Objective To investigate the toxic reaction,toxic organs or target tissues of protamine recombinant human insulin (Insulin NPH),and provide basis for clinical trials by single dose toxicity test in mice,repeated toxicity and immunogenicity of Beagle's dogs,and systemic active allergy in guinea pig.Methods ① Using maximum dose method,mice in single dose toxicity test were sc injected with normal saline (NS),vehicle,and Insulin NPH (2092-2488 IU/kg),the toxic reactions after injection were monitored.② In repeated toxicity study,Beagle's dogs were sc administrated with vehicle,the original (Humulin NPH,1.5 IU/kg)and different doses of Insulin NPH (0.5,1.0 and 1.5 IU/kg) for 30 d continuously,followed by a 14-d recovery.During the administration and recovery period,general observation,local irritation,body weight,anus temperature,blood glucose,and electrocardiogram (ECG) were checked,moreover,hematology,serum biochemistry and urine were detected.Also,organic weights and histopathological examination were conducted.Binding antibodies in dog serum were measured by indirect ELISA method in immunogenicity test.③ In systemic active allergy study,cavies were sc injected with low-and high-dose (4 and 12 IU/kg) Insulin NPH,normal saline and vehicle.Besides,ova as positive control was also included.After five times of sensitization test with above doses,the excitation reactions of iv injection with tripled sensitizing doses were observed.Results No obvious toxicity was observed in mice after injected with 165 times of usual clinical dose of Insulin NPH.Repeated toxicity study of Beagle's dogs revealed that 1.0 IU/kg was the no-toxic-effect dose (NOAEL) for Insulin NPH,which was equivalent to 2 times of clinical dose.No bindingantibodies were found in immunogenicity test.There was no obvious allergic symptom in the active systemic allergy study of guinea pig.Conclusion Under the experimental conditions,no serious toxicity of Insulin NPH is found.

Objective:To study the feasibility, safety and technique for laparoscopic anatomical liver resection of segment 8.Methods:The clinical data of 9 patients who underwent laparoscopic anatomical liver resection of segment 8 from January 2015 to December 2019 at Hunan Provincial People's Hospital were retrospectively analyzed. There were 6 males and 3 females, with age ranging from 29 to 67 years (average 53.6 years). The operation time, intraoperative blood loss , postoperative hospital stay, postoperative complications, and long-term survival and recurrence rates on follow-up were analysed.Results:Laparoscopic anatomical liver resection of segment 8 was successfully carried out in these patients. The mean operative time was 188.9 min(range 140-240 min). The mean estimated intraoperative blood loss was 117.8 ml (range 20-300 ml). The postoperative hospital stay was 6.9 days (range 3-12 days). One patient developed pleural effusion after operation and responded to conservative treatment. Another patients developed ascites with delayed extubation. The patient was successfully treated with conservative treatment. No patients developed complications above Clavien Dindo Ⅲa. There were no perioperative deaths. The postoperative pathological results showed hepatocellular adenoma ( n=2), hepatocellular carcinoma ( n=4), cholangiocarcinoma ( n=1), and metastatic liver cancer ( n=2). On follow-up for 12-58 months (median 22 months) one patient with hepatocellular carcinoma developed recurrence at 18 months after operation and was treated with microwave ablation. The other patients were well on follow-up. Conclusions:With adequate preoperative evaluation, reasonable case selection, rigorous surgical planning, and skilled laparoscopic techniques, laparoscopic anatomical liver resection of segment 8 was safe and feasible, and the short-term efficacy was good in this study.

Objective:To explore the genetic basis for a child featuring facial dysmorphism and intellectual disabilities.Methods:A child who was diagnosed at Linyi People′s Hospital on January 5 2023 due to "mental retardation" was selected as the study subject. Peripheral blood samples of the child and his parents, in addition with an amniotic fluid sample from the his mother were collected for the extraction of genomic DNA. Whole exome sequencing was carried out for the child, and candidate variant was verified by Sanger sequencing of his family members.Results:The child was found to harbor a hemizygous c. 1123dupG (p. E375Gfs*4) variant of the NEXMIF gene, for which both of his parents and the fetus were of the wild type. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was predicted to be pathogenic (PVS1+ PS2-P+ PM2-P). A healthy infant was subsequently born. Conclusion:The hemizygous c. 1123dupG (p. E375Gfs*4) variant of the NEXMIF gene probably underlay the disease in this child. Based on his clinical phenotype and genotype, the child was ultimately diagnosed with X-linked intellectual developmental disorder-98. Above finding has also enriched the mutational spectrum of the NEXMIF gene.

This paper reported the genetic analysis of a pedigree in which three affected fetuses with short limbs were revealed by first-trimester ultrasonography in three consecutive pregnancies. Tissues of the second aborted fetus were collected and analyzed by chromosome karyotype analysis and whole exome sequencing. The results indicated compound heterozygous mutations of EX64-EX83 Del and c.8190G>T in the DYNC2H1 gene. Real-time fluorescence quantitative polymerase chain reaction and Sanger sequencing further confirmed that the two variants were inherited from the father and the mother with normal phenotypes, respectively. EX64-EX83 Del was a likely pathogenic variant and c.8190G>T was a variant of uncertain significance. Based on the above results and the medical history, it was highly suspected that the fetus had autosomal recessive short rib polydactyly syndrome type Ⅲ caused by compound heterozygous variants. Real-time fluorescent quantitative polymerase chain reaction and Sanger sequencing results of the third aborted fetus were consistent with the second fetus. Given the same phenotypes of fetuses in the second and third pregnancy, it was strongly suggested that the heterozygous variations of EX64-EX83 Del and c.8190G>T in the DYNC2H1 gene were the pathogenic variants in this pedigree.

OBJECTIVE: To explore whether thrombopoietin (TPO) can rescue megakaryopoiesis by protecting bone marrowderived endothelial progenitor cells (BM-EPCs) in patients receiving chemotherapy for hematological malignancies. METHODS: Bone marrow samples were collected from 23 patients with hematological malignancies 30 days after chemotherapy and from 10 healthy volunteers. BM-EPCs isolated from the samples were identified by staining for CD34, CD309 and CD133, and their proliferation in response to treatment with TPO was assessed using CCK8 assay. DiL-Ac-LDL uptake and FITC-UEA-I binding assay were performed to evaluate the amount of BM-EPCs from the subjects. Tube-formation and migration experiments were used for functional assessment of the BM-EPCs. The BM-EPCs with or without TPO treatment were co-cultured with human megakaryocytes, and the proliferation of the megakaryocytes was detected with flow cytometry. RESULTS: Flow cytometry indicated that the TPO-treated cells had high expressions of CD34, CD133, and CD309. CCK8 assay demonstrated that TPO treatment enhanced the proliferation of the BM-EPCs, and the optimal concentration of TPO was 100 μg/L. Double immunofluorescence assay indicated that the number of BM-EPC was significantly higher in TPO-treated group than in the control group. The TPO-treated BM-EPCs exhibited stronger tube-formation and migration abilities ( < 0.05) and more significantly enhanced the proliferation of co-cultured human megakaryocytes than the control cells ( < 0.05). CONCLUSIONS TPO can directly stimulate megakaryopoiesis and reduce hemorrhage via protecting the function of BM-EPCs in patients following chemotherapy for hematological malignancies.
Bone Marrow ; Bone Marrow Cells ; Cells, Cultured ; Hematologic Neoplasms ; Humans ; Megakaryocytes ; Thrombopoietin