Human lactoferrin efficiently targeted into caprine beta-lactoglobulin locus with transcription activator-like effector nucleases

Yu-Guo Yuan,Shao-Zheng Song,Meng-Ming Zhu,Zheng-Yi He,Rui Lu,Ting Zhang,Fei Mi,Jin-Yu Wang,Yong Cheng 아세아·태평양축산학회 2017 Animal Bioscience Vol.30 No.8

Objective: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion: This finding signifies the combined use of TALENs and SCNT can generate bi-allelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

Complete ¹H-NMR and <SUP>13</SUP>C-NMR spectral assignment of five malonyl ginsenosides from the fresh flower buds of Panax ginseng

Yu-Shuai Wang,Yin-Ping Jin,Wei Gao,Sheng-Yuan Xiao,Yu-Wei Zhang,Pei-He Zheng,Jia Wang,Jun-Xia Liu,Cheng-He Sun,Ying-Ping Wang 고려인삼학회 2016 Journal of Ginseng Research Vol.40 No.3

Background: Ginsenosides are the major effective ingredients responsible for the pharmacological effects of ginseng. Malonyl ginsenosides are natural ginsenosides that contain a malonyl group attached to a glucose unit of the corresponding neutral ginsenosides. Methods: Medium-pressure liquid chromatography and semipreparative high-performance liquid chromatography were used to isolate purified compounds and their structures determined by extensive one-dimensional- and two-dimensional nuclear magnetic resonance (NMR) experiments. Results: A new saponin, namely malonyl-ginsenoside Re, was isolated from the fresh flower buds of Panax ginseng, along with malonyl-ginsenosides Rb1, Rb2, Rc, Rd. Some assignments for previously published ¹H- and <SUP>13</SUP>C-NMR spectra were found to be inaccurate. Conclusion: This study reports the complete NMR assignment of malonyl-ginsenoside Re, Rb₁, Rb₂, Rc, and Rd for the first time.

Single Nucleotide Polymorphisms of the GnRHR Gene Associated with Reproductive Traits of Japanese Flounder (Paralichthys olivaceus)

He, Feng,Wen, Hai-Shen,Li, Ji-Fang,Yu, Da-Hui,Ma, Rui-Qin,Shi, Dan,Mu, Wei-Jie,Zhang, Yuan-Qing,Hu, Jian,Liu, Miao,Han, Wei-Guo,Zhang, Jia-Nan,Wang, Qing-Qing,Yuan, Yu-Ren,Liu, Qun Asian Australasian Association of Animal Productio 2011 Animal Bioscience Vol.24 No.4

Gonadotropin-releasing hormone receptor (GnRHR) gene is expressed at the anterior pituitary gland and plays a key role in gonad development. This study aimed to investigate molecular genetic characteristics of the GnRHR gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of GnRHR gene on sex steroid level in Japanese flounder (Paralichthys olivaceus). We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the GnRHR gene in 75 individuals. We identified three SNPs in the GnRHR gene: P1 locus (C759A and C830T) in the coding region of exon2 which were both linked together and P2 locus (G984T) in the coding region of exon3, which added a new transcript factor (ADR1) and a new methylation site (CG). Only C830T of P1 leads to amino acid changes Thr266Ile. Statistical analysis showed that P1 was significantly associated with $17{\beta}$-estradiol ($E_2$) level (p<0.01) and gonadosomatic index (GSI) (p<0.05). Individuals with genotype BB of P1 had significantly higher serum $E_2$ levels (p<0.01) and GSI (p<0.05) than those of genotype AA or AB. Another SNP, P2, synonymous mutation, was significantly associated with GSI (p<0.05). Individuals with genotype AB of P2 had significantly higher GSI (p<0.05) than that of genotype AA. In addition, there was a significant association between one diplotype based on three SNPs and reproductive traits. The genetic effects for both serum $E_2$ level and GSI of diplotype D4 were super diplotypes (p<0.05). These results suggest that the SNPs in Japanese Flounder GnRHR are associated with $E_2$ level and GSI.

Practical Fixed-time Distributed Average-tracking with Input Delay Based on Event-triggered Method

Yuan-Jun Yu,Cheng-Lin Liu,Yu-Ling Li,He Li 제어·로봇·시스템학회 2023 International Journal of Control, Automation, and Vol.21 No.3

This paper addresses the distributed average-tracking (DAT) problem of multiagent networks (MANs) with same input delay under an undirected topology. A distributed fixed-time average-tracking algorithm driven by an event-triggered method is constructed to ensure the agents reach the average value of multiple time-varying reference input signals (TVRISs) within the fixed time. By using Lyapunov function method, sufficient conditions on estimating the fixed settling time and the bounded average-tracking errors are obtained. In addition, it is proved that the event-triggered control strategy avoids the Zeno behavior. Finally, a few numerical simulations are given to illustrate the effectiveness of the proposed algorithm.

Association between SNPs within Prolactin Gene and Milk Performance Traits in Holstein Dairy Cattle

He, Feng,Sun, Dongxiao,Yu, Ying,Wang, Yachun,Zhang, Yuan Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.10

Prolactin plays an important role in mammary gland development, milk section initiation and maintenance of lactation, so the bovine prolactin gene is considered as a potential quantitative trait locus affecting milk performance traits in dairy cattle. In this study, to determine the association between prolactin and milk performance traits, the genetic polymorphisms of a part of the prolactin gene were detected in a population of 649 cows of Chinese Holstein Dairy Cattle. Three SNPs in the promoter and one SNP in the intron1 of prolactin were identified, which was A/C (-767), G/T (-485), C/A (-247), and C/T (427), respectively. Statistical results indicated that one of SNP within promote, CHBP2, was significantly associated with milk yield (p<0.01), fat yield (p<0.05), protein yield (p<0.01), and protein percentage (p<0.05). The cows with genotype BB of CHBP2 had significantly higher milk yield (p<0.01), fat yield (p<0.05), and protein yield (p<0.01) than those of cows with genotype AA, while cows with genotype AA showed the highest protein percentage (p<0.05). In addition, based on the nine major haplotypes constructed from the four SNPs, the association analysis between diplotypes and milk performance trait was carried out. Results showed that the least square mean for fat yield of diplotype H2H8 was significantly higher than those of other eleven diplotypes (p<0.05). Our findings implied that CHBP2 and H2H8 of prolactin would be useful genetic markers in selection program on milk performance traits in Holstein Dairy Cattle.

Molecular cloning and expression analysis of the first two key genes through 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway from Pyropia haitanensis(Bangiales, Rhodophyta)

Yu Du,Jian Guan,Ruijun Xu,Xin Liu,Weijie Shen,Yafeng Ma,Yuan He,Songdong Shen 한국조류학회I 2017 ALGAE Vol.32 No.4

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata is one of the most commercially usefulmacroalgae cultivated in southeastern China. In red algae, the biosynthesis of terpenoids through 2-C-methyl-Derythritol4-phosphate (MEP) pathway can produce a direct influence on the synthesis of many biologically importantmetabolites. In this study, two genes of cDNAs, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-Dxylulose-5-phosphatereductase (DXR), which encoding the first two rate-limiting enzymes among MEP pathway werecloned from P. haitanensis. The cDNAs of P. haitanensis DXS (PhDXS) and DXR (PhDXR) both contained complete openreading frames encoding polypeptides of 764 and 426 amino acids residues, separately. The expression analysis showedthat PhDXS was significant differently expressed between leafy thallus and conchocelis as PhDXR been non-significant. Additionally, expression of PhDXR and its downstream gene geranylgeranyl diphosphate synthase were both inhibitedby fosmidomycin significantly. Meanwhile, we constructed types of phylogenetic trees through different algae and higherplants DXS and DXR encoding amino acid sequences, as a result we found tree clustering consequences basically in linewith the "Cavalier-Smith endosymbiotic theory." Whereupon, we speculated that in red algae, there existed only completeMEP pathway to meet needs of terpenoids synthesis for themselves; Terpenoids synthesis of red algae derivativesthrough mevalonate pathway came from two or more times endosymbiosis of heterotrophic eukaryotic parasitifer. Thisstudy demonstrated that PhDXS and PhDXR could play significant roles in terpenoids biosynthesis at molecular levels. Meanwhile, as nuclear genes among MEP pathway, PhDXS and PhDXR could provide a new way of thinking to researchthe problem of chromalveolata biological evolution.

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes

Yu, Qin Ping,Feng, Ding Yuan,He, Xiao Jun,Wu, Fan,Xia, Min Hao,Dong, Tao,Liu, Yi Hua,Tan, Hui Ze,Zou, Shi Geng,Zheng, Tao,Ou, Xian Hua,Zuo, Jian Jun Asian Australasian Association of Animal Productio 2017 Animal Bioscience Vol.30 No.11

Objective: This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula's extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods: In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results: The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber Ι, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type Ι fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC Ι, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC Ι and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor ${\gamma}$ $coactivator-1{\alpha}$ and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by $4{\mu}g/mL$ and $20{\mu}g/mL$ TCMF water extraction (p<0.05). Both $1{\mu}g/mL$ and $5{\mu}g/mL$ of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC Ι mRNA expression in porcine myocytes was decreased by $5{\mu}g/mL$ TCMF water extraction (p<0.05). Porcine myocyte MyHC Ι and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by $5{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). MyHC Ι and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by $1{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by $5{\mu}g/mL$ TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by $1{\mu}g/mL$ in a TCMF petroleum ether extraction (p<0.05). Conclusion: These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.

Benefit of Post-mastectomy Radiotherapy of the Supra-/infraclavicular Lymphatic Drainage Area in Breast Cancer Patients

He, Zhen-Yu,Wu, San-Gang,Zhou, Juan,Sun, Jia-Yuan,Li, Feng-Yan,Lin, Qin,Guo, Ling,Lin, Huan-Xin Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.14

Background: This study investigated the survival benefit of radiotherapy (RT) of the supra- and infraclavicular lymphatic drainage area in Chinese women with T1-2N1M0 breast cancer receiving mastectomy. Methods: A total of 593 cases were retrospectively reviewed from 1998 to 2007. The relationship between supra- or infraclavicular fossa relapse (SCFR) and post-operative RT at the supra-/infraclavicular lymphatic drainage area was evaluated. Results: The majority of patients (532/593; 89. 8%) received no RT while 61 patients received RT. The median follow-up was 85 months. Among patients without RT, 54 (10. 2%) developed recurrence in the chest wall or ipsilateral SCFR. However, none of the 61 patients who underwent RT demonstrated SCFR. One patient who received RT (1. 6%) experienced recurrence in the chest wall. Univariate analysis revealed that age and molecular subtype (both P < 0. 05) were two prognostic factors related to supraclavicular and infraclavicular fossa relapse-free survival (SFRFS). Multivariate analysis revealed that only Her-2 positive status (P = 0. 011) was an independent predictor of SFRFS. RT had no influence on distant metastasis (P = 0. 328) or overall survival (P = 0. 541). SCFR significantly affected probability of distant metastasis (P < 0. 001) and overall survival (P < 0. 001). Conclusion: Although RT was not significantly associated with SFRFS, postoperative RT was significantly associated with a lower locoregional (i. e., supraclavicular/infraclavicular and chest wall) recurrence rate. SCFR significantly influenced distant metastasis-free survival, which significantly influenced the overall survival of T1-2N1M0 breast cancer patients after mastectomy. Thus, prophylactic RT is recommended in T1-2N1M0 breast cancer patients, especially those who have Her-2 positive lesions.

American ginseng significantly reduced the progression of high-fat-diet-enhanced colon carcinogenesis in Apc^Min/+ mice

Yu, Chunhao,Wen, Xiao-Dong,Zhang, Zhiyu,Zhang, Chun-Feng,Wu, Xiaohui,He, Xin,Liao, Yang,Wu, Ningning,Wang, Chong-Zhi,Du, Wei,He, Tong-Chuan,Yuan, Chun-Su The Korean Society of Ginseng 2015 Journal of Ginseng Research Vol.39 No.3

Background: Colorectal cancer (CRC) is a leading cause of death worldwide. Chronic gut inflammation is recognized as a risk factor for tumor development, including CRC. American ginseng is a very commonly used ginseng species in the West. Methods: A genetically engineered $Apc^{Min/+}$ mouse model was used in this study. We analyzed the saponin composition of American ginseng used in this project, and evaluated its effects on the progression of high-fat-diet-enhanced CRC carcinogenesis. Results: After oral ginseng administration (10-20 mg/kg/d for up to 32 wk), experimental data showed that, compared with the untreated mice, ginseng very significantly reduced tumor initiation and progression in both the small intestine (including the proximal end, middle end, and distal end) and the colon (all p < 0.01). This tumor number reduction was more obvious in those mice treated with a low dose of ginseng. The tumor multiplicity data were supported by body weight changes and gut tissue histology examinations. In addition, quantitative real-time polymerase chain reaction analysis showed that compared with the untreated group, ginseng very significantly reduced the gene expression of inflammatory cytokines, including interleukin-$1{\alpha}$ (IL-$1{\alpha}$), IL-$1{\beta}$, IL-6, tumor necrosis factor-${\alpha}$, granulocyte-colony stimulating factor, and granulocyte-macrophage colony-stimulating factor in both the small intestine and the colon (all p < 0.01). Conclusion: Further studies are needed to link our observed effects to the actions of the gut microbiome in converting the parent ginsenosides to bioactive ginseng metabolites. Our data suggest that American ginseng may have potential value in CRC chemoprevention.

American ginseng attenuates azoxymethane/dextran sodium sulfate-induced colon carcinogenesis in mice

Yu, Chunhao,Wen, Xiao-Dong,Zhang, Zhiyu,Zhang, Chun-Feng,Wu, Xiao-Hui,Martin, Adiba,Du, Wei,He, Tong-Chuan,Wang, Chong-Zhi,Yuan, Chun-Su The Korean Society of Ginseng 2015 Journal of Ginseng Research Vol.39 No.1

Background: Colorectal cancer is a leading cause of cancer-related death, and inflammatory bowel disease is a risk factor for this malignancy. We previously reported colon cancer chemoprevention potential using American ginseng (AG) in a xenograft mice model. However, the nude mouse model is not a gut-specific colon carcinogenesis animal model. Methods: In this study, an experimental colitis and colitis-associated colorectal carcinogenesis mouse model, chemically induced by azoxymethane/dextran sodium sulfate (DSS) was established and the effects of oral AG were evaluated. The contents of representative ginseng saponins in the extract were determined. Results: AG significantly reduced experimental colitis measured by the disease activity index scores. This suppression of the experimental colitis was not only evident during DSS treatment, but also very obvious after the cessation of DSS, suggesting that the ginseng significantly promoted recovery from the colitis. Consistent with the anti-inflammation data, we showed that ginseng very significantly attenuated azoxymethane/DSS-induced colon carcinogenesis by reducing the colon tumor number and tumor load. The ginseng also effectively suppressed DSS-induced proinflammatory cytokines activation using an enzyme-linked immunosorbent assay array, in which 12 proinflammatory cytokine levels were assessed, and this effect was supported subsequently by real-time polymerase chain reaction data. Conclusion: AG, as a candidate of botanical-based colon cancer chemoprevention, should be further investigated for its potential clinical utility.