Objective To explore the feasibility and reference value of allogeneic lung transplantation and postoperative monitoring in miniature pigs for lung transplantation research. Methods Two miniature pigs (R1 and R2) underwent left lung allogeneic transplantation. Complement-dependent cytotoxicity tests and blood cross-matching were performed before surgery. The main operative times and partial pressure of arterial oxygen (PaO₂) after opening the pulmonary artery were recorded during surgery. Postoperatively, routine blood tests, biochemical blood indicators and inflammatory factors were detected, and pathological examinations of multiple organs were conducted. Results The complement-dependent cytotoxicity test showed that the survival rate of lymphocytes between donors and recipients was 42.5%-47.3%, and no agglutination reaction occurred in the cross-matching. The first warm ischemia times of D1 and D2 were 17 min and 10 min, respectively, and the cold ischemia times were 246 min and 216 min, respectively. Ultimately, R1 and R2 survived for 1.5 h and 104 h, respectively. Postoperatively, in R1, albumin (ALB) and globulin (GLB) decreased, and alanine aminotransferase increased; in R2, ALB, GLB and aspartate aminotransferase all increased. Urea nitrogen and serum creatinine increased in both recipients. Pathological results showed that in R1, the transplanted lung had partial consolidation with inflammatory cell infiltration, and multiple organs were congested and damaged. In R2, the transplanted lung had severe necrosis with fibrosis, and multiple organs had mild to moderate damage. The expression levels of interleukin-1β and interleukin-6 increased in the transplanted lungs. Conclusions The allogeneic lung transplantation model in miniature pigs may systematically evaluate immunological compatibility, intraoperative function and postoperative organ damage. The data obtained may provide technical references for subsequent lung transplantation research.

Owing to their high genetic and physiological similarities to humans,non-human primates(NHPs)have become pivotal animal models in life sciences research and biomedical development.NHP laboratory animals are not only an ideal platform for exploring the mechanisms of neurological diseases and infectious diseases,but they are also widely used in preclinical safety evaluations of macromolecular drugs,which are considered the"gold standard".Nevertheless,this biological similarity increases the risk of zoonotic disease transmission to personnel working with NHP laboratory animals,their tissues,and body fluids.In light of recent domestic and international outbreaks of zoonotic diseases as well as the implementation of the Biosafety Law,this study examines the occupational risk factors encountered by personnel working with NHPs.This includes biological,chemical,and physical factors.This paper also covers common zoonoses,classification of the corresponding pathogens,transmission routes,risk severity levels,and protocols for post-exposure management.A multidimensional prevention and control framework is proposed,which includes the following components.(1)Risk Assessment and Emergency Response:Regularly identify hazards through an Occupational Health and Safety Committee(OHSC)and develop post-exposure emergency protocols.(2)Optimization of Management Systems:Improve facility design,optimize the allocation of personal protective equipment,and enhance health surveillance and vaccination programs.(3)Technical Training and Standardized Operations:Provide specialized training in NHP laboratory animal ethology and biosafety practices.Additionally,implement intelligent monitoring technologies to reduce the occurrence of aggressive incidents.This paper outlines measures designed to enhance health and safety awareness among personnel working with NHP laboratory animals.It emphasizes the need for strengthened guidance on the use of personal protective equipment(PPE)and the standardization of professional operational practices.The goal is to safeguard personnel health and safety,reduce occupational exposure rates,and effectively prevent occupational diseases related to laboratory animals.

Objective Three different doses of diethylnitrosamine(DEN)were used to establish a rat primary liver cancer(PLC)model to establish an efficient,stable,and economical animal model of PLC.Methods Forty-five male SD rats were randomly divided into four groups:normal group,DEN 50 mg/kg dose group(low dose group),70 mg/kg dose group(medium dose group),and 200 mg/kg dose group(high dose group).There were 6 animals in the normal group and 13 animals in each of the other groups.The normal control group received no treatment.The model group and low dose groups were injected intraperitoneally twice a week during weeks 1～4 and once a week during weeks 5～12;the medium dose group was injected intraperitoneally once a week for 16 consecutive weeks;and the high dose group was administered only once in the first week.The rats in each group were then followed for 16 weeks.The establishment of the model and optimal evaluation were verified by survival rate,pathological tests,biochemical tests,liver and spleen index calculation,immunohistochemistry,enzyme-linked immunosorbent assay(ELISA),and other assays.Results The survival rate was 100％in the normal group,46.15％in the low dose group,69.23％in the medium dose group,and 84.61％in the high dose group.The liver tissues of the rats in the normal group showed no abnormality to the naked eye;the liver of the rats in the low dose group became darker in color,rougher in surface,with a small number of cancerous nodules and slightly hard texture;the liver of the rats in the medium dose group was rough in surface,with several small cancerous nodules and scattered massive occupying nodules and hard texture;The liver of rats in the high dose group became lighter in color,slightly rougher in surface,with no obvious cancerous nodules;HE staining showed that the liver tissues of rats in the low and medium dose groups were structurally disorganized,with large cellular heterogeneity and tumor cells.HE staining showed that the liver tissues of rats in the low and medium dose groups were structurally disorganized,with large cellular heterogeneity and tumor cell formation,while the structure of the liver lobules of the high dose group was unclear,with different degrees of edema,degeneration and necrosis of liver cells,and no obvious tumor cell formation was seen.Compared with the normal group,serum liver function alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBIL)were elevated in the low,medium,and high dose groups;ALT and AST were significantly elevated in the low dose group(P<0.05),the difference was statistically significant,ALT,AST and TBIL were significantly elevated in the medium dose group(P<0.05),the difference was statistically significant,and the difference was statistically significant,although liver function in the high dose group was elevated,he increase was not significant,the difference was not statistically significant(P>0.05);compared with the normal group,the international normalized ratio(INR)of coagulation function was significantly higher in the low dose group,with a statistically significant difference(P<0.05),and the activated partial thromboplastin time(APTT),prothrombin time(PT),and alpha-fetoprotein(AFP)levels were increased(P<0.05),and the difference was not statistically significant;serum APTT,PT,INR,and AFP levels were significantly increased in the medium dose group(P<0.05),and the difference was statistically significant;serum PT and AFP levels were increased in the high dose group(P<0.05),the difference was statistically significant,and plasma APTT levels were slightly increased(P>0.05),the difference was not statistically significant;liver and spleen indexes were increased in the medium dose group(P<0.05),the spleen index increased in the low dose group(P<0.05),and the liver index increased in the high dose group(P<0.05),the difference was statistically significant;the optical density value of liver tissue AFP increased significantly in the low,medium and high dose groups(P<0.05),the difference was statistically significant.Conclusions Both the low and medium dose groups could successfully induce the PLC rat model,but the pathological changes and biochemical findings of the medium dose group were more consistent with the pathogenesis of human liver tissue from liver injury to hepatic fibrosis to cirrhosis to hepatocellular carcinoma,and the number of administrations of the drug is less,and the survival rate of the rats is higher so that a more cost-effective and superior PLC model can be established.

Owing to their high genetic and physiological similarities to humans,non-human primates(NHPs)have become pivotal animal models in life sciences research and biomedical development.NHP laboratory animals are not only an ideal platform for exploring the mechanisms of neurological diseases and infectious diseases,but they are also widely used in preclinical safety evaluations of macromolecular drugs,which are considered the"gold standard".Nevertheless,this biological similarity increases the risk of zoonotic disease transmission to personnel working with NHP laboratory animals,their tissues,and body fluids.In light of recent domestic and international outbreaks of zoonotic diseases as well as the implementation of the Biosafety Law,this study examines the occupational risk factors encountered by personnel working with NHPs.This includes biological,chemical,and physical factors.This paper also covers common zoonoses,classification of the corresponding pathogens,transmission routes,risk severity levels,and protocols for post-exposure management.A multidimensional prevention and control framework is proposed,which includes the following components.(1)Risk Assessment and Emergency Response:Regularly identify hazards through an Occupational Health and Safety Committee(OHSC)and develop post-exposure emergency protocols.(2)Optimization of Management Systems:Improve facility design,optimize the allocation of personal protective equipment,and enhance health surveillance and vaccination programs.(3)Technical Training and Standardized Operations:Provide specialized training in NHP laboratory animal ethology and biosafety practices.Additionally,implement intelligent monitoring technologies to reduce the occurrence of aggressive incidents.This paper outlines measures designed to enhance health and safety awareness among personnel working with NHP laboratory animals.It emphasizes the need for strengthened guidance on the use of personal protective equipment(PPE)and the standardization of professional operational practices.The goal is to safeguard personnel health and safety,reduce occupational exposure rates,and effectively prevent occupational diseases related to laboratory animals.

Objective:To investigate the value of anlotinib combined with radiotherapy in second-line targeted drug-resistant advanced lung adenocarcinoma with epidermal growth factor receptor(EGFR) mutation.Methods:A total of 150 patients with advanced lung adenocarcinoma with EGFR mutation who were resistant to second-line targeted therapy and admitted to the Affiliated Hospital of Shaoxing University from July 2021 to July 2023 were prospectively selected as the study objects, and they were divided into the control group and the study group by random number table method, with 75 cases in each group. The control group received radiotherapy, and the study group received anrotinib combined radiotherapy. The recent clinical effects, tumor indexes, cellular immune indexes and the occurrence of adverse reactions were compared between the two groups.Results:After treatment, the objective response rate (ORR) and disease control rate (DCR) in the study group were higher than those in the control group: 53.33%(40/75) vs. 34.67%(26/75), 86.67%(65/75) vs. 64.00%(48/75), there were statistical differences ( χ2 = 5.30, 10.37, P<0.05). After treatment, the levels of VEGF, cytokeratin 21-1 fragment (CYFRA21-1) and carcinoembryonic antigen (CEA) in the study group were lower than those in the control group: (472.93 ± 45.71) ng/L vs. (510.26 ± 50.49) ng/L, (4.82 ± 1.13) μg/L vs. (5.25 ± 1.34) μg/L, (5.65 ± 1.24) μg/L vs. (6.17 ± 1.42) μg/L, there were statistical differences ( P<0.05). After treatment, the levels of CD 3+, CD 4+ and CD 4+/CD 8+ in the study group were higher than those in the control group, and the level of CD 8+ was lower than that in the control group: 0.532 ± 0.044 vs. 0.508 ± 0.041, 0.321 ± 0.030 vs. 0.305 ± 0.027, 1.02 ± 0.19 vs. 0.93 ± 0.16, 0.303 ± 0.040 vs. 0.320 ± 0.044, there were statistical differences ( P<0.05). There was no significant difference in the occurrence of adverse reactions between the two groups ( P>0.05). Conclusions:Anlotinib combined with radiotherapy can enhance the clinical efficacy of second-line targeted drug resistant advanced lung adenocarcinoma with EGFR mutation, improve tumor indexes, promote the recovery of immune function, without significantly increasing adverse reactions.

Objectives:To explore whether short-course radiotherapy (SCRT)-based total neoadjuvant therapy (TNT) combined with PD-1 inhibitors could further promote tumor regression and improve the prognosis.Methods:This is a prospective, multicenter, two-arm randomized controlled, seamless phase Ⅱ/Ⅲ trial for proficient mismatch repair or microsatellite stable (pMMR/MSS) locally advanced rectal cancer (LARC). Eligible patients were randomly assigned to the iTNT (TNT+PD-1) group or the TNT group. Patients in the TNT group received SCRT (5 Gy×5) followed by 4 cycles of CAPOX or 6 cycles of mFOLFOX chemotherapy, with the iTNT group receiving SCRT followed by the same regime in combination with 4 cycles of Sintilimab. Total mesorectal excision (TME) surgery or watch and wait (W&W) was performed after neoadjuvant therapy and then 2 cycles of same regimen as before were recommended. The primary endpoints are the complete response (CR) rate for phase Ⅱ trial and 3-year disease-free survival (DFS) for phase Ⅲ trial. A total of 588 patients will be enrolled for the phase Ⅱ/Ⅲ trial. Short-term efficacy and safety data from the initial 100 treated patients were analyzed as planned.Results:From 2022-8-31 to 2023-5-24 the initial 100 patients were enrolled from 10 hospitals in China, 76.0%(76/100) patients were male, and the median age was 61 years (21-74 years). More patients had tumors located in the lower rectum (78.0%, 78/100), staged T3-4 (97.0%, 97/100) and N1-2 (93.0%, 93/100), and about half of the tumors invaded the mesorectal fascia (52.0%, 52/100) and with extramural vascular invasion (51.0%, 51/100). Analyses were performed according to the per-protocal (PP) set. All patients in the iTNT group ( n=52) and the TNT group ( n=48) completed SCRT; The 4-cycle chemotherapy±Sintilimab completion rates were 86.5% and 100.0% in the iTNT and TNT groups, respectively. In the iTNT group, 82.7% (43/52), 11.5% (6/52), and 5.8% (3/52) of the patients received 4, 3, and 2 cycles of PD-1 inhibitor. After TNT, 68 patients underwent radical surgery and 15 patients achieved cCR and adopted W&W. The pathological complete response (pCR) rates were 48.5% (16/33) and 17.1% (6/35) in the iTNT and TNT groups, with CR rates of 50.0% (25/50) and 26.1% (12/46), respectively. The incidence of treatment-related grade 3-4 adverse events was 26.9% (14/52, iTNT group) and 18.8% (9/48, TNT group), with thrombocytopenia and leukopenia being the most common. Among patients receiving immunotherapy, grade 3 immunotherapy-related adverse events occurred in 2 (3.8%, 2/52) patients: one case was pancreatitis, another case was hepatitis combined with myositis and myocarditis. Conclusion:The preliminary results show that SCRT-based TNT combined with PD-1 inhibitors could further improve the CR rate for LARC without unexpected serious adverse events.

BACKGROUND:Angiogenesis is the main treatment target of cardiovascular diseases.Bone morphogenetic protein 2 can modulate angiogenesis,but the regulatory effect of endothelial cell-specific bone morphogenetic protein 2 on angiogenesis is unclear. OBJECTIVE:To investigate the effect of endothelial-specific bone morphogenetic protein 2 on angiogenesis. METHODS:(1)Bioinformatics analysis:Cellular expression specificity and abundance of bone morphogenetic protein 2 were meta-analyzed by the PanglaoDB single-cell transcriptome database.The endothelial cell transcriptome sequencing dataset of the mouse hindlimb model and endocardial transcriptome dataset of mice overexpressing bone morphogenetic protein 2 were reanalyzed to evaluate the effect of endothelial cell bone morphogenetic protein 2 on the angiogenesis pathway.(2)Validation in vivo:After establishing the mouse hindlimb model,we compared the blood perfusion between the affected and sham limb at 7,14,and 21 days.The expression of the colocation of bone morphogenetic protein 2 and CD31 was explored by immunofluorescence and immunohistochemical staining.(3)Validation in vitro:The cultured human umbilical vein endothelial cells in vitro were divided into a control group,a hypoxia group,and a bone morphogenetic protein 2 inhibitor Noggin intervention group.After being cultured for 24 hours,the angiogenesis of endothelial cells in each group was observed. RESULTS AND CONCLUSION:(1)Endothelial cells are an important cell subgroup expressing bone morphogenetic protein 2.Both in the mouse hindlimb ischemia model and endocardial cells overexpressing bone morphogenetic protein 2,bone morphogenetic protein 2 was significantly up-regulated,and the angiogenesis pathway was significantly activated.(2)In the mouse hindlimb model,bone morphogenetic protein 2-positive blood vessels around neoangiogenesis increased significantly at 7 days of ischemia(P<0.05),and decreased significantly after 2 weeks of ischemia(P<0.001).(3)In umbilical vein endothelial cells cultured in vitro,after hypoxic intervention,the migration and sprouting of endothelial cells increased significantly,and the expression of angiogenesis factors vascular endothelial growth factor and platelet-derived growth factor was significantly increased.Noggin significantly reduced hypoxia-induced endothelial cell angiogenesis(P<0.001)and down-regulated the expression of vascular endothelial growth factor and platelet-derived growth factor(P<0.01).(4)These findings verify that endothelial cell-specific bone morphogenetic protein 2 can regulate angiogenesis,and targeting endothelial cell bone morphogenetic protein 2 is a promising way to improve angiogenesis.

OBJECTIVE: To investigate the clinical phenotype and genetic diagnosis process of fetuses with 21 hydroxylase deficiency (21-OHD) caused by compound heterozygous variant of the CYP21A2 gene . METHODS: A fetus who was diagnosed at Taizhou Hospital in Zhejiang Province on December 4, 2020 due to unclear characteristics of external genitalia on ultrasound was selected as the study subject. Chromosome copy number variation sequencing (CNV-seq) and whole exome sequencing (WES) were performed on amniotic fluid samples. Candidate variants were validated by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA), and short tandem repeat (STR) analysis was used to exclude maternal blood contamination. The pathogenic mechanism of the variants was further explored. The procedure followed by this study was approved by the Medical Ethics Committee of Taizhou Hospital (Ethics No.: K20201009). RESULTS: The MRI examination of the fetal external genitalia showed thickening of labia minora and enlargement of the clitoris. The CNV-seq results of the fetus showed no significant abnormality. The WES results showed that the fetus had a homozygous c.293-13C>G variant in the CYP21A2 gene (NM-000500.9). STR testing excluded maternal blood contamination. Sanger sequencing verified the presence of heterozygous c.293-13C>G variant of the CYP21A2 gene in the fetus and its mother, while its father did not detect this mutation. Further MLPA testing results showed that the fetus and its father had heterozygous deletion (I2G-C locus) mutations in exon 1~7 of the CYP21A2 gene. Based on the "Standards and Guidelines for Interpretation of Sequence Variants" jointly developed by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP), both variants of the CYP21A2 gene carried by the fetus were predicted to be pathogenic. According to the imaging and genetic testing results of the external genitalia of the fetus, the fetus was prenatally diagnosed as 21-OHD caused by the CYP21A2 gene variant. Follow-up after prenatal diagnosis showed that the couple had opted to terminate the pregnancy at a local hospital at 31⁺ weeks of gestation, and the clinical phenotype of the abortion fetus was consistent with the imaging and molecular genetic diagnosis. CONCLUSION The imaging features of this fetus are suspected to be congenital adrenal hyperplasia (CAH). Combined with WES, Sanger sequencing, and MLPA testing results, the fetus was diagnosed with 21-OHD caused by compound heterozygous variants of the CYP21A2 gene, which provided a basis for prenatal diagnosis.
Humans ; Steroid 21-Hydroxylase/genetics* ; Female ; Pregnancy ; Adrenal Hyperplasia, Congenital/diagnosis* ; Heterozygote ; Prenatal Diagnosis/methods* ; Adult ; Fetus ; DNA Copy Number Variations ; Mutation ; Genetic Testing

OBJECTIVE: To investigate the clinical characteristics and genetic etiology of a male with a normal phenotype and SRY gene-positive 46,XX/46,XY tetrazoospermia chimerism. METHODS: A male patient with an abnormal peripheral blood chromosomal karyotype detected at the Infertility Center of Taizhou Hospital of Zhejiang Province on December 2, 2013 was selected as the study subject. Peripheral venous blood samples were collected from the proband and his family members, together with a semen sample from the proband. Chromosomal karyotype analysis, red blood cell blood group identification, chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH), sex-determining region Y (SRY) gene detection, and short tandem repeat (STR) microsatellite marker analysis were performed on the peripheral venous blood sample from the proband. Routine semen analysis, sperm FISH, and STR testing were also conducted. STR verification was performed on both parents. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: k20201009). RESULTS: The proband, a 37-year-old male, had normal secondary sexual characteristics and external genitalia development. The chromosomal karyotype of his peripheral blood sample was 46,XX[94]/46,XY[6]. ABO blood group typing was positive for Rh(D) type O and negative for Rh(D) type A, indicating the presence of two red blood cell populations. CMA result was arr[GRCh37](1-22)×2,(XX)×1. Autosomal and X chromosome SNP genotypes were BB-BB, AB-AB, and AA-AA, making it impossible to identify homozygous/heterozygous chimerism. FISH detection of interphase nuclei showed nuc ish XX[92]/XY[8]. Testing of the SRY gene was positive. STR analysis showed a single X peak (no Y peak) at the AMEL locus, 10/12 at the Penta D locus, and no third allele at other loci. Routine semen analysis were normal. Sperm FISH detection showed haploid nuclei nuc ish X[53]/Y[47]. Sperm STR analysis revealed an X/Y bimodal distribution at the AMEL locus and a 9/14 distribution at the Penta D locus, with no third allele observed at other loci. Above results suggested that the proband's blood and germ cell lines had originated from a heterozygous chimera formed by the fusion of two different zygotes. CONCLUSION Combined genetic techniques confirmed that the proband's peripheral blood AMEL genotype is X/X, while the sperm is X/Y. The Penta D locus showed a bi-allelic heterozygous pattern of 10/12 in the peripheral blood sample and 9/14 in the sperm sample, suggesting that the proband is a tetrazygotic chimera resulted from the fusion of 46,XX/46,XY zygotes.
Humans ; Male ; Adult ; Chimerism ; Microsatellite Repeats ; Sex-Determining Region Y Protein/genetics* ; Phenotype ; Genes, sry ; In Situ Hybridization, Fluorescence ; Karyotyping

Alzheimer's disease (AD) is characterized by cognitive and functional deterioration, with pathological features such as amyloid-beta (Aβ) aggregates in the extracellular spaces of parenchymal neurons and intracellular neurofibrillary tangles formed by the hyperphosphorylation of tau protein. Despite a thorough investigation, current treatments targeting the reduction of Aβ production, promotion of its clearance, and inhibition of tau protein phosphorylation and aggregation have not met clinical expectations, posing a substantial obstacle in the development of drugs for AD. Recently, artificial intelligence (AI), computational biology (CB), and systems biology (SB) have emerged as promising methodologies in AD research. Their capacity to analyze extensive and varied datasets facilitates the identification of intricate patterns, thereby enriching our comprehension of AD pathology. This paper provides a comprehensive examination of the utilization of AI, CB, and SB in the diagnosis of AD, including the use of imaging omics for early detection, drug discovery methods such as lecanemab, and complementary therapies like phototherapy. This review offers novel perspectives and potential avenues for further research in the realm of translational AD studies.