Objective:To explore the potential of the novel fibroblast activation protein (FAP)-targeted theranostic agent 68Ga/ 177Lu-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-2P (FAP inhibitor (FAPI)) 2 in microsatellite stable (MSS) colorectal cancer, and to evaluate the efficacy and underlying mechanism of 177Lu-DOTA-2P(FAPI) 2 in combination with immune checkpoint inhibitors (ICIs). Methods:This study was a randomized, parallel-group design. DOTA-2P(FAPI) 2 was labeled with 68Ga or 177Lu respectively. The binding performance of DOTA-2P(FAPI) 2 to FAP was validated through in vitro cell experiments. FAP-positive CT26-FAP tumor-bearing mouse model was constructed, and microPET imaging and biodistribution were performed. The in vivo antitumor efficacy was assessed for the 177Lu-DOTA-2P(FAPI) 2 monotherapy, α programmed death-ligand 1 (PD-L1) monotherapy, and the combination of 177Lu-DOTA-2P(FAPI) 2 with αPD-L1 therapy groups. Changes in the tumor microenvironment were analyzed using single-cell RNA sequencing to elucidate the mechanism of the combined treatment. Independent-sample t test was used to analyze data. Survival analysis was performed using the log-rank test. Results:The labeling yields of 68Ga/ 177Lu-DOTA-2P(FAPI) 2 were both >90%, with the radiochemical purities both >95%. In vitro cellular uptake and blocking assays showed that FAPI-46 significantly inhibited the binding of 68Ga-DOTA-2P(FAPI) 2 to FAP in CT26-FAP cells, with the cellular uptake values at 60min of (51.5±0.8)% and (1.0±0.3)%, respectively ( t=102.40, P<0.001). MicroPET imaging showed that the tumor uptake of 68Ga-DOTA-2P(FAPI) 2 remained stable even at 4 h post-injection, with a significantly higher uptake value compared to 68Ga-FAPI-46 ((7.3±1.6) vs (3.7±0.2) percentage activity of injection dose per gram of tissue (%ID/g); t=3.87, P=0.018). The biodistribution results indicated significant tumor uptake of 177Lu-DOTA-2P(FAPI) 2 even at 24 h post-injection ((4.30±0.52)%ID/g). The combination of 177Lu-DOTA-2P(FAPI) 2 and αPD-L1 achieved the 30-day survival rate of 100%, which was significantly superior to that of the control group (saline injection; χ2=9.53, P=0.002). Further mechanistic studies revealed that the combination therapy reprogramed the tumor microenvironment, enhanced anti-tumor intercellular communication, and activated signaling pathways such as Fas-FasL between T cells/natural killer (NK) cells and tumor cells, thereby synergistically inhibiting tumor progression. Conclusions:68Ga/ 177Lu-DOTA-2P(FAPI) 2 exhibits theranostic potential for MSS colorectal cancer. The combination of 177Lu-DOTA-2P(FAPI) 2 with ICIs may significantly prolong survival, demonstrating significant potential for clinical translation.

Objective:To explore the predictive value of the difference in average CT attenuation values between renal pelvis urine and bladder urine on the risk of pyonephrosis, and to establish a nomogram model in combination with other parameters and to verify its predictive ability.Methods:The clinical data of patients with obstructive hydronephrosis who came to our hospital for emergency percutaneous nephrostomy (PCN) between January 2020 and December 2022 were retrospectively analyzed. There were 16 men and 33 women, with the age of (57.59, 14.67) years. Body mass index was (23.34, 3.11) kg/m 2. Urine nitrite was positive in 14 cases. Urine culture was positive in 21 cases. In the routine blood test, the median white blood cell count was 10.96 (7.21, 15.15) ×10 9/L, haemoglobin (115.08, 22.71) g/L and platelets (263.00, 97.20)×10 9/L. The difference in average CT attenuation values between renal pelvis urine and bladder urine (mean CT value of pyelonephritis-mean CT value of cysturia) was -2.19 (-7.04, 4.05) HU. Patients were divided into pyonephrosis group and hydronephrosis group according to whether the drainage fluid after PCN was pus. Single-factor and multi-factor logistic regression models were used to analyze the independent predictors of pyonephrosis and to construct nomograms. The discrimination of the model was assessed by the area under the curve (AUC) of the receiver operating characteristics (ROC), the accuracy by the calibration curve and the clinical efficacy by the decision curve analysis (DCA). Results:There were 49 cases in this study, 31 cases in the hydronephrosis group and 18 cases in the pyonephrosis group. The average CT attenuation values of renal pelvis urine were 3.35(0.56, 8.96) HU and 7.78 (3.75, 18.38) HU, respectively, and the difference between the two groups was statistically significant ( P=0.023). The average CT attenuation values of bladder urine were (7.81±6.15)HU and (7.22±7.50)HU, respectively, and there was no significant difference between the two groups ( P=0.780). The difference in average CT attenuation values between renal pelvis urine and bladder urine were -3.98(-7.54, 0.60)HU and 2.13 (-5.15, 9.36)HU, respectively, and the difference between the two groups was not statistically significant ( P=0.059); the white blood cells were 9.82(7.04, 12.46) ×10 9/L and 13.99(9.75, 18.44) ×10 9/L, respectively, and the difference between the two groups was statistically significant ( P=0.048). Platelet counts were (248.06±87.87)×10 9/L and (288.72±109.29)×10 9/L, respectively, and there was no statistical difference between the two groups ( P=0.189). The proportion of urine protein positive was higher in the pyonephrosis group (17 vs. 20, P=0.048). Between the two groups, sex, age, body mass index, clinical symptoms (with or without low back pain), surgical history of upper urinary tract stones, underlying diseases (including hypertension, diabetes, coronary heart disease, etc.), location of hydronephrosis (left, right, and both sides), reasons of obstruction [upper urinary tract stones, other factors (such as tumor, ureteral stricture, etc.)], haemoglobin, were not statistical different. There were no significant difference in blood glucose, blood potassium, blood sodium, urine leukocytes, urine erythrocytes, urine nitrite and urine culture ( P>0.05). Multivariate logistic regression analysis showed that the difference in average CT attenuation values between renal pelvis urine and bladder urine ( OR=1.196, 95% CI 1.055-1.437, P=0.018), white blood cells ( OR=1.252, 95% CI 1.036-1.615, P=0.038), and platelets ( OR=1.014, 95% CI 1.003-1.030, P=0.025) were independent predictors of pyonephrosis. According to the above indicators, the nomogram model was established and the AUC value of the model was 0.767 (95% CI 0.616-0.918), the sensitivity was 0.611 and the specificity was 0.935. The calibration curve showed that there is a good fit between the observed value and the predicted value. The DCA analysis showed that the nomogram model has a net gain in a wide threshold range, demonstrating its predictive accuracy and clinical practicality in predicting the risk of pyonephrosis. When the cut-off value of the difference between the average CT values of pyelonephritis and cystourethrosis was 6.54 HU, the AUC value of the independent prediction of pyonephrosis was 0.690(95% CI 0.564-0.816), the sensitivity was 0.444 and the specificity was 0.935. Conclusions:The difference in average CT attenuation values between renal pelvis urine and bladder urine is an independent risk factor for predicting pyonephrosis, and the nomogram constructed by combining it with white blood cells and platelets has a good predictive effect for predicting the risk of pyonephrosis. If the difference in average CT attenuation values between renal pelvis urine and bladder urine is greater than 6.54HU, it should be alert to the occurrence of pyonephrosis.

Objective:To explore the evaluation and relationship of hepatitis B virus(HBV)surface antigen(HBsAg)in clinical laboratory in different detection systems,further scientifically and reasonably to explain the test results for serving clinical practices.Methods:During the period from June 2021 to July 2022,100 425 specimens of patients with screened,suspected and confirmed HBV infections were collected from the clinical departments(mainly infectious hepatology)of Nanyang Central Hospital.Detection methodology included quantitative(electrochemiluminescence and chemiluminescence),qualitative(gold standard),semi-quantita-tive(ELISA),and highly sensitive HBV-DNA(RT-PCR)methods,and then analyzed the strengths and weaknesses and closeness be-tween each methodology.The relationship between the two Roche HBsAg detection systems was analyzed by correlation analysis.The HBsAg efficacy analysis was validated using Cut/Off value setting and detection limit,which in turn analyzed the distribution of false-positive and false-negative reporting models.Results:Detection results for low-and medium-concentration HBsAg showed a correla-tion between the electrochemical luminescence semi-quantitative method and ELISA method.ELISA method still had advantages in terms of sensitivity and specificity when detecting HBsAg,and there were no significant differences compared to domestic and interna-tional HBsAg quantitative detection systems.Performance validation conducted in accordance with the CNAS-GL038 document showed that the minimum detection limit for HBsAg calculated using the ELISA method in this laboratory was 0.1 U/ml.When the ROC curve Cut/Off value was set to 0.105,the area under the curve was the largest(AUC=0.986).Based on Roche's semi-quantitative electroche-miluminescence detection and patient medical history,in the common reporting model for hepatitis B five-item detection using ELISA method,HBsAg false positives occurred most frequently when HBsAg was positive alone,and HBsAg occurred most frequently in the false positive range when the OD value was less than 0.5.In ELISA method for detecting HBsAg,as the OD value increased from 0.01 to 0.10,the number of false-negative results also increased.Roche Elecsys HBsAg Ⅱ Quant Ⅱ and Elecsys HBsAg Ⅱ testing systems exhibited good linearity under certain conditions,with a ratio of approximately 1/0.18.In Elecsys HBsAg Ⅱ Quant Ⅱ detection sys-tem,the test results of HBsAg samples diluted 400 times were highly consistent with the original test results with a coefficient of deter-mination R2=0.993 8.Conclusion:There was a certain relationship between various detection systems of HBsAg at a suitable concen-tration.The detection of HBsAg by ELISA can meet the needs of clinical detection.

Objective:To explore the evaluation and relationship of hepatitis B virus(HBV)surface antigen(HBsAg)in clinical laboratory in different detection systems,further scientifically and reasonably to explain the test results for serving clinical practices.Methods:During the period from June 2021 to July 2022,100 425 specimens of patients with screened,suspected and confirmed HBV infections were collected from the clinical departments(mainly infectious hepatology)of Nanyang Central Hospital.Detection methodology included quantitative(electrochemiluminescence and chemiluminescence),qualitative(gold standard),semi-quantita-tive(ELISA),and highly sensitive HBV-DNA(RT-PCR)methods,and then analyzed the strengths and weaknesses and closeness be-tween each methodology.The relationship between the two Roche HBsAg detection systems was analyzed by correlation analysis.The HBsAg efficacy analysis was validated using Cut/Off value setting and detection limit,which in turn analyzed the distribution of false-positive and false-negative reporting models.Results:Detection results for low-and medium-concentration HBsAg showed a correla-tion between the electrochemical luminescence semi-quantitative method and ELISA method.ELISA method still had advantages in terms of sensitivity and specificity when detecting HBsAg,and there were no significant differences compared to domestic and interna-tional HBsAg quantitative detection systems.Performance validation conducted in accordance with the CNAS-GL038 document showed that the minimum detection limit for HBsAg calculated using the ELISA method in this laboratory was 0.1 U/ml.When the ROC curve Cut/Off value was set to 0.105,the area under the curve was the largest(AUC=0.986).Based on Roche's semi-quantitative electroche-miluminescence detection and patient medical history,in the common reporting model for hepatitis B five-item detection using ELISA method,HBsAg false positives occurred most frequently when HBsAg was positive alone,and HBsAg occurred most frequently in the false positive range when the OD value was less than 0.5.In ELISA method for detecting HBsAg,as the OD value increased from 0.01 to 0.10,the number of false-negative results also increased.Roche Elecsys HBsAg Ⅱ Quant Ⅱ and Elecsys HBsAg Ⅱ testing systems exhibited good linearity under certain conditions,with a ratio of approximately 1/0.18.In Elecsys HBsAg Ⅱ Quant Ⅱ detection sys-tem,the test results of HBsAg samples diluted 400 times were highly consistent with the original test results with a coefficient of deter-mination R2=0.993 8.Conclusion:There was a certain relationship between various detection systems of HBsAg at a suitable concen-tration.The detection of HBsAg by ELISA can meet the needs of clinical detection.

Objective:To explore the potential of the novel fibroblast activation protein (FAP)-targeted theranostic agent 68Ga/ 177Lu-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA)-2P (FAP inhibitor (FAPI)) 2 in microsatellite stable (MSS) colorectal cancer, and to evaluate the efficacy and underlying mechanism of 177Lu-DOTA-2P(FAPI) 2 in combination with immune checkpoint inhibitors (ICIs). Methods:This study was a randomized, parallel-group design. DOTA-2P(FAPI) 2 was labeled with 68Ga or 177Lu respectively. The binding performance of DOTA-2P(FAPI) 2 to FAP was validated through in vitro cell experiments. FAP-positive CT26-FAP tumor-bearing mouse model was constructed, and microPET imaging and biodistribution were performed. The in vivo antitumor efficacy was assessed for the 177Lu-DOTA-2P(FAPI) 2 monotherapy, α programmed death-ligand 1 (PD-L1) monotherapy, and the combination of 177Lu-DOTA-2P(FAPI) 2 with αPD-L1 therapy groups. Changes in the tumor microenvironment were analyzed using single-cell RNA sequencing to elucidate the mechanism of the combined treatment. Independent-sample t test was used to analyze data. Survival analysis was performed using the log-rank test. Results:The labeling yields of 68Ga/ 177Lu-DOTA-2P(FAPI) 2 were both >90%, with the radiochemical purities both >95%. In vitro cellular uptake and blocking assays showed that FAPI-46 significantly inhibited the binding of 68Ga-DOTA-2P(FAPI) 2 to FAP in CT26-FAP cells, with the cellular uptake values at 60min of (51.5±0.8)% and (1.0±0.3)%, respectively ( t=102.40, P<0.001). MicroPET imaging showed that the tumor uptake of 68Ga-DOTA-2P(FAPI) 2 remained stable even at 4 h post-injection, with a significantly higher uptake value compared to 68Ga-FAPI-46 ((7.3±1.6) vs (3.7±0.2) percentage activity of injection dose per gram of tissue (%ID/g); t=3.87, P=0.018). The biodistribution results indicated significant tumor uptake of 177Lu-DOTA-2P(FAPI) 2 even at 24 h post-injection ((4.30±0.52)%ID/g). The combination of 177Lu-DOTA-2P(FAPI) 2 and αPD-L1 achieved the 30-day survival rate of 100%, which was significantly superior to that of the control group (saline injection; χ2=9.53, P=0.002). Further mechanistic studies revealed that the combination therapy reprogramed the tumor microenvironment, enhanced anti-tumor intercellular communication, and activated signaling pathways such as Fas-FasL between T cells/natural killer (NK) cells and tumor cells, thereby synergistically inhibiting tumor progression. Conclusions:68Ga/ 177Lu-DOTA-2P(FAPI) 2 exhibits theranostic potential for MSS colorectal cancer. The combination of 177Lu-DOTA-2P(FAPI) 2 with ICIs may significantly prolong survival, demonstrating significant potential for clinical translation.

Objective:To explore the predictive value of the difference in average CT attenuation values between renal pelvis urine and bladder urine on the risk of pyonephrosis, and to establish a nomogram model in combination with other parameters and to verify its predictive ability.Methods:The clinical data of patients with obstructive hydronephrosis who came to our hospital for emergency percutaneous nephrostomy (PCN) between January 2020 and December 2022 were retrospectively analyzed. There were 16 men and 33 women, with the age of (57.59, 14.67) years. Body mass index was (23.34, 3.11) kg/m 2. Urine nitrite was positive in 14 cases. Urine culture was positive in 21 cases. In the routine blood test, the median white blood cell count was 10.96 (7.21, 15.15) ×10 9/L, haemoglobin (115.08, 22.71) g/L and platelets (263.00, 97.20)×10 9/L. The difference in average CT attenuation values between renal pelvis urine and bladder urine (mean CT value of pyelonephritis-mean CT value of cysturia) was -2.19 (-7.04, 4.05) HU. Patients were divided into pyonephrosis group and hydronephrosis group according to whether the drainage fluid after PCN was pus. Single-factor and multi-factor logistic regression models were used to analyze the independent predictors of pyonephrosis and to construct nomograms. The discrimination of the model was assessed by the area under the curve (AUC) of the receiver operating characteristics (ROC), the accuracy by the calibration curve and the clinical efficacy by the decision curve analysis (DCA). Results:There were 49 cases in this study, 31 cases in the hydronephrosis group and 18 cases in the pyonephrosis group. The average CT attenuation values of renal pelvis urine were 3.35(0.56, 8.96) HU and 7.78 (3.75, 18.38) HU, respectively, and the difference between the two groups was statistically significant ( P=0.023). The average CT attenuation values of bladder urine were (7.81±6.15)HU and (7.22±7.50)HU, respectively, and there was no significant difference between the two groups ( P=0.780). The difference in average CT attenuation values between renal pelvis urine and bladder urine were -3.98(-7.54, 0.60)HU and 2.13 (-5.15, 9.36)HU, respectively, and the difference between the two groups was not statistically significant ( P=0.059); the white blood cells were 9.82(7.04, 12.46) ×10 9/L and 13.99(9.75, 18.44) ×10 9/L, respectively, and the difference between the two groups was statistically significant ( P=0.048). Platelet counts were (248.06±87.87)×10 9/L and (288.72±109.29)×10 9/L, respectively, and there was no statistical difference between the two groups ( P=0.189). The proportion of urine protein positive was higher in the pyonephrosis group (17 vs. 20, P=0.048). Between the two groups, sex, age, body mass index, clinical symptoms (with or without low back pain), surgical history of upper urinary tract stones, underlying diseases (including hypertension, diabetes, coronary heart disease, etc.), location of hydronephrosis (left, right, and both sides), reasons of obstruction [upper urinary tract stones, other factors (such as tumor, ureteral stricture, etc.)], haemoglobin, were not statistical different. There were no significant difference in blood glucose, blood potassium, blood sodium, urine leukocytes, urine erythrocytes, urine nitrite and urine culture ( P>0.05). Multivariate logistic regression analysis showed that the difference in average CT attenuation values between renal pelvis urine and bladder urine ( OR=1.196, 95% CI 1.055-1.437, P=0.018), white blood cells ( OR=1.252, 95% CI 1.036-1.615, P=0.038), and platelets ( OR=1.014, 95% CI 1.003-1.030, P=0.025) were independent predictors of pyonephrosis. According to the above indicators, the nomogram model was established and the AUC value of the model was 0.767 (95% CI 0.616-0.918), the sensitivity was 0.611 and the specificity was 0.935. The calibration curve showed that there is a good fit between the observed value and the predicted value. The DCA analysis showed that the nomogram model has a net gain in a wide threshold range, demonstrating its predictive accuracy and clinical practicality in predicting the risk of pyonephrosis. When the cut-off value of the difference between the average CT values of pyelonephritis and cystourethrosis was 6.54 HU, the AUC value of the independent prediction of pyonephrosis was 0.690(95% CI 0.564-0.816), the sensitivity was 0.444 and the specificity was 0.935. Conclusions:The difference in average CT attenuation values between renal pelvis urine and bladder urine is an independent risk factor for predicting pyonephrosis, and the nomogram constructed by combining it with white blood cells and platelets has a good predictive effect for predicting the risk of pyonephrosis. If the difference in average CT attenuation values between renal pelvis urine and bladder urine is greater than 6.54HU, it should be alert to the occurrence of pyonephrosis.

Objective: To construct a hit-deficient mutant strain of S. mutans ATCC25175 and verify its cell cycle regulatory function. Method : Genomic DNA was extracted from S. mutans ATCC25175 strains, and then the upstream and downstream DNA fragments of the hit gene were cloned into the pFW5 vector (spectinomycin resistant) to construct recombinant plasmids using PCR amplification. Third, employed by natural genetic transformation in S. mutans ATCC25175 strains, the linearized recombinant plasmids were transformed into their genetic competence, induced by the synthesized competence-stimulating peptide (CSP), and then, homologous recombination was utilized to produce crossover and noncrossover products. Fourth, the hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin-resistance marker and identified by the electrophoresis of PCR products and PCR Sanger sequencing. Finally, its growth rate in vegetative BHI medium was also investigated. Results : The upstream (856 bp) and downstream (519 bp) DNA fragments of the hit gene from the genomic DNA materials of S. mutans ATCC25175 were cloned into two multiple cloning sites (MCS-I and MCS-II) of the pFW5 vector, respectively, and the recombinant plasmid pFW5_hit_Up_Down was constructed and identified by double-emzyme digestion and PCR Sanger sequencing. The linearized recombinant plasmids were transformed into their genetic competence, induced by the synthetic CSP, and then, homologous recombination was utilized to produce various products. The hit-deficient mutant strains of S. mutans ATCC25175 were screened through the spectinomycin resistance marker and identified by the electrophoresis of PCR products and Sanger sequencing. The growth rate of the hit-deficient mutant strains versus their parental S. mutans ATCC25175 strains was increased greatly (P<0.001). Conclusion The hit-deficient mutant strains of S. mutans ATCC25175, having heritable traits, were successfully constructed, and the encoding Hit protein is growth-phase regulated in the cell cycle.

【Objective】 To investigate the changes of platelet, white blood cell, red blood cell counts, Hb and PCV before and after plateletpheresis donation. 【Methods】 10 444 platelet donors who donated platelet from Aug 1, 2019 to Jul 31, 2020 were selected as subjects, among which first-time donors were selected as control group and donors who donated twice or more as donation group. After matching gender and age using Propensity Score Matching method in the two groups, the changes of platelet, white blood cell, red blood cell counts, Hb and PCV of platelet blood donors were compared by t test. 【Results】 There were a slight increase in Plt (t=-6.651, P<0.05), a decrease in WBC (t=2.312, P<0.05), a decrease in hemoglobin(t=4.643, P<0.05), and no change in RBC (t=1.439, P>0.05) and in PCV (t=0.030, P>0.05) after plateletpheresis donation. 【Conclusion】 Plateletpheresis donation has an effect on some peripheral blood parameters, but within the normal reference range.

Identification of components and metabolites of traditional Chinese medicines (TCMs) employing liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) techniques with information-dependent acquisition (IDA) approaches is increasingly frequent. A current drawback of IDA-MS is that the complexity of a sample might prevent important compounds from being triggered in IDA settings. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) is a data-independent acquisition (DIA) method where the instrument deterministically fragments all precursor ions within the predefined m/z range in a systematic and unbiased fashion. Herein, the superiority of SWATH on the detection of TCMs' components was firstly investigated by comparing the detection ef-ficiency of SWATH-MS and IDA-MS data acquisition modes, and sanguisorbin extract was used as a mode TCM. After optimizing the setting parameters of SWATH, rolling collision energy (CE) and variable Q1 isolation windows were found to be more efficient for sanguisorbin identification than the fixed CE and fixed Q1 isolation window. More importantly, the qualitative efficiency of SWATH-MS on sanguisorbins was found significantly higher than that of IDA-MS data acquisition. In IDA mode, 18 kinds of sangui-sorbins were detected in sanguisorbin extract. A total of 47 sanguisorbins were detected when SWATH-MS was used under rolling CE and flexible Q1 isolation window modes. Besides, 26 metabolites of sangui-sorbins were identified in rat plasma, and their metabolic pathways could be deduced as decarbonylation, oxidization, reduction, methylation, and glucuronidation according to their fragmental ions acquired in SWATH-MS mode. Thus, SWATH-MS data acquisition could provide more comprehensive information for the component and metabolite identification for TCMs than IDA-MS.

Ataxins ; Humans ; Male ; Middle Aged ; Nerve Tissue Proteins ; genetics ; Spinocerebellar Ataxias ; genetics ; Trinucleotide Repeats