless cloning via in vitro ligation kit,the HPV16 E6E7 fusion protein antigen gene constructed in our lab was spliced to the complement plasmid pCWgfp-LM dal-Amp that contained the nutritional gene dal.Then,we replaced the ampicillin(Amp)resistance gene of the complement plasmid with the asd nutrition gene.The ligation reaction mixture was transformed into Escherichia coli(E.coli)recipient bacteria DH5аΔasd and the complement plasmid pCWgfp-E6E7-LM dal-Ampfree,which expressed cervical cancer antigens and had no Amp resistance,was obtained by nutrition screening from the E.coli DH5аΔasd.The plasmid pCWgfp-E6E7-LM dal-Ampfree was complemented into LMΔdd and LIΔdd,the attenuated nutrition-deficient Listeria strains with the virulence genes actA and plcB and nutrition genes dal and dat deleted by electroporation,thereby obtaining LM and LI balanced lethal systems expressing cervical cancer antigen genes.The in vitro growth of the strains was observed.Western blot was performed to examine the status of antigen protein expression.PCR was performed to measure the in vitro passage stability of complement plasmid pCWgfp-E6E7-LM dal-Ampfree.Their basic biological characteristics were examined by biochemical reaction tests and hemolysis assay.Results Two Listeria balanced lethal systems expressing cervical cancer antigen were successfully constructed.The HPV16 type E6E7 fusion protein was successfully expressed in the two Listeria balanced lethal systems.pCWgfp-E6E7-LM dal-Ampfree,the positive plasmid expressing cervical cancer antigen,maintained stable existence in the two Listeria balanced lethal systems.The two Listeria balanced lethal systems expressing cervical cancer antigen showed significantly better recovery growth in comparison with Listeria nutrition deficiency strains.The results of biochemical reaction tests showed that most of the biochemical reaction of the two Listeria balanced lethal systems expressing cervical cancer antigen were consistent with those of Listeria attenuated strains.The two Listeria balanced lethal systems expressing cervical cancer antigen still maintained the hemolytic ability,although their hemolytic ability was slightly inferior to that of the Listeria balanced lethal systems not expressing cervical cancer antigen and the Listeria attenuated strains.Conclusion The two Listeria balanced lethal systems expressing cervical cancer antigen genes are constructed successfully.They display normal in vitro growth.The complement plasmid pCWgfp-E6E7-LM dal-Ampfree can maintain stable existence in vitro,showing little change in its biochemical characteristics and hemolytic ability.Further research should be conducted to investigate the potential of these two recombinant strains to be used as candidate strains for cervical cancer therapeutic vaccine.

Nowadays hospitals have been at the forefront fighting against novel coronavirus pneumonia, with diagnosing and treating of patients as a top priority. In order to ensure the smooth progress of diagnosis and treatment, and prevent the occurrence of nosocomial infection, logistics support needs to make allowances for the isolation ward in time from the perspectives of logistics, facilities and equipment, and to transform the in-and-out double channels of ward access as required, thus setting up the partition of the three zones. Secondly, logistics support needs to optimize the logistics service workflow, including the medical waste management, the environmental disinfection isolation, and to optimize the catering service within hospitals to reduce the gathering and flow of personnel. Thirdly, logistics support needs to increase personnel training, and to eliminate psychological panic as well as to stabilize the logistics support team by putting logistics management cadres on the front line. Meanwhile, the logistics department needs to take over the hospital access screening work, strictly manage those who enter the hospital, maximize the safety and reliability of the logistics support within the hospital, and ensure the smooth progress of the epidemic prevention work.

At present, we are fighting against the outbreak of novel coronavirus pneumonia (NCP) in China. For the purposes of diagnosis and treatment of NCP patients, Hangzhou Xixi Hospital, as a designated hospital, make available the wards quickly, initiated the management system of public health emergencies, and established a "tolerate admission- strict discharge" patients management program. Meanwhile, the hospital has established an emergency supply and coordinated distribution mechanism for medical protection materials, and a full-system and multi-model training system, ensuring smooth progress of the diagnosis and treatment work.

Public hospitals in the face of COVID-19, should prioritize medical services of patients as the topmost task. In order to ensure the smooth progress of diagnosis and treatment, and prevent the occurrence of nosocomial infection, the hospital took an overall response strategy featuring " logistics support mode 3+ 1" . This strategy requires to make facilities ready by transforming isolation wards, overall management and deployment of protection supplies, optimizing logistics service flow, strict sterilization and isolation of medical wastes and environment, optimizing catering service within the hospital to reduce the gathering and flow of personnel. It also enhanced personnel training, to eliminate staff panic and to stabilize the logistics support team. Meanwhile, the logistics department took over the hospital access screening work for tight access control, which maximize the safety and reliability of the logistics support within the hospital, and ensure the smooth progress of the epidemic prevention work.

At present, we are fighting against the outbreak of COVID-19 in China.For the purposes of diagnosis and treatment of these patients, Hangzhou Xixi Hospital, as a designated hospital, made available the wards quickly, initiated the management system of public health emergencies, and established a " tolerate admission-strict discharge" patients management program. Meanwhile, the hospital has established an emergency supply and coordinated distribution mechanism for medical protection materials, and a full-system and multi-model training system, ensuring smooth progress of the diagnosis and treatment work.