Rational design peptide inhibitors of Cyclophilin D as a potential treatment for acute pancreatitis.

Cyclophilin D (CypD) is a mitochondrial matrix peptidyl prolidase that regulates the mitochondrial permeability transition pore. Inhibition of CypD was suggested as a therapeutic strategy for acute pancreatitis. Peptide inhibitors emerged as novel binding ligand for blocking receptor activity. In this study, we present our computational approach for designing peptide inhibitors of CypD. The 3-D structure of random peptides were built, and docked into the active center of CypD using Rosetta script integrated FlexPepDock module. The peptide displayed the lowest binding energy against CypD was further selected for virtual iterative mutation based on virtual mutagenesis and molecular docking. Finally, the top 5 peptides with the lowest binding energy was selected for validating their affinity against CypD using inhibitory assay. We showed 4 out of the selected 5 peptides were capable for blocking the activity of CypD, while WACLQ display the strongest affinity against CypD, which reached 0.28 mM. The binding mechanism between WACLQ and CypD was characterized using molecular dynamics simulation. Here, we proved our approach can be a robust method for screening peptide inhibitors.

Primary support optimization of large-span and shallow buried hard rock tunnels based on the active support concept.

Considering the large-span underground excavation subway station of Qingdao Metro Line 6 of China for analysis, it is necessary to optimize the traditional support system by investigating relevant codes and other tunnel projects. Based on the active support concept, a high prestressed rock bolt support system is proposed, and the optimization direction is defined to apply a high prestress force to rock bolts, increasing the appropriate spacing between supporting arches and strengthening support at key parts such as the large arch foot area, sidewalls and junctions. Numerical calculations and field monitoring are performed to analyze and evaluate the new support system. Numerical simulation results show that the new support system can effectively improve the stress state of the surrounding rock; the tensile stress area markedly decreases in size or disappears; and the plastic area also decreases in size. Field monitoring results show that the settlement of the arch crown is concentrated at 2-5 mm and the deformation rates are less than 0.5 mm/day. The supporting arches, shotcrete and rock bolts are all less than the yield strength and a high safety reserve. These results verify the safety and rationality of the proposed support system, which can be used as a reference for similar projects.

Dynamics of parenting styles of adolescent students from the perspective of intergenerational conflict / 中国学校卫生

Objective@#To explore dynamics of parenting styles of adolescents from 1999 to 2019 from the perspective of intergenerational conflict, to provide support for family education and adolescent healthy development.@*Methods@#Using a multistage stratified cluster random sampling method, the unified questionnaire was administered to 2 590 students in the same sampling junior and senior high schools in 1999, 2009, and 2019 using the Egna Minnen av Barndoms Uppfostran own memories of parental rearing practices in childhood(EMBU).@*Results@#Overall there were differences in the nine factors of parenting styles across generations ( F = 12.07-72.52, P <0.01), with decreasing ratings of warmth and understanding of father and mother (F1, M1), over interference of father (F3) over generations(F1:46.72±9.41, 45.87±11.33, 43.61±11.27; M1:51.56±9.38, 51.03±11.59, 46.23± 12.27 ; F3:19.03±4.00, 18.29±4.32, 17.95±4.51), and all other parenting styles rated higher in 2019 than in 2009 and 1999(except for the over protection and over interference of mother, and punishment, firm control of mother). Parenting styles across generations (except for the rejection and denial of father among girls) showed gender difference.The overall gender trend coincided with the total population trend. Parenting styles across generations varied significantly among middle and high school students( F =3.92-47.27, P <0.05 ), changes in F1 and F3 factors coincided with the overall decreasing trend. Factor analysis showed that parenting styles could be classified into two dimensions, with varied factor loading across generation.@*Conclusion@#Intergenerational decreases in parental emotional warmth and paternal interfering are observed in a sex and grade specific manner. Based on the diversity of needs and population differentiation, optimal intervention for comprehensive health development of adolescents are in great need to keep pace with the times and promoting the high quality development of adolescents.

CRISPR/Cas9-mediated correction of MITF homozygous point mutation in a Waardenburg syndrome 2A pig model.

Gene therapy for curing congenital human diseases is promising, but the feasibility and safety need to be further evaluated. In this study, based on a pig model that carries the c.740T>C (L247S) mutation in MITF with an inheritance pattern and clinical pathology that mimics Waardenburg syndrome 2A (WS2A), we corrected the point mutation by the CRISPR-Cas9 system in the mutant fibroblast cells using single-stranded oligodeoxynucleotide (ssODN) and long donor plasmid DNA as the repair template. By using long donor DNA, precise correction of this point mutation was achieved. The corrected cells were then used as the donor cell for somatic cell nuclear transfer (SCNT) to produce piglets, which exhibited a successfully rescued phenotype of WS2A, including anophthalmia and hearing loss. Furthermore, engineered base editors (BEs) were exploited to make the correction in mutant porcine fibroblast cells and early embryos. The correction efficiency was greatly improved, whereas substantial off-targeting mutations were detected, raising a safety concern for their potential applications in gene therapy. Thus, we explored the possibility of precise correction of WS2A-causing gene mutation by the CRISPR-Cas9 system in a large-animal model, suggesting great prospects for its future applications in treating human genetic diseases.

Ribophorin II promotes cell proliferation, migration, and invasion in esophageal cancer cells in vitro and in vivo.

Esophageal cancer is a common digestive tract cancer, which is a serious threat to human health. Ribophorin II (RPN2) is a part of an N-oligosaccharyltransferase complex, which is excessively expressed in many kinds of cancers. In the present study, we explore the biological role of RNP2 in esophageal cancer. First, we found that the expression of RPN2 was higher in esophageal cancer tissues than in adjacent non-tumor tissues, and negatively correlated with E-cadherin expression. RPN2 expression levels in esophageal cancer tissues were positively associated with differentiation and tumor node metastasis (TNM) stage. Furthermore, the expression of RPN2 was increased significantly in esophageal cancer cell lines compared with normal cells. The effect of RPN2 down-regulation on cell proliferation, cell migration, and cell invasion was examined by cell counting kit-8 (CCK8), wound healing assay, and Transwell assay, respectively. Silencing RPN2 effectively inhibited cell proliferation of esophageal cancer cells in vitro and in vivo Cell migration and invasion were also weakened dramatically by siRPN2 treatment of esophageal cancer cells. In addition, protein expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP-2), and E-cadherin in esophageal cancer cells was determined by Western blot analysis. PCNA, MMP-2, E-cadherin, Snail and phosphorylation-Smad2/3 expression was also regulated notably by siRPN2 treatment. These findings indicate that RPN2 exhibits oncogenetic capabilities in esophageal cancer, which could provide novel insights into esophageal cancer prevention and treatment.

A harlequin ichthyosis pig model with a novel ABCA12 mutation can be rescued by acitretin treatment.

Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.

An exonic splicing enhancer mutation in DUOX2 causes aberrant alternative splicing and severe congenital hypothyroidism in Bama pigs.

Pigs share many similarities with humans in terms of anatomy, physiology and genetics, and have long been recognized as important experimental animals in biomedical research. Using an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we previously identified a large number of pig mutants, which could be further established as human disease models. However, the identification of causative mutations in large animals with great heterogeneity remains a challenging endeavor. Here, we select one pig mutant, showing congenital nude skin and thyroid deficiency in a recessive inheritance pattern. We were able to efficiently map the causative mutation using family-based genome-wide association studies combined with whole-exome sequencing and a small sample size. A loss-of-function variant (c.1226 A>G) that resulted in a highly conserved amino acid substitution (D409G) was identified in the DUOX2 gene. This mutation, located within an exonic splicing enhancer motif, caused aberrant splicing of DUOX2 transcripts and resulted in lower H2O2 production, which might cause a severe defect in thyroid hormone production. Our findings suggest that exome sequencing is an efficient way to map causative mutations and that DUOX2D409G/D409G mutant pigs could be a potential large animal model for human congenital hypothyroidism.

MicroRNA-182 promoted esophageal squamous cell carcinoma cell growth and metastasis via targeting YWHAG.

PURPOSE: To study the functioning mode of miR-182 on esophageal squamous cell carcinoma (ESCC) cell growth and metastasis and provide therapeutic targets for ESCC. METHODS: miR-182 expression level in ESCC cell lines was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Using miR-182 inhibitor, we analyzed the effects of miR-182 down-expression on cell proliferation, invasion, cell apoptosis and cell cycle. Dual-luciferase activity assay was used to examine the potential target gene YWHAG which was predicted by several databases. Protein level was studied using western blotting. RESULTS: Decreased expression of miR-182 in ESCC cells was accompanied by decreased cell invasion and proliferation, promotion of cell apoptosis and cell cycle arrest at G0/G1 phase. Dual-luciferase and western blot confirmed YWHAG as a target gene of miR-182. Furthermore, silence of YWHAG counteracted the suppressive effect of miR-182 down-expression on cell growth and metastasis. CONCLUSIONS: miR-182 could suppress ESCC cell proliferation and metastasis via regulating YWHAG, which might provide a new target for ESCC diagnosis and therapy.

Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

Thyroid hormone regulates hematopoiesis via the TR-KLF9 axis.

Congenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 (DUOX2) gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (H2O2) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 (KLF9) was predominantly downregulated in hypothyroid mutants. KLF9 was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of klf9 in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.

Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity.

Uncoupling protein 1 (UCP1) is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity. Pigs (Artiodactyl family Suidae) lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production. Here, we report application of a CRISPR/Cas9-mediated, homologous recombination (HR)-independent approach to efficiently insert mouse adiponectin-UCP1 into the porcine endogenous UCP1 locus. The resultant UCP1 knock-in (KI) pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure (DEE). Furthermore, ectopic UCP1 expression in white adipose tissue (WAT) dramatically decreased fat deposition by 4.89% (P < 0.01), consequently increasing carcass lean percentage (CLP; P < 0.05). Mechanism studies indicated that the loss of fat upon UCP1 activation in WAT was linked to elevated lipolysis. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Transplantation of neonatal cardiomyocytes plus fibrin sealant restores myocardial function in a rat model of myocardial infarction.

BACKGROUND: Most cardiac regenerative approaches can restore injured heart muscles. In this study, we investigated if fibrin sealant could help neonatal cardiomyocytes restore myocardial function in a rat model of myocardial infarction. METHODS: The left anterior descending artery in adult female Sprague-Dawley (SD) rats was ligated to make a myocardial infarction model. Neonatal ventricular cardiomyocytes from one-day male SD rats were isolated, labeled and cultured. The cells were injected into the infarcted area three weeks later. The animals were randomized into four recipient groups: (1) cardiomyocytes plus fibrin sealant (group CF, n = 10); (2) cardiomyocytes alone (group C, n = 10); (3) fibrin sealant recipients alone (group F, n = 10); (4) control group (n = 10). Four weeks after transplantation, echocardiography and Langerdoff model were used to assess heart function. Immunohistochemical staining and polymerase chain reaction (PCR) were performed to track the implanted cardiomyocytes and detect the sex-determining region Y gene on Y chromosome. RESULTS: Echocardiography showed the fraction shortening (FS) in groups CF, C, F and control group was (27.80 +/- 6.32)%, (22.29 +/- 4.54)%, (19.24 +/- 6.29)% and (20.36 +/- 3.29)% respectively with statistically significant differences in group CF compared with the other groups (P < 0.05). The Langendoff model revealed that the left ventricular development of peak pressure (LVDPmax, mmHg) in groups CF, C, F and control group was 104.81 +/- 17.05, 80.97 +/- 21.60, 72.07 +/- 26.17 and 71.42 +/- 17.55 respectively with statistically significant differences in group CF compared with the other groups (P < 0.05). Pathological examination and PCR indicated that transplanted cardiomyocytes in group CF survived better than those in the other groups. CONCLUSION: Transplanted neonatal cardiomyocytes plus fibrin sealant can survive in myocardial infarctioned area and improve heart function greatly in rat models.