Traditional development of small protein scaffolds has relied on display technologies and mutation-based engineering, which limit sequence and functional diversity, thereby constraining their therapeutic and application potential. Protein design tools have significantly advanced the creation of novel protein sequences, structures, and functions. However, further improvements in design strategies are still needed to more efficiently optimize the functional performance of protein-based drugs and enhance their druggability. Here, we extended an evolution-based design protocol to create a novel minibinder, BindHer, against the human epidermal growth factor receptor 2 (HER2). It not only exhibits super stability and binding selectivity but also demonstrates remarkable properties in tissue specificity. Radiolabeling experiments with ^99mTc, ⁶⁸Ga, and ¹⁸F revealed that BindHer efficiently targets tumors in HER2-positive breast cancer mouse models, with minimal nonspecific liver absorption, outperforming scaffolds designed through traditional engineering. These findings highlight a new rational approach to automated protein design, offering significant potential for large-scale applications in therapeutic mini-protein development.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

Objective To analyze the risk factors and survival status of Epstein-Barr virus(EBV)infection in pa-tients with aplastic anemia(AA)after haploid allogeneic hematopoietic stem cell transplantation(Haplo-HSCT).Methods Clinical data of 78 AA patients who underwent Haplo-HSCT in the hematology department of a hospital from January 1,2019 to October 31,2022 were analyzed retrospectively.The occurrence and onset time of EBV viremia,EBV-related diseases(EBV diseases),and post-transplant lymphoproliferative disorders(PTLD)were ob-served,risk factors and survival status were analyzed.Results Among the 78 patients,38 were males and 40 were females,with a median age of 33(9-56)years old;53 patients experienced EBV reactivation,with a total inci-dence of 67.9％,and the median time for EBV reactivation was 33(13,416)days after transplantation.Among pa-tients with EBV reactivation,49 cases(62.8％)were simple EBV viremia,2 cases(2.6％)were possible EBV di-seases,and 2 cases(2.6％)were already confirmed EBV diseases(PTLD).Univariate analysis showed that age 1＜40 years old at the time of transplantation,umbilical cord blood infusion,occurrence of acute graft-versus-host disease(aGVHD)after transplantation,and concurrent cytomegalovirus(CMV)infection were independent risk fac-tors for EBV reactivation in AA patients after Haplo-HSCT.Multivariate analysis showed that concurrent CMV in-fection was an independent risk factor for EBV reactivation in A A patients after Haplo-HSCT(P=0.048).Ritu-ximab intervention before stem cell reinfusion was a factor affecting the duration of EBV reactivation(P＜0.05).The mortality of EBV viremia,EBV diseases,and PTLD alone were 8.2％,50.0％,and 100％,respectively.The 2-year overall survival rate of patients with and without EBV reactivation were 85.3％,and 90.7％,respectively,difference was not statistically significant(P=0.897).However,patients treated with rituximab had 2-year lower survival rate than those who did not use it,with a statistically significant difference(P=0.046).Conclusion EBV reactivation is one of the serious complications in AA patients after Haplo-HSCT,which affects the prognosis and survival of patients.

Abstract: Objective To develop an ELISA kit to detect IgG antibodies of Chikungunya virus (CHIKV), providing a new method for epidemiological investigation and detection in the field for CHIKV infection. Methods Using the CHIKV-specific recombinant protein pMal-chik23 as diagnostic antigen, HRP-labeled anti-IgG antibody as color-developing antibody, and the working concentration of diagnostic antigen, serum to be tested and second antibody were optimized using orthogonal. The reaction conditions of ELISA reaction, such as coating, blocking, incubation, and color-developing were systematically optimized. The cut-off value for ELISA detection was established based on the assessment of a large clinical sample set. On this basis, the specificity, sensitivity, and stability of the ELISA response were evaluated to develop and assemble a rapid ELISA kit for the detection of Chikungunya fever IgG antibodies. Results On the basis of systematic conditions optimization, an indirect ELISA kit for the detection of IgG antibodies against CHIKV was developed and assembled. The optimal reaction conditions were identified as 1.0 μg/mL antigen was coated using carbonate buffer at 4 ℃ for 24 hours. Then the microplate was blocked using HBV blocking solution at 37 ℃ for 4 hours. 100 μL/well samples to be tested were diluted at 1∶101, reacted at 37 ℃ for 40 minutes, and washed 4 times with PBST. Thus, HRP-labeled rabbit anti-human IgG was diluted at 1∶20 000, HRP-labeled sheep anti-mouse IgG was diluted at 1∶10 000, reaction at 37 ℃ for 30 minutes, and washed 5 times with PBST. Finally, 100 μL/well TMB solution was added and incubated at 37 ℃ for 10 minutes. Then terminate the reaction with 50 μL of 20% H2SO4 and measure the A450 value at dual wavelengths of 450/630 nm (A450) . The evaluation results showed that ELISA A450 of Chikungunya fever-positive samples were more than 0.43, while the ELISA A450 of negative samples was less than 0.04, and the S/N ratio > 10. Specificity test showed that the developed kit had no cross-reaction with 9 other similar arbovirus species such as Sindbis, Geta, Ross River, and Dengue virus. The stability evaluation of the reagent kit indicated that it had high stability, with a coefficient of variation (CV) within the microplate ranging from 0.76% to 2.12%, the coefficient of variation between the microplate ranged from 0.64% to 1.85%, and the coefficient of variation between batches ranged from 0.83% to 2.31%, all of which were less than 3%. The sensitivity of the kit did not decrease significantly after being stored at 4°C for 1 year. Conclusions A rapid indirect ELISA kit for the detection of Chikungunya fever IgG antibodies was successfully developed, exhibiting good sensitivity, specificity, and stability.

Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

Objective: To compare the effects and safety of dydrogesterone (DG) and medroxyprogesterone acetate (MPA) on the treatment in patients with endometrial hyperplasia without atypia (EH). Methods: This was a single-center, open-label, prospective non-inferior randomized controlled phase Ⅲ trial. From February 2019 to November 2021, patients with EH admitted to the Obstetrics and Gynecology Hospital of Fudan University were recruited. Enrolled patients were stratified according to the pathological types of simple hyperplasia (SH) or complex hyperplasia (CH), and were randomised to receive MPA or DG. Untill May 14, 2022, the median follow-up time after complete response (CR) was 9.3 months (1.1-17.2 months). The primary endpoint was the 6-month CR rate (6m-CR rate). The secondary endpoints included the 3-month CR rate (3m-CR rate), adverse events rate, recurrence rate, and pregnancy rate in one year after CR. Results: (1) A total of 292 patients with EH were enrolled in the study with the median age of 39 years (31-45 years). A total of 135 SH patients were randomly assigned to MPA group (n=67) and DG group (n=68), and 157 CH patients were randomly assigned to MPA group (n=79) and DG group (n=78). (2) Among 292 patients, 205 patients enrolled into the primary endpoint analysis, including 92 SH patients and 113 CH patients, with 100 patients in MPA group and 105 in DG group, respectively. The 6m-CR rate of MPA group and DG group were 90.0% (90/100) and 88.6% (93/105) respectively, and there were no statistical significance (χ²=0.11, P=0.741), with the rate difference (RD) was -1.4% (95%CI:-9.9%-7.0%). Stratified by the pathology types, the 6m-CR rate of SH patients was 93.5% (86/92), and MPA group and DG group were respectively 91.1% (41/45) and 95.7% (45/47); and the 6m-CR rate of CH patients was 85.8% (97/113), and MPA group and DG group were 89.1% (49/55) and 82.8% (48/58) respectively. The 6m-CR rates of the two treatments had no statistical significance either (all P>0.05). A total of 194 EH patients enrolled into the secondary endpoint analysis, including 88 SH patients and 106 CH patients, and 96 patients in MPA group and 98 in DG group, respectively. The 3m-CR rate of SH patients were 87.5% (77/88), while the 3m-CR rates of MPA group and DG group were 90.7% (39/43) and 84.4% (38/45), respectively; the 3m-CR rate of CH patients was 66.0% (70/106), and MPA group and DG group had the same 3m-CR rate of 66.0% (35/53). No statistical significance was found between the two treatments both in SH and CH patients (all P>0.05). (3) The incidence of adverse events between MPA group and DG group had no statistical significance (P>0.05). (4) A total of 93 SH patients achieved CR, and the cumulative recurrence rate in one year after CR were 5.9% and 0 in MPA group and DG group, respectively. While 112 CH patients achieved CR, and the cumulative recurrence rate in one year after CR were 8.8% and 6.5% in MPA group and DG group, respectively. There were no statistical significance between two treatment groups (all P>0.05). Among the 93 SH patients, 10 patients had family planning but no pregnancy happened during the follow-up period. Among the 112 CH patients, 21 were actively preparing for pregnancy, and the pregnancy rate and live-birth rate in one year after CR in MPA group were 7/9 and 2/7, while in DG group were respectively 4/12 and 2/4, and there were no statistical significance in pregnancy rate and live-birth rate between the two treatment groups (all P>0.05). Conclusions: Compared with MPA, DG is of good efficacy and safety in treating EH. DG is a favorable alternative treatment for EH patients.
Female ; Humans ; Adult ; Medroxyprogesterone Acetate/adverse effects* ; Endometrial Hyperplasia/pathology* ; Dydrogesterone/adverse effects* ; Hyperplasia ; Prospective Studies

BackgroundGender and age differences in the clinical manifestations of patients with bipolar disorder can affect the clinical diagnosis and treatment process. The current treatment effect of bipolar disorder in adolescents is not ideal， which has become the main reason for disability during the learning period. ObjectiveTo analyze the clinical features and medication therapy status of bipolar disorder in adolescents， and to provide references to support for personalized diagnosis and treatment. MethodsOn January 16， 2023， 1 169 patients with bipolar disorder who were hospitalized at Beijing Anding Hospital Affiliated Capital Medical University from January 1， 2014 to December 31， 2017 were retrospectively enrolled. Medical records were collected and analyzed to compare the clinical features among patients of different gender and age groups， and to explore the differences in medication use among patients of different genders in different types of seizures. ResultsMale patients reported a larger proportion of manic episodes， and a smaller proportion of depressive episodes than female patients （P<0.05）.Female patients reported a larger proportion of non-suicidal self-harm behaviors than male patients （χ²=7.761， P<0.01）.And patients in low-age group featured a larger proportion of mixed seizures， impulsive behaviors and family history of bipolar disorders along with a smaller proportion of manic episodes than those in high-age group （P<0.05 or 0.01）. High-age group had a longer average length of hospital stay than low-age group （t=-2.930， P<0.01）. In manic episode patients， males were found to have a larger proportion of valproate and atypical antipsychotic drug administration than females （P<0.01）. Among atypical antipsychotic drugs， males accounted for a larger proportion of administration of risperidone and olanzapine （χ²=26.957） than females （P<0.05 or 0.01）， while females constituted a larger proportion of administration of quetiapine （χ²=14.865） and aripiprazole than males （P<0.01）. In depressive episode patients， females had a larger proportion of administration of olanzapine than males （P<0.01）. In patients with mixed seizures， females occupied a larger proportion of administration of lithium carbonate than males （χ²=9.253， P<0.01）， and males exhibited a larger proportion of administration of valproate than females （P<0.05）. ConclusionDifferences have been shown in diagnostic classification and concomitant symptoms among adolescent bipolar disorder of different genders and ages. Furthermore， medications of lithium carbonate， valproate， atypical antipsychotic and other drugs differ by gender among adolescents of different subtypes of bipolar disorder. ［Funded by Key Special Project of the National Key R&D Program of the Ministry of Science and Technology for "Major Chronic Non communicable Disease Prevention and Control Research" （number， 2017YFC1311101）］

Objective:To explore the treatment experience of holmium laser enucleation of prostate (HoLEP) with preservation of longitudinal urethral mucosa at 12 o’clock for benign prostatic hyperplasia (BPH) with small-medium gland.Method:From October 2018 to April 2021, 256 patients diagnosed BPH with small-medium gland(prostate volume 30-60 ml)were retrospectively analyzed, including general information, way of operation, intraoperative parameters and follow-up data. From October 2018 to June 2020, 186 BPH patients underwent conventional HoLEP, which did not retain longitudinal urethral mucosa at 12 o’clock as a conventional operation group. From July 2020 to April 2021, 70 BPH patients underwent modified HoLEP, which retained longitudinal urethral mucosa at 12 o’clock as a modified operation group. There was no significant difference between the two groups( P>0.05) in term of the age[(70.5±4.4)years old vs.(68.5±3.2)years old], Q max[(7.5±2.8)ml/s vs.(7.5±2.1)ml/s], IPSS[(20.3±4.6)vs.(21.4±3.7)], QOL[(4.5±1.0)vs.(4.2±1.4)], postvoid residual volume[(126.9±29.36)ml vs.(132.2±32.3)ml], PSA[(1.5±1.3)ng/ml vs.(1.8±1.1)ng/ml] and prostate volume[(48.1±11.1)ml vs.(48.0±12.7)ml]. Both groups were treated with "trefoil" enucleation of prostate. The modified group was improved compared with the conventional group by retaining a 12 o’clock longitudinal urethra mucosa from the bladder neck to the apex of the prostate. The technical improvements were as follows: ①the left lobe of prostate was removed from at 5 o’clock at the verumontanum to 1 o’clock at the prostate apex along the gap between the hyperplasia gland and the surgical envelope; ②the right lobe was removed from 7 o’clock at the verumontanum to 11 o’clock at the apex; ③the urethra mucous membrane was cut vertically from 1 and 11 o’clock at the bladder neck to 1 and 11 o’clock at the apex respectively, and retaining the longitudinal mucous membrane between 11 and 1 o’clock (including 12 o’clock). Efficacy and postoperative complications of the two groups were compared. Results:The difference between the conventional group and the modified group in operation time[(36.5±10.4)min vs.(40.7±9.7)min], enucleated glandular weight[(35.5±12.2)g vs.(31.6±10.4)g], hemoglobin decline[(6.1±2.2)g/L vs.(5.6±2.5) g/L], postoperative hospitalization time [(1.2±0.2)d vs.(1.5±0.4)d]and catheter indwelling duration[(2.3±1.3)d vs.(2.0±1.0)d] were not statistically significant ( P>0.05). There were 252 patients for follow-up, including 183 cases in the conventional group and 69 cases in the modified group, and 4 cases were lost to follow-up. Mean time of follow-up was 8.4 months. In both groups, postoperative IPSS were 5.4±2.3 and 5.9±1.2 respectively, QOL1.5±0.3 and 2.0±1.0 respectively, Q max(24.3±9.2)ml/s and (22.5±11.3)ml/s respectively and postvoid residual volume (8.3±4.5)ml and (7.7±2.9)ml respectively, which were significantly different from that before the operation ( P<0.05). However, there was not significant difference between the two groups ( P>0.05). The postoperative immediate urinary continence rate of the conventional group and modified group were 85.2% (156/183), 98.6% (68/69), respectively, and two groups had statistical differences ( P<0.05). Incidence of postoperative bladder neck contraction were 4.4% (8/183) and 0 respectively in the conventional and modified group, whose difference was significant( P<0.05). Conclusions:HoLEP with preservation of longitudinal urethral mucosa at 12 o'clock is the same effective as conventional operation in the treatment of BPH with small-medium gland, likewise it could significantly improve immediate urinary continence rate and reduce the incidence of bladder neck contraction.

Objective: To evaluate the efficacy of in-situ full size split liver transplantation(fSLT) for adult recipients using the living donor liver transplantation(LDLT) technique and to compare the characteristics of the left hemiliver graft (LHG) and the right hemiliver graft(RHG)transplantation. Methods: Deceased donor and recipient data of 25 consecutive cases of fSLT at Department of Hepatopancreatobiliary Surgery, Ningbo Medical Center Lihuili Hospital from March to December 2021 was retrieved and the patients divided into two groups:LHG group and RHG group. Among the 13 donors,11 were male and 2 were female,aged (M(IQR))38(19) years(range: 25 to 56 years),with height of 168(5) cm(range:160 to 175 cm) and weight of 65(9) kg(range: 50 to 75 kg). The median age of the 25 recipients was 52(14) years(range:35 to 71 years),17 were male and 8 were female,15 had primary liver cancer and 10 had benign end-stage liver disease,model for end-stage liver disease score was 10(9) points(range:7 to 23 points). Of the 25 recipients,10 recipients had previously undergone hepatobiliary surgery. The follow-up period was to January 2022. Demographic,clinicopathological,surgical outcomes and postoperative complications were evaluated and compared between the two groups. Continuous quantitative data were compared using Mann-Whitney U test. Classification data were expressed as frequencies,and were compared between groups using χ² test or Fisher exact probability method. Results: Using LDLT technique,in-situ full-left/full-right liver splitting was performed and 13 viable pairs of hemiliver grafts were harvested with acquisition time of 230(53) minutes(range:125 to 352 minutes) and blood loss of 250(100) ml(range:150 to 1 000 ml). A total of 25 hemiliver grafts(13 LHG and 12 RHG) were allocated to patients listed for liver transplantation in our center by China Organ Transplant Response System. In the LHG group(13 cases),there were more females and more patients with benign end-stage liver disease than in the RHG group(12 cases)(P<0.05). The body weight and graft weight of recipients in the LHG group were lower than that in RHG group(both P<0.05). There were no significant differences in other baseline data between the two groups(all P>0.05). The graft to recipient weight ratio(GRWR) was 1.2(0.4)%(range:0.7% to 1.9%) for 25 recipients,1.1(0.5)%(range:0.7% to 1.6%)for the LHG group and 1.3(0.5)%(range:0.9% to 1.9%)for the RHG group. There was no significant difference between the two groups (P>0.05). Sharing patterns of hepatic vessels and the common bile duct are as follows:all the trunk of middle hepatic vein were allocated to the LHG group. The proportion of celiac trunk,main portal vein and common bile duct assigned to LHG and RHG was 10∶3 (P=0.009), 9∶4 (P>0.05) and 4∶9 (P=0.027),respectively. The vena cava of 12 donors in early stage retained in LHG and that of last one was shared between LHG and RHG (P<0.01). The median cold ischemia time of 25 hemiliver grafts was 240(90) minutes(range:138 to 420 minutes). For the total of 25 fSLT,the median anhepatic phase was 50(16) minutes(range:31 to 98 minutes) and the operation time was 474(138)minutes(range:294 to 680 minutes) with blood loss of 800(640) ml(range:200 to 5 000 ml). There were no significant differences in all of operation data between two groups. In the LHG group,3 patients with GRWR≤0.8% had postoperative small-for-size syndrome which improved after treatment. Postoperative Clavien-Dindo grade≥Ⅲ complications were observed in 6 cases(24.0%),4 cases(4/13) in the LHG group and 2 cases(2/12) in the RHG group,respectively. The difference was not statistically significant. Among them,5 cases improved after re-operation and intervention,1 case in LHG group died of secondary infection 2 weeks after operation,and the mortality was 4.0%. Analysis of serious postoperative complications and death has suggested that conventional caval interposition should not be used for LHG transplantation. Conclusion: Relying on accurate donor-recipient evaluation and the apply of LDLT technique,the morbidity and mortality of in-situ fSLT in adults is acceptable.
Adult ; Aged ; End Stage Liver Disease/surgery* ; Female ; Humans ; Liver/surgery* ; Liver Transplantation/methods* ; Living Donors ; Male ; Middle Aged ; Postoperative Complications ; Retrospective Studies ; Severity of Illness Index ; Treatment Outcome