Objective:To observe and analyze the clinical phenotype and genetic characteristics of COL2A1 and COL11A1 de novo mutation (DNM) related Stickler syndrome type Ⅰ and Ⅱ patients. Methods:A family-based cohort study. From December 2023 to November 2024, 4 patients (all probands) with Stickler syndrome diagnosed by clinical and genetic testing in Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region and their parents (8 cases) were included in the study. The patients came from 4 unrelated families. A detailed medical history was taken, and the patients underwent best-corrected visual acuity (BCVA), refraction, and fundus color photography examinations. Systemic examinations included the oral and facial regions, skeletal, joints, and hearing. Peripheral venous blood samples were collected from the patients and their parents, and genomic DNA was extracted. Whole-exome sequencing was used to screen for pathogenic genes and their loci, which were then validated by Sanger sequencing and combined with segregation analysis in the families to identify candidate gene mutation sites. The candidate variants were assessed for pathogenicity according to the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines for the classification of genetic variants. Additionally, cross-species conservation analysis was performed to determine the evolutionary conservation of wild-type amino acids, and protein three-dimensional modeling techniques were used to characterize the spatial conformational changes of the variant proteins and the alterations in their local hydrogen bond networks.Results:Among the 4 patients, there were 2 males and 2 females; their ages ranged from 3 to 12 years. There were 2 cases of Stickler syndrome type Ⅰ (proband of families 1 and 2) and 2 cases of type Ⅱ (proband of families 3 and 4). The diopters ranged from -8.00 to-18.00 D. BCVA ranged from no light perception to 0.6 -. There were 2 cases each of vitreous membrane-like and "bead-like" opacity. Three cases showed peripapillary atrophy arcs and leopard pattern changes in the retina; one case had bilateral retinal detachment with a large macular hole in the left eye, which had previously been treated with vitrectomy surgery. One case had bilateral sensorineural hearing loss. There were 3 cases of simple micrognathia; one case had a flat nasal bridge, short nose, midface depression, and micrognathia. Two cases had excessive elbow joint extension. The phenotypes of the parents of the 4 patients were normal. Genetic testing results revealed that the probands of families 1 and 2 carried COL2A1 gene c.85+1G>C (M1) splice site variant and c.3950_3951insA (p.M1317Ifs*48) (M2) frameshift variant, respectively; the probands of families 3 and 4 carried COL11A1 gene (NM_001854.4) c.2549 G>T (p.G850V) (M3) missense variant and c.3816+6T>C (M4) splice site variant, respectively. The parents did not carry the related gene variants. Among them, M2, M3, and M4 are newly reported DNM. According to the ACMG guidelines, they were all considered likely pathogenic. The cross-species conservation analysis results showed that the wild-type amino acid of the COL11A1 gene M3 missense variant was highly conserved across multiple different species. Protein local structure modeling analysis revealed that the COL2A1 gene M2 frameshift variant and the COL11A1 gene M3 missense variant significantly altered the tertiary structure conformation of the protein, leading to abnormal spatial arrangement and hydrogen bond network in the key functional domains Conclusion:The COL2A1 gene M1 splice site variant, M2 frameshift variant, and the COL11A1 gene M3 missense variant, M4 splice site variant are respectively the potential pathogenic genes for families 1, 2, and families 3, 4; leading to the onset of Stickler syndrome type Ⅰ in families 1 and 2, and type Ⅱ in families 3 and 4.

Objective:To observe changes in intestinal flora in children with anorexia and the effects of pediatric Tuina(Chinese therapeutic massage)on their intestinal flora,and to explore the relationship between alterations in intestinal flora and anorexia,as well as the therapeutic mechanisms of pediatric Tuina in treating children with anorexia.Methods:A total of 60 healthy children who underwent physical examinations were recruited as the blank group.One hundred and twenty children with anorexia were randomly divided into a Tuina group and a medication group,with 60 children in each group,according to the random number table method.The blank group received no intervention;the Tuina group was treated with pediatric Tuina therapy;the medication group was treated with Jian Wei Xiao Shi(stomach-invigorating and digestion-promoting)tablets.Both groups underwent continuous treatment for 7 d as one course,with a 1-day rest period between courses,for a total of 4 courses.Fecal samples were collected from the three groups.The intestinal flora was detected using the 16S rDNA method.Results:Before treatment,compared to the blank group,the abundance of Firmicutes in the Tuina and medication groups was significantly lower(P<0.05).After treatment,the abundance of Firmicutes in the Tuina group was significantly increased compared to before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05);in the medication group,there was a trend of increased abundance of Firmicutes,but there was no significant difference compared to that before treatment in the same group(P>0.05),and the difference compared to the blank group remained significant(P<0.05).Before treatment,compared to the blank group,the abundance of Faecalibacterium prausnitzii(F.prausnitzii)in the Tuina and medication groups was significantly lower(P<0.05).After treatment,the abundance of F.prausnitzii in the Tuina group was significantly increased compared to before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05);in the medication group,there was a trend of increased abundance of F.prausnitzii,but no significant difference was showed compared to before treatment in the same group(P>0.05),and the differences compared to the blank and Tuina groups were significant(P<0.05).Before treatment,compared to the blank group,the abundance of Eubacterium in the Tuina and medication groups was significantly lower(P<0.05);after treatment,the abundance of Eubacterium in both Tuina and medication groups was significantly increased compared to that before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05).Before treatment,compared to the blank group,the abundance of Roseburia in the Tuina and medication groups was significantly lower(P<0.05);after treatment,the abundance of Roseburia in both Tuina and medication groups was significantly increased compared to that before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05).Conclusion:The reduction of beneficial intestinal flora may be involved in the pathogenesis of pediatric anorexia.Pediatric Tuina can promote the recovery of intestinal flora balance by increasing the abundance of beneficial flora.

Objective To analyze the impact of sarcopenia on the efficacy and adverse reactions of immunotherapy combined with chemotherapy in patients with advanced gastric cancer.Methods Patients with locally advanced or metastatic gastric cancer confirmed by pathology who were not eligible for radical surgery were selected as study subjects.A body composition analyzer was used to measure the appendicular muscle mass of the patients and calculate the skeletal muscle mass index(SMI).Based on the SMI,the patients were divided into sarcopenia group and non-sarcopenia group.On the basis of nutritional intervention and comprehensive exercise therapy,the patients were administered immu-notherapy combined with chemotherapy.The efficacy and adverse reactions were evaluated.The primary endpoint was progression-free survival(PFS),and the secondary endpoints were the objec-tive response rate(ORR)and treatment-related adverse reactions.Results A total of 52 gastric cancer patients were included,with 23 in the sarcopenia group and 29 in the non-sarcopenia group.The median PFS in the non-sarcopenia group was 9.8 months(95％CI,8.9 to 12.4),and was 5.4 months in the sarcopenia group(95％CI,4.9 to 8.1).The median PFS in the non-sarcopenia group was longer than that in the sarcopenia group,and the difference was statistically significant[HR(95％CI)=0.41(0.23 to 0.73),P=0.003].The results of the multivariate Cox propor-tional hazards regression model showed that comorbidities,treatment cycles,and sarcopenia were all independent prognostic factors affecting the PFS of gastric cancer patients(P＜0.05).The ORR in the non-sarcopenia group was 48.28％(14/29),and was 17.39％(4/23)in the sarcopenia group(x2=5.276,P＜0.05).Treatment-related adverse reactions with grading ≥3 in both groups were mainly hematological toxicities.In the non-sarcopenia group,the incidence of grading ≥ 3 treat-ment-related adverse reactions was 27.59％(8/29),and the incidence of grading＜3 treatment-re-lated adverse reactions(including those with no adverse reactions)was 72.41％(21/29).In the sarcopenia group,the incidence of grading ≥3 treatment-related adverse reactions was 56.52％(13/23),and the incidence of grading＜3 treatment-related adverse reactions(including those without adverse reactions)was 43.48％(10/23).The incidence of grading ≥3 treatment-related adverse reactions in the non-sarcopenia group was lower than that in the sarcopenia group(P=0.035).Conclusion For patients with locally advanced or metastatic gastric cancer complicated with sarcopenia,the median PFS of immunotherapy combined with chemotherapy is shorter,the ORR is lower,and the incidence of treatment-related adverse reactions is increased.Therefore,ear-ly intervention for sarcopenia should be implemented to improve the quality of life of patients with advanced gastric cancer.

OBJECTIVE To investigate the stability of 5-fluorouracil （5-FU） in human blood and to establish a standardized clinical sampling and delivery procedure for therapeutic drug monitoring （TDM） of 5-FU. METHODS The EDTA-anticoagulated whole blood was used as the matrix to prepare stability assessment samples of 5-FU at both low （200 ng/mL） and high （5 000 ng/mL） concentrations （with groups without stabilizer and with 1% volume ratio of stabilizer）. The stability assessment samples were placed under room temperature （[ 25±2） ℃] and refrigerated （2-8 ℃） conditions， with sampling at 0， 0.5， 1， 2， 4， 7， and 24 h. After vortexing and centrifugation， the upper plasma layer was collected； proteins were precipitated using methanol， and the concentration of 5-FU in plasma was determined by liquid chromatography-tandem mass spectrometry. Based on the whole blood stability results， clinical sampling and delivery procedures were established. RESULTS The concentration of 5-FU in blank whole blood samples without stabilizers was significantly lower than that in samples with stabilizers （P＜0.05）. However， varying volumes （10， 25， 50 μL） of stabilizers had no significant effect on the measured concentrations of 5-FU in stability assessment samples with low and high concentrations （P＞0.05）. Without the addition of a stabilizer， low- and high-concentration 5-FU whole blood samples remained stable at room temperature for 0.5 h and 1 h， respectively， and under refrigeration for 2 h and 7 h， respectively. After the addition of a 1% stabilizer， the whole blood samples remained stable for up to 24 h under both room temperature and refrigerated conditions. Based on these findings， the following procedure was established： after collection， whole blood samples could be temporarily stored at room temperature （≤0.5 h） or at 4　℃ （≤2 h）， and transported at 2-8　℃. Upon delivery to the laboratory， a 1% volume ratio of stabilizer must be added immediately， followed by centrifugation within 24 h. The resulting plasma should be stored at －20 ℃ . CONCLUSIONS 5-FU in whole blood exhibits poor stability at room temperature. Refrigeration at 2-8　℃ slightly improves stability ， but degradation still occurs rapidly. Adding a stabilizer at a 1% volume ratio significantly prolongs the refrigerated storage time. The established sampling and transport procedure for 5-FU TDM innovatively introduces the stabilizer addition step at the laboratory sample reception stage （rather than immediately after blood draw）. This approach ensures analytical quality while offering greater adaptability to real-world clinical sampling conditions， significantly improving practical feasibility.

Objective Using meta-analysis to identify the risk factors for slow-flow or no-reflow during percutaneous coronary intervention(PCI)in patients with ST-segment elevation acute myocardial infarction(AMI).Methods A computerized retrieval of academic papers concerning the risk factors for slow-flow or no-reflow during PCI in patients with ST-segment elevation AMI from the databases of CNKI,Wanfang Database,VIP,SinoMed,PubMed,Web of Science,Embase,and Cochrane Library was conducted.The retrieval time period was from the establishment of the database to January 2024.In order to ensure the accuracy and reliability of the study,two independent reviewers screened the literature according to the preset inclusion and exclusion criteria,extracted key data,and strictly evaluated the quality of the literature.RevMan5.4 software was used to make meta-analysis.Results A total of 23 articles with a total of 9 780 cases were included in this analysis.The results of meta-analysis showed that reperfusion time ≥6 h(OR=1.52),preoperative TIMI blood flow≤level-Ⅰ(OR=1.12),heavy thrombus burden(OR=1.60),advanced age(OR=1.56),diabetes(OR=1.83),preoperative Killip grade≥Ⅲ(OR=2.52),long target vessel disease(OR=1.95),and collateral flow≤level-Ⅰ(OR=1.61)were the risk factors for slow-flow or no-reflow during PCI in patients with ST-segment elevation AMI.Preoperative systolic blood pressure＜90 mmHg(OR=1.17)and high white blood cell(WBC)count(OR=1.27)were not the risk factors for slow-flow or no-reflow during PCI in patients with ST-segment elevation AMI.Conclusion Reperfusion time ≥ 6 h,preoperative TIMI blood flow≤level-Ⅰ,heavy thrombus burden,advanced age,diabetes,preoperative Killip grade≥level-Ⅲ,long target vessel lesion,and collateral blood flow≤level-Ⅰ are the independent risk factors for slow-flow or no-reflow during PCI in patients with ST-segment elevation AMI.

Objective:To investigate the impact of different luteal phase support protocols on pregnancy outcomes in patients aged ≤35 years undergoing modified natural cycle frozen-thawed embryo transfer (mNC-FET).Methods:A retrospective cohort study was conducted to analyze 2 086 cycles of patients aged ≤35 years who received mNC-FET cycles in Reproductive Health Hospital of the Third Affiliated Hospital of Zhengzhou University from January 2018 to December 2020. The cycles were divided into three groups based on luteal phase support protocols used. The patients received a combination of progesterone soft capsule and dydrogesterone in the group A (446 cycles), the patients received dydrogesterone in the group B (439 cycles), and the patients received a combination of progesterone vaginal sustained-release gel and dydrogesterone in the group C (1 201 cycles). The pregnancy and perinatal outcomes were compared between groups A and B, groups C and B after matching the baseline data in a ratio of 1∶1 using the propensity score matching (PSM). The effect of different luteal phase support on live birth rate was analyzed after adjusting for confounding factors affected by univariate and multivariate generalized estimating equation (GEE).Results:After PSM, there were no significant differences between groups A and B, groups C and B in human chorionic gonadotropin positive rate, clinical pregnancy rate, ectopic pregnancy rate, live birth rate in transplant cycle, incidence of low weight, macrosomia, premature delivery rate, pregnancy complication rate and incidence of birth defects (all P>0.05). GEE analysis showed that three different luteal phase support regimens were not associated with live birth rate. Conclusion:In the mNC-FET cycle, patients aged ≤35 years who chose dydrogesterone alone as luteal phase support drug, had no difference in live birth rate and perinatal outcome between progesterone soft capsules or progesterone vaginal sustained-release gel combined with dydrogesterone, but the outcome still needs to be confirmed by large sample prospective studies.

Objective:To investigate the impact of different luteal phase support protocols on pregnancy outcomes in patients aged ≤35 years undergoing modified natural cycle frozen-thawed embryo transfer (mNC-FET).Methods:A retrospective cohort study was conducted to analyze 2 086 cycles of patients aged ≤35 years who received mNC-FET cycles in Reproductive Health Hospital of the Third Affiliated Hospital of Zhengzhou University from January 2018 to December 2020. The cycles were divided into three groups based on luteal phase support protocols used. The patients received a combination of progesterone soft capsule and dydrogesterone in the group A (446 cycles), the patients received dydrogesterone in the group B (439 cycles), and the patients received a combination of progesterone vaginal sustained-release gel and dydrogesterone in the group C (1 201 cycles). The pregnancy and perinatal outcomes were compared between groups A and B, groups C and B after matching the baseline data in a ratio of 1∶1 using the propensity score matching (PSM). The effect of different luteal phase support on live birth rate was analyzed after adjusting for confounding factors affected by univariate and multivariate generalized estimating equation (GEE).Results:After PSM, there were no significant differences between groups A and B, groups C and B in human chorionic gonadotropin positive rate, clinical pregnancy rate, ectopic pregnancy rate, live birth rate in transplant cycle, incidence of low weight, macrosomia, premature delivery rate, pregnancy complication rate and incidence of birth defects (all P>0.05). GEE analysis showed that three different luteal phase support regimens were not associated with live birth rate. Conclusion:In the mNC-FET cycle, patients aged ≤35 years who chose dydrogesterone alone as luteal phase support drug, had no difference in live birth rate and perinatal outcome between progesterone soft capsules or progesterone vaginal sustained-release gel combined with dydrogesterone, but the outcome still needs to be confirmed by large sample prospective studies.

Objective:To observe changes in intestinal flora in children with anorexia and the effects of pediatric Tuina(Chinese therapeutic massage)on their intestinal flora,and to explore the relationship between alterations in intestinal flora and anorexia,as well as the therapeutic mechanisms of pediatric Tuina in treating children with anorexia.Methods:A total of 60 healthy children who underwent physical examinations were recruited as the blank group.One hundred and twenty children with anorexia were randomly divided into a Tuina group and a medication group,with 60 children in each group,according to the random number table method.The blank group received no intervention;the Tuina group was treated with pediatric Tuina therapy;the medication group was treated with Jian Wei Xiao Shi(stomach-invigorating and digestion-promoting)tablets.Both groups underwent continuous treatment for 7 d as one course,with a 1-day rest period between courses,for a total of 4 courses.Fecal samples were collected from the three groups.The intestinal flora was detected using the 16S rDNA method.Results:Before treatment,compared to the blank group,the abundance of Firmicutes in the Tuina and medication groups was significantly lower(P<0.05).After treatment,the abundance of Firmicutes in the Tuina group was significantly increased compared to before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05);in the medication group,there was a trend of increased abundance of Firmicutes,but there was no significant difference compared to that before treatment in the same group(P>0.05),and the difference compared to the blank group remained significant(P<0.05).Before treatment,compared to the blank group,the abundance of Faecalibacterium prausnitzii(F.prausnitzii)in the Tuina and medication groups was significantly lower(P<0.05).After treatment,the abundance of F.prausnitzii in the Tuina group was significantly increased compared to before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05);in the medication group,there was a trend of increased abundance of F.prausnitzii,but no significant difference was showed compared to before treatment in the same group(P>0.05),and the differences compared to the blank and Tuina groups were significant(P<0.05).Before treatment,compared to the blank group,the abundance of Eubacterium in the Tuina and medication groups was significantly lower(P<0.05);after treatment,the abundance of Eubacterium in both Tuina and medication groups was significantly increased compared to that before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05).Before treatment,compared to the blank group,the abundance of Roseburia in the Tuina and medication groups was significantly lower(P<0.05);after treatment,the abundance of Roseburia in both Tuina and medication groups was significantly increased compared to that before treatment(P<0.05),with no significant difference compared to the blank group(P>0.05).Conclusion:The reduction of beneficial intestinal flora may be involved in the pathogenesis of pediatric anorexia.Pediatric Tuina can promote the recovery of intestinal flora balance by increasing the abundance of beneficial flora.

Objective:To observe and analyze the clinical phenotype and genetic characteristics of COL2A1 and COL11A1 de novo mutation (DNM) related Stickler syndrome type Ⅰ and Ⅱ patients. Methods:A family-based cohort study. From December 2023 to November 2024, 4 patients (all probands) with Stickler syndrome diagnosed by clinical and genetic testing in Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region and their parents (8 cases) were included in the study. The patients came from 4 unrelated families. A detailed medical history was taken, and the patients underwent best-corrected visual acuity (BCVA), refraction, and fundus color photography examinations. Systemic examinations included the oral and facial regions, skeletal, joints, and hearing. Peripheral venous blood samples were collected from the patients and their parents, and genomic DNA was extracted. Whole-exome sequencing was used to screen for pathogenic genes and their loci, which were then validated by Sanger sequencing and combined with segregation analysis in the families to identify candidate gene mutation sites. The candidate variants were assessed for pathogenicity according to the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines for the classification of genetic variants. Additionally, cross-species conservation analysis was performed to determine the evolutionary conservation of wild-type amino acids, and protein three-dimensional modeling techniques were used to characterize the spatial conformational changes of the variant proteins and the alterations in their local hydrogen bond networks.Results:Among the 4 patients, there were 2 males and 2 females; their ages ranged from 3 to 12 years. There were 2 cases of Stickler syndrome type Ⅰ (proband of families 1 and 2) and 2 cases of type Ⅱ (proband of families 3 and 4). The diopters ranged from -8.00 to-18.00 D. BCVA ranged from no light perception to 0.6 -. There were 2 cases each of vitreous membrane-like and "bead-like" opacity. Three cases showed peripapillary atrophy arcs and leopard pattern changes in the retina; one case had bilateral retinal detachment with a large macular hole in the left eye, which had previously been treated with vitrectomy surgery. One case had bilateral sensorineural hearing loss. There were 3 cases of simple micrognathia; one case had a flat nasal bridge, short nose, midface depression, and micrognathia. Two cases had excessive elbow joint extension. The phenotypes of the parents of the 4 patients were normal. Genetic testing results revealed that the probands of families 1 and 2 carried COL2A1 gene c.85+1G>C (M1) splice site variant and c.3950_3951insA (p.M1317Ifs*48) (M2) frameshift variant, respectively; the probands of families 3 and 4 carried COL11A1 gene (NM_001854.4) c.2549 G>T (p.G850V) (M3) missense variant and c.3816+6T>C (M4) splice site variant, respectively. The parents did not carry the related gene variants. Among them, M2, M3, and M4 are newly reported DNM. According to the ACMG guidelines, they were all considered likely pathogenic. The cross-species conservation analysis results showed that the wild-type amino acid of the COL11A1 gene M3 missense variant was highly conserved across multiple different species. Protein local structure modeling analysis revealed that the COL2A1 gene M2 frameshift variant and the COL11A1 gene M3 missense variant significantly altered the tertiary structure conformation of the protein, leading to abnormal spatial arrangement and hydrogen bond network in the key functional domains Conclusion:The COL2A1 gene M1 splice site variant, M2 frameshift variant, and the COL11A1 gene M3 missense variant, M4 splice site variant are respectively the potential pathogenic genes for families 1, 2, and families 3, 4; leading to the onset of Stickler syndrome type Ⅰ in families 1 and 2, and type Ⅱ in families 3 and 4.

Malignant adenomyoepithelioma(MAME)of the breast is a rare tumor with an incidence less than 1%of primary breast cancer.The low incidence and diverse histomorphology pose challenges to the ac-curate diagnosis and clinical management of MAME.This paper reports a case of MAME of the breast with an in-traductal papillary growth pattern and summarizes the clinical features,pathological features,diagnosis,treat-ment,and prognosis of MAME of the breast in the last 5 years.