To construct a recombinant expression system for a single-domain antibody targeting the urease of Helicobacter pylori(Hp),this study employed several strategies.First,using artificial intelligence(AI)auxiliary tools such as Pymol,I-TASSER,and ClussPro2,the molecular interactions between different antibodies and Hp urease subunit B(UreB)were analyzed.The fully humanized single-domain antibody UreBAb was identified as the primary research target.Next,the UreBAb gene sequence was optimized based on Escherichia coli codon preferences,and was inserted into expression vectors such as pET28a and pE-SUMO.The resulting recombinant expression strains were obtained by transforming Escherichia coli Rosetta(DE3).Recombinant antibody proteins were prepared through IPTG induction,and its activity was detected using extracted Hp urease as the antigen.SDS-PAGE analysis confirmed the correct expression of both UreBAb and SUMO-UreBAb,with protein yields of 0.34 mg/mL and 0.41 mg/mL,respectively.Unidirectional immunodiffusion experiments further confirmed that both recombinant antibodies exhibited strong affinity for Hp UreB antigen,with inhibition rates of 51.27%and 74.07%,respectively.Additionally,leveraging artificial intelligence tools such as AlphaFold2,cluspro2,mCSM-AB,OSPREY,and FoldX,the study evaluated and analyzed key binding sites and mutational strategies affecting the stability of the antigen-antibody complex.Subsequently,nine UreBAb evolution mutants were constructed,and their binding activities with the antigen were enhanced.Among these,the I107W mutant showed the most significant improvement,achieving a 24.95%increase compared to the wild-type UreBAb.This research lays a solid foundation for the development of fully humanized single-domain antibodies against Hp.

Objective To explore the effect of glioma stem cells with high expression of protein tyrosin phosphatase receptor type Z1 (PTPRZ1 )on the phenotypic polarization and phagocytosis of tumor-associated macrophages and its regulatory mechanism.Methods GSCs and non-stem tumor cells (NSTCs) were screened out from human glioblastoma (GBM) specimens using flow cytometry,and the PTPRZ1 expression in paired GSCs and NSTCs were detected.Human peripheral blood mononuclear cells (PBMC)-derived CD14+monocytes were exposed to the conditioned medium from glioma cells or recombinant chemokine C-C motif ligand 20 (CCL20)for TAM polarization.Stable PTPRZ1 knockout GSCs (PTPRZ1-KO GSCs) were constructed using CRISPR/Cas9. TAM phagocytosis to GSCs,NSTCs,PTPRZ1-Control GSCs (PTPRZ1-Ctrl GSCs)and PTPRZ1-KO GSCs and the expression of immunosuppressive phenotype (M2) polarization marker CD163 were examined using flow cytometry.Differentially expressed genes (DEGs ) between paired GSCs and NSTCs were determined using a bulk RNA-sequencing dataset (GSE54791 )from Gene Expression Omnibus (GEO).A gene set informing worse outcome of patients with GBM was generated using The Cancer Genome Atlas (TCGA)-GBM cohort.By intersecting the aforementioned gene set with the gene set that encodes for human membrance proteins,the PTPRZ1 gene is obtained.Gene set enrichment analysis (GSEA)was used for pathway enrichment analysis to compare the differentially regulated pathways between GBMs with high or low PTPRZ1 expression.Bulk RNA sequencing,qRT-PCR and Western blotting were used to identify the DEGs between PTPRZ1-KO GSCs and PTPRZ1-Ctrl GSCs.Results GSCs were more capable of escaping from TAM phagocytosis than NSTCs (P<0.05 )and had specifically up-regulated PTPRZ1 expression.PTPRZ1-KO significantly suppressed GSCs escaping from TAM phagocytosis (P<0.01 ). GBMs with high PTPRZ1 expression showed significant inhibition of pathways mediating phagocytosis (P<0.05).The expression of CCL20 as a M2 TAM polarization chemokine was significantly down-regulated in PTPRZ1-KO GSCs (P<0.05 ).Treatment with recombinant CCL20 up-regulated the expression of CD163 as a M2 TAM marker in TAM.Conclusion PTPRZ1+GSCs mediate M2 TAM polarization and inhibit TAM phagocytosis,which may be related to the up-regulation of CCL20 in PTPRZ1+GSCs.

Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl₄+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl₄+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl₄+PHx group. Compared with the CCl₄+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl₄+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl₄+PHx group. Compared with the CCl₄+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl₄+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl₄+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl₄+PHx+MSC-EV group was higher than that in the CCl₄+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.

Objective:To establish surgical difficulty scoring system of partial nephrectomy based on holographic imaging and explore its application value in partial nephrectomy.Methods:A total of 184 patients including 110 males and 74 females with renal tumors diagnosed as stage cT 1 to cT 2 before surgery in the First Affiliated Hospital of Xiamen University from October 2019 to January 2022 were included. Among the 184 patients, 141 patients were treated with partial nephrectomy and 43 patients were treated with radical nephrectomy (3 partial nephrectomies were finally changed to radical nephrectomies due to vascular and tumor location). 60 patients had hypertension. 24 patients had diabetes. 7 patients had hyperuricemia. The median age was 55(47, 62) years. The median BMI was 23.7(21.8, 26.4) kg/m 2. The median maximum tumor diameter was 3.9(2.9, 5.2) cm. The median preoperative eGFR was 99.7(83.4, 114.2) ml/(min·1.73m 2). The median R. E.N.A.L. score was 8(6, 9). The median PADUA score was 9(8, 10). 153 patients were diagnosed as stage cT 1 and 31 patients were diagnosed as stage cT 2. The hologram reconstruction was performed according to preoperative CT or MRI examination. The maximum diameter of the tumor in the kidney (D), the compression degree of the renal segmental vessels by tumor(C), the area of the renal sinus occupied by tumor(O) and the mass of exophytic rate(M) were comprehensively considered and finally constituted the difficulty scoring system named DCOM score for partial nephrectomy. The DCOM score divided the complexity of tumor surgery into mild (4-6 points), moderate (7-8 points) and high (≥ 9 points). Meanwhile, the MIC (surgical margins are negative, WIT is <20 min, and no major complications)was used to evaluate the overall surgical effect. The DCOM, R. E.N.A.L. and PADUA scores were performed on all patients and compared with each other to evaluate the surgical effect of DCOM score in partial nephrectomy. Results:All surgeries in this study were successfully completed, including 141 partial nephrectomies and 43 radical nephrectomies. The DCOM score was 10(9, 11) for radical nephrectomy and 6(5, 8) for partial nephrectomy, and the difference was statistically significant ( P=0.001). There were 23 patients (37.7%) in highly complex group, 39 patients (88.6%) in moderately complex group and 79 patients (100.0%) in mildly complex group underwent partial nephrectomy, respectively. According to multifactorial analysis, patients in highly and moderately complex group of DCOM score had 8.88 times ( P=0.001) and 1.76 times ( P=0.005) less reach MIC than those in mildly complex group, respectively. Patients in highly and moderately complex group of PADUA score had 4.86 times ( P=0.005)and 3.41 times ( P=0.006)less reach MIC than patients in mildly complex group of DCOM score, respectively. What’s more, patients in moderately complex group of R. E.N.A.L. score had 3.11 times ( P=0.003) less reach MIC than patients in mildly complex group of DCOM score. In the ROC curves to predict MIC achievement, the AUC values of R. E.N.A.L., PADUA and DCOM scores were 0.657, 0.655 and 0.746, respectively. Comparing:R. E.N.A.L. score with DCOM score, the AUC value was statistically significant ( P=0.025). Conclusions The surgical difficulty scoring system (DCOM score) based on holographic imaging can predict the outcome of partial nephrectomy, but further verification is needed.

Objective:To explore the efficacy of endoscopic retrograde cholangiopancreatography (ERCP) biliary stent implantation in patients with unresectable malignant biliary stricture (MBS) and the influencing factors of overall survival.Methods:The clinical data of 346 patients who underwent ERCP biliary stent implantation due to MBS from May 2013 to October 2016 in Xijing Digestive Disease Hospital of Air Force Military Medical University, Shanxi Bethune Hospital and Mengchao Hepatobiliary Hospital of Fujian Medical University were retrospectively analyzed, and the efficacy, complications and risk factors affecting overall survival were also analyzed.Results:After ERCP biliary stent implantation, the levels of total bilirubin, γ-glutamyl transpeptidase, alkaline phosphatase and alanine aminotransferase were lower than those before surgery (all P < 0.01). The incidence of infection after operation was 14.7% (51/346), and the incidence of biliary infection was 13.0% (45/346). The incidence of post-ERCP pancreatitis (PEP) was 4.6% (16/346). The median survival time after ERCP was 131.0 d (70.3 d, 246.5 d). Multivariate Cox regression analysis showed that the independent risk factors affecting the overall survival patients included the hilar bile duct stenosis ( HR = 1.85, 95% CI 1.44-2.38, P < 0.01), preoperative bilirubin level exceeding the upper limit of normal level by 5 times ( HR = 1.75, 95% CI 1.30-2.36, P < 0.01), carbohydrate antigen 199 level exceeding the upper limit of normal level by 10 times ( HR = 1.27, 95% CI 1.00-1.61, P = 0.050), vascular and organ metastasis ( HR = 1.32, 95% CI 1.04-1.69, P = 0.023), and the poor jaundice decreasing level ( HR = 1.37, 95% CI 1.02-1.85, P = 0.037) . Conclusions:The ERCP biliary stent implantation is a safe and effective therapy for MBS. ERCP biliary stent implantation MBS patients with hilar bile duct stenosis, preoperative bilirubin levels more than 5 times of the upper limit of normal level, carbohydrate antigen 199 levels more than 10 times of the upper limit of normal level, vascular and organ metastasis, and poor jaundice decreasing level may have poor overall survival.

【Objective】 To investigate the unqualified rate of anti-HIV detection of blood screening laboratories in Beijing-Tianjin-Hebei region, and explore the differences in anti-HIV detection ability and influencing factors in each laboratory. 【Methods】 Through filling questionnaires via e-mail, the anti-HIV ELISA unqualified rate and confirmed (WB) positive results (data) from January to December 2018 from 15 blood screening laboratories in Beijing-Tianjin-Hebei region were collected. Our laboratory was responsible for data collection and confirmation, and statistics software SPSS22.0 was used for analysis. 【Results】 1) There was a statistically significant difference among the unqualified rate of anti-HIV ELISA(6.77‱~35.71‱) and confirmed positive rate(0.60‱~3.56‱) in 15 blood screening laboratories in Beijing-Tianjin-Hebei region (P<0.05); 2) There were significant differencse among the ELISA unqualified rate and the confirmed positive rate of 8 reagents for anti-HIV detection(P<0.01), and the sensitivity of the 4th generation detection reagent and the imported reagent was higher than that of the 3rd generation reagent and the domestic reagent. The anti-HIV ELISA unqualified rate of R5 was the highest (19.08‱). 3)There were significant differences in the anti-HIV ELISA unqualified rate of R1, R2, R3, R5 and R7 reagents among different blood station laboratories(P<0.05), and there were no significant differences in the anti-HIV ELISA unqualified rate of R4, R6 and R8 reagents among different blood station laboratories(P>0.05). 4)The unqualified rate of anti-HIV ELISA of laboratories using different regents showed significant differences(P<0.05), except H, J, M. The unqualified rate of imported reagent was significantly higher than that of domestic reagents of laboratories using imported and domestic reagents combinations(P<0.05), except O. 62.5% (5/8) laboratories using domestic 3^rd and 4^th generation reagent combination showed significant differences in the unqualified rates among different reagents(P<0.05); 5) The positive rate of single-reagent(62.02%~95.45%)in 15 blood screening laboratories showed significant difference(P<0.001), and A was the lowest (62.02%). 【Conclusion】 The anti-HIV detection ability among 15 blood screening laboratories in Beijing-Tianjin-Hebei region is quite different. The application of different reagents is the main factor for the difference, and other factors such as personnel, instruments and test strategies also has a great impact on the detection of anti-HIV. It is still necessary to promote the process of homogenization of blood testing quality among blood screening laboratories in Beijing-Tianjin-Hebei region.

OBJECTIVE: To investigate the sensing volume of open-ended coaxial probe technique for measurement of dielectric characteristics. METHODS: A measurement model combining macro- measurement device with a layer model of dielectric properties parameters was established for evaluating the sensing volume of open-ended coaxial probe technique. We defined sensing depth and sensing diameter to describe the distance that could be detected in vertical and horizontal direction. Using a variety of materials with different dielectric properties (Teflon, deionized water, ethanol, and gradient concentration sodium chloride solution), a layered model of dielectric properties differentiation was established. The total combined uncertainties (TCU) were calculated for different output power, and the output power was controlled to increase from -50 dBm to 15 dBm to calibrate the error range of the dielectric properties measurement system. The optimal output power range was determined based on the results of TCU test. In sensing volume measurement experiment, we set the control groups based on measurement parameters that potentially affect the sensing volume including output power (-10, -5, 0, 3, 6, and 9 dBm), frequency (1-500 MHz), Teflon, deionized water, and ethanol to form a dielectric constant difference between high and low contrast groups. Different concentrations of sodium chloride solution and Teflon were used to generate a conductivity difference between high and low contrast groups. These groups were tested in the sensing depth and sensing diameter measurement experiments. RESULTS: The result of TCU test indicated that accurate and stable measurement results could be obtained when the output power was greater or equal to-10 dBm (TCU < 2%). Sensing volume measurement experiment revealed a positive correlation between the sensing depth and output power ( < 0.05). As the measured power increased, the sensing depth gradually increased in deionized water and ethanol, and the difference reached 70 μm. The sensing depth was negatively correlated frequency ( < 0.05). As the concentration of sodium chloride solution increased, the corresponding sensing depth gradually decreased, with a difference reaching 270 μm. The sensing depth of high dielectric materials was greater than that pf low dielectric materials. The results of sensing diameter measurement were not obviously affected by the measurement parameters, and the sensing diameter was stable in a fixed range (1.0 to 1.8 mm) between the diameter of the inner conductor and the diameter of the insulation layer, and was less than the diameter of the probe. CONCLUSIONS The sensing volume of open-ended coaxial probe technique is affected by measurement parameters and dielectric properties of materials, which significantly affect the sensing depth.
Algorithms ; Electric Conductivity ; Electrochemistry ; instrumentation

OBJECTIVE: To investigate the sensing volume of open-ended coaxial probe technique for measurement of dielectric characteristics. METHODS: A measurement model combining macro- measurement device with a layer model of dielectric properties parameters was established for evaluating the sensing volume of open-ended coaxial probe technique. We defined sensing depth and sensing diameter to describe the distance that could be detected in vertical and horizontal direction. Using a variety of materials with different dielectric properties (Teflon, deionized water, ethanol, and gradient concentration sodium chloride solution), a layered model of dielectric properties differentiation was established. The total combined uncertainties (TCU) were calculated for different output power, and the output power was controlled to increase from -50 dBm to 15 dBm to calibrate the error range of the dielectric properties measurement system. The optimal output power range was determined based on the results of TCU test. In sensing volume measurement experiment, we set the control groups based on measurement parameters that potentially affect the sensing volume including output power (-10, -5, 0, 3, 6, and 9 dBm), frequency (1-500 MHz), Teflon, deionized water, and ethanol to form a dielectric constant difference between high and low contrast groups. Different concentrations of sodium chloride solution and Teflon were used to generate a conductivity difference between high and low contrast groups. These groups were tested in the sensing depth and sensing diameter measurement experiments. RESULTS: The result of TCU test indicated that accurate and stable measurement results could be obtained when the output power was greater or equal to-10 dBm (TCU < 2%). Sensing volume measurement experiment revealed a positive correlation between the sensing depth and output power ( < 0.05). As the measured power increased, the sensing depth gradually increased in deionized water and ethanol, and the difference reached 70 μm. The sensing depth was negatively correlated frequency ( < 0.05). As the concentration of sodium chloride solution increased, the corresponding sensing depth gradually decreased, with a difference reaching 270 μm. The sensing depth of high dielectric materials was greater than that pf low dielectric materials. The results of sensing diameter measurement were not obviously affected by the measurement parameters, and the sensing diameter was stable in a fixed range (1.0 to 1.8 mm) between the diameter of the inner conductor and the diameter of the insulation layer, and was less than the diameter of the probe. CONCLUSIONS The sensing volume of open-ended coaxial probe technique is affected by measurement parameters and dielectric properties of materials, which significantly affect the sensing depth.
Algorithms ; Electric Conductivity

OBJECTIVE: To investigate the effects of millimeter wave (MMW) exposure on apoptosis of human melanoma A375 cells and explore the mechanisms. METHODS: Through electromagnetic field calculation we simulated MMW exposure in cells and calculated the specific absorption rate (SAR). The optimal irradiation parameters were determined according to the uniformity and intensity of the SAR. A375 cells were then exposed to MMV for 15, 30, 60, or 90 min, with or without pretreatment with the caspase-3 inhibitor AC-DEVD-fmk (10 μmol/L) for 1 h at 90 min before the exposure. CCK-8 assay was used to assess the changes in the viability and Annexin-V/ PI staining was used to detect the apoptosis of the cells following the exposures; Western blotting was used to detect the expression of caspase-3 in the cells. RESULTS: The results of electromagnetic field calculation showed that for optimal MMV exposure, the incident field needed to be perpendicular to the bottom of the plastic Petri dish with the antenna placed below the dish. CCk-8 assay showed that MMW exposure significantly inhibited the cell viability in a time-dependent manner ( < 0.05); exposures for 15, 30, 60, and 90 min all resulted in significantly increased apoptosis of the cells ( < 0.05). The cells with MMW exposure showed significantly increased expression of caspase-3. The inhibitory effect of MMW on the cell viability was antagonized significantly by pretreatment of the cells with AC-DEVD-fmk ( < 0.05), which increased the cell viability rate from (36.7±0.09)% to (59.8±0.06)% ( < 0.05). CONCLUSIONS 35.2 GHz millimeter wave irradiation induces apoptosis in A375 cells by activating the caspase-3 protein.
Apoptosis ; Caspase 3 ; metabolism ; Caspase Inhibitors ; pharmacology ; Cell Line, Tumor ; Cell Survival ; Electromagnetic Fields ; Enzyme Activation ; Humans ; Magnetic Field Therapy ; Melanoma ; enzymology ; pathology ; therapy ; Time Factors

OBJECTIVE: To investigate the application of high-frequency ultrasound (HFUS) and optical coherence tomography (OCT) for monitoring the progression of cutaneous melanoma (CM) in nude mice. METHODS: Twenty 4-week-old nude mice were randomly divided into CM group (=16) and control group (=4). In CM group, A375 cell suspension were injected subcutaneously on the back of the nude mice, and only culture medium was injected in the control group. The tumor growth was monitored by gross observation and with HFUS and OCT on a daily basis. The tumor formation rate, time, and size were recorded and melanoma visibility was assessed quantitatively using the contrast- to-background-noise ratio (CNR). Twentyfour days after cell implantation, the tumors were dissected for pathological examination. RESULTS: The tumor formation rate was 87.5% in CM group. OCT detected tumor formation at an earlier time than HFUS. With the growth of the tumor, HFUS detected spots or bands of strong echoes, and flattening of the upper dermis could be observed in OCT; the of HFUS and OCT were obviously enhanced over time ( < 0.05). The tumor growth curve showed that OCT was more sensitive for measurement of the tumor thickness than HFUS. The length, depth and volume of the tumors measured by HFUS were significantly greater than those measured by OCT ( < 0.05). CONCLUSIONS HFUS and OCT can monitor the dynamic development of cutaneous melanoma in nude mice, and their imaging performance differs in different stages of tumor development.
Animals ; Melanoma ; Mice ; Mice, Nude ; Skin Neoplasms ; Tomography, Optical Coherence ; Ultrasonography