Objective To establish a simple technical procedure for preparing doggybone DNA(dbDNA),and to discuss the distinctions in gene expression and immune response between dbDNA and conventional plasmid DNA.Induced pluripotent stem cell(iPSC)was generated from human peripheral blood mononuclear cells(PBMCs)by using dbDNA.To investigate a protocol for inserting ultra-long DNA fragments into the iPSC adeno-associated virus site 1(AAVS1),concurrently with the integration of the Cas9 expression frame point into the dbDNA-derived iPSC AAVS1 safe harbor,so as to establish a stable cell line that can achieve iPSC gene editing exclusively through the introduction of the single guide RNA(sgRNA)for the targeted gene.Methods A simple dbDNA preparation technique was established through utilization of Phi29 DNA polymerase multiple displacement amplification,protelomerase TelN digestion and covalent ligation,as well as the subsequent removal of the back-bone loop through the action of a restriction enzyme and exonuclease.The efficiency of green fluorescent protein(GFP)was eval-uated following electroporation of the dbDNA-GFP vector and the pMax-dbDNA-GFP traditional plasmid vector into PB-MCs.The mRNA expression of interferon-γ(IFN-γ)in the two groups was quantified by qPCR.The dbDNA reprogramming vectors dbDNA-MOS,dbDNA-KLF4,dbDNA-MYC and dbDNA-BCL were prepared and subsequently transferred into PBMCs to induce reprogramming.The dbDNA-iPSC cell line was identified through morphological analysis,alkaline phosphatase stai-ning,RT-PCR,and detection of pluripotent stem cell markers.The ZFN and TALEN gene editing techniques were employed to insert an 11.5 kb ultra-long DNA fragment containing the Cas9 expression cassette,EGFP reading frame with stop codons in the sequence,a eukaryotic cell screening marker and other elements at the iPSC AAVS1.The identification of the iPSC lines with Cas9 integrated into AAVS1 was conducted through the utilization of red fluorescence detection,genomic PCR reaction and Western blot.The EGFP single-strand homologous repair template and EGFP sgRNA were co-transfected into positive iPSC cell lines to repair the erroneous stop codons in the EGFP gene and to express green fluorescent protein correctly.This approach was undertaken in order to verify the gene editing ability of the Cas9 protein.Results The dbDNA vector was successfully pre-pared and employed for gene expression.The GFP gene expression efficiency of dbDNA was similar to that of traditional plas-mid DNA.The expression level of IFN-γ mRNA in the dbDNA group was lower than that of traditional plasmid DNA group.The iPSC cell lines resembling human embryonic stem cells were successfully obtained,iPSC cell lines with positive alka-line phosphatase staining,pluripotency related gene expression and stem cell pluripotency markers.The ultra-long fragment CRISPR-Cas9 gene editing reporting system was successfully inserted into the safe harbor AAVS1 site of the cell line,and the integrated fragment was successfully inserted into the genome by genomic PCR.Red fluorescent protein was expressed.Cas9 protein expression and EGFP gene recovery was confirmed by Western blot.Green fluorescence was observed.Conclusion A simple technical procedure for the preparation of dbDNA is successfully established.The gene expression efficiency of the dbD-NA vector is comparable to that of traditional plasmid DNA,with less immune response.The dbDNA-derived iPSCs are success-fully obtained,which is confirmed by morphological analysis,alkaline phosphatase staining,RT-PCR,and stem cell pluripotency markers.An 11.5 kb ultra-long DNA fragment is successfully inserted into the iPSC AAVS1 safe harbor to establish a stable iPSC cell line with Cas9-mediated gene editing.

Objective To establish a simple technical procedure for preparing doggybone DNA(dbDNA),and to discuss the distinctions in gene expression and immune response between dbDNA and conventional plasmid DNA.Induced pluripotent stem cell(iPSC)was generated from human peripheral blood mononuclear cells(PBMCs)by using dbDNA.To investigate a protocol for inserting ultra-long DNA fragments into the iPSC adeno-associated virus site 1(AAVS1),concurrently with the integration of the Cas9 expression frame point into the dbDNA-derived iPSC AAVS1 safe harbor,so as to establish a stable cell line that can achieve iPSC gene editing exclusively through the introduction of the single guide RNA(sgRNA)for the targeted gene.Methods A simple dbDNA preparation technique was established through utilization of Phi29 DNA polymerase multiple displacement amplification,protelomerase TelN digestion and covalent ligation,as well as the subsequent removal of the back-bone loop through the action of a restriction enzyme and exonuclease.The efficiency of green fluorescent protein(GFP)was eval-uated following electroporation of the dbDNA-GFP vector and the pMax-dbDNA-GFP traditional plasmid vector into PB-MCs.The mRNA expression of interferon-γ(IFN-γ)in the two groups was quantified by qPCR.The dbDNA reprogramming vectors dbDNA-MOS,dbDNA-KLF4,dbDNA-MYC and dbDNA-BCL were prepared and subsequently transferred into PBMCs to induce reprogramming.The dbDNA-iPSC cell line was identified through morphological analysis,alkaline phosphatase stai-ning,RT-PCR,and detection of pluripotent stem cell markers.The ZFN and TALEN gene editing techniques were employed to insert an 11.5 kb ultra-long DNA fragment containing the Cas9 expression cassette,EGFP reading frame with stop codons in the sequence,a eukaryotic cell screening marker and other elements at the iPSC AAVS1.The identification of the iPSC lines with Cas9 integrated into AAVS1 was conducted through the utilization of red fluorescence detection,genomic PCR reaction and Western blot.The EGFP single-strand homologous repair template and EGFP sgRNA were co-transfected into positive iPSC cell lines to repair the erroneous stop codons in the EGFP gene and to express green fluorescent protein correctly.This approach was undertaken in order to verify the gene editing ability of the Cas9 protein.Results The dbDNA vector was successfully pre-pared and employed for gene expression.The GFP gene expression efficiency of dbDNA was similar to that of traditional plas-mid DNA.The expression level of IFN-γ mRNA in the dbDNA group was lower than that of traditional plasmid DNA group.The iPSC cell lines resembling human embryonic stem cells were successfully obtained,iPSC cell lines with positive alka-line phosphatase staining,pluripotency related gene expression and stem cell pluripotency markers.The ultra-long fragment CRISPR-Cas9 gene editing reporting system was successfully inserted into the safe harbor AAVS1 site of the cell line,and the integrated fragment was successfully inserted into the genome by genomic PCR.Red fluorescent protein was expressed.Cas9 protein expression and EGFP gene recovery was confirmed by Western blot.Green fluorescence was observed.Conclusion A simple technical procedure for the preparation of dbDNA is successfully established.The gene expression efficiency of the dbD-NA vector is comparable to that of traditional plasmid DNA,with less immune response.The dbDNA-derived iPSCs are success-fully obtained,which is confirmed by morphological analysis,alkaline phosphatase staining,RT-PCR,and stem cell pluripotency markers.An 11.5 kb ultra-long DNA fragment is successfully inserted into the iPSC AAVS1 safe harbor to establish a stable iPSC cell line with Cas9-mediated gene editing.

Objective To develop and validate risk prediction models utilizing five machine learning algorithms for assessing postoperative pulmonary infection(PPI)risk in lung cancer patients undergoing grade Ⅳ thoracoscopic surgery.Methods A retrospective cohort study included 2,380 lung cancer patients who underwent grade Ⅳ thoracoscopic surgery at a tertiary hospital in Shanghai(January 2022 to June 2024).Patients were stratified into training(n=1,665)and validation(n=715)cohorts.Five machine learning algorithms—Logistic regression(LR),artificial neural network(ANN),support vector machine(S VM),random forest(RF),and extreme gradient boosting(XGB)—were employed to construct predictive models.A nomogram was developed for clinical utility.Results Among 2,380 patients,226(9.5％)developed PPI.The Least Absolute Shrinkage and Se-lection Operator(LASSO)regression identified eight predictive variables:daily cigarette consumption,diabetes history,preoperative diffusing capacity,maximal tumor diameter,24-hour postoperative chest drainage volume,perioperative oral nutritional supplementation(ONS),postoperative urinary cathe-terization,and intraoperative pleural adhesion severity.All models demonstrated robust discrimina-tion,with area under the curve(AUC)values ranging from 0.862 to 0.947.The XGB model a-chieved superior performance(AUC=0.947,95％CI,0.937 to 0.962),followed closely by the LR model(AUC=0.926,95％CI,0.918 to 0.933).Conclusion Machine learning-based algo-rithms models effectively stratify PPI risk in lung cancer patients following grade Ⅳ thoracoscopic surgery.The derived nomogram provides a practical tool for perioperative risk management by healthcare providers.

ObjectiveTo develop a scientifically rigorous and contextually appropriate instrument for evaluating the health service experience of pulmonary tuberculosis patients in China, to enable systematic assessment of core medical care dimensions, and to provide quantitative evidence for service improvement. MethodsGrounded in the theoretical framework of healthcare accessibility and the clinical care pathway for tuberculosis patients, the tool was developed through a systematic literature review and the Delphi expert consultation method. A multi-stage cluster sampling strategy was employed to survey pulmonary tuberculosis patients who had been receiving treatment for more than two months, aimed to explore the scale’s applicability in real-world settings. Reliability was assessed using Cronbach’s α and split-half reliability coefficients. Validity was evaluated through content validity, structural validity, convergent validity, and discriminant validity. ResultsThe tool was composed of 21 items across four dimensions: awareness, accessibility, affordability, and acceptability of tuberculosis medical care. It demonstrated a Cronbach’s α coefficient of 0.838 and a split-half reliability coefficient of 0.859. Exploratory factor analyses extracted six factors: satisfaction with healthcare services, supportive role of nurses, affordability of treatment costs, doctor-patient communication, waiting time for medical appointments, and transportation cost. The goodness-of-fit index (GFI) and other indices met the recommended standards, with the loading matrix indicating robust structural validity of the tool. The constructed factor model exhibited satisfactory content validity and discriminant validity. ConclusionThe scale for assessing patients’ experiences with tuberculosis-related medical care developed in this study demonstrates good reliability and validity and serves as a practical tool for evaluating patient experiences of tuberculosis medical care in China.

Objective:To analyze the clinical characteristics and outcomes of patients with invasive fungal sinusitis (invasive fungal rhinosinusitis, IFR) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and explored the risk factors for IFR after allo-HSCT.Methods:Nineteen patients with IFR after allo-HSCT at Peking University People’s Hospital from January 2012 to December 2021 were selected as the study group, and 95 patients without IFR after allo-HSCT during this period were randomly selected as the control group (1:5 ratio) .Results:Nineteen patients, including 10 males and 9 females, had IFR after allo-HSCT. The median age was 36 (10–59) years. The median IFR onset time was 68 (9–880) days after allo-HSCT. There were seven patients with acute myeloid leukemia, five with acute lymphoblastic leukemia, two with myelodysplastic syndrome, two with chronic myeloid leukemia, one with acute mixed-cell leukemia, one with multiple myeloma, and one with T-lymphoblastic lymph node tumor. There were 13 confirmed cases and 6 clinically diagnosed cases. The responsible fungus was Mucor in two cases, Rhizopus in four, Aspergillus in four, and Candida in three. Five patients received combined treatment comprising amphotericin B and posaconazole, one patient received combined treatment comprising voriconazole and posaconazole, nine patients received voriconazole, and four patients received amphotericin B. In addition to antifungal treatment, 10 patients underwent surgery. After antifungal treatment and surgery, 15 patients achieved a response, including 13 patients with a complete response and 2 patients with a partial response. Multivariate analysis revealed that neutropenia before transplantation ( P=0.021) , hemorrhagic cystitis after transplantation ( P=0.012) , delayed platelet engraftment ( P=0.008) , and lower transplant mononuclear cell count ( P=0.012) were independent risk factors for IFR after allo-HSCT. The 5-year overall survival rates in the IFR and control groups after transplantation were 29.00%±0.12% and 91.00%±0.03%, respectively ( P<0.01) . Conclusion:Although IFR is rare, it is associated with poor outcomes in patients undergoing allo-HSCT. The combination of antifungal treatment and surgery might be effective.

Objective To analyze the risk factors for the occurrence of rhegmatogenous retinal detach-ment(RRD)in the fellow eye of the patients with monocular RRD.Methods A single-center retrospective cohort study was conducted.The information such as the gender,age,laterality,bilateral best corrected visual acuity(BCVA),bilateral refractive error,whether the fellow eye implanting an intraocular lens,and location distribution of retinal tears or degenerative areas(such as lattice,cystic,and snail track degeneration)in the fellow eye of 331 patients with unilateral RRD surgery in this hospital from January 2018 to December 2020 was analyzed.The patients were followed up for one year,during this period,the prophylactic laser photocoag-ulation was performed if retinal tears or degenerative areas occurred in the fellow eye.The risk factors for RRD occurrence in the fellow eye were analyzed by the logistic regression.The Kappa value was calculated.The location distribution of retinal tears or degenerative areas in the affected eye and the fellow eye conducted the consistency analysis.Results The influencing factors for RRD occurrence of the fellow eye in the two groups mainly included the age,preoperative BCVA and preoperative refractive error(P＜0.05).Logistic re-gression analysis revealed that the age 40 to＜60 years old(OR=6.906,P＜0.001),preoperative BCVA＜0.05(OR=3.015,P＜0.001)and refractive error-3.00 to＜-6.00 D(OR=5.511,P＜0.001)were sig-nificant independent risk factors for the occurrence of retinal tears or degenerative areas in the fellow eye.The age＜40 years old(OR=0.101,P＜0.001)and refractive error+0.50 D-＜-0.50 D(OR=0.160,P=0.001)were the protective factors.The numbers of cases in the fellow eye with retinal tears or degenerative areas in the 4 quadrants were 59,14,27,and 8,respectively.In the affected eye,the numbers of cases with reti-nal tears or degenerative areas in the same quadrants were 43,6,10,and 3,respectively.The Kappa value was 0.296,P＜0.001.All patients with RRD or lattice degeneration in the fellow eye received prophylactic laser photocoagulation,and no further retinal detachment occurred during the one-year follow-up period.Conclusion Ai-ming at the patients with unilateral RRD,the contralateral eye should be examined in detail,especially in mid-dle-aged and elderly patients with BCVA＜0.50 and diopter-3.00-＜-6.00 D,focusing on the retinal quadrant of the contralateral eye that is the same as the affected eye hole,and the incidence of contralateral RRD should be effectively reduced through regular and comprehensive eye examination.

Objective To investigate the effects of intrauterine perfusion with granulocyte colony-stimulating factor(G-CSF)on the endometrial thickness,volume,and blood flow parameters of patients with thin endometrium and their clinical outcomes.Methods We designed a prospective non-randomized synchronous controlled trial and recruited patients with thin endometrium who underwent frozen-thawed embryo transfer(FET)at Mianyang Central Hospital between September 1,2021 and September 1,2023.They were divided into two groups,an experimental group of patients who received the experimental treatment of intrauterine perfusion with G-CSF and a control group of patients who did not receive the experimental treatment.The general data and the clinical outcomes of the two groups were analyzed and compared.The endometrial thickness,volume and blood flow parameters of patients in the experimental group before and after intrauterine perfusion with G-CSF were analyzed.Results The clinical data of 83 patients were included in the study.The experimental group included 51 cases,while the control group included 31 cases.There were no significant differences in the baseline data between the two groups.The clinical pregnancy rate of the experimental group(56.86% )was higher than that of the control group(50.00% )and the rate of spontaneous abortion in the experimental group(27.59% )was lower than that in the control group(37.50% ),but the differences were not statistically significant(P>0.05).In the experimental group,the postperfusion endometrial thickness([0.67±0.1]cm)was greater than the preperfusion endometrial thickness([0.59±0.09]cm),the postperfusion([1.84±0.81]cm3)was greater than the preperfusion endometrial volume([1.54±0.69]cm3),and the postperfusion vascularization flow index(VFI)(1.97±2.82)was greater than the preperfusion VFI(0.99±1.04),with all the differences being statistically significant(P<0.05).Conclusion Intrauterine perfusion with G-CSF can enhance the endometrial thickness,volume,and some blood flow parameters in patients with thin endometrium.

With the development of molecular biology, biomaterials and tissue engineering, regenerative treatment of pulpal and periradicular diseases is facing new opportunities. At present, a large number of studies on dental pulp regeneration reveal that cytokines are essential for promoting migration, proliferation and osteogenic differentiation of dental pulp stem cells. In this paper, we review several kinds of cytokines related to dental pulp regeneration, and analyze their roles and regulatory mechanisms in dental pulp regeneration.

Pancytopenia is one of the serious complications of Graves disease, and its clinical treatment is quite challenging. Based on traditional Chinese medicine theory and combining with literature reports and clinical practice in China, we discuss the etiology, pathogenesis, and syndromes-based treatment of pancytopenia, hoping to open up new treatment approaches, guide clinical practice, and improve treatment effectiveness.

Background: and Purpose We aimed to determine the clinical features of Miller Fisher syndrome (MFS) in southern China and compare them with those presenting in other countries. Methods: We collected the medical records of patients diagnosed with MFS during 2013–2016.We analyzed the age, sex, onset season, precursor events, clinical symptoms and signs, findings of nerve conduction studies (NCS), cerebrospinal fluid (CSF), therapeutic remedies, nadir time, and length of hospital stay of patients with MFS in southern China. We concurrently compared the differences between urban and rural areas and between patients with incomplete ophthalmoplegia (IO) and complete ophthalmoplegia (CO). Results: The study enrolled 72 patients: 36 from rural areas and 36 from urban areas, and 50 males and 22 females. The mean age at onset was 47.72 years, and 30 (41.7%) and 21 (29.2%) patients developed MFS in spring and winter, respectively. The typical triad of ophthalmoplegia, ataxia, and areflexia was observed in 50 (69.4%) patients. A history of upper respiratory tract infection 1 week before onset was found in 52.8% of the patients, while 5.6% experienced gastrointestinal infections and 48 (73.8%) exhibited albuminocytological dissociation in the CSF study. Only 26 (36.1%) patients presented abnormalities in NCS. Moreover, restricted outward eyeball movement presented in 83.5% of the patients with classic MFS and acute ophthalmoplegia, and bilateral symmetrical ophthalmoplegia presented in 64.2%. With the exception of the higher proportion of NCS abnormalities in urban areas (47.2% vs. 25.0%), urban and rural differences were insignificant regarding sex ratio, age at onset, high-incidence season, precursor events, disease characteristics, and albuminocytological dissociation in the CSF. Furthermore, patients with CO were older than those with IO (64.53±7.69 vs. 43.19±14.40 years [mean±standard deviation], p<0.001). Conclusions The patients with MFS were mostly male and middle-aged, and most presented in winter and (especially) spring. More than half of the patients had clear precursor events, most of which were classic MFS with the typical triad. More than 70% of the patients presented albuminocytological dissociation in the CSF. NCS abnormalities were uncommon in MFS. The age at onset was lower in patients with IO than in patients with CO; bilateral symmetrical extraocular muscle paralysis was the most common symptom, and the external rectus was the most frequently involved muscle.