Objective To develop a simplified bisulfite genomie sequencing(BGS)method for DNA methylation marker scanning.Methods According to modified BGS protocol,the desalt DNA treated with bisulfite were directly used for bisulfite-PCR(BSP)without alkali treatmenL Complement of the bisulfite modification Wag accomplished by a prolonged pre-denaturation stage.After BSP,a second round PCR was performed with a pair of GC tagged primers to adjust the GC content of the amplieon for direct sequencing.To assess this improved protocol,promotor methylation of TNF-α gene in 3T3-L1 cell and androgen receptor(AR)gene in Hela cell was investigated.The real time BSP for Alu was also used to compare the sensitivity of the modified assay with traditional assay.Results Both the hypermethylated TNF-α promotor and hypomethylated AR promotor were successfully sequenced by improved BGS method,and the results were consistent with that of the traditional assay.The conversion rate reached 100%,while the conversion specificity was higher than 93.75%.The sensitivity of improved BGS method inereaged significantly(t=2.978 2,P＜0.05)and showed good reproducibility.Condusion The improved BGS method is simple and sensitive,facilitating more ambitious genomic methyhtion mapping studies.

Objective:To explore the effectiveness of medial malleolus screw epiphyseodesis for pediatric ankle valgus correction and calculate the correction rate, and analyse the influence factors on deformity correction and the risk factors of deformity recurrence after screw removal.Methods:Medical records and radiographs of patients undergoing screw hemiepiphyseodesis of the medial physis of the distal tibia for ankle valgus between Jan 2011 and Dec 2020, at a single pediatric orthopedic department were retrospectively analyzed. A total of 41 patients (49 ankles) were included in following study, including 28 male patients and 13 female patients; with 24 left sides and 25 right sides. Median age at surgery was 10.75 (4.5, 13.9) years, and median follow-up time was 27 (12, 64) months. According to the clinical diagnosis: hereditary multiple exostoses were 23 ankles, fibula hemimelia were 5 ankles, tibia hemimelia were 2 ankles, endochondromatosis were 5 ankles, neurofibromatosis were 5 ankles, traumatic fracture were 6 ankles, and fibrous dysplasia were 3 ankles. The lateral distal tibial angle (LDTA) was measured on ankle weight-bearing X-ray to evaluate the deformity correction. Malhotra classification was used to describe shortening of the fibula at the ankle.Results:Eventually effective correction were obtained in 36 ankles, with an overall effective rate of 73.5%. Screw type (partial-thread or full-thread), screw length (whether to the contralateral cortex), washer used, clinical diagnosis, and age are not independent risk factors for effective correction. Multiple linear regression analysis ( R 2=0.67) was applied for postoperative correction amplitude in effective correction cases, and the length of correction time ( P<0.001), clinical diagnosis ( P=0.013) and preoperative LDTA ( P=0.002) were significant predictive factors after adjusting for age differences. One-way ANOVA were used to compare data between different clinical diagnosis, showed a significant difference in the mean postoperative correction rate ( F=5.05, P=0.003). Conclusion:Medial malleolus screw epiphyseodesis is an effective and reliable method for the treatment of ankle valgus deformity in children; different clinical diagnosis can produce significant differences in the correction rate of ankle valgus deformity.

Objective:To optimize and evaluate the pretreatment method for high-throughput sequencing of fecal and tissue samples to improve the sensitivity of high-throughput sequencing in the study of virome.Methods:For fecal samples, five virus-positive samples that have been detoxified from feces were selected, mixed as the simulated samples, and filters made of different materials were used, different processing times were set for nucleases, and different kits were used to extract nucleic acid. For tissue samples, two virus-positive samples that have been detected in animal tissues, filter and nuclease treatment were used, and different extraction method were used to extract nucleic acid. TaqMan real-time PCR was used to quantitatively evaluate the impact of each treatment on the virus and the advantages and disadvantages were compared. We used the SYBR Green real-time PCR quantitative method to evaluate the removal effect of the above method on bacteria 16S rRNA and host 12S rRNA genome.Results:For fecal samples, the 0.22 μm PES filter showed a better filtering effect, and the PVDF material filter reduces the sample volume; 2 h nuclease digestion was better than 1 h digestion to remove bacteria, and the virus loss was less; the use of RPMK kits can effectively reduce bacteria, but the effect of extracting some viruses was poor, and the MVSK kit has a better effect of extracting viral nucleic acid. For tissue samples, 0.22 μm PES filter filtration, nuclease digestion for 1 h and VNAEK II kit extraction of nucleic acid were the best, Trizol LS combined with the RPMK method was better for gDNA removal, but the virus loss was larger. The virus loss of the whole process of the pretreatment method of feces and tissue samples was (1.7-3.0) Ct and (1.6-2.5) Ct, respectively.Conclusions:The optimal method for fecal samples was to filter with a PES filter, then digest with nuclease for 2 hours, and then extract nucleic acids using the MVSK kit; the optimal method for tissue samples was to filter with a PES filter, then perform 1 h nuclease digestion, and then use VNAEK II kit to extract nucleic acids.

Objective:To understand the prevalence of human parechovirus (HPeV) in neonates of Hunan Provincial People’s Hospital, and analyze genetic evolutionary characteristics.Methods:From June to September 2021, fecal samples of inpatient neonates were collected in Hunan Provincial People′s Hospital. TaqMan real-time qPCR and RT-PCR were used for HPeV screening and genotyping. High-throughput sequencing and PCR were used to obtain whole genomes. Phylogenetic analysis was performed after sequencing.Results:A total of 123 fecal samples of neonates were collected, of which 22 were HPeV positive, with 17.89% positive rate. All the strains belonged to the HPeV-1 genotype. One full-length genomic sequence of 7 269 bp were obtained, and provisionally named Hunan/HPeV/2021, which has the highest nucleotide identity with known HPeV-1 genotype, with 86.6%-91.9% nucleotide identity. The nucleotide and amino acid identity of open reading frame (ORF) with known similar sequences were 90.3%-92.6% and 97.3%-98.3%, respectively. The phylogenetic analysis showed that Hunan/HPeV/2021 belongs to the HPeV-1 genotype, which is clustered into the same clade as the popular HPeV-1 strains in China.Conclusions:HPeV has a high prevalence in inpatient neonates of Hunan Provincial People’s Hospital and belong to the HPeV-1 genotype.

Objective:To compare the clinical efficacy between arthroscopically assisted reduction versus open reduction in the treatment of paediatric humeral lateral condylar fractures with Kirschner wire fixation.Methods:A retrospective study was conducted to analyze the data of 28 children with humeral lateral condylar fracture who had undergone surgery at Department of Pediatric Orthopedics, Beijing Jishuitan Hospital from August 2023 to September 2023. There were 24 males and 4 females with an age of (6.5±1.6) years, 24 cases of type Ⅱ and 4 cases of type Ⅲ fractures according to the Jacob classification, and 1 case of type Ⅰ and 27 cases of type Ⅱ fractures according to the Milch classification. The time from injury to surgery averaged (71.6±21.3) hours. The children were divided into 2 groups according to their reduction methods: an arthroscopic group of 16 cases treated by arthroscopically assisted reduction and K-wire fixation and an open reduction group of 12 cases treated by open reduction and K-wire fixation. The operation time, incision length, Kirschner wire removal time, Mayo elbow performance score (MEPS) and the incidence of lateral bony spur on the X ray at the last follow-up were compared between the 2 groups; the flexion-extension and carrying angle of the elbow were compared between the healthy side and the affected side within each group at the last follow-up.Results:There was no significant difference in the preoperative general data between the 2 groups, indicating comparability ( P>0.05). The 28 pediatric patients were followed up for (4.9±0.4) months after surgery. The incision length [(1.3±0.4) cm] and Kirschner wire removal time [(42.8±3.5) d] in the arthroscopic group were significantly shorter than those in the open reduction group [(4.8±0.5) cm and (95.5±16.4) d] ( P<0.05). There was no significant difference in the operation time between the 2 groups [(70.6±15.5) min versus (61.7±14.5) min] ( P>0.05). There was no significant difference in the flexion-extension or in the carrying angle between the healthy and affected sides within each group at the last follow-up ( P>0.05). There was no significant difference either in the MEPS score or in the incidence of lateral bony spur between the 2 groups at the last follow-up ( P>0.05). Follow-up revealed pin infection in 1 patient in the arthroscopic group, and exposure of Kirschner wire tail in 1 patient in the open reduction group. Conclusions:In the treatment of humeral lateral condylar fractures, arthroscopically assisted reduction and Kirschner wire fixation can allow for debridement of the fracture ends in a minimally invasive way, visual fracture evaluation and reduction. Compared with open reduction, arthroscopically assisted reduction can reduce operative trauma without significantly increasing operation time.

Objective:To explore the blocking effect of temporary epiphysiodesis technique with "8"-plate in the treatment of leg length discrepancy (LLD) in children.Methods:From January 2013 to July 2020, 49 children (27 males and 22 females) with leg length discrepancy treated with "8"-plate in Pediatric Orthopedics Department of Beijing Jishuitan hospital were analyzed retrospectively. The average age at the time of surgery was 8.8±2.6 years (ranging from 3.8 to 13.1 years). The length difference between femur and tibia was measured before operation on full-length X-ray films of lower limbs, and the "8"-plate was placed on both medial and lateral sides of the distal femur and/or the proximal tibia. During the follow-up, the shape of epiphyseal plate and internal fixation change were evaluated on X-ray films of the knee joint, and the length difference before and after the operation was compared to evaluate the blocking effect. The blocking rate was compared according to the following conditions: the blocking position (femur, tibia), classification of LLD (constant or increasing), screw diameter (3.5 mm or 4.5 mm) and the age at operation (≥8 or <8 years).Results:49 patients were successfully operated. The follow-up time was 39.6±18.5 months (range 16-91 months) with the fixing time of 24.3±11.8 months (range 7-66 months). A total of 76 independent data of single bone were obtained. The correction range was 8.6±6.4 mm (range -8-37 mm). The correction rate was 0.35±0.31 mm/month. The correction rate of 52 femurs was 0.41±0.32 mm/month, which was significant higher than that of 24 tibias with 0.20±0.14mm/month ( t=5.323, P=0.008). The correction rate of the constant group with 0.54±0.32 mm/month was obviously better than the increasing group with 0.26±0.21 mm/month ( t=7.362, P=0.001). The average correction rate of 4.5 mm diameter screw group was 0.46±0.23 mm/month, which was significantly better than that of 3.5 mm diameter screw group with 0.26±0.24 mm/month ( t=3.467, P=0.022). The correction rate in the group of <8 years old was 0.32±0.25 mm/month, which was not significantly different from that in the group of ≥8 years old with 0.37±0.31 mm/month ( t=1.026, P=0.548). Conclusion:The blocking effect of "8" -plate temporary epiphysiodesis technique in the treatment of leg length discrepancy was related to the LLD classification and blocking position. Distal femoral block was better than proximal tibia block. Block effect of patients with constant unequal distance between lower limbs was better than that of patients with increased distance. 4.5 mm diameter screw was better than 3.5mm. Because the blocking effect was slow, early treatment and a firm stick to indications was of great importance.

Objective: During the 2016 winter season, GII.2 norovirus(NoV) suddenly emerged in China. To elucidate its mechanism of epidemic, this study focused on characteristics of binding between the P protein of capsid and histo-blood group antigens (HBGAs). Methods: The research object was GⅡ.2 ZTX strain which had an outbreaks by the end of 2016 in Beijing. Recombinant prokaryotic expression plasmid was constructed, and the expression of virus P protein was determined and purified. The P protein characteristics of binding to HBGAs was studied through saliva and oligosaccharide binding experiments. Results: Soluble P protein was successfully obtained, and combined with type A, B, AB saliva. Conclusions The result illuminate the combination with new outbreaks of NoV and salivary types, which provided a basis for its pathogenic mechanism and prevention and control measures.

ObjectiveTo explore the therapeutic effect and mechanism of Yiqi Huoxue Tongbian prescription on slow transit constipation （STC） in rats. MethodThe rat model of STC was established by gavage of loperamide hydrochloride. Rats were assigned into control， model， mosapride， low-， medium-， and high-dose （3.51， 7.02， and 14.04 g·kg^-1， respectively） Yiqi Huoxue Tongbian prescription groups. The changes of general signs， fecal moisture content， and intestinal propulsion rate were measured after model establishment and drug administration. The colonic mucosal changes were observed by hematoxylin eosin staining. Enzyme-linked immunosorbent assay was employed to determine the content of substance P （SP） and vasoactive intestinal peptide （VIP） in the colon of rats in each group. The gray values of aquaporin （AQP） 3， AQP4， AQP8， and c-Kit in rat colon tissue were measured by immunohistochemistry and Western blot， and the changes of intestinal flora were detected by 16S rRNA high-throughput sequencing. ResultCompared with the model group， 10 days of treatment with Yiqi Huoxue Tongbian prescription increased the fecal moisture content and intestinal propulsion rate （P<0.01）. The medium- and high-dose Yiqi Huoxue Tongbian prescription groups and the mosapride group showed no obvious mucosal inflammation and neat arrangement of goblet cells with a large number in the colon tissue. Moreover， the three groups showed increased SP content （P<0.01） and decreased VIP content （P<0.01） in the serum. The medium- and high-dose Yiqi Huoxue Tongbian prescription groups showed down-regulated protein levels of AQP3， AQP4， and AQP8 （P<0.01） and up-regulated protein level of c-Kit （P<0.01）. The drug administration groups presented slightly increased observed species， Chao1， ACE， and Shannon， Simpson， and PD whole tree. The principal component analysis showed that the control group had a short distance with the high- and medium-dose Yiqi Huoxue Tongbian prescription groups， indicating that high- and medium-dose Yiqi Huoxue Tongbian prescription can recover the intestinal flora to that in the control group. ConclusionYiqi Huoxue Tongbian prescription can alleviate the defecation status of rats with slow transit constipation by down-regulating the expression of AQP3， AQP4， and AQP8 to reduce the absorption of water in the intestine， up-regulating the expression of c-Kit to increase the number and distribution of Cajal interstitial cells， and regulating the balance of flora in the colon tissue.

ObjectiveTo clarify the spatial distribution characteristics of medicinal plant resources in Gansu province， analyze the causes， changing trends， and driving factors of the spatial differentiation， and thus lay a scientific basis for the rational development and sustainable development of medicinal plant resources in this province. MethodBased on the data of The Fourth National Survey of Chinese Medicine Resources， the richness and spatial distribution difference of medicinal plant resources in 87 counties （districts） of Gansu province were analyzed via the global spatial autocorrelation analysis， trend surface analysis， local spatial autocorrelation analysis， and hotspot analysis. Moreover， the correlation of vegetation type， soil texture， annual average temperature， annual average precipitation， and altitude with the spatial distribution pattern of the medicinal plant resources was discussed. ResultCounties （districts） with high or low richness of medicinal plant resources in Gansu province were respectively clustered together. To be specific， counties （districts） with high richness of the medicinal resources were mainly in southeastern Gansu， while those with low richness in northwestern Gansu. The leading driving factors affecting the cold and hot spots included vegetation type， soil texture， and average annual rainfall. ConclusionThe species richness of medicinal plant resources in Gansu province rises from west to east and from north to south. The natural driving factors are the key to the diversity and spatial distribution pattern of medicinal plant resources， which show significant influence on them.

Dopamine D₃ receptor (D₃R) is implicated in multiple psychotic symptoms. Increasing the D₃R selectivity over dopamine D₂ receptor (D₂R) would facilitate the antipsychotic treatments. Herein, novel carbazole and tetrahydro-carboline derivatives were reported as D₃R selective ligands. Through a structure-based virtual screen, ZLG-25 (D₃R K_i = 685 nmol/L; D₂R K_i > 10,000 nmol/L) was identified as a novel D₃R selective bitopic ligand with a carbazole scaffold. Scaffolds hopping led to the discovery of novel D₃R-selective analogs with tetrahydro-β-carboline or tetrahydro-γ-carboline core. Further functional studies showed that most derivatives acted as hD₃R-selective antagonists. Several lead compounds could dose-dependently inhibit the MK-801-induced hyperactivity. Additional investigation revealed that 23j and 36b could decrease the apomorphine-induced climbing without cataleptic reaction. Furthermore, 36b demonstrated unusual antidepressant-like activity in the forced swimming tests and the tail suspension tests, and alleviated the MK-801-induced disruption of novel object recognition in mice. Additionally, preliminary studies confirmed the favorable PK/PD profiles, no weight gain and limited serum prolactin levels in mice. These results revealed that 36b provided potential opportunities to new antipsychotic drugs with the multiple antipsychotic-like properties.