Objective To develop a size exclusion-high-performance liquid chromatography(SEC-HPLC) method for the simultaneous determination of free proteins and residual 4-dimethylaminopyridine(DMAP) in the bulk solution of pneumococcal polysaccharide-protein conjugate, and to verify and preliminarily apply the method.Methods By studying the characteristic ultraviolet absorption peak of DMAP and optimizing the separation method of chromatographic column, the SEC-HPLC method was established to realize the retention and separation of free proteins and DMAP on the same chromatographic column and quantify them. The method was verified for specificity(system suitability), limit of quantification(LOQ), accuracy, precision, linearity and range, and robustness, and was used to detect the free proteins and DMAP residues in the test sample.Results The chromatographic conditions were determined as follows: TSKgel G5000 PWXL analytical column was used as the column(7. 8 mm × 300 mm, 10 ??m), the column temperature was set at 35 ℃, the mobile phase was0. 85% sodium chloride solution, the pH was(6. 2 ± 0. 2), the flow rate was 0. 5 mL/min, and the detection wavelength was280 nm. The resolution of free protein peaks of diphtheria toxoid(DT) or tetanus toxoid(TT) and DMAP peak from adjacent peaks was greater than 1. 5. The LOQs of TT, DT and DMAP were 0. 50, 1. 20, and 0. 02 ??g/mL, respectively. The spiked recovery rates of TT, DT and DMAP were 98. 11%-104. 60%, 95. 43%-103. 26% and 97. 11%-98. 42%, respectively.The relative standard deviations(RSDs) of intermediate precision of TT, DT and DMAP were all less than 5%. The R~2 values of TT and DT were greater than 0. 999 0 in the range of 6-24 ??g/mL, and the R~2 value of DMAP was 1. 000 in the range of2-10 ??g/mL. The RSDs of TT, DT and DMAP content were all less than 3% at the injection temperature of 30, 35 and 40 ℃.The established method was used to determine the content of free proteins and DMAP in the test sample, and rapid and accurate results were obtained.Conclusion The established SEC-HPLC method has good system suitability, accuracy,precision, linear relationship and robustness, which can realize the dual quantification of free proteins and DMAP in the same experiment, and can be used to monitor the impurities in the binding process.

Objective:To explore the clinical manifestations and genetic basis for a rare case of Generalized arterial calcification of infancy (GACI).Methods:A 44-day-old female infant who was treated at Baoding Hospital of Beijing Children′s Hospital Affiliated to Capital Medical University on August 26, 2022 was selected as the study subject. Clinical data of the child was collected, and Trio-whole exome sequencing (Trio-WES), whole genome copy number variation sequencing (CNV-seq) and minigene splicing assay were carried out to analyze the pathogenicity of the variants.Results:The child had presented with fever and high inflammatory indicators, for which treatment with various antibiotics was ineffective. Ultrasound had revealed extensive arterial calcification and arterial wall thickening. The child was suspected for GACI with arteritis related to the primary disease. Her fever was relieved by treatment with glucocorticoid and biological agents. Trio-WES revealed that she has harbored compound heterozygous variants of the ABCC6 gene, namely c. 4404-1G>A and c. 4041+ 5G>T, for which the latter was unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the variants were classified as likely pathogenic (PVS1+ PM2_Supporting) and variant of unknown significance (PM2_Supporting+ PM3+ PP3), respectively. The result of CNV-seq was negative. And the minigene splicing assay has further verified that both variants can result in alternative splicing. Conclusion:For pyrexia with unknown causes and refractory to conventional treatment, it is necessary to recommend early genetic testing to avoid missed diagnosis of GACI.

[Objective] To explore the expression of Nectin-4 in invasive bladder urothelial carcinoma (BUC) tissue and its clinical significance, so as to provide reference for clinical diagnosis and treatment of BUC. [Methods] Nectin-4 expression in 60 cases of invasive BUC and 40 cases of chronic inflammation of bladder mucosa was detected with immunohistochemical staining (IHC) and RNAscope.The results of the two methods were analyzed and compared, and the relationship between the two methods and the clinicopathological characteristics of invasive BUC was discussed.The correlation between the protein expression of Nectin-4 in BUC tissues, human epidermal growth factor receptor 2 (Her-2) and programmed death factor ligand 1 (PD-L1) was analyzed. [Results] The positive protein expression rates of Nectin-4 detected by IHC were 78.33%(47/60) and 17.50% (7/40) in the invasive BUC group and inflammatory group, respectively, while the positive mRNA expression rates of Nectin-4 detected by RNAscope were 83.33% (50/60) and 12.50% (5/40), respectively.The Kappa values of Nectin-4 in the invasive BUC group and inflammatory group were 0.732 and 0.610, respectively, with general consistency.The protein expression of Nectin-4 in invasive BUC was correlated with muscular invasion, histological grade, vascular thrombus, lymph node metastasis and clinical stage (P<0.05). The mRNA expression of Nectin-4 in invasive BUC was correlated with max tumor diameter, muscular invasion, histological grade, vascular thrombus, lymph node metastasis and clinical stage (P<0.05). The high expression of Nectin-4 in invasive BUC was positively correlated with the expression of Her-2 (P=0.002), but not with the expression of PD-L1 (P>0.05). [Conclusion] Nectin-4 is highly expressed in invasive BUC, and is usually associated with the pathological parameters of poor prognosis.Detection of Nectin-4 expression will help to guide clinical diagnosis and treatment.

Objective:To investigate the clinical features and therapeutic efficacy of patients with hereditary leiomyomatosis and renal cell carcinoma(RCC) syndrome-associated RCC (HLRCC-RCC) in China.Methods:The clinical data of 119 HLRCC-RCC patients with fumarate hydratase (FH) germline mutation confirmed by genetic diagnosis from 15 medical centers nationwide from January 2008 to December 2021 were retrospectively analyzed. Among them, 73 were male and 46 were female. The median age was 38(13, 74) years. The median tumor diameter was 6.5 (1.0, 20.5) cm. There were 38 cases (31.9%) in stage Ⅰ-Ⅱand 81 cases (68.1%) in stage Ⅲ-Ⅳ. In this group, only 11 of 119 HLRCC-RCC patients presented with skin smooth muscle tumors, and 44 of 46 female HLRCC-RCC patients had a history of uterine fibroids. The pathological characteristics, treatment methods, prognosis and survival of the patients were summarized.Results:A total of 86 patients underwent surgical treatment, including 70 cases of radical nephrectomy, 5 cases of partial nephrectomy, and 11 cases of reductive nephrectomy. The other 33 patients with newly diagnosed metastasis underwent renal puncture biopsy. The results of genetic testing showed that 94 patients had FH gene point mutation, 18 had FH gene insertion/deletion mutation, 4 had FH gene splicing mutation, 2 had FH gene large fragment deletion and 1 had FH gene copy number mutation. Immunohistochemical staining showed strong 2-succinocysteine (2-SC) positive and FH negative in 113 patients. A total of 102 patients received systematic treatment, including 44 newly diagnosed patients with metastasis and 58 patients with postoperative metastasis. Among them, 33 patients were treated with tyrosine kinase inhibitor (TKI) combined with immune checkpoint inhibitor (ICI), 8 patients were treated with bevacizumab combined with erlotinib, and 61 patients were treated with TKI monotherapy. Survival analysis showed that the median progression-free survival (PFS) of TKI combined with ICI was 18 (5, 38) months, and the median overall survival (OS) was not reached. The median PFS and OS were 12 (5, 14) months and 30 (10, 32) months in the bevacizumab combined with erlotinib treatment group, respectively. The median PFS and OS were 10 (3, 64) months and 44 (10, 74) months in the TKI monotherapy group, respectively. PFS ( P=0.009) and OS ( P=0.006) in TKI combined with ICI group were better than those in bevacizumab combined with erlotinib group. The median PFS ( P=0.003) and median OS ( P=0.028) in TKI combined with ICI group were better than those in TKI monotherapy group. Conclusions:HLRCC-RCC is rare but has a high degree of malignancy, poor prognosis and familial genetic characteristics. Immunohistochemical staining with strong positive 2-SC and negative FH can provide an important basis for clinical diagnosis. Genetic detection of FH gene germ line mutation can confirm the diagnosis. The preliminary study results confirmed that TKI combined with ICI had a good clinical effect, but it needs to be confirmed by the results of a large sample multi-center randomized controlled clinical study.

Objective:To investigate the epidemiological and genetic characteristics of human metapneumovirus (hMPV) in Tianjin during two influenza epidemic seasons from October 2020 to March 2021 and from October 2021 to March 2022, and enrich the whole genome database of hMPV in China.Methods:A total of 1 040 pharyngeal swab samples collected from patients with influenza-like illness (ILI) were analyzed using microfluidic chip fluorescence quantitative PCR. RT-PCR was used to amplify the whole genome in hMPV-positive samples, and the second-generation sequencing was performed for complete genome sequencing. Bioinformatics software including CLC, DNAStar, and MEGA was used for sequence assembly, nucleotide and amino acid homology analysis, and phylogenetic tree mapping.Results:Among the 1 040 samples, 25 were positive for hMPV with a positive rate of 2.40%. The highest positive rate was observed in the age group of 3 to 5 years, reaching 3.71% (16/431). During the influenza epidemic seasons, the detection rate of hMPV peaked in December, reaching 6.67% (12/180). Twelve strains were successfully sequenced, and there were seven of type B2, four of type A2b, and one of type B1. More variations were detected in the G gene, with 111nt-dup sequence repeats observed in the G gene of three A2b strains.Conclusions:The prevalence of hMPV peaks in December during the influenza epidemic seasons in Tianjin, with Type B2 being the predominant type. Except for the G gene with more mutations, other genes remain stable.

Objective:To analyze the clinical effect of low-frequency repetitive transcranial magnetic stimulation(rTMS)combined with neuromuscular joint facilitation(NJF)technique in improving the enhancement of muscle tone after stroke.Methods:From October 2020 to October 22,a total of 114 patients with increased muscle tone at lower limb during the recovery period of stroke who admitted to Beijing Rehabilitation hospital were selected,and they were randomly divided into the observation group(57 cases)and the control group(57 cases)according to the random number table method.The control group received routine rehabilitation treatment.The observation group added low-frequency rTMS and NJF training on the basis of the routine rehabilitation treatment.The differences of clinical efficacy and various indicators of muscle tone of lower limb before and after treatment between two groups were compared,which included modified Ashwonh score(MAS)scale,functional ambulation category(FAC)scale of the motor of lower limb,Fugl-Meyer motor function scale-Lower limbs(FMA-L),clinical spasmodic index(CSI),modified Barthel index(MBI)of activity ability of daily life,integrated electromyogram(IEMG)and root mean square(RMS)of tibialis anterior muscle of lower limb in electromyogram.Results:After intervention,29 cases appeared significant effect,and 23 cases appeared valid result,and 5 cases appeared invalid result in the observation group,and 20 cases appeared significant effect,and 20 cases appeared valid result,and 17 cases appeared invalid result in the control group.The total effective rate of the observation group was 91.23%,which was significantly higher than 70.18%of the control group,and the difference was statistically significant(x2=8.111,P<0.05).Before intervention,there was no statistically significant difference in each indicator between the two groups(P>0.05).The MAS score,CSI score,IEMG and RMS of two groups after intervention were significantly lower than those before intervention,while the FAC score,FMA-L score and MBI score were significantly higher than those before intervention,and the differences were significant(t=3.377,4.398,5.554,P<0.05),respectively.At the same time,the MAS score,CSI score,IEMG and RMS of the observation group were significantly higher than those of the control group,and the differences were statistically significant(t=11.160,9.625,16.993,8.095,P<0.05),respectively.Conclusion:The combined application of low-frequency rTMS and NJF training can significantly improve the enhancement of muscle tone after stroke,and effectively reduce the muscle tone of lower limb,and improve the motor function of lower limb and the degree of spasticity,and improve the activity ability of daily life.

Objective:To study the association of frailty status with nutritional risk and the effect on clinical outcomes among elderly surgical inpatients.Methods:Elderly inpatients from the surgery department of Beijing Hospital were enrolled from January to June 2021. Frail scale and nutritional risk screening 2002 (NRS 2002) were used for frailty evaluation and nutrition risk screening. The influence of frailty and associated nutrition risk in elderly surgical inpatients was analyzed.Results:487 elderly surgical patients were included, of whom 131 cases were in the non-frailty group, 279 cases were in the pre-frailty group and 77 cases were in the frailty group, according to the Frail scale score. 146 cases were at nutritional risk, of whom 8 (6.1% of 131) were in the non-frailty group, 87 (31.2% of 279) in the pre-frailty group and 51 (66.2% of 77) were in the frailty group. According to univariate/multivariate logistic regression analysis of frailty in elderly surgical patients, a higher NRS 2002 score, older age, and the presence of multiple concurrent diseases (≥ 5) were significantly associated with frailty ( P < 0.001). The Frail scale score was positively correlated with NRS 2002 score ( r = 0.448, P < 0.01). Multiple comparisons showed that frailty had statistically significant effects on hospital stay and medical costs in elderly surgical patients ( P < 0.05). Conclusions:The prevalence of frailty is higher in elderly surgical patients, and the prevalence of nutritional risk increases with the progression of frailty. Frailty can lead to prolonged hospital stays and increased hospital costs in elderly surgical patients.

Objective:To investigate the prevalence of malnutrition in elderly patients with neurological diseases and the of nutrition, and to explore their association with clinical outcomes.Methods:A retrospective study was conducted to analyze 566 elderly patients with neurological diseases in the database of the "National Multicenter Survey on the Dynamic Changes of Nutritional Status of Hospitalized Patients" by using the Global leadership Initiative on Malnutrition(GLIM)criteria and subjective global assessment(SGA). The two diagnostic tools for malnutrition were compared to explore the correlation between malnutrition and clinical outcomes.Results:Based on the GLIM criteria, 83 cases were diagnosed with malnutrition and the incidence of malnutrition was 14.66%(83/566), with 14.72%(48/326)in men and 14.58%(35/240)in women.Patients with moderate malnutrition accounted for 8.30%(47/566)and patients with severe malnutrition accounted for 6.36%(36/566). According to the SGA, the incidence of moderate malnutrition(SGA Grade B)was 15.55%(88/566), the incidence of severe malnutrition(SGA Grade C)was 1.94%(11/566), and all cases of malnutrition(SGA Grade B+ C)accounted for 17.49% of the participants(99/566). The total length of hospital stay was(15.46±6.49)days in the malnutrition group and(13.55±5.09)days in the non-malnutrition group, with a statistical difference between the two groups( t=-3.02, P<0.01). The body weight of the malnutrition group was significantly lower than non-malnutrition group[(52.0±8.5)kg vs.(65.2±9.6)kg, t=12.92, P<0.01]. There were also statistically significant differences in BMI(19.1±2.7 kg/m 2vs.23.9±2.6 kg/m 2, t=15.48, P<0.01), upper arm circumference[(22.3±2.5)cm vs.(28.3±3.9)cm, t=7.01, P<0.01], and lower leg circumference[(28.9±3.4)cm vs.(32.5±3.3)cm, t=6.81, P<0.01]between the two groups.Laboratory tests showed that there were significant differences in lymphocytes[(5.0±8.5)×10 9/L vs.(9.4±11.8)×10 9/L, t=3.61, P<0.01]and albumin[(38.5±4.4)g/L vs.(40.7±5.1)g/L, t=3.18, P<0.01]between the malnutrition group and the non-malnutrition group.The correlation between GLIM and SGA was good, and the consistency was reasonable(AUC=0.711). Conclusions:The incidence of malnutrition in elderly patients with neurological diseases is relatively high; The GLIM criteria are suitable for the diagnosis of malnutrition in elderly patients with neurological diseases, and the diagnostic results have a good correlation with those of SGA.Malnutrition is associated with anthropometric measurements, laboratory indicators, and clinical outcomes.

Objective:To study the correlation between nutritional status and length of hospital stay in patients with digestive disorders.Methods:The data were collected from the database of a multi-center investigation on the dynamic changes of nutritional status of hospitalized patients in China, a project led by the Geriatric Nutrition Support Group, Society of Parenteral and Enteral Nutrition, Chinese Medical Association. The enrolled patients were screened for malnutrition and possible sarcopenia using Global Leadership Initiative on Malnutrition criteria, and the dynamic changes of serum biochemical indexes during hospital stay and the effects of malnutrition and possible sarcopenia on the length and cost of hospital stay were analyzed.Results:A total of 1 180 patients were enrolled, with an average age of (56.3±16.1) years, the average height of (164.65±8.29) cm, and the average weight of (62.12±12.12) kg. There were significant differences in body weight, body mass index, calf circumference, lymphocyte count, triglyceride, hemoglobin, albumin and total protein between at discharge and at admission ( P<0.001). There might be a correlation between post-admission malnutrition and sarcopenia. There was neither significant difference in the proportion of patients with malnutrition at admission among different age groups ( P=0.438), nor in that at discharge among different age groups ( P=0.439). The proportion of patients with malnutrition showed no significant difference between subgroups with patients<65 years old and ≥ 65 years old, at admission and discharge ( P>0.05). However, comparison of the proportion of patients with sarcopenia between subgroups with patients<65 years old and ≥65 years old displayed significant differences at admission and discharge ( P<0.001), but not the comparison of the proportion of patients with possible sarcopenia ( P>0.05). The length of hospital stay in patients with malnutrition was significantly longer than that in patients without malnutrition [(13.22±6.24) days vs. (12.08±5.25) days, P<0.001]. The length of hospital stay of patients with and without sarcopenia was also significantly different [(12.87±5.93) days vs. (12.02±5.22) days, P<0.001). Patients with concurrent malnutrition and sarcopenia had longer hospital stay [(14.57±7.15) days vs. (12.07±5.22) days, P<0.001], and higher medical cost [(2.78±2.19) ten thousand Chinese Yuan vs. (2.24±2.33) ten thousand Chinese Yuan, P<0.05)] compared with those without concurrent malnutrition and sarcopenia. Conclusions:A large proportion of patients with digestive disorders were diagnosed with malnutrition and/or possible sarcopenia during hospitalization. There is possible correlation between malnutrition and possible sarcopenia, and both can lead to a longer hospital stay and higher medical cost.

Objective To evaluate the influence of stress distributions on bone-anchored maxillary protraction at different protraction sites, so as to guide patients to choose an optimal protraction site in clinic. Methods A three-dimensional (3D) finite element model of child head with implant anchorages was establised. Four protraction sites were set according to the position of implant installation. Working condition 1: the alveolar bone at the intersection of distal 2 mm of primary lateral incisor crown distal surface and gingival cervical margin to 5 mm. Working condition 2: the alveolar bone at the intersection of mesial 2 mm of maxillary first primary molar crown mesial surface and gingival cervical margin to 5 mm. Working condition 3: the alveolar bone at the intersection of mesial 2 mm of maxillary first molar crown mesial surface and gingival cervical margin to 5 mm. Working condition 4: the alveolar bone at the intersection of distal 2 mm of maxillary first molar crown distal surface and gingival cervical margin to 5 mm. The finite element models were loaded with 500 g protraction force at each side with 30° forward direction to the occlusal plane. Stress distributions on each suture were analysed. Results The maximum stress of frontomaxillary suture was in working condition 2 (1 477-28 190 Pa). The maximum stress of nasomaxillary suture was in working condition 1 (5.296-924 Pa). The maximum stress of zygomaticomaxillary suture was in working condition 4(394.7-13 130 Pa). The maximum stress of zygomaticofrontalis suture was in working condition 4 (495.2-31 690 Pa). The maximum stress of zygomaticotemporal suture was in working condition 3 (1 148-15 870 Pa). The maximum stress of medianpalatine suture was in working condition I (6.479-730 Pa). Conclusions When the protraction sites are set in distal maxillary primary lateral incisor and mesial maxillary first primary molar, it is of positive significance to improve the concave profile, especially in nose root. When the protraction sites are set in mesial or distal maxillary first molar, it is of positive significance to improve the concave profile, especially in maxillary basal bone of the midface.