In protein engineering, while computational models are increasingly used to predict mutation effects, their evaluations primarily rely on high-throughput deep mutational scanning (DMS) experiments that use surrogate readouts, which may not adequately capture the complex biochemical properties of interest. Many proteins and their functions cannot be assessed through high-throughput methods due to technical limitations or the nature of the desired properties, and this is particularly true for the real industrial application scenario. Therefore, the desired testing datasets, will be small-size (∼10-100) experimental data for each protein, and involve as many proteins as possible and as many properties as possible, which is, however, lacking. Here, we present VenusMutHub, a comprehensive benchmark study using 905 small-scale experimental datasets curated from published literature and public databases, spanning 527 proteins across diverse functional properties including stability, activity, binding affinity, and selectivity. These datasets feature direct biochemical measurements rather than surrogate readouts, providing a more rigorous assessment of model performance in predicting mutations that affect specific molecular functions. We evaluate 23 computational models across various methodological paradigms, such as sequence-based, structure-informed and evolutionary approaches. This benchmark provides practical guidance for selecting appropriate prediction methods in protein engineering applications where accurate prediction of specific functional properties is crucial.

Objective:To investigate the protective effect and mechanism of low-intensity pulsed ultrasound on human periodontal ligament stem cells (hPDLSCs) injured by high glucose.Methods:hPDLSCs were cultured in vitro and randomly divided into normal control group, high glucose model group and three low intensity pulsed ultrasound groups (30, 60, 90 mW/cm 2). Cells in the high glucose model group and three low intensity pulsed ultrasound groups were treated with high glucose medium (glucose 40 mmol/L) for 48 h to establish high glucose model. Cells in the normal control group were treated with normal medium (glucose 11 mmol/L). After modeling, three low intensity pulsed ultrasound groups were treated with low-frequency pulse for 20 min each day for 7 days at a dose of 30, 60 and 90 mW/cm 2 respectively. After 7 days, the proliferation ability of each group was determined by MTT assay. The changes of cell cycle and apoptosis were detected by flow cytometry. The level of Caspase-3, Caspase-6, and Caspase-9 was detected by colorimetry. The levels of Wnt1, Wnt10b and β-catenin genes and proteins were detected by reverse transcription PCR and Western Blot respectively. Results:Compared with the hPDLSCs in the normal control group, the hPDLSCs in the high glucose model group have the following changes except for the cell cycles (all P>0.05), i.e. the activity of the cells was decreased and the apoptosis was increased (all P<0.05), the activities of Caspase-3, Caspase-6, and Caspase-9 were increased (all P<0.05), and the expressions of Wnt1, Wnt10b and β-catenin genes and proteins were decreased (all P<0.05). Compared with the hPDLSCs in the high glucose model group, the hPDLSCs in low-intensity pulsed ultrasound groups have the following changes expect for the cell cycles (all P>0.05), i.e. cell viabilities were increased (all P<0.05), proliferation abilities were increased (all P<0.05), apoptosis rates were decreased (all P<0.05), the activities of Caspase-3, Caspase-6, Caspase-9 were decreased (all P<0.05), and the expressions of Wnt1, Wnt10b and β-catenin genes and proteins were up-regulated ( P<0.05). Among the three low-intensity pulsed ultrasound groups, when the ultrasonic intensity is 90 mW/cm 2, the above-mentioned index changes most obviously (all P<0.05). Conclusions:Low-intensity pulsed ultrasound can significantly alleviate the hPDLSCs damage induced by high glucose, which may be achieved by reducing the activities of Caspase-3, Caspase-6 and Caspase-9, and up-regulating the expression of Wnt1, Wnt10b and β-catenin.

Objective:To summarize the clinical features, treatment and outcomes of cervical neuroblastoma (NB) subjected to multi-disciplinary treatment.Methods:The clinical features of cervical NB patients who were admitted to Beijing Children′s Hospital, Capital Medical University from February 2015 to October 2018, were retrospectively analyzed.The tumor makers [lactate dehydrogenase(LDH), neuron-specific enolase(NSE), urine homovanillic acid/creatinine(HVA/Crn), and urine vanillyl-mandelic acid/creatinine(VMA/Crn)], index of tumor burden(KTB), bone marrow examination, histopathologic types, N- MYC, gene amplification and 11q23 depletion type, staging and grouping, treatment and outcomes were analyzed.Follow-up was ended on March 31 st, 2019. Results:The 13 cervical NB patients aged from 1 month to 47 months (median age: 10 months), and 8 patients (61.5%) were younger than 18 months old.The course of disease ranged from 0.5 to 24.0 months (median course: more than 1 month). Seven patients (53.8%) presented with cervical masses.According to International Neuroblastoma Staging System (INSS), 8 patients (61.5%) were identified as stage Ⅱ, 3 patients (23.1%) as stage Ⅲ, and 2 patients (15.4%) as stage Ⅳ.There were 8 patients (61.1%) at low risk, 4 patients (30.8%) at intermediate risk, 1 patient (7.7%) at high risk.As for the laboratory examinations, LDH was increased in 7 patients (53.8%), and normal in 5 patients (38.5%). NSE was increased in 9 patients (69.2%), and normal in 4 patients (30.8%). Urine VMA/Crn was increased in 5 patients (38.5%), and normal in 8 patients (61.5%). HVA/Crn was increased in 8 patients (61.5%), and normal in 5 patients (38.5%). KTB was increased in 5 patients (38.5%), and normal in 5 patients (38.5%). No NB cell was detected in bone marrow of 13 patients.The pathologic type was NB in 9 patients (69.2%), and ganglioneuroma in 4 patients (30.8%). N-MYC gene amplification and 11q depletion were not detected.All the 13 patients accepted regular chemotherapy, radiotherapy and primary tumor resection in accordance with the staging and clinical risk grouping.The range of follow-up time was 5 to 48 months (median: 24 months). All of the patients fi-nished their treatment and were followed up regularly.Nine patients (69.2%) achieved complete remission, 4 patients (30.8%) achieved partial remission, and none of the patients had progression of disease.Conclusions:Cervical NB subjected to multi-disciplinary treatment has a smaller age at admission, mostly presented with cervical masses and rarely accompanied with distant metastasis.They are mostly at low risk or intermediate risk, and can achieve good outcomes after regular treatment.

Objective: To analyze the clinical characteristics of children with neuroblastoma (NB) complica-ted with lung or pleural metastasis, further to explore the correlation between characteristics and short-term outcome of NB, so as to provide a basis for clinical diagnosis and treatment. Methods: A retrospective analysis was performed concerning the age of onset, clinical features, treatment and outcome of 36 patients with NB who were admitted at Blood Tumor Center, Beijing Children′s Hospital of Capital Medical University from December 2007 to December 2017.The diagnostic criteria, therapeutic regimen and therapeutic efficacy criteria of the enrolled children were all based on the NB protocol of Beijing Children′s Hospital of Capital Medical University (BCH-NB-2007), the clinical stage was based on international clinical stage of neuroblastoma (INSS stage), and stratified treatment was conducted according to the BCH-NB risk grouping standard.The follow-up period lasted till October 31, 2018. Results: (1)The common clinical features of grouped children: 36 patients were selected into the group, accounted for 5.99% (36/601 cases) in total hospitalized NB children, they were less than 10 years old, 10 cases under 18 months, and the median age was 29.5 months (9-105 months); 20 cases were male, and 16 cases were female; the primary tumor was located in the retroperitoneal site in 19 cases, accounting for 52.78%, 9 cases in adrenal site, accounting for 25.00%, and 8 cases in mediastinal site, accounting for 22.22%.Risk groups: 29 cases were in high-risk group, 6 cases were in medium-risk group and 1 case was in low-risk group.The main symptoms were of pain onset in 8 cases, fever in 6 cases, local mass in 6 cases, abdominal mass in 4 cases, mediastinal mass in 3 cases, paleness in 3 cases, subcutaneous nodules in 2 cases, abdominal distension in 2 cases, lower limb swelling in 1 case, and diarrhea in 1 case. Among them, 16 cases had respiratory system symptoms first, accounting for 44.4%.(2)Laboratory examination: there were 35 patients of neuron-specific enolase (NSE) ≥25 μg/L on the initial diagnosis, of which 11 cases were more than 370 μg/L, the value of serum lactate dehydrogenase (LDH) ≥717.5 U/L in 25 patients, accounting for 69.44%, and 10 cases were accompanied by N-myc gene amplification.(3)Imaging examination: on the first diagnosis, chest/abdomen CT showed 75.75%(24/33 cases) of pleural or lung involvement, PET-CT showed 81.8% (27/33 cases) of pleural or lung involvement, B-ultrasound showed 41.67%(5/12 cases) of pleural or lung involvement; 3 imaging examinations showed: 1 positive in 16 cases, 2 positive in 16 cases, and 3 positive in 4 cases.(4)Comparison of clinical features of pleural and pulmonary involvement: among the 30 children with pleural involvement, 6 cases had respiratory symptoms, 3 cases had respiratory symptoms on the 6 cases with pulmonary involvement only, and 4 cases with pleural and pulmonary involvement had no respiratory symptoms at first diagnosis.(5)Treatment and outcome: 2 cases died because of critical condition after diagnosis, 5 cases didn′t receive the law treatment, 29 cases accepted law stratification treatment, among them, 1 case in low risk group, INSS-Ⅳ, alleviate current (CR); 4 cases in the moderate group, INSS-Ⅳ, CR in 2 cases, partial response (PR) in 1 case, progress in 1 case, new tumor foci occurring after chemotherapy discontinuation for 13 months. Twenty-four cases were in the high-risk group, event occurred in 7 cases (29.17%) of them, 1 case had progression by postoperative evaluation, 4 cases of progression at 1.5, 2.0, 3.0 and 6.0 months after cessation of chemotherapy, and 2 cases had recurrence at 11 and 17 months after cessation of chemotherapy.Overall survival rate (OS) was 41.4% for all children analyzed by Kaplan-Merier, and 32.9% of them were predicted to have 3-year event-free survival. Conclusions Children with pulmonary or pleural metastasis of neuroblastoma have no specific respiratory symptoms.CT scan might be a useful method for diagnosing the group Ⅳ children with pulmonary or pleural metastasis of neuroblastoma.Moreover, there seemed to be no significant correlation between the N-myc gene expression and survival prognosis of these children.

OBJECTIVE To optimize the parameters of passive cutaneous anaphylaxis(PCA)in rats immunized by ovalbumin(OVA). METHODS 1-2 month-old Sprague-Dawley rats were immu?nized by ip injection of OVA(0.2,1.0 and 5.0 mg per rat)mixed with complete Freund′s adjuvant once every other day 3 times. Serum was collected on the 12th-16th days after final immunization. Then the rats were intracutaneously injected with sensitized serum and then stimulated by iv injection of the same dose of OVA mixed with Evans blue after a latent period of 0.5,1.5,3,6,12,24,36,48 and 60 h. Finally,the diameters of blue spots in the skin were measured at stimulation. RESULTS Serum total-IgE(T-IgE)and OVA-specific IgE(sIgE)levels increased significantly and reached the peak on the 3rd-7th days and 12th-16th days after final immunization,respectively. There was no correlation between the serum T-IgE level and OVA-sIgE level when the rats were immunized with OVA at OVA 0.2-5.0 mg per rat. The rats experienced PCA after injection of OVA 1.0 and 5.0 mg per rat. Diameters of blue spots in the skin reached the maximum value after rats were sensitized for 0.5-3 h. Moreover,the shape,color and size of blue spots were better 30-60 min after stimulation. CONCLUSION Optimized PCA is as follows:1-2 month-old rats are immunized on the 1st,3rd and 5th days by ip injection of OVA 1.0-5.0 mg. The immunizing serum is collected at 12-16 d after final immunization. The rats are stimulated by OVA and Evans blue after a latent period of 0.5-3 h. Diameters of blue spots in rats′ skin are then measured 30-60 min after stimulation.

BACKGROUND:The biomechanical effect of the implant-bone interface is one of the most important factors for bone resorption. The new structure of the periodontal-ligament-like implants may improve the distribution of the interfacial stress. OBJECTIVE:To discuss the effect of the internal structure changes of traditional implants on the cortical bone stress distribution and peak at the implant-bone interface under different occlusal load conditions, so as to provide a theoretical basis for the optimization design and clinical application of new structure implants. METHODS:Two kinds of digital models, new structure implant (model A) and non-threaded cylindrical implant (model B), were established by Pro/ENGINEER software. Variations of the stress peak and stress distribution of implant-bone interface cortical bone area under the same bone and force environment were analyzed using Ansys software. RESULTS AND CONCLUSION: Under a vertical loading, the stress peak under different forces was reduced by 17.54% in model A compared with model B; under a 45° loading, the stress peak of model A was reduced by 2.59% compared with model B, and it showed an evident tendency of high stress area focusing to the buccal side of model B. Under the chew-simulation loading, the stress peak of model A was lower than that of model B. The biggest difference (0.353 2 MPa) appeared atβ=12°(β is the angle of force direction and the implant axis), and it gradualy reduced atβ > 12°. At the same time, model A had a wider range of application degree compared with model B in two quantitative indicators, including optimal peak stress of promoting bone tissue growth and stress peak of maintaining healthy bone tissue. These results suggest that the optimized structure of implants contributes to improve the cortical bone stress distribution at the implant-bone interface, decrease the peak stress, and reduce the risk of cortical bone absorption in a wider range.