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.

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.

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.

This study reported two cases of distal 18q deletions identified through non-invasive prenatal testing (NIPT). Case 1 harbored a de novo 20.4 Mb deletion of 46, XY, del(18) (q21.32q23), classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Follow-up at five years of age revealed global developmental delay, congenital heart disease, and distinct facial features. Case 2 had a 5.71 Mb paternal-origin deletion of 46,XX,del(18)(q22.1q22.2), with only mild tricuspid regurgitation detected at eight months of age. These phenotypic discrepancies demonstrated that the clinical manifestations of 18q deletion syndrome were closely associated with the size of the deleted segment and the involved critical genes. Therefore, individualized genetic counseling and long-term follow-up were necessary for the affected children.

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.

Objectives:To evaluate the effect of extended single prone positioning ventilation on survival and weaning rate of acute respiratory distress syndrome (ARDS) patients supported by VV-ECMO.Methods:ARDS patients supported by VV-ECMO admitted to Jinhua Central Hospital, the Fourth Affiliated Hospital of Zhejiang University School of Medicine and the First Hospital of Jiaxing from September 2014 to May 2025 were retrospectively enrolled into the study. The clinical data, ECMO and ventilator related parameters and outcomes of the patients were collected. The patients were divided into the extended prone positioning group and prone positioning group according to whether the duration of prone position ventilation was greater than 24 h. The clinical data of the two groups were compared to explore the effects on 30-day survival in-hospital survival and ECMO withdraw rate of these patients. Multivariate logistic regression analysis was used to explore the relationship between the duration of single prone position ventilation and the success of ECMO weaning, 30-day survival and hospital survival.Results:Total of 163 ARDS patients supported by VV-ECMO receiving prone positioning ventilation were included in study, 64 in extended prone positioning group and 72 in prone positioning group. The 30-day survival (54.7% vs. 52.8%) in-hospital survival (51.6% vs. 48.6%) and ECMO withdraw rate (57.8% vs. 61.1%) between the two groups were not statistically different ( P>0.05) as well as the duration of ECMO support [12(10,15)d vs. 11(10,13)d] the duration of ventilation [16(13,18)d vs. 16(12,18)d] the duration of ICU stay [26(15,32)d vs. 26(19,29)d] and the duration of hospital stay [32(15,42)d vs. 34(28,35)d]. Logistic regression analysis revealed that the duration of each prone position ventilation was not associated with successful weaning ( OR=0.979, 95% CI:0.952-1.006), 30-day survival ( OR=1.015, 95% CI: 0.975-1.056) and hospital survival ( OR=1.014, 95% CI: 0.974-1.055) even after adjusting for the severity of illness, age, and type of pneumonia. Conclusions:For ARDS patients supported by VV-ECMO, extended single prone positioning ventilation for more than 24 hours neither increase 30-day survival in-hospital survival and successful ECMO weaning rate, nor shorten ECMO support duration.

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

Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

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.