ObjectiveTo evaluate the effect of Ningying Formula （宁瘿方） combined with low-dose antithyroid drugs （ATDs） on the relapse risk for patients with Graves' hyperthyroidism （GH） during the remission phase， and to analyze the related factors between GH relapse and thyrotropin receptor antibody （TRAb） negativity， so as to provide evidence for the standardized management of GH in remission stage. MethodsA single-center retrospective cohort study was conducted， including 269 GH patients in the remission stage. After propensity score matching （PSM）， 102 matched pairs （204 patients） were established. The control group received low-dose ATDs as maintenance therapy， while the exposure group received the core Ningying Formula in addition to low-dose ATDs. The primary outcome was the GH recurrence rate； the secondary outcome was the thyrotropin receptor antibody （TRAb） negativity rate （TRAb＜1.75 IU/L）. Safety outcomes included treatment-related adverse events. Differences between groups were assessed using Cox regression models and Kaplan-Meier curves， with sensitivity analysis performed using inverse probability of treatment weighting （IPTW）. ResultsThe median follow-up in the matched cohort was 28.07 months. Regarding the GH recurrence outcome， the recurrence rate in the exposure group （18/102， 17.6%） was significantly lower than that in the control group （31/102， 30.4%； χ²=4.539， P=0.033）； regarding the TRAb negativity outcome， the TRAb negativity rate in the exposure group （50/102， 49.0%） was significantly higher than that in the control group （23/102， 22.5%； χ²=15.551， P＜0.001）. Multivariate Cox regression analysis for recurrence showed that Ningying Formula treatment reduced the risk of recurrence ［HR=0.324， 95%CI（0.170， 0.617）， P＜0.001］. Male ［HR=2.209， 95%CI（1.079， 4.520）， P=0.030］， higher initial TRAb level ［per 1 IU/L increase： HR=1.033， 95%CI（1.003， 1.064）， P=0.032］， and larger thyroid volume ［per 1 ml increase： HR=1.045， 95%CI（1.003， 1.088）， P=0.035］ were identified as independent risk factors for recurrence； multivariate Cox regression analysis for TRAb negativity indicated that Ningying Formula treatment promoted TRAb negativity ［HR=1.826， 95%CI（1.091， 3.056）， P=0.022］， while a higher initial TRAb level was associated with a lower probability of negativity ［HR=0.974， 95%CI（0.950， 0.998）， P=0.032］. Survival analysis showed significant differences in relapse rate between groups （Log-Rank P=0.003） and in TRAb outcomes （Log-Rank P=0.034）. The incidence of treatment-related adverse events was similar between groups （P=0.757）. The IPTW sensitivity analysis was consistent with the primary analysis， indicating robust results. ConclusionThe Ningying Formula combined with low-dose ATDs can significantly reduce the risk of recurrence and can improve the TRAb negativity rate in GH patients during the remission stage， without increasing common adverse events， making it an optional strategy for reducing relapse risk during remission. Male gender， higher baseline TRAb level， and larger thyroid volume indicate a higher risk of recurrence， warranting focused follow-up and stratified management.

Objective:To investigate the anemia conditions and characteristics of iron metabolism during different stages of pregnancy in women with different genotypes of thalassemia.Methods:This cohort study selected 3 303 singleton pregnant women who underwent regular prenatal examinations and genetic tests of thalassemia and were delivered at Maternal & Child Health Hospital of Guangxi Zhuang Autonomous Region from January 2019 to December 2023. According to the results of thalassemia gene testing, the women were divided into groups: those without thalassemia genes served as the control group (1 539 cases), and those with thalassemia genes (1 764 cases) were further divided based on genotype into the -α/αα group (326 cases), --/αα or -α/-α group (649 cases), point mutation α-thalassemia group (201 cases), β 0-thalassemia group (368 cases), β +-thalassemia group (91 cases), and α combined with β-thalassemia group (129 cases). Hemoglobin (Hb) and serum ferritin (SF) levels were measured in the first, second, and third trimester of pregnancy. Differences in anemia and iron reserves among the groups at different pregnancy stages were compared using repeated measures analysis of variance, LSD test, Kruskal-Wallis rank-sum test, and Bonferroni correction. Results:Compared to the first trimester, Hb levels decreased in the second and third trimester across all groups (LSD test, all P<0.05), and the severity of anemia increased (Bonferroni correction, all P<0.017). The severity of anemia varied among the groups at the same pregnancy stage ( Hfirst trimester=918.20, Hsecond trimester=1 224.50, Hthird trimester=980.19; all P<0.001), and Hb levels also differed ( Ffirst trimester=282.54, Fsecond trimester=352.31, Fthird trimester=239.02; all P<0.001). The β 0-thalassemia group had higher rates of moderate anemia in the first, second, and third trimester of pregnancy [38.6% (142/368), 85.3% (314/368), and 73.6% (271/368)] compared to other groups (Bonferroni correction, all P<0.002), and lower Hb levels [(102.1±8.9), (92.0±7.3), and (94.6±7.7) g/L] than other groups (LSD test, all P<0.05). As pregnancy progresses, SF levels in each group of pregnant women gradually decreased (LSD test, all P<0.05), and the degree of iron deficiency worsened (Bonferroni correction, all P<0.05). The iron deficiency rate in thalassemia pregnant women during the third trimester ranges from 21.5% (79/368) to 46.0% (150/326). The degree of iron deficiency varies among groups within the same gestational period ( Hfirst trimester=79.13, Hsecond trimester=203.98, Hthird trimester=130.55; all P<0.001), and SF levels also differ ( Ffirst trimester=17.28, Fsecond trimester=44.60, Fthird trimester=31.87; all P<0.001). Among them, the β 0-thalassemia group had the lowest iron deficiency rates in the second, and third trimesters [9.8% (36/368), and 21.5% (79/368)] (Bonferroni correction, all P<0.002). SF levels in the β 0-thalassemia and β +-thalassemia groups were higher than those in other groups during each gestational period (LSD test, all P<0.05). Conclusions:Pregnant women with thalassemia may experience varying degrees of iron deficiency during pregnancy, with the severity of iron deficiency and anemia increasing with gestational age. The degree of iron deficiency and anemia during pregnancy varies among pregnant women with different genotypes of thalassemia. Clinically, individualized management should be provided for pregnant women with thalassemia based on their genotypes, with dynamic monitoring of anemia and iron metabolism changes.

Objective:To explore the value of dual-layer spectral CT virtual monoenergetic images (VMI) in contrast-enhanced abdominal CT scans with reduced contrast medium volume in pediatric tumor patients.Methods:The study is a self-matched case-control study. From January to October 2024, pediatric patients admitted to Shandong Cancer Hospital with abdominal tumors who underwent low contrast dose spectral CT contrast-enhanced scans during follow-up were prospectively included. A total of 47 patients aged (6.2±2.2) years (4-14 years) were enrolled. Usual contrast dose enhanced CT served as the conventional-dose group, while the follow-up low-dose spectral CT scans employed a protocol with half the contrast agent dose (low-dose group). Images were reconstructed as conventional CT images and VMI at 45, 55, and 65 keV. Using muscle as the reference background, differences in CT values and contrast-to-noise ratio (CNR) in the aorta, kidneys, liver, and spleen were compared between the low-dose group and conventional-dose group. Multi-group comparisons were performed using the Friedman test. Post-hoc pairwise comparisons were conducted with Bonferroni correction for P-values. Results:CT values and CNRs for all measured regions progressively increased with decreasing keV levels in spectral CT VMI. Significant overall differences were found in CT values and CNRs for the aorta, kidneys, liver, and spleen among the low-dose group (all VMIs) and the conventional-dose group (all P<0.001). At 65 keV VMI in the low-dose group, both CT values and CNRs (except for the liver CNR) were significantly lower than those in the conventional-dose group (all adjusted P<0.05). At 55 keV VMI in the low-dose group, CT values and CNRs for all regions did not show statistically significant differences compared to the conventional-dose group (all adjusted P>0.05). At 45 keV VMI in the low-dose group, CT values for all structures and CNR for the spleen were significantly higher than those in the conventional-dose group (all adjusted P<0.05). However, no statistically significant difference was found in CNRs for the aorta, kidneys, and liver (adjusted P=1.000, 0.313, and 0.503, respectively). Conclusion:When the contrast dose is halved, spectral CT 45 keV VMI enhances CT attenuation values and CNR in the abdomen of pediatric tumor patients, while 55 keV VMI provides image quality comparable to that of conventional-dose CT.

Objective To establish a DNA quantitation method based on dithiothreitol(DTT)-crystal vio-let.Methods DTT was used to decolorize crystal violet,mixed with different concentrations of λ-DNA and salmon sperm DNA standard samples or concentration standard samples,and the absorbance was read at 595 nm wavelength by microplate reader,and compared with the results of ultraviolet absorbance method.DTT-crystal violet method and ultraviolet absorbance method were used to compare the concentration of plasmid samples and the concentration of genomic DNA samples of cervical exfoliated cells.The protein tolerance of the two methods was assessed by simulating protein contaminants with bovine serum albumin(BSA).Results In the quantification of λ-DNA and salmon sperm DNA,the DTT-crystal violet method had a robust linear correlation between the absorbance at 595 nm and DNA concentration(r2＞0.95),and the measured concentrations of the standard samples were not significantly different from the theoretical concentrations of the prepared standard samples(P＞0.05).There was no significant difference in the concentration of plasmid samples measured by DTT-crystal violet method and ultraviolet absorption method(P＞0.05).The concen-tration of DNA samples from cervical exfoliated cells measured by ultraviolet absorption method was positive-ly correlated with that by DTT-crystal violet method(r=0.94,P＜0.01).The concentration of the standard sample containing BSA 1 μg/μL measured by ultraviolet absorption method was higher than that of the con-trol sample,and the difference was statistically significant(P＜0.01),whereas the DTT-crystal violet method was not significantly affected(P＞0.05).Conclusion DTT-crystal violet method has obvious advantages over the existing DNA quantitation method,and is suitable for DNA quantitative analysis in scientific research and clinic.

[This corrects the article DOI: 10.1016/j.apsb.2021.10.012.].

Objective·To investigate the molecular mechanisms and biological functions of the E3 ubiquitin ligase membrane-associated RING-CH 9(MARCH9)in regulating the ubiquitination of receptor protein tyrosine phosphatase alpha(RPTPα).Methods·Western blotting was employed to identify the ubiquitination type of RPTPα and to evaluate the regulatory effect of MARCH9 on its ubiquitination level;Comparative analysis of RPTPα protein stability was performed among wild-type MARCH9,catalytically inactive MARCH9 mutants(MARCH9 S198A or MARCH9-HC/CC),and endogenous MARCH9 knockdown via shRNA.Proteasome inhibitor MG132,autophagy inhibitor 3-MA,and lysosomal inhibitor chloroquine(CQ)were used to determine the degradation pathway of MARCH9-mediated RPTPα ubiquitination.The mechanism underlying 43℃heat shock-induced RPTPα degradation was explored.Stable lung cancer cell lines with MARCH9 single-knockdown(H1299-sh MARCH9)and MARCH9/RPTPα double-knockdown(H1299-sh MARCH9-sh RPTPα)were established using lentiviral vectors.CCK-8 proliferation assay,colony formation assay,and soft agar assay were conducted to evaluate the effects of MARCH9 or RPTPα on lung cancer cell proliferation and clonogenicity.Vasculogenic mimicry formation assay and scratch wound healing assay were performed to assess the impacts on tumor cell invasion and migration.Subcutaneous xenograft models in nude mice were established to examine in vivo tumorigenicity.Bioinformatics analysis was used to compare the expression differences and prognostic correlations of MARCH9 and RPTPα in lung cancer patients.Results·RPTPα predominantly underwent K63-linked poly-ubiquitination,which was significantly enhanced by MARCH9 overexpression.Wild-type MARCH9,but not its catalytic mutants,markedly reduced RPTPα protein stability,while endogenous MARCH9 knockdown increased RPTPα levels.CQ,not MG132 or 3-MA,restored RPTPα stability,indicating that MARCH9 mediated lysosomal degradation of RPTPα through ubiquitination.Heat shock at 43℃specifically enhanced MARCH9-RPTPα interaction,promoting RPTPα degradation.Functional assays revealed that,compared to control H1299 cells,MARCH9-knockdown cells exhibited elevated RPTPα levels,accelerated proliferation,enhanced clonogenicity and invasive capacity,and increased tumorigenicity in nude mice.These phenotypes could be reversed by double knockdown of MARCH9/RPTPα.Bioinformatics analysis demonstrated that high RPTPα expression correlated with poor prognosis and tumor metastasis in lung cancer patients,while MARCH9 showed inverse correlations.Conclusion·MARCH9 mediates K63-linked ubiquitination-dependent lysosomal degradation of phosphatase RPTPα,providing new insights into developing RPTPα-targeted cancer therapeutic strategies.

Objective To explore the diagnostic efficacy of MR with three-dimensional sampling perfection with application opti-mized contrast using different flip angle evolutions(3D-SPACE)and two-dimensional fast spin echo(2D-FSE)techniques in knee meniscus injury.Methods The imaging data of 100 patients who underwent arthroscopy for knee pain or trauma were collected.All patients underwent arthroscopy,conventional 2D-FSE sequence and 3D-SPACE sequence examination.Using the results of arthroscopy as the gold standard,the classification of meniscus injury and the classification of grade Ⅲ injury morphology were observed in two MR sequences.The diagnostic efficacy of meniscus injury was measured,and the contrast-to-noise ratio(CNR)of meniscus tissue was compared.Results Arthroscopy revealed 92 meniscus injury,and 58 grade Ⅲ meniscus injury,among 100 patients with suspected knee meniscus injury.The accuracy of classification diagnosis with the 3D-SPACE sequence(95.65%)was significantly better than that of the 2D-FSE sequence classification diagnosis acuracy(80.43%)(P<0.05).Specifically,the diagnostic accuracy of gradeⅡ(96.15%)and grade Ⅲ(94.83%)with the 3D-SPACE sequence was significantly higher than that of the 2D-FSE sequence(76.92%for grade Ⅱ and 81.03%for grade Ⅲ)(P<0.05).The diagnostic accuracy of mixed-type tears with the 3D-SPACE sequence(94.74%)was significantly better than that of the 2D-FSE sequence(63.16%)(P<0.05).Consistency analysis confirmed that the sensitivity,specificity,positive predictive value,negative predictive value and accuracy of the 2D-FSE and 3D-SPACE sequences for overall detec-tion of meniscus injury were 78.26%,75.00%,97.30%,23.08%and 78.00%(Kappa value=0.262)and 94.57%,87.50%,98.86%,58.33%,94.00%(Kappa value=0.646)(P<0.05),respectively.The meniscus/ligament CNR of the 3D-SPACE sequence was higher than that of the 2D-FSE sequence(P<0.05).Conclusion The 3D-SPACE sequence exhibits high sensitivity and accuracy in the diagnosis of knee meniscus injury.It has significant advantages in determining the classification of meniscus injury and preoperative location of partial tears,providing a more reliable basis for the diagno-sis and treatment of patients with suspected knee meniscus injury.

Objective:To explore the value of dual-layer spectral CT virtual monoenergetic images (VMI) in contrast-enhanced abdominal CT scans with reduced contrast medium volume in pediatric tumor patients.Methods:The study is a self-matched case-control study. From January to October 2024, pediatric patients admitted to Shandong Cancer Hospital with abdominal tumors who underwent low contrast dose spectral CT contrast-enhanced scans during follow-up were prospectively included. A total of 47 patients aged (6.2±2.2) years (4-14 years) were enrolled. Usual contrast dose enhanced CT served as the conventional-dose group, while the follow-up low-dose spectral CT scans employed a protocol with half the contrast agent dose (low-dose group). Images were reconstructed as conventional CT images and VMI at 45, 55, and 65 keV. Using muscle as the reference background, differences in CT values and contrast-to-noise ratio (CNR) in the aorta, kidneys, liver, and spleen were compared between the low-dose group and conventional-dose group. Multi-group comparisons were performed using the Friedman test. Post-hoc pairwise comparisons were conducted with Bonferroni correction for P-values. Results:CT values and CNRs for all measured regions progressively increased with decreasing keV levels in spectral CT VMI. Significant overall differences were found in CT values and CNRs for the aorta, kidneys, liver, and spleen among the low-dose group (all VMIs) and the conventional-dose group (all P<0.001). At 65 keV VMI in the low-dose group, both CT values and CNRs (except for the liver CNR) were significantly lower than those in the conventional-dose group (all adjusted P<0.05). At 55 keV VMI in the low-dose group, CT values and CNRs for all regions did not show statistically significant differences compared to the conventional-dose group (all adjusted P>0.05). At 45 keV VMI in the low-dose group, CT values for all structures and CNR for the spleen were significantly higher than those in the conventional-dose group (all adjusted P<0.05). However, no statistically significant difference was found in CNRs for the aorta, kidneys, and liver (adjusted P=1.000, 0.313, and 0.503, respectively). Conclusion:When the contrast dose is halved, spectral CT 45 keV VMI enhances CT attenuation values and CNR in the abdomen of pediatric tumor patients, while 55 keV VMI provides image quality comparable to that of conventional-dose CT.

Objective·To investigate the molecular mechanisms and biological functions of the E3 ubiquitin ligase membrane-associated RING-CH 9(MARCH9)in regulating the ubiquitination of receptor protein tyrosine phosphatase alpha(RPTPα).Methods·Western blotting was employed to identify the ubiquitination type of RPTPα and to evaluate the regulatory effect of MARCH9 on its ubiquitination level;Comparative analysis of RPTPα protein stability was performed among wild-type MARCH9,catalytically inactive MARCH9 mutants(MARCH9 S198A or MARCH9-HC/CC),and endogenous MARCH9 knockdown via shRNA.Proteasome inhibitor MG132,autophagy inhibitor 3-MA,and lysosomal inhibitor chloroquine(CQ)were used to determine the degradation pathway of MARCH9-mediated RPTPα ubiquitination.The mechanism underlying 43℃heat shock-induced RPTPα degradation was explored.Stable lung cancer cell lines with MARCH9 single-knockdown(H1299-sh MARCH9)and MARCH9/RPTPα double-knockdown(H1299-sh MARCH9-sh RPTPα)were established using lentiviral vectors.CCK-8 proliferation assay,colony formation assay,and soft agar assay were conducted to evaluate the effects of MARCH9 or RPTPα on lung cancer cell proliferation and clonogenicity.Vasculogenic mimicry formation assay and scratch wound healing assay were performed to assess the impacts on tumor cell invasion and migration.Subcutaneous xenograft models in nude mice were established to examine in vivo tumorigenicity.Bioinformatics analysis was used to compare the expression differences and prognostic correlations of MARCH9 and RPTPα in lung cancer patients.Results·RPTPα predominantly underwent K63-linked poly-ubiquitination,which was significantly enhanced by MARCH9 overexpression.Wild-type MARCH9,but not its catalytic mutants,markedly reduced RPTPα protein stability,while endogenous MARCH9 knockdown increased RPTPα levels.CQ,not MG132 or 3-MA,restored RPTPα stability,indicating that MARCH9 mediated lysosomal degradation of RPTPα through ubiquitination.Heat shock at 43℃specifically enhanced MARCH9-RPTPα interaction,promoting RPTPα degradation.Functional assays revealed that,compared to control H1299 cells,MARCH9-knockdown cells exhibited elevated RPTPα levels,accelerated proliferation,enhanced clonogenicity and invasive capacity,and increased tumorigenicity in nude mice.These phenotypes could be reversed by double knockdown of MARCH9/RPTPα.Bioinformatics analysis demonstrated that high RPTPα expression correlated with poor prognosis and tumor metastasis in lung cancer patients,while MARCH9 showed inverse correlations.Conclusion·MARCH9 mediates K63-linked ubiquitination-dependent lysosomal degradation of phosphatase RPTPα,providing new insights into developing RPTPα-targeted cancer therapeutic strategies.

Objective:To investigate the anemia conditions and characteristics of iron metabolism during different stages of pregnancy in women with different genotypes of thalassemia.Methods:This cohort study selected 3 303 singleton pregnant women who underwent regular prenatal examinations and genetic tests of thalassemia and were delivered at Maternal & Child Health Hospital of Guangxi Zhuang Autonomous Region from January 2019 to December 2023. According to the results of thalassemia gene testing, the women were divided into groups: those without thalassemia genes served as the control group (1 539 cases), and those with thalassemia genes (1 764 cases) were further divided based on genotype into the -α/αα group (326 cases), --/αα or -α/-α group (649 cases), point mutation α-thalassemia group (201 cases), β 0-thalassemia group (368 cases), β +-thalassemia group (91 cases), and α combined with β-thalassemia group (129 cases). Hemoglobin (Hb) and serum ferritin (SF) levels were measured in the first, second, and third trimester of pregnancy. Differences in anemia and iron reserves among the groups at different pregnancy stages were compared using repeated measures analysis of variance, LSD test, Kruskal-Wallis rank-sum test, and Bonferroni correction. Results:Compared to the first trimester, Hb levels decreased in the second and third trimester across all groups (LSD test, all P<0.05), and the severity of anemia increased (Bonferroni correction, all P<0.017). The severity of anemia varied among the groups at the same pregnancy stage ( Hfirst trimester=918.20, Hsecond trimester=1 224.50, Hthird trimester=980.19; all P<0.001), and Hb levels also differed ( Ffirst trimester=282.54, Fsecond trimester=352.31, Fthird trimester=239.02; all P<0.001). The β 0-thalassemia group had higher rates of moderate anemia in the first, second, and third trimester of pregnancy [38.6% (142/368), 85.3% (314/368), and 73.6% (271/368)] compared to other groups (Bonferroni correction, all P<0.002), and lower Hb levels [(102.1±8.9), (92.0±7.3), and (94.6±7.7) g/L] than other groups (LSD test, all P<0.05). As pregnancy progresses, SF levels in each group of pregnant women gradually decreased (LSD test, all P<0.05), and the degree of iron deficiency worsened (Bonferroni correction, all P<0.05). The iron deficiency rate in thalassemia pregnant women during the third trimester ranges from 21.5% (79/368) to 46.0% (150/326). The degree of iron deficiency varies among groups within the same gestational period ( Hfirst trimester=79.13, Hsecond trimester=203.98, Hthird trimester=130.55; all P<0.001), and SF levels also differ ( Ffirst trimester=17.28, Fsecond trimester=44.60, Fthird trimester=31.87; all P<0.001). Among them, the β 0-thalassemia group had the lowest iron deficiency rates in the second, and third trimesters [9.8% (36/368), and 21.5% (79/368)] (Bonferroni correction, all P<0.002). SF levels in the β 0-thalassemia and β +-thalassemia groups were higher than those in other groups during each gestational period (LSD test, all P<0.05). Conclusions:Pregnant women with thalassemia may experience varying degrees of iron deficiency during pregnancy, with the severity of iron deficiency and anemia increasing with gestational age. The degree of iron deficiency and anemia during pregnancy varies among pregnant women with different genotypes of thalassemia. Clinically, individualized management should be provided for pregnant women with thalassemia based on their genotypes, with dynamic monitoring of anemia and iron metabolism changes.