scholarly journals Seamless Ligation Cloning Extract (SLiCE) Cloning Method

2013 ◽  
pp. 235-244 ◽  
Author(s):  
Yongwei Zhang ◽  
Uwe Werling ◽  
Winfried Edelmann
Keyword(s):  
Cloning Method

scholarly journals Identification and Target-Modification of SL-BBI: A Novel Bowman–Birk Type Trypsin Inhibitor from Sylvirana latouchii

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1254 ◽  
Author(s):  
Xi Chen ◽  
Dong Chen ◽  
Linyuan Huang ◽  
Xiaoling Chen ◽  
Mei Zhou ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Membrane Damage ◽  
Antiproliferative Effects ◽  
Docking Simulations ◽  
Cell Membrane Damage ◽  
Action Mode ◽  
Trypsin Inhibition ◽  
Cloning Method

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

A new cloning method for antibody-forming lymphoblastoid cells

1986 ◽  
Vol 94 (1-2) ◽  
pp. 201-208 ◽  
Author(s):  
Slawomir Kosinski ◽  
Ulrich Hämmerling
Keyword(s):  
Lymphoblastoid Cells ◽  
Cloning Method

Piglets born from handmade cloning, an innovative cloning method without micromanipulation

2007 ◽  
Vol 68 (8) ◽  
pp. 1104-1110 ◽  
Author(s):  
Y. Du ◽  
P.M. Kragh ◽  
Y. Zhang ◽  
J. Li ◽  
M. Schmidt ◽  
...  
Keyword(s):  
Cloning Method ◽  
Handmade Cloning

The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene

1993 ◽  
Vol 13 (3) ◽  
pp. 1962-1970
Author(s):  
T D Moore ◽  
J C Edman
Keyword(s):  
Cryptococcus Neoformans ◽  
Mating Type ◽  
Cell Types ◽  
Yeast Cells ◽  
Mating Types ◽  
Type Locus ◽  
Reading Frame ◽  
Cloning Method ◽  
Opportunistic Fungal Pathogen ◽  
Specific Fragment

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.

scholarly journals Improving the Learning of Self-driving Vehicles Based on Real Driving Behavior using Deep Neural Network Techniques

2020 ◽  
Author(s):  
Nayere Zaghari ◽  
Mahmood Fathy ◽  
Seyed Mahdi Jameii ◽  
Mohammad Sabokrou ◽  
Mohammad Shahverdy
Keyword(s):  
Neural Network ◽  
Deep Neural Network ◽  
Autonomous Vehicle ◽  
Driving Behavior ◽  
Obstacle Detection ◽  
Convolutional Network ◽  
The Real ◽  
Cloning Method ◽  
Vehicle Acceleration ◽  
Performance Results

Considering the significant advancements in autonomous vehicle technology, research in this field is of interest to researchers. To drive vehicles autonomously, controlling steer angle, gas hatch, and brakes need to be learned. The behavioral cloning method is used to imitate humans’ driving behavior. We created a dataset of driving in different routes and conditions and using the designed model, the output used for controlling the vehicle is obtained. In this paper, the Learning of Self-driving Vehicles Based on Real Driving Behavior Using Deep Neural Network Techniques (LSV-DNN) is proposed. We designed a convolutional network which uses the real driving data obtained through the vehicle’s camera and computer. The response of the driver is during driving is recorded in different situations and by converting the real driver’s driving video to images and transferring the data to an excel file, obstacle detection is carried out with the best accuracy and speed using the Yolo algorithm version 3. This way, the network learns the response of the driver to obstacles in different locations and the network is trained with the Yolo algorithm version 3 and the output of obstacle detection. Then, it outputs the steer angle and amount of brake, gas, and vehicle acceleration. The LSV-DNN is evaluated here via extensive simulations carried out in Python and TensorFlow environment. We evaluated the network error using the loss function. By comparing other methods which were conducted on the simulator’s data, we obtained good performance results for the designed network on the data from KITTI benchmark, the data collected using a private vehicle, and the data we collected.

scholarly journals Growth and Antioxidant-Related Effects of the Reestablished Ascorbic Acid Pathway in Zebrafish (Danio rerio) by Genomic Integration of L-Gulonolactone Oxidase From Cloudy Catshark (Scyliorhinus torazame)

2021 ◽  
Vol 12 ◽  
Author(s):  
K. A. S. N. Shanaka ◽  
Sumi Jung ◽  
N. D. Janson ◽  
J. R. P. Jayasingha ◽  
K. P. Madushani ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
System Analysis ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Wild Type ◽  
Cloning Method ◽  
Production Growth ◽  
Gulonolactone Oxidase ◽  
Acid Pathway ◽  
Species Production

Loss of L-gulonolactone oxidase (GULO), which catalyzes the last step of the ascorbic acid (AA) biosynthesis pathway, results in a complete lack of AA in several Osteichthyes fish species, including zebrafish. In this study, sGULO, the active GULO gene from cloudy catshark (Scyliorhinus torazame) was cloned into zebrafish using the Gateway cloning method. The resulting Tg(b-actin:sGULO:mCherry) fish were analyzed for the effects of a reestablished AA pathway. Fluorescent microscopy and PCR were used to analyze the integration of the construct into the zebrafish genome. Catalytic activity of sGULO, AA production, growth-related characteristics, and gene expression were investigated to evaluate the effects of AA production in Tg fish. The mCherry fluorescent protein indicated the proper integration and expression of the sGULO construct in zebrafish. The sGULO gene was ubiquitously expressed in all the studied tissues and the enzyme activity indicated an increased AA production in Tg fish. The growth of Tg fish was also increased, and antioxidant system analysis suggests that reactive oxygen species production was reduced in Tg fish compared with wild type. Expression of the AA transporter slc23a1 was significantly downregulated in Tg homozygous fish. These results collectively indicate the effects of reestablished AA synthesis in zebrafish.

scholarly journals Fluorescence Imaging of Mobility Shifts: An Expression Cloning Method for Identification of Cell Signaling Targets

2002 ◽  
Vol 82 (12) ◽  
pp. 1631-1636
Author(s):  
James W Mandell ◽  
Ri-ichiroh Manabe ◽  
Alan F Horwitz ◽  
Joel P Baumgart
Keyword(s):  
Cell Signaling ◽  
Fluorescence Imaging ◽  
Expression Cloning ◽  
Cloning Method
Sign in / Sign up

Export Citation Format

Share Document