ObjectiveTo generate cynomolgus and rhesus monkey models carrying the Booroola fecundity (FecB) variant of bone morphogenetic protein receptor type 1B (BMPR1B) gene (designated BMPR1B^FecB) using adenine base edit technology and to explore genetic improvement strategies for enhancing the reproductive performance of laboratory macaques. MethodsFour single-guide RNAs (sgRNAs) were designed to target the BMPR1B c.746A>G locus in cynomolgus and rhesus monkeys. The sgRNAs were combined with the ABE8e-SpRY base editor, and the resulting editing complexes were microinjected into zygotes of both macaque species. Following in vitro culture of the injected embryos, nested PCR amplification was performed to detect the base-editing efficiency at the target site to screen for highly efficient sgRNAs. Subsequently, normally developing gene-edited embryos were transferred into the oviducts of surrogates. Seven days after birth, ear skin or peripheral blood samples were collected from the newborn monkeys for genotyping and mosaicism analysis, and the growth, development, and health status of the gene-edited monkeys were monitored. ResultsAmong the four candidate sgRNAs, sgRNA4 was identified as achieving 100% editing efficiency at the c.746A>G site. A total of 17 live monkey offspring were obtained via embryo transfer, including 8 cynomolgus monkeys and 9 rhesus monkeys. Genotyping results showed that all 8 newborn cynomolgus monkeys carried the c.746A>G point mutation, with an editing efficiency of 100% (8/8), of which 37.50% (3/8) were mosaics. Among the 9 newborn rhesus monkeys, 8 individuals carried the c.746A>G mutation, with an editing efficiency of 88.89% (8/9), of which 75.00% (6/8) were mosaics. ConclusionThe ABE8e-SpRY base-editing system enables precise editing at the c.746A>G (p.Q249R) locus of the BMPR1B gene in both cynomolgus and rhesus monkey embryos, resulting in the successful establishment of laboratory monkey models carrying the BMPR1B^FecB variant. These models provide a valuable foundation for future studies of the regulatory role of the BMPR1B^FecB variant in reproductive performance at the non‑human primate level, as well as for exploring genetic improvement strategies to enhance reproductive performance in laboratory monkeys.

BACKGROUND: Disease-modifying therapies have been approved for the treatment of relapsing multiple sclerosis (RMS). The present study aims to examine the safety of teriflunomide in Chinese patients with RMS. METHODS: This non-randomized, multi-center, 24-week, prospective study enrolled RMS patients with variant (c.421C>A) or wild type ABCG2 who received once-daily oral teriflunomide 14 mg. The primary endpoint was the relationship between ABCG2 polymorphisms and teriflunomide exposure over 24 weeks. Safety was assessed over the 24-week treatment with teriflunomide. RESULTS: Eighty-two patients were assigned to variant ( n  = 42) and wild type groups ( n  = 40), respectively. Geometric mean and geometric standard deviation (SD) of pre-dose concentration (variant, 54.9 [38.0] μg/mL; wild type, 49.1 [32.0] μg/mL) and area under plasma concentration-time curve over a dosing interval (AUC tau ) (variant, 1731.3 [769.0] μg∙h/mL; wild type, 1564.5 [1053.0] μg∙h/mL) values at steady state were approximately similar between the two groups. Safety profile was similar and well tolerated across variant and wild type groups in terms of rates of treatment emergent adverse events (TEAE), treatment-related TEAE, grade ≥3 TEAE, and serious adverse events (AEs). No new specific safety concerns or deaths were reported in the study. CONCLUSION: ABCG2 polymorphisms did not affect the steady-state exposure of teriflunomide, suggesting a similar efficacy and safety profile between variant and wild type RMS patients. REGISTRATION NCT04410965, https://clinicaltrials.gov .
Humans ; Crotonates/adverse effects* ; Toluidines/adverse effects* ; Nitriles ; Hydroxybutyrates ; Female ; Male ; Adult ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics* ; Middle Aged ; Multiple Sclerosis, Relapsing-Remitting/genetics* ; Prospective Studies ; Young Adult ; Neoplasm Proteins/genetics* ; East Asian People

BACKGROUND: Spatiotemporal disparities exist in the disease burden of non-communicable diseases (NCDs) attributable to kidney dysfunction, which has been poorly assessed. The present study aimed to evaluate the spatiotemporal trends of the global burden of NCDs attributable to kidney dysfunction and to predict future trends. METHODS: Data on NCDs attributable to kidney dysfunction, quantified using deaths and disability-adjusted life-years (DALYs), were extracted from the Global Burden of Diseases Injuries, and Risk Factors (GBD) Study in 2019. Estimated annual percentage change (EAPC) of age-standardized rate (ASR) was calculated with linear regression to assess the changing trend. Pearson's correlation analysis was used to determine the association between ASR and sociodemographic index (SDI) for 21 GBD regions. A Bayesian age-period-cohort (BAPC) model was used to predict future trends up to 2040. RESULTS: Between 1990 and 2019, the absolute number of deaths and DALYs from NCDs attributable to kidney dysfunction increased globally. The death cases increased from 1,571,720 (95% uncertainty interval [UI]: 1,344,420-1,805,598) in 1990 to 3,161,552 (95% UI: 2,723,363-3,623,814) in 2019 for both sexes combined. Both the ASR of death and DALYs increased in Andean Latin America, the Caribbean, Central Latin America, Southeast Asia, Oceania, and Southern Sub-Saharan Africa. In contrast, the age-standardized metrics decreased in the high-income Asia Pacific region. The relationship between SDI and ASR of death and DALYs was negatively correlated. The BAPC model indicated that there would be approximately 5,806,780 death cases and 119,013,659 DALY cases in 2040 that could be attributed to kidney dysfunction. Age-standardized death of cardiovascular diseases (CVDs) and CKD attributable to kidney dysfunction were predicted to decrease and increase from 2020 to 2040, respectively. CONCLUSION NCDs attributable to kidney dysfunction remain a major public health concern worldwide. Efforts are required to attenuate the death and disability burden, particularly in low and low-to-middle SDI regions.
Humans ; Noncommunicable Diseases/epidemiology* ; Global Burden of Disease ; Disability-Adjusted Life Years ; Male ; Female ; Risk Factors ; Middle Aged ; Kidney Diseases/epidemiology* ; Bayes Theorem ; Adult ; Aged ; Global Health ; Quality-Adjusted Life Years

ObjectiveTo investigate the risk factors for pyogenic liver abscess （PLA） comorbid with sepsis by analyzing clinical features， and to construct a predictive model. MethodsA retrospective analysis was performed for 489 patients who were hospitalized and diagnosed with PLA in The Affiliated Hospital of Xuzhou Medical University from January 2019 to December 2023， and according to the presence or absence of sepsis， they were divided into sepsis group with 306 patients and non-sepsis group with 183 patients. Related data were collected， including general information， laboratory markers， and outcome measures. The patients were further divided into a training set of 342 patients and a validation set of 147 patients at a ratio of 7∶3， and the training set was used for screening of variables and construction of a predictive model， while the validation set was used to test the performance of the model. An LASSO regression analysis was used for the screening of variables， and a multivariate Logistic regression analysis was used to construct the predictive model and plot a nomogram. The calibration curve， the receiver operating characteristic （ROC） curve， and the decision curve analysis were used for the validation of the model， and internal validation was performed for assessment. The independent-samples t test was used for comparison of normally distributed continuous data between two groups， and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups； the chi-square test was used for comparison of categorical variables between groups. ResultsThere were significant differences between the sepsis group and the non-sepsis group in pulse rate， mean arterial pressure， duration pf symptoms， comorbidity of liver cirrhosis or malignant tumor， leukocyte count， neutrophil count， lymphocyte count， platelet count （PLT）， activated partial thromboplastin time， fibrinogen， C-reactive protein， aspartate aminotransferase， alanine aminotransferase， albumin， total bilirubin （TBil）， creatinine， potassium， and prognostic nutritional index （PNI） （all P<0.05）. In the training set， the LASSO regression analysis identified four predictive factors of pulse rate， PLT， TBil and PNI， and the multivariate Logistic regression analysis showed that pulse rate （odds ratio ［OR］=1.033， 95% confidence interval ［CI］： 1.006‍ ‍—‍ ‍1.061， P=0.018）， PLT （OR=0.981， 95%CI： 0.975‍ ‍—‍ ‍0.987， P<0.001）， TBil （OR=1.086， 95%CI： 1.053‍ ‍—‍ ‍1.125， P<0.001）， and PNI （OR=0.935， 95%CI： 0.882‍ ‍—‍ ‍0.988， P=0.019） were independent influencing factors for the risk of sepsis in patients with PLA. The model constructed based on these factors showed a good predictive ability， with an area under the ROC curve of 0.948 （95%CI： 0.923‍ ‍—‍ ‍0.973） in the training set and 0.912 （95%CI： 0.848‍ ‍—‍ ‍0.976） in the validation set. The decision curve analysis showed that the model has a good net benefit within the range of 0.3‍ ‍—‍ ‍0.9 for threshold probability. ConclusionThe nomogram prediction model constructed based on pulse rate， PLT， TBil， and PNI has a certain clinical value and can well predict the risk of sepsis in patients with PLA.

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

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BACKGROUND:Some research has suggested that regulation of gut microbiota may influence the course of intervertebral disc degeneration.However,the causal relationship of gut microbiota on intervertebral disc degeneration is unknown.OBJECTIVE:To assess the potential causal relationship between gut microbiota and intervertebral disc degeneration using a Mendelian randomization method.METHODS:Genome-wide association analysis summary statistics for 473 gut microbiota and genome-wide association analysis summary data for intervertebral disc degeneration from the R11 of the FinnGen database(46 205 cases of intervertebral disc degeneration and 322 314 controls)from the most recent publicly available publication were applied.Inverse variance weighting,MR-Egger regression,weighted median,weighted modeling,and simple modeling were used to investigate the causal relationship between gut microbiota and intervertebral disc degeneration.Sensitivity analyses were used to test whether the results of Mendelian randomization analyses were reliable.Reverse Mendelian randomization was performed with all gut microbiota as the outcomes for effect analysis and sensitivity analysis.RESULTS AND CONCLUSION:(1)The results of the inverse variance weighting method of the forward Mendelian randomization method showed that the order Trichosporonaceae,the family UBA-6960,the family Anaerobes thermophilus,the family Salmonellaceae,the genus Pseudomonas tufts,the species Gordonella and the species Euclidia showed a positive correlation with intervertebral disc degeneration.The order Spirochaetes,the order Pseudomonas,the family Spirochaetaceae,the genus CAG-776,the genus Helicobacter,the species CAG-448 sp003150135,the species CAG-776 sp000438195,the species Brautella-A sp000285855 and the species Hanson's Brautella showed a negative correlation with intervertebral disc degeneration.(2)The results of reverse Mendelian randomization showed that intervertebral disc degeneration was positively correlated with the genus Bartonella rosea,the genus Geobacillus C,the species Escherichia fumigatus,the species Propionibacterium fumigatus,the species UBA-1777 sp900319835,the species Pseudomonas aeruginosa and the species Bacillus subtilis,while negatively correlated with the species Streptomyces mingoldii,the species Prevotella sp000434975,the species Brault's A sp000285855,the species CAG-194 sp002441865 and the species CAG-590 sp000431135.(3)No heterogeneity or horizontal pleiotropy was found in the two-way sensitivity analysis.(4)The results described above indicate that the causal relationship between gut microbiota and intervertebral disc degeneration based on the Finnish database contributes to the exploration on new biomarkers for the early prediction and treatment of intervertebral disc degeneration in clinical practice.In addition,the establishment of a large database and the integration of medical data from multiple centers can be drawn upon in biomedical research in China to provide a solid foundation for studying the relationship between gut microbiota and intervertebral disc degeneration.We will strengthen communication and cooperation with research teams in other countries to jointly promote the research on the relationship between gut microbiota and diseases and contribute to the development of global medicine.

Objective:To investigate the impact of different treatment sequences of immunotherapy combined with chemoradiotherapy (CRT) as the first-line therapy on the prognosis of patients with stage III non-small cell lung cancer (NSCLC).Methods:Clinical data of 112 patients with stage III NSCLC treated at the Fourth Hospital of Hebei Medical University from January 2019 to December 2021 were retrospectively collected, with follow-up continued until December 31, 2023. According to the sequence of CRT and immune checkpoint inhibitors (ICIs) therapy, patients were divided into 3 groups: ICIs simultaneous with CRT (sICR, n=20), chemotherapy combined with ICIs followed by CRT (CI-CR, n=53), and CRT followed by consolidative ICIs (CR-I, n=39). Analyses were performed before and after propensity score matching (PSM). Survival outcomes were assessed using the Kaplan-Meier method and compared by log-rank tests, and prognostic factors were identified through multivariate Cox regression analysis. Results:The median overall survival (OS) and progression-free survival (PFS) for the entire cohort were 30.1 months (95% CI: 21.4-38.9) and 12.8 months (95% CI: 9.14-16.1), respectively. Before PSM: No significant differences were observed in OS and PFS among the 3 groups ( χ2=0.18, 1.05; P=0.669, 0.305). However, OS in the sICR and CR-I groups was significantly better than that in the CI-CR group ( χ2=4.43, 6.11; P=0.035, 0.013). After PSM: Each group included 17 patients. There were no significant differences in OS or PFS among the 3 groups ( χ2=2.50, 2.74; P=0.287, 0.254), and pairwise comparisons also showed no significant differences. Multivariate Cox regression analysis revealed that clinical stage ( HR=3.392, 95% CI: 1.215-9.470, P=0.020), number of immunotherapy cycles ( HR=0.312, 95% CI: 0.100-0.972, P=0.044), and treatment response ( HR=6.566, 95% CI: 1.705-25.284, P=0.006) were independent prognostic factors for OS. After PSM, the numbers of patients with grade ≥2 treatment-related adverse events were 13 in the sICR group, 10 in the CI-CR group, and 9 in the CR-I group, with no significant differences among them ( χ2=2.181, P=0.336). Conclusions:First-line immunotherapy combined with chemoradiotherapy showed favorable clinical efficacy in locally advanced NSCLC compared to other studies, but the treatment sequence did not significantly affect prognosis. It is recommended that immunotherapy be administered for at least four cycles.

In order to establish a method for the detection of serum antibodies to donkey-derived e-quine coronavirus(ECoV),recombinant ECoV N protein was expressed in E.coli system,purified by nickel column affinity chromatography and identified by Western blot.After optimizing the re-action conditions,the indirect ELISA(iELISA)detection method was established using the puri-fied recombinant protein as coating antigen and used to detect 143 clinical serum samples.The re-sults showed that the recombinant N protein,which has good reaction activity with serum antibod-y,was successfully expressed.The optimum conditions of the established iELISA method were as follows:the amount of antigen coated was 0.2 μg/well and overnight at 4 ℃,10％skimmed milk powder solution was sealed at 37℃ for 1.5 h,the dilution concentration of serum was 1∶200,and the enzyme-labeled secondary antibody diluted at 1∶10 000.The sensitivity test results showed that the positive serum could be diluted to 1∶6 400.The specificity test results showed that all an-tibodies to several donkey pathogens were negative.The repetitive test results showed that the in-tra-and inter-batch coefficients of variation were 2.90％-6.12％and 2.29％-7.88％respectively.The positive rate of clinical donkey serum was 57.3％.The iELISA established in this study pro-vides a technical support for epidemiological investigation and antibody surveillance.