AIM: To develop a novel gene-delivery therapeutic based on CRISPR/Cas9 genome editing technology capable of specifically targeting and knocking out the VEGFA gene, thereby achieving sustained suppression of VEGFA expression in retinal pigment epithelial(RPE)cells and providing a new strategy for gene therapy in retinal neovascular diseases.METHODS:Single guide RNAs targeting the human VEGFA gene for knockout were designed, and corresponding recombinant plasmids were constructed. A novel polymer(PTEE)was used to encapsulate the plasmids to prepare a PTEE-loaded anti-VEGFA plasmid(PLAP)gene delivery system. PTEE materials at concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 μg/μL were co-incubated with ARPE-19 cells, and the biocompatibility of PTEE was evaluated using the cell counting kit-8(CCK-8)assay. Recombinant plasmids expressing green fluorescent protein(GFP)were constructed. Lipofectamine 3000 and jetOPTIMUS®DNA transfection reagents were used as control groups, and PTEE nanomaterials were used as the experimental group to encapsulate the plasmids. When the cell confluence reached 80%, the formulations were transfected into ARPE-19 and 293T cells. GFP expression was observed under light microscopy, and the transfection efficiencies of each group were compared. ARPE-19 cells were induced under hypoxia, and PLAP was transfected into the cells. The expression level of VEGFA was detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the efficacy of this novel gene delivery system.RESULTS: After co-incubation of ARPE-19 cells with different concentrations of PTEE for 24 h and 48 h, no significant effect on cell viability was observed in any group. The transfection efficiency of PLAP in ARPE-19 cells was higher than that in the Lipo3000 and jetOPTIMUS groups, with statistically significant differences(P<0.01). Hypoxia for 6 h significantly induced the upregulation of VEGFA mRNA expression in ARPE-19 cells, and under hypoxic conditions, the PTEE group exhibited a significant inhibitory effect on VEGFA expression(P<0.01).CONCLUSION:PLAP exhibits favorable biocompatibility and prominent VEGFA inhibitory effects in vitro, making it a potential candidate drug for gene therapy of retinal neovascular diseases.

Diabetes is one of the major chronic non-communicable diseases in China. This article discusses the progress of diabetes prevention and control under the leadership of the government and how to integrate the National Diabetes Prevention and Control Center (DPCC) into the work of primary medical and health institutions in Yunnan Province, providing reference and experience for diabetes prevention and control efforts in the central and western regions of China.

Objective Aimed at investigating the effect and molecular mechanism of artemisinin on hemo-dynamics and vascular remodeling in monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH)rats.Methods 30 male SD rats were randomly divided into 3 groups(n=10):control group,MCT-induced PAH group(MCT group,60 mg/kg)and artemisinin intervention group(50 mg/kg).At 28 days after modeling,the right ventricular systolic pressure(RVSP),mean pulmonary artery pressure(mPAP),heart rate and right ventricular hypertrophy index(RVHI)were measured to evaluate the development of PAH.HE staining and α-SMA immuno-histochemistry were used to observe the morphology and assess muscularization of pulmonary arterioles,and the percentage of medial wall thickness(WT%),the percentage of vascular wall area(WA%)and the proportion of muscular vessels were calculated to evaluate the degree of pulmonary vascular remodeling.The mRNA and protein levels of HIF-1α and LDHA were detected by real-time PCR and Western blot,respectively.Pyruvate and lactate concentration in lung tissue was measured using pyruvate and lactateassay kit.Results Compared with the control group,the RVSP,mPAP,heart rate and RVHI were significantly increased in MCT-induced PAH rats(all P<0.05).Histological analysis showed that the increasedmedial wall thickness of small pulmonary arteries and vascular muscularization were observed in MCT-treated rats compared with control rats.WT%,WA%and muscularization degrees of pulmonary arterioles were higher in MCT-treated rats than those in the control group(all P<0.05),suggesting successful construction of PAH model.Compared with the MCT group,the RVSP,mPAP,heart rate and RVHI decreased in the rats treated with artemisinin(all P<0.05),accompanied with lower WT%and WA%(P<0.05),and muscularization of pulmonary arterioles was improved(P<0.05).Further study showed the mRNA and protein levels ofHIF-1α and LDHA in lung tissue of MCT-induced PAH rats were higher than those in the control group,the content of lactate and pyruvate and the ratio of lactate to pyruvate were higher than that in the control group(all P<0.05).However,the mRNA and protein levels of HIF-1α and LDHA in lung tissue of rats treated with artemisinin were lower than those in the MCT group,the content of lactate and pyruvate and the ratio of lactate to pyruvate were lower than that in the MCT group(all P<0.05).Conclusion Artemisinin improves hemodynamic and pulmonary vascular remodeling in PAH rats through inhibiting HIF-1α/LDHA signaling pathway-mediated glycolysis.

To verify whether chaperones can promote the soluble expression of PlpE in Escherichia coli and whether the expressed protein is active,prokaryotic expression and Western blot detection were performed.The results showed that:The PlpE prokaryotic expression vector pET-32a(+)-plpE was expressed as inclusion body,and the expression form was not changed by changing the concentration of inducer,induction time and temperature.The companion proteins pG-KJE8,pGro7,pKJE7 and pG-Tf2 were co-expressed with pET-32a(+)-plpE in Eschierichia coli expres-sion system,respectively.When the final concentration of IPTG of 0.5 mmol/L,L-arabinose of 0.5 g/L or tetracycline of 5.0 μg/L were added as inducers and induced at 37 ℃ for 8 h,the results showed that the molecular companion pGro7 could change the expression of rp-PlpE from inclu-sion body to soluble expression.pG-KJE8,pKJE7 and pG-Tf2 had no effect on the expression of rp-PlpE.The soluble rp-PlpE can react specifically with the positive serum of Mannheimia haemolyti-ca.Therefore,the study showed that the co-expression of the chaperone protein pGro7 can make the rp-PlpE protein express in a soluble form,and the purified protein exhibits reactogenicity.These findings lay the foundation for the establishment of a subunit vaccine and serological diagno-sis methods for Mannheimia haemolytica.

Purpose To evaluate the diagnostic value of three-dimensional amide proton transfer weighted imaging(APTWI)in distinguishing benign from malignant bone tumors and the diagnostic efficacy of APTWI combined with diffusion-weighted imaging(DWI).Materials and Methods Sixty-nine patients with pelvic and lower extremity bone tumors,confirmed by puncture or surgical pathology at the First Affiliated Hospital of Zhengzhou University,were prospectively collected from April 2022 to July 2023.On the relevant parameter maps of APTWI and DWI,the asymmetric magnetization transfer rate at a chemical shift of 3.5 ppm[MTRasym(3.5 ppm)]and apparent diffusion coefficient(ADC)values corresponding to the lesions were measured,respectively.Comparative analysis of differences in each parameter was conducted,and the diagnostic efficacy of these parameters alone and in combination was compared.Results Among 69 bone tumor,24 were benign cases and 45 malignant cases.MTRasym(3.5 ppm)of malignant bone tumors was higher than that of benign tumors[(2.974±0.630)%vs.(2.215±0.963)%],while ADC values of malignant bone tumors were lower than those of benign tumors[(1.143±0.406)×10-3 s/mm2 vs.(1.757±0.449)×10-3 s/mm2],with significant differences between the two groups(t=3.532,-5.645;P<0.001).Receiver operating characteristic curve showed that MTRasym(3.5 ppm),ADC values,and their combination exhibited good performance in the differential diagnosis of benign and malignant bone tumors,with the area under the curve being 0.765,0.841 and 0.874,respectively.There were no statistically significant differences in the area under the curve between the combined APTWI-DWI and either APTWI or DWI alone in diagnosing benign and malignant bone tumors(combined vs.APTWI:Z=1.873,P=0.064;combined vs.DWI:Z=1.333,P=0.167).The diagnostic specificity of combined APTWI-DWI was significantly higher than that of APTWI alone(96%vs.56%,P=0.001),but there was no statistically significant difference compared with DWI alone(96%vs.80%,P=0.189).Conclusion APTWI and DWI,either alone or in combination,could serve as effective imaging indicators for distinguishing benign from malignant bone tumors.The combination of APTWI and DWI shows a trend of achieving higher diagnostic efficacy and diagnostic specificity in differentiating benign from malignant bone tumors,thus holding potential clinical application value.

Diabetes is one of the major chronic non-communicable diseases in China. This article discusses the progress of diabetes prevention and control under the leadership of the government and how to integrate the National Diabetes Prevention and Control Center (DPCC) into the work of primary medical and health institutions in Yunnan Province, providing reference and experience for diabetes prevention and control efforts in the central and western regions of China.

Objective Aimed at investigating the effect and molecular mechanism of artemisinin on hemo-dynamics and vascular remodeling in monocrotaline(MCT)-induced pulmonary arterial hypertension(PAH)rats.Methods 30 male SD rats were randomly divided into 3 groups(n=10):control group,MCT-induced PAH group(MCT group,60 mg/kg)and artemisinin intervention group(50 mg/kg).At 28 days after modeling,the right ventricular systolic pressure(RVSP),mean pulmonary artery pressure(mPAP),heart rate and right ventricular hypertrophy index(RVHI)were measured to evaluate the development of PAH.HE staining and α-SMA immuno-histochemistry were used to observe the morphology and assess muscularization of pulmonary arterioles,and the percentage of medial wall thickness(WT%),the percentage of vascular wall area(WA%)and the proportion of muscular vessels were calculated to evaluate the degree of pulmonary vascular remodeling.The mRNA and protein levels of HIF-1α and LDHA were detected by real-time PCR and Western blot,respectively.Pyruvate and lactate concentration in lung tissue was measured using pyruvate and lactateassay kit.Results Compared with the control group,the RVSP,mPAP,heart rate and RVHI were significantly increased in MCT-induced PAH rats(all P<0.05).Histological analysis showed that the increasedmedial wall thickness of small pulmonary arteries and vascular muscularization were observed in MCT-treated rats compared with control rats.WT%,WA%and muscularization degrees of pulmonary arterioles were higher in MCT-treated rats than those in the control group(all P<0.05),suggesting successful construction of PAH model.Compared with the MCT group,the RVSP,mPAP,heart rate and RVHI decreased in the rats treated with artemisinin(all P<0.05),accompanied with lower WT%and WA%(P<0.05),and muscularization of pulmonary arterioles was improved(P<0.05).Further study showed the mRNA and protein levels ofHIF-1α and LDHA in lung tissue of MCT-induced PAH rats were higher than those in the control group,the content of lactate and pyruvate and the ratio of lactate to pyruvate were higher than that in the control group(all P<0.05).However,the mRNA and protein levels of HIF-1α and LDHA in lung tissue of rats treated with artemisinin were lower than those in the MCT group,the content of lactate and pyruvate and the ratio of lactate to pyruvate were lower than that in the MCT group(all P<0.05).Conclusion Artemisinin improves hemodynamic and pulmonary vascular remodeling in PAH rats through inhibiting HIF-1α/LDHA signaling pathway-mediated glycolysis.

Objective:To explore the relationship between programmed death-1(PD-1)inhibitors and cancer-related fatigue(CRF)in patients with malignant melanoma,and to identify associated influencing factors.Methods:A total of 100 patients with malignant melanoma treated at Jinan People's Hospital between April 2019 and April 2024 were included as study subjects.The Chinese version of the Piper Fatigue Scale was used to evaluate patients'fatigue levels within three months before and after the initiation of PD-1 inhibitor therapy.Results:There was a significant difference in CRF score before and after PD-1 inhibitor treatment(P<0.001).Univariate analysis showed no significant association between fatigue severity and factors such as gender,smoking history,tumor site,or PD-1 inhibitor type(all P>0.05).However,age,tumor stage,anemia,leukopenia,secondary hypothyroidism,secondary adrenal insufficiency(AI),and secondary adrenocorticotropic hormone deficiency(P<0.05 or P<0.01 or P<0.001)were significantly associated with CRF.Multivariate regression analysis identified secondary hypothyroidism,secondary AI,anemia,and leukopenia as independent risk factors for severe CRF in patients with malignant melanoma(all P<0.05).Conclusion:Adverse reactions of PD-1 inhibitors,including secondary hypothyroidism,secondary AI,anemia,and leukopenia,are independent risk factors for CRF in patients with malignant melanoma.

To verify whether chaperones can promote the soluble expression of PlpE in Escherichia coli and whether the expressed protein is active,prokaryotic expression and Western blot detection were performed.The results showed that:The PlpE prokaryotic expression vector pET-32a(+)-plpE was expressed as inclusion body,and the expression form was not changed by changing the concentration of inducer,induction time and temperature.The companion proteins pG-KJE8,pGro7,pKJE7 and pG-Tf2 were co-expressed with pET-32a(+)-plpE in Eschierichia coli expres-sion system,respectively.When the final concentration of IPTG of 0.5 mmol/L,L-arabinose of 0.5 g/L or tetracycline of 5.0 μg/L were added as inducers and induced at 37 ℃ for 8 h,the results showed that the molecular companion pGro7 could change the expression of rp-PlpE from inclu-sion body to soluble expression.pG-KJE8,pKJE7 and pG-Tf2 had no effect on the expression of rp-PlpE.The soluble rp-PlpE can react specifically with the positive serum of Mannheimia haemolyti-ca.Therefore,the study showed that the co-expression of the chaperone protein pGro7 can make the rp-PlpE protein express in a soluble form,and the purified protein exhibits reactogenicity.These findings lay the foundation for the establishment of a subunit vaccine and serological diagno-sis methods for Mannheimia haemolytica.

Purpose To evaluate the diagnostic value of three-dimensional amide proton transfer weighted imaging(APTWI)in distinguishing benign from malignant bone tumors and the diagnostic efficacy of APTWI combined with diffusion-weighted imaging(DWI).Materials and Methods Sixty-nine patients with pelvic and lower extremity bone tumors,confirmed by puncture or surgical pathology at the First Affiliated Hospital of Zhengzhou University,were prospectively collected from April 2022 to July 2023.On the relevant parameter maps of APTWI and DWI,the asymmetric magnetization transfer rate at a chemical shift of 3.5 ppm[MTRasym(3.5 ppm)]and apparent diffusion coefficient(ADC)values corresponding to the lesions were measured,respectively.Comparative analysis of differences in each parameter was conducted,and the diagnostic efficacy of these parameters alone and in combination was compared.Results Among 69 bone tumor,24 were benign cases and 45 malignant cases.MTRasym(3.5 ppm)of malignant bone tumors was higher than that of benign tumors[(2.974±0.630)%vs.(2.215±0.963)%],while ADC values of malignant bone tumors were lower than those of benign tumors[(1.143±0.406)×10-3 s/mm2 vs.(1.757±0.449)×10-3 s/mm2],with significant differences between the two groups(t=3.532,-5.645;P<0.001).Receiver operating characteristic curve showed that MTRasym(3.5 ppm),ADC values,and their combination exhibited good performance in the differential diagnosis of benign and malignant bone tumors,with the area under the curve being 0.765,0.841 and 0.874,respectively.There were no statistically significant differences in the area under the curve between the combined APTWI-DWI and either APTWI or DWI alone in diagnosing benign and malignant bone tumors(combined vs.APTWI:Z=1.873,P=0.064;combined vs.DWI:Z=1.333,P=0.167).The diagnostic specificity of combined APTWI-DWI was significantly higher than that of APTWI alone(96%vs.56%,P=0.001),but there was no statistically significant difference compared with DWI alone(96%vs.80%,P=0.189).Conclusion APTWI and DWI,either alone or in combination,could serve as effective imaging indicators for distinguishing benign from malignant bone tumors.The combination of APTWI and DWI shows a trend of achieving higher diagnostic efficacy and diagnostic specificity in differentiating benign from malignant bone tumors,thus holding potential clinical application value.