Purification and Functional Characterization of Recombinant Human ADAM8 protease

From recent studies in different tumor entities it became apparent that the membrane-anchored metalloprotease-disintegrin ADAM8 plays an active role in tumor progression and consequently in patient prognosis. It is therefore desirable to understand the functional role of ADAM8 in vitro by characterizing the soluble extracellular part of ADAM8, which is called the ADAM8 ectodomain. The human ectodomain ADAM8 (hEctoA8) protein consists of the prodomain, metalloprotease domain (MP), a disintegrin domain (DIS), a cysteine-rich domain (Cys) and a EGF-like domain (EGF), whilst removal of the prodomain by autocatalysis leads to the active form of hEctoA8. Like the full-length ADAM8, hEctoA8 contains the catalytic consensus sequence HEXXHXXGXXHD in the metalloprotease domain and is therefore predicted to be proteolytically active. Up to now, functional studies on ADAM8 in vitro were hampered by the lack of sufficient quantities of folded, biologically active, and purified recombinant forms of hEctoA8. In our study, we successfully purified the recombinant hEctoA8 protein from supernatants of transfected HEK cells. Purified recombinant hEctoA8 containing the catalytic function of ADAM8 can cleave CD23 in cells in trans and FN in vitro; CD23 cleavage resulted in fragments of 37, 33, 25 and 16 kDa and FN cleavage by recombinant hEctoA8 resulted in 9 fragments, one fragment of 38 kDa contains a RGD motif essential for cell adhesion. Functionally, ADAM8-dependent cell adhesion of pancreatic tumor cells Panc1 was affected by FN cleavage and resulted in reduced cell adhesion. Concomitantly, a decreased expression of integrin α5; was found, whereas cleavage of FN by recombinant hEctoA8 had no effect on the expression levels of integrin β1, and on the activation of p-ERK1/2 and p-Akt

Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established

Cardioprotection of Electroacupuncture for Enhanced Recovery after Surgery on Patients Undergoing Heart Valve Replacement with Cardiopulmonary Bypass: A Randomized Control Clinical Trial

We attempted to investigate cardioprotection of electroacupuncture (EA) for enhanced recovery after surgery on patients undergoing heart valve replacement with cardiopulmonary bypass. Forty-four patients with acquired heart valve replacement were randomly allocated to the EA group or the control group. Patients in the EA group received EA stimulus at bilateral Neiguan (PC6), Ximen (PC4), Shenting (GV24), and Baihui (GV20) acupoints twenty minutes before anesthesia induction to the end of surgery. The primary end point was cardioprotection effect of electroacupuncture postoperatively and the secondary endpoints were quality of recovery and cognitive functioning postoperatively. The present study demonstrated that electroacupuncture reduced the occurrence of complications and played a role of cardioprotective effect on patients after heart valve replacement surgery with cardiopulmonary bypass, and it benefits patients more comfortable and contributes to recovery after surgery. This trial is registered with ChiCTR-IOC-16009123

Purification and Functional Characterization of Recombinant Human ADAM8 protease

From recent studies in different tumor entities it became apparent that the membrane-anchored metalloprotease-disintegrin ADAM8 plays an active role in tumor progression and consequently in patient prognosis. It is therefore desirable to understand the functional role of ADAM8 in vitro by characterizing the soluble extracellular part of ADAM8, which is called the ADAM8 ectodomain. The human ectodomain ADAM8 (hEctoA8) protein consists of the prodomain, metalloprotease domain (MP), a disintegrin domain (DIS), a cysteine-rich domain (Cys) and a EGF-like domain (EGF), whilst removal of the prodomain by autocatalysis leads to the active form of hEctoA8. Like the full-length ADAM8, hEctoA8 contains the catalytic consensus sequence HEXXHXXGXXHD in the metalloprotease domain and is therefore predicted to be proteolytically active. Up to now, functional studies on ADAM8 in vitro were hampered by the lack of sufficient quantities of folded, biologically active, and purified recombinant forms of hEctoA8. In our study, we successfully purified the recombinant hEctoA8 protein from supernatants of transfected HEK cells. Purified recombinant hEctoA8 containing the catalytic function of ADAM8 can cleave CD23 in cells in trans and FN in vitro; CD23 cleavage resulted in fragments of 37, 33, 25 and 16 kDa and FN cleavage by recombinant hEctoA8 resulted in 9 fragments, one fragment of 38 kDa contains a RGD motif essential for cell adhesion. Functionally, ADAM8-dependent cell adhesion of pancreatic tumor cells Panc1 was affected by FN cleavage and resulted in reduced cell adhesion. Concomitantly, a decreased expression of integrin α5; was found, whereas cleavage of FN by recombinant hEctoA8 had no effect on the expression levels of integrin β1, and on the activation of p-ERK1/2 and p-Akt

Single Variable-Constrained NDT Matching in Traffic Data Collection Using a Laser-Based Detector

As indispensable components of intelligent transportation systems, traffic detection and surveillance technologies deliver speed monitoring, traffic counting, and vehicle identification and classification. This paper proposes a normal distribution transform (NDT) algorithm to improve the speed accuracy and robustness of a laser-based detector. This method can deliver more accurate estimation of vehicle speed, enabling computation of the parameters of length and height. The results of simulation with different detector update rates suggest that the average estimation errors of vehicle parameters can be reduced using the NDT matching method, especially for the low detector update rate. The study also implemented a series of field experiments using the proposed detector prototype to verify the detector\u27s measurements of vehicle parameters. The proposed method is a promising way in which to improve the laser-based traffic detector. In simulation test, initial experiments show that the accuracy of speed estimation can reach 95%, given the update rate of 1000 Hz for detector, the average length error can be reduced by approximately 60%. Even for speeding vehicles traveling at 150 km/h, the estimated speed error is limited to 10 km/h. In field test, for a vehicle at the speed of 80km/h, the estimation errors are within the threshold of the maximum errors of simulation, that is, 32 cm for length error and 5.71 km/h for speed error results