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Aiming at the problem that XY motion platform cannot realize high-speed and high-precision machining simultaneously, a high-speed and high-precision laser processing algorithm is proposed based on the macro-micro structure of laser cutter with a laser galvanometer scanner. The velocity planning of macro platform and composite motion is carried out by applying finite impulse response (FIR) filters three times, and velocity planning of micro platform is obtained by vector subtraction method. The motion decomposition of the macro-micro platform is realized accurately. The macro platform is responsible for large-format movement, and the micro platform is responsible for fast-speed turning. Velocity overlap of adjacent segments is employed to improve the machining efficiency. The constraints are established to determine the overlapping time, so that the algorithm can realize high-speed machining without introducing contour error, and ensure that the velocity, acceleration, and jerk of the macro platform can be processed within the limit range. Experiment and simulation analysis show that compared with using only the XY platform, the total machining time of using the macro-micro platform with the algorithm proposed in this paper is reduced by more than 35%.

The underwater glider (UG) is a new type of long-endurance marine vehicle that is currently widely used in marine environment surveillance. However, the limited capability of underwater communication and the uncertainty of the marine environment leads to a large deviation between the dead-reckoned trajectory and the real trajectory of UG. Therefore, a localization and navigation method based on ocean current data is proposed in this study. Firstly, the working principle and configuration of UG are illustrated, and the dynamic model is also analyzed. Then, the depth-averaged current is derived with motion parameters of UG, and the ocean current data from the Hybrid Coordinate Ocean Model is also introduced and processed. Meanwhile, to make an accurate localization, the DAC is stratified according to the layer weight extracted from the processed ocean current model to obtain layered DAC (LDAC). Subsequently, because the sample intervals of sensors are asynchronous with LDAC, a new matching algorithm based on sample time difference is presented to match the information of sensors and LDAC. Furthermore, the matched data is applied to localization for accurate dead reckoning. Finally, based on the data collected from two experiments, the LDAC-aided localization method proposed in this paper is compared with other benchmark positioning methods, including dynamic model-aided localization, sea current-aided localization, and acoustic beacon-aided localization methods. The results indicate that the proposed new method achieves better localization performance than other benchmark methods. In addition, the proposed method could be applied to other autonomous vehicles with communication constraints.

Background: β-mannanase, an enzyme can randomly hydrolyze mannan, has been applied in different fields such as agriculture, food, and paper industries. Although various β-mannanase with different properties have been characterized, few of them meet the high demand in the biorefinery industry.Results: A β-mannanase mutant with higher activity was achieved and applied to prepare manoligosaccharides. To improve β-mannanase activity, the directed evolution of β-mannanase gene from Bacillus licheniformis KD-1 was carried out by error-prone PCR and expressed in Bacillus subtilis. Two loci (I91N and L211I) located at outside of the β-mannanase catalytic center influence the enzyme activity at great degree. For the biosafety of β -mannanase application, the B. subtilis food-grade expression system was constructed. Firstly, the d-alanine racemase (dal) gene of the bacteria was deleted by CRISPR/Cpf1 genome editing without scars. Secondly, the corresponding expression vector was constructed by deleting the antibiotics marker genes replaced with dal gene to realize auxotrophic complementation screening. The results showed that the β-mannanase mutant, ManBl (I91N/L211I) had the enzyme activity of 17601.3 U/mL and its specific activity was 15554.7 U/mg, which was 4.2 times of the wild-type ManBl. The food-grade expressed β-mannanase was applied to degrade konjac glucomannan for preparing manoligosaccharides and the results showed that the formed manoligosaccharides accounted for 85% of the hydrolysate. Conclusions: Our findings provide evidence of two loci located at outside the β-mannanase ManBl catalytic center influence the enzyme activity at great degree. The food-grade expressed β-mannanase (I91N/L211I) mutant with higher activity can be used to prepare manoligosaccharides, which lays the basis for transforming the low-value materials to high-value products.

Water-saturated column experiments were conducted to study the effect of nonwoven geotextiles on bacteria transport and deposition through two sandy porous media with grain sizes 1.05 and 3.25 mm. The breakthrough curves (BTCs) of tracer for the all porous media exhibited an asymmetrical shape with a substantial tailing, indicating that non-equilibrium and dispersive flow patterns in these porous media. The mass recovery of the bacteria from the effluent (Meff) increased with grain size. The retention profiles (RPs) exhibited hyper-exponential behavior, especially in the finer sand. The presence of the geotextiles increased bacteria retention rate. For a given geotextile, greater retention was observed in the surrounding region close to the geotextile. Moreover, the retention of bacteria became more significant in the geotextile with a lower porosity. Results demonstrated that model simulations of bacteria transport and fate need to accurately account for both observed BTC and RP behaviors and also the geotextile placement can impact mechanisms of retention.

Growing evidence supports that LKB1‐deficient KRAS‐driven lung tumors represent a unique therapeutic challenge, displaying strong cancer plasticity that promotes lineage conversion and drug resistance. Here we find that murine lung tumors from the KrasLSL‐G12D/+; Lkb1flox/flox (KL) model show strong plasticity, which associates with up‐regulation of stem cell pluripotency genes such as Nanog. Deletion of Nanog in KL model initiates a gastric differentiation program and promotes mucinous lung tumor growth. We find that NANOG is not expressed at a meaningful level in human lung adenocarcinoma (ADC), as well as in human lung invasive mucinous adenocarcinoma (IMA). Gastric differentiation involves activation of Notch signaling, and perturbation of Notch pathway by the γ‐secretase inhibitor LY‐411575 remarkably impairs mucinous tumor formation. In contrast to non‐mucinous tumors, mucinous tumors are resistant to phenformin treatment. Such therapeutic resistance could be overcome through combined treatments with LY‐411575 and phenformin. Overall, we uncover a previously unappreciated plasticity of LKB1‐deficient tumors and identify the Nanog‐Notch axis in regulating gastric differentiation, which holds important therapeutic implication for the treatment of mucinous lung cancer., This study reveals the plasticity of LKB1‐deficient tumors, and identifies the Nanog‐Notch axis in regulating gastric differentiation. Combinational treatment of γ‐secretase inhibitor LY‐411575 and phenformin effectively blocked invasive mucinous adenocarcinoma IMA formation.

Background：Neuromyelitis optica spectrum disorder (NMOSD) is a severe relapsing and disabling inflammatory autoimmune disease of the central nervous system. Despite the progress made in understanding the pathogenesis of the disease, the optimal first line treatment to reduce relapse rate and ameliorate neurological disability remains unclear. Mesenchymal stem cells (MSC) are known for having anti-inflammatory and regenerative properties and there are a number of studies on the use of human umbilical cord MSCs (hUC-MSCs) in NMOSD patients. Therefore, we will conduct a prospective, multicenter, randomized, placebo-controlled clinical trial to study the safety and effectiveness of hUC-MSCs in the treatment of NMOSD.Methods：The trial is planned to recruit 430 AQP4-IgG seropositive NMOSD patients. The multicenter study will be conducted in six clinical centers. The whole clinical trial consists of three consecutive stages. The first stage will be carried out in the leading center (Ren Ji Hospital) only and it will last 24 months. In the first stage the primary objective is to evaluate the safety of hUC-MSCs. Patients (n=30) will be treated with three different doses of hUC-MSC: the low dose group (n=10, 1×106 MSC / kg·weight), the medium dose group (n=10, 2×106 MSC / kg·weight) and the high dose group (n=10, 5×106 MSC / kg·weight). The second stage, which aims to find the optimal dosage, will last 24 months and will be carried out in six centers (one leading center and five branch centers). Patients (n=160) will be randomized into four groups by 1:1:1:1 allocation ratio: the low, medium, high dose groups and the controlled group (n=40). The third stage, which aims to evaluate the effectiveness, will lasts for 24 months and will be developed in six centers. Patients (n=320) will be 1:1 randomized into two groups: the optimal dose group (n=160) and the controlled group (n=160). HUC-MSC infusion will be given four times to the intervention groups and stem cell solution will be given four times to the control group every 3 months; besides the primary drugs will be provided to both groups. Primary endpoint is the first recurrent time and secondary endpoints are the recurrent times, EDSS scores, MRI lesion numbers, OSIS scores, Hauser walking index and SF-36 (quality of life short Form-36) scores. Exploratory endpoints will include: serum lymphocyte subsets, cytokines, complements, serum anti-AQP4 antibody titers etc. Endpoint events and side-effects will be evaluated at baseline and every 3 months for a total of 24 months follow-up.Discussion：Although hUC-MSC has shown promising treatment effect of NMOSD in preclinical study, there is still a lack of well-designed clinical trials to evaluate the safety and effectiveness of hUC-MSC among NMOSD patients. As far as we know, this trial will be the first one to systematically demonstrate the clinical safety and efficacy of hUC-MSC in treating NMOSD and might be able to determine the optimal dose of hUC-MSC for NMOSD patients.Trial registration：The study was registered with Chinese Clinical Trial Registry (CHICTR.org.cn) on Mar 2, 2016 (registration No. ChiCTR-INR-16008037), and the revised trial protocol (Protocol version 1.2.1) was released on March 16, 2020.

Non-contact vibration measurement based on tip timing has been widely used in aero-engine blade health monitoring. However, it is different to inspect the vibration of turbine blades with small size in high rotating speeds, temperature and pressure operation. To overcome the difficulty of aero-engine turbine blade measurement, firstly, the optical sensor with cooling channel is designed and a novel air-cooling method is proposed. Secondly, a disk for once-per-revolution (OPR) sensor placement is designed and the black paint is sprayed on the shaft. The OPR signal can be achieved by detecting the intensity change of light as the shaft rotates. Thirdly, the non-contact vibration measurement based on tip timing is conducted to monitor turbine blades, and the resonance frequencies, blade tip vibration amplitude and rotational speeds of blades are obtained. The test results show that the 1st bending mode resonance of the turbine blades occurred at about 8200r/min excited by engine order 12 and 9100r/min excited by engine order 11. Furthermore, the identified vibration parameters from the test have a good match with the results of the numerical calculation. It demonstrates the promise of the proposed non-contact vibration measurement technique based on blade tip timing for health monitoring of aero-engine turbine blades.

In the process of multi axis machine tool, sometimes it is necessary to switch between the rotating spindle and the rotary feed shaft. In order to satisfy the request mentioned above, this article makes a brief introduction to the upper software and analyzes the control principle of spindle by using PMAC, combined with that, a method to deliver the automatic switching function between spindle mode and C-axis mode has been put forward, which has already used in the independent R&D project single-point diamond ultra-precision CNC system. In addition, on the basis of this method, a more advanced function called constant linear speed mode will be explored, which can both ensure the quality of workpiece and improving efficiency enormously.

Compensation of the positioning error caused by external uncontrollable factors is an important issue for studying the hydrostatic rotary table in the closed loop control of 5-axis ultra-precision machine tool. In order to solve the problem, improve the surface roughness and the dimensional accuracy of the parts, the paper proposes a hardware platform based on the UMAC (Universal Motion and Automation Controller), drive and IPC (Industrial Personal Computer). High-precision detection method with laser interferometer is used to measure and assess the positioning error of the turntable under closed loop control so as to establish error compensation table. The algorithm of UMAC inserted is used to compensate the positioning error in real time. The experimental results show that the UMAC error compensation function improves the hydrostatic rotary table positioning accuracy (up to ±2").

As a high-precision multi-axis motion controller designed by Delta Tau, PMAC has been widely used in industrial fields. Mirror image is a function through which can machine the same trace in other place of the workpiece, not only saving the tool setting time but also simplifying the edit of program. By this way, product could have good accuracy and interchangeability. However, researches aimed at mirror function based on PMAC is rare. This paper analyzed the principle of mirror image, combined with coordinate transformation and G-code development way, a method to deliver the mirror function G-code has been put forward, which has already used in the independent R&D project single-point diamond ultra-precision CNC system. In addition, this article introduced an upper computer software of CNC system based on Qt and listed the program code of mirror function. The method is available to any CNC system based on PMAC, offering good versatility.

Advances in genomic sequencing and insights into molecular leukemogenesis are opening the door to using targeted agents to tailor treatment for acute myeloid leukemia (AML) in individual patients. Although this shift away from traditional cytotoxic therapies represents an innovative approach to AML therapy, a number of obstacles stand in the way of widespread adoption of targeted therapy into daily practice. For example, the effects of single agents are marginal, and the degree of variability among patients is great. Some have advocated incorporation of newly identified biomarkers into clinical trials to guide patient-specific treatment, but the relevance of these biomarkers to clinical response is uncertain and requires further validation. Combining targeted agents with other targeted agents or with conventional chemotherapy to overcome the biological heterogeneity of AML may enhance treatment efficacy; however, drug toxicities also are increased and drug resistance continues to occur. Overall survival is an impractical endpoint for clinical trials of AML, which may be addressed by using the endpoint of event-free survival to evaluate novel targeted agents. Another barrier to implementation is the high cost and limited availability of targeted agents. Herein, we address the above practical questions and propose potential strategies for the future evaluation of targeted treatments.

ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.

Electrogenerated chemiluminescence (ECL) of a novel phenyleneethynylene derivatives [4,4’-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid; P-acid] possessing carboxylic acid groups at the para positions in aqueous solutions and its first immunosensing application are studied. Nanotubular mesoporous Pt–Ag alloy nanoparticles are first used to fabricate the ECL immunosensor as a signal amplification carrier. Absorption, photoluminescence, cyclic voltammetry, scanning electron microscopy, transmission electron microscopy, and electrochemical impedance spectroscopy are used as powerful tools to characterize P-acid, Pt–Ag alloy nanoparticles, and the fabrication process of the immunosensor. P-acid exhibits two ECL peaks at –1.07 (ECL-1) and 0.64 (ECL-2) V in air-saturated pH 7.4, 0.1 M PBS containing 0.1 M KCl during the cyclic sweep between 1.51 and –2.67 V at 0.1 V s−1; the ECL mechanism of these two ECL peaks is demonstrated. The electron-transfer reaction between electrochemically oxidized P-acid and tri-n-propylamine (TPrA) radical cation is also demonstrated to produce high ECL emissions and used to develop an ultrasensitive ECL immunosensor. In addition, a Pt–Ag alloy nanoparticles amplification carrier for protein ECL analysis is applied for improvement of the detection sensitivity. Thus, this ECL immunosensor exhibits high sensitivity, good reproducibility, rapid response, and long-term stability.

Thin films of molecularly imprinted sol–gel polymer with specific binding sites for trichlorfon were prepared, fixed on glassy carbon electrodes and used as recognition material. The binding characteristic of the imprinted films to trichlorfon was evaluated by equilibrium binding experiments; and, the morphology was studied by scanning electronic microscope. A novel electrochemical sensor for determination of trichlorfon was developed based on the reaction between trichlorfon and the molecularly imprinted sol–gel film, which was a modified glassy carbon electrode. The sensor displayed excellent selectivity and high sensitivity. The linear response range of the sensor was 10−8 –10−6 g mL−1, and the limit of detection was 2.8 × 10−9 g mL−1. The relative standard deviation for the determination of 10−7 g mL−1 trichlorfon was 3.5%. The sensor was applied to the determination of trichlorfon in vegetables with satisfactory results.

Si-Cu alloys have been prepared by electroless depositions with different process. Si-Cu/MCMB materials have been prepared by carbonization of the mixture of Si-Cu, MCMB and mesophase pitch. The influences of electroless deposition condition and heat-treatment temperature on the anodic performance of Cu-Si/MCMB have been studied. Compared with Si/MCMB, Si-Cu/MCMB has more stable cyclical performance. Furthermore, the Si-Cu/MCMB sample produced by cleaning and then electroless deposition on Si shows the better anodic performance than the Si-Cu/MCMB sample produced by direct electroless deposition on Si, because the former sample has more Cu contents than the latter sample.

A novel polyfluorene derivative, poly[(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7- fluorene)-alt-2,7-(9,9- p-divinylbenzene)] (P-1) was synthesized by the palladium-catalyzed Suzuki coupling reaction and it’s quaternized ammonium polyelectrolyte derivatives was obtained through a postpolymerization treatment on the terminal amino groups. The electrochemical and optical properties of the resulting copolymers was fully investigated. The results showed that the new polyfluorene derivative had potential useful in used as materials for electronic conductivity, chemical and biological sensors that derive from its highly conductive and strong fluorescence.

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr1605-Met1606 bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal α-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln1624 and Arg1668, and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln1624-Arg1641 minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4′-P18′ residues on either side of the Tyr1605-Met1606 bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.

Hearing loss is currently an incurable degenerative disease characterized by a paucity of hair cells (HCs), which cannot be spontaneously replaced in mammals. Recent technological advancements in gene therapy and local drug delivery have shed new light for hearing loss. Atoh1, also known as Math1, Hath1, and Cath1, is a proneural basic helix-loop-helix (bHLH) transcription factor that is essential for HC differentiation. At various stages in development, Atoh1 activity is sufficient to drive HC differentiation in the cochlea. Thus, Atoh1 related gene therapy is the most promising option for HC induction. DAPT, an inhibitor of Notch signaling, enhances the expression of Atoh1 indirectly, which in turn promotes the induction of a HC fate. Here, we show that DAPT cooperates with Atoh1 to synergistically promote HC fate in ependymal cells in vitro and promote hair cell regeneration in the cultured basilar membrane (BM) which mimics the microenvironment in vivo. Taken together, our findings demonstrated that DAPT is sufficient to induce HC-like cells via enhancing of the expression of Atoh1 to inhibit the progression of HC apoptosis and to induce new HC formation.

Aortic aneurysms are characterized by vascular inflammation, neovascularization, and extracellular matrix destruction of the aortic wall. Although experimental studies indicate a potential role of CD248 in microvessel remodeling the functions of CD248 in human vascular pathologies remain unexplored. Here we aimed to study how CD248 interferes with pathological vascular remodeling of human aortic aneurysms. Immunofluorescent staining showed that CD248 expression was mainly localized in the CD8+ T cells infiltrating in the adventitia and media of aortic walls of patients with ascending thoracic aortic aneurysms (TAA). qPCR and immunofluorescent staining analyses revealed increased aortic CD248 expression and infiltrating CD248+CD8+ T cells in aortic aneurysms than in nonaneurysmal aortas. Flow cytometry analysis of human peripheral blood further identified a fraction of circulating CD248+ cells which was confined in the CD8+ T-cell compartment. The increased infiltrating of CD248+CD8+ T cells was coincident with reduced circulating CD248+CD8+ T cells in patients with ascending TAA when compared with patients with coronary artery diseases (CAD) and healthy donors. The CD248+CD8+ T cells were characterized by upregulated IL-10 and downregulated IL-1β/INF-γ expression when compared with CD248-CD8+ T cells. Moreover, when co-cultured with human aortic endothelial cells, the CD248+CD8+ T cells not only downregulated endothelial expression of ICAM1/VCAM1 and MMP2/3 but also suppressed endothelial migration. Thus, this study shows that CD248 reduces pathological vascular remodeling via anti-inflammatory CD248+CD8+ T cells, revealing a CD248-mediated cellular mechanism against human aortic aneurysms.

Cardiac tissue engineering using biomaterials with or without a combination of cardiac stem cell therapy offers a new therapeutic option for repairing infarcted heart. So far, cardiac tissue scaffold is designed mainly based on natural and synthetic biomaterials, which do not mimic human myocardial extracellular matrix. This raises a fundamental issue about the biocompatibility of currently used biomaterials with myocardial tissue. Here we hypothesized that human heart valve-derived scaffold (hHVS) may provide a clinically relevant novel biomaterial for cardiac repair. In this study, human heart valve tissue was sliced into 100 μm tissue sheet by frozen-sectioning and then decellularized to form the hHVS. Upon anchoring onto the hHVS, post-infarct murine BM c-kit+ cells exhibited an increased capacity for proliferation and cardiomyogenic differentiation in vitro. When used to patch infarcted heart in a murine model of myocardial infarction, either implantation of the hHVS alone or c-kit+ cell-seeded hHVS significantly improved cardiac function, as shown by transthoracic echocardiography as well as by hemodynamic measurements via a Millar catheter, and by reduced infarct size; while c-kit+ cell-seeded hHVS was even superior to the hHVS alone. Thus, we have successfully developed a hHVS for cardiac repair. Our in vitro and in vivo observations provide the first evidence for translating the hHVS-based cardiac tissue engineering into clinical strategies to treat myocardial infarction.

Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr 1605 –Met 1606 scissile bond in the VWF A2 domain depends on a Glu 1660 –Arg 1668 segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme–substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage ≈20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.

Introduction: Neuronal and vascular regeneration is important for post-ischemic recovery. In this study we investigated whether co-transplantation of neural stem cells (NSCs) and vascular progenitor cells (VPCs) could promote the therapeutic efficacy of stem cell based therapy after transient middle cerebral artery occlusion (tMCAO). Methods: tMCAO was generated in adult male SD rats. One day after MCAO, NPCs (1x106), VPCs (1x106) or NPCs (5 x105) and VPCs (5x105) were transplanted into the cortical perifocal region. Neurobehavioral outcomes, infarct volume, cell differentiation, angiogenesis and neurogenesis were examined within 14 days following tMCAO. Results: MRI analysis and behavioral tests showed that co-transplantation of NSCs and VPCs significantly decreased infarct volume and improved neurobehavioral recovery at 7 and 14 days after ischemia compared to NPCs group and VPCs group (p Conclusion: We demonstrated that co-transplantation of NPCs and VPCs had great benefits for post-ischemic recovery. Our findings suggest that combined transplantation of different types of stem cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.

Most ADAMTS13 assays use non-physiological conditions (low ionic strength, low pH, barium chloride), are subject to interference from plasma proteins, hemoglobin and bilirubin, and have limited sensitivity, especially for inhibitors.We addressed these constraints by designing a substrate that can be used in undiluted plasma.A polypeptide was expressed in E. coli that corresponds to von Willebrand factor Gln(1599) -Arg(1668) , with mutations N1610C and K1617R and an N-terminal Gly. Substrate FRETS-rVWF71 was prepared by modifying Cys(1610) with DyLight 633 (abs 638 nm, em 658 nm) and the N-terminus with IRDye QC-1 (abs 500-800 nm). Assays were performed at pH 7.4 in 150 mm NaCl, 10 mm CaCl2 .Serum and plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity with FRETS-rVWF71. Neither bilirubin (≤ 20 mg dL(-1) ) nor hemoglobin (≤ 20 g L(-1) ) interfered with product detection. Assays with FRETS-rVWF71 and FRETS-VWF73 gave similar results (R(2 ) = 0.95) for plasma from 80 subjects with thrombotic microangiopathy, 22 subjects with other causes of thrombocytopenia, and 20 healthy controls. The limit of detection with FRETS-rVWF71 for ADAMTS13 activity was ≤ 0.3%. Inhibitor assays with FRETS-rVWF71 gave titers ~2.5-fold higher than with FRETS-VWF73 and clearly distinguished patients with and without inhibitors.FRETS-rVWF71 is suitable for ADAMTS13 assays in minimally diluted plasma or serum without interference from proteins, bilirubin or free hemoglobin in plasma. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses during the treatment of thrombotic thrombocytopenic purpura.

A novel, simple and highly sensitive amplified fluorescence label-free immunosensor by using p-acid-encapsulated silica nanomaterials has been developed for the first time. 4,4'-(2,5-Dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl)dibenzoic acid (p-acid) and p-acid-encapsulated silica were prepared, and characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet visible spectroscopy (UV-vis) and fluorescence spectroscopy. In layer-by-layer self-assembling processes using (3-aminopropyl)triethoxysilane, p-acid@SiO(2) was assembled on the glass substrate. Antibody was immobilized on the surface of p-acid@SiO(2) with N,N'-carbonyldiimidazole. The functional nanomaterials present good analytical properties with a calibration range of 0.1-100 ng mL(-1), and allow the detection of carcinoembryonic antigen (CEA) at a concentration as low as 0.04 ng mL(-1). What is important is that the as-synthesized p-acid@SiO(2) nanomaterials could be further extended for the detection of other biomarkers or biocompounds.

A novel water-soluble polyfluorene derivative, poly[(9,9-bis(3′-((N,N-dimethylamino)N-ethylammonium)propyl)-2,7-fluorene)-alt-2,7-(9,9-p-divinylbenzene)]dibromide (P-2) was synthesized by the palladium-catalyzed Suzuki coupling reaction and it’s quaternized ammonium polyelectrolyte derivatives was obtained through a postpolymerization treatment on the terminal amino groups. The electrochemical and optical properties of the copolymers was fully investigated. The results showed that the new polyfluorene derivative had high electronic conductivity and strong fluorescence, therefore it had good potential to be used in chemical and biological sensors, as shown in optical sensing of bovine albumin (BSA) in this study.

A simple and convenient on-line molecular imprinted solid-phase extraction flow-injection fluorescence sensor was developed in this study. Florfenicol (FF)-imprinted polymer was prepared by self-assembly with acrylamide (AM) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The binding characteristics of the imprinted polymer to florfenicol were evaluated by equilibrium binding experiments, and the morphology of the imprinted polymer was studied by scanning electron microscope (SEM). Fluorescence intensity of 3-p-nitrylphenyl-5-(2'-sulfonophenylazo) rhodanine (4NRASP) and bovine serum albumin (BSA) was inhibited by FF. Under optimum conditions the intensity of fluorescence shows a linear relationship with the concentration of FF over the range of 1.2x10(-6) to 2.6x10(-5)gmL(-1) with a regression equation of DeltaI=23.54+17.86c (c 10(-6)gmL(-1)) (r=0.9965, n=6). The low detection limit of FF was found to be 3.4x10(-7)gmL(-1) according to 11 parallel determinations for the blank solution. The relative standard deviation for the determination of 2.0x10(-6)gmL(-1) FF solution was 3.5% (n=11). This proposed sensor could be satisfactorily applied to the determination of FF in liver and meat samples.

To develop three-dimensional (3D) porous nanofiber scaffold of PLGA-silk fibroin-collagen and to investigate its cytocompatibility in vitro.Method of electrostatic spinning was used to prepare 3D porous nanofiber scaffold of PLGA-silk fibroin-collagen (the experimental group) and 3D porous nanofiber scaffold of PLGA (the control group). The scaffold in each group was observed by scanning electron microscope (SEM). The parameters of scaffold fiber diameter, porosity, water absorption rate, and tensile strength were detected. SC harvested from the bilateral brachial plexus and sciatic nerve of 8 SD suckling rats of inbred strains were cultured. SC purity was detected by S-100 immunohistochemistry staining. The SCs at passage 4 (5 x 10(4) cells/mL) were treated with the scaffold extract of each group at a concentration of 25%, 50%, and 100%, respectively; the cells treated with DMEM served as blank control group. MTT method was used to detect absorbance (A) value 1, 3, 5, and 7 days after culture. The SC at passage 4 were seeded on the scaffold of the experimental and the control group, respectively. SEM observation was conducted 2, 4, and 6 days after co-culture, and laser scanning confocal microscope (LSCM) observation was performed 4 days after co-culture for the growth condition of SC on the scaffold.SEM observation: the scaffold in two groups had interconnected porous network structure; the fiber diameter in the experimental and the control group was (141 +/- 9) nm and (205 +/- 11) nm, respectively; the pores in the scaffold were interconnected; the porosity was 87.4% +/- 1.1% and 85.3% +/- 1.3%, respectively; the water absorption rate was 2 647% +/- 172% and 2 593% +/- 161%, respectively; the tensile strength was (0.32 +/- 0.03) MPa and (0.28 +/- 0.04) MPa, respectively. S-100 immunohistochemistry staining showed that the SC purity was 96.5% +/- 1.3%. MTT detection: SC grew well in the different concentration groups and the control group, the absorbance (A) value increased over time, significant differences were noted among different time points in the same group (P0.05), and there was no significant difference between the different concentration groups and the blank control group at different time points (P0.05). SEM observation: in the experimental group, SC grew well on the scaffold, axon connection occurred 4 days after co-culture, the cells proliferated massively and secreted matrix 6 days after co-culture, and the growth condition of the cells was better than the control group. The condition observed by LSCM 4 days after co-culture was the same as that of SEM.The 3D porous nanofiber scaffold PLGA-silk fibroin-collagen prepared by the method of electrostatic spinning is safe, free of toxicity, and suitable for SC growth, and has good cytocompatibility and proper aperture and porosity. It is a potential scaffold carrier for tissue engineered nerve.

To study the outcomes of nerve defect repair with the tissue engineered nerve, which is composed of the complex of SCs, 30% ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable poly (D, L-lactic acid) (PDLLA) catheters.SCs were cultured and purified from the sciatic nerves of 1-day-old neonatal SD rats. The 1st passage cells were compounded with bFGF-PLGA sustained release microspheres and ECM gel, and then were injected into permeable PDLLA catheters with PLGA microfilaments inside. In this way, the tissue engineered nerve was constructed. Sixty SD rats were included. The model of 15-mm sciatic nerve defects was made, and then the rats were randomly divided into 5 groups, with 12 rats in each. In group A, autograft was adopted. In group B, the blank PDLLA catheters with PBS inside were used. In group C, PDLLA catheters, with PLGA microfilaments and 30% ECM gel inside, were used. In group D, PDLLA catheters, with PLGA microfilaments, SCs and 30% ECM gel inside, were used. In group E, the tissue engineered nerve was applied. After the operation, observation was made for general conditions of the rats. The sciatic function index (SFI) analysis was performed at 12, 16, 20 and 24 weeks after the operation, respectively. Electrophysiological detection and histological observation were performed at 12 and 24 weeks after the operation, respectively.All rats survived to the end of the experiment. At 12 and 16 weeks after the operation, group E was significantly different from group B in SFI (P0.05). At 20 and 24 weeks after the operation, group E was significantly different from groups B and C in SFI (P0.05). At 12 weeks after the operation, electrophysiological detection showed nerve conduct velocity (NCV) of group E was bigger than that of groups B and C (P0.05), and compound amplitude (AMP) as well as action potential area (AREA) of group E were bigger than those of groups B, C and D (P0.05). At 24 weeks after the operation, NCV, AMP and AREA of group E were bigger than those of groups B and C (P0.05). At 12 weeks after the operation, histological observation showed the area of regenerated nerves and the number of myelinated fibers in group E were significantly differents from those in groups A, B and C (P0.05). The density and diameter of myelinated fibers in group E were smaller than those in group A (P0.05), but bigger than those in groups B, C and D (P0.05). At 24 weeks after the operation, the area of regenerative nerves in group E is bigger than those in group B (P0.05); the number of myelinated fibers in group E was significantly different from those in groups A, B, C (P0.05); and the density and diameter of myelinated fibers in group E were bigger than those in groups B and C (P0.05).The tissue engineered nerve with the complex of SCs, ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable PDLLA catheters promote nerve regeneration and has similar effect to autograft in repair of nerve defects.

To evaluate the effectiveness of percutaneous fixation with iliosacral screw in the management of unstable pelvic fractures.From March 2003 to January 2007, 15 patients with unstable pelvic fractures were treated by percutaneous fixation with iliosacral screws. There were 6 males and 9 females, aged 21-56 years. Fractures were caused by traffic accident in 8 cases, high falling in 6 cases and crushing injury in 1 case. The disease course ranged from 4 hours to 3 days. Of 15 patients, 7 had fractures of pubium and sacrum; 2 had fractures of ilium and sacrum; 4 had dislocation of sacroiliac joint with fractures of pubic rami; and 2 had dislocation sacroiliac joint with fracture of sacrum of pubium. Anterior fixation was performed by means of plating in 3 cases, by external fixation in 7 cases, and by both in 5 cases.The blood loss was from 1,000 to 1,500 mL in 4 patients with acetabular fracture and about 50 mL in the others. The average operative time was 153.6 minutes (range, 60-305 minutes). Fifteen patients were followed up 6 months to 3 years with an average of 18 months. According to Matta criterion for fracture reduction, the results were excellent in 14 cases and good in 1 case. After a follow-up of 28.8 months, fracture union was achieved in all patients. Screw loosening occurred in 1 case, pain of lumbosacral region in 3 cases after tiredness and mild claudication in 2 cases. Nine patients returned to their occupation, three patients changed occupation, and 3 patients could not work at last follow-up. According to Majeed functional scoring, the results were excellent in 11 cases and good in 4 cases.Percutaneous iliosacral screw fixation is a useful method in treatment of unstable pelvic fracture.

Reportedly, complexes between factor XI and ADAMTS13 are detected with a commercial ADAMTS13/FXI ELISA kit in plasma and are decreased in thrombotic thrombocytopenic purpura (TTP). Using this kit, control and TTP patient plasma contained varying amounts of signal (25-670% of a reference plasma) but no signal was observed for mixtures of recombinant enzymes, suggesting little interaction. ADAMTS13/FXI complexes were undetectable by immunoprecipitation or gel filtration chromatography in control plasma or mixtures of recombinant proteins. These results suggest that ADAMTS13/FXI complexes are insignificant in plasma and unlikely to affect the function of either protein during normal hemostasis or in TTP.

To investigate the method for isolating greater epithelial ridge by use of thermolysin digestion combined with dissection under microscope.The basement membrane prepared from postnatal day 0 to postnatal day 3 rat was placed into D-Hanks solution with thermolysin and DNase, then incubated at 37 degrees C for 25 minute and dissected under microscope. After GER had been isolated, morphological comparison, immunohistochemistry, RT-PCR and GER culture were performed for the purpose of identification of GER.Immunohistochemistry indicated that the isolated GER was purely epithelial tissue. RT-PCR analysis certified that the isolated GER expressed Hes1 which was selectively expressed in GER at early postnatal in cochlea. The isolated GER could be successfully cultured in the presence of 5% serum and could survive at least 12 day in the serum-free medium.GER isolation can be successfully performed by use of thermolysin combined with microdissection. This method may provide a good technique to establish the GER cell lines or perform series of gene transfection experiments.

In this paper, a novel amino-functionalized polyfluorene derivative has been successfully designed and synthesized. Polyfluorene derivatives are commonly used as signal-transducing elements for sensor applications due to their signal amplification properties and the versatility of the molecular design process. Hence, an electrogenerated chemiluminescence (ECL) label-free immunosensor based on a novel ECL reagent was also prepared by chemical modification of a polyfluorene derivative on an indium tin oxide (ITO) glass electrode. The ECL and X-ray photoelectron measurements demonstrated the covalent attachment of the polyfluorene derivative to the ITO glass electrode. It can be found that the ECL of the polyfluorene derivative at a low positive potential (ca. 0.3 V vs. Ag/AgCl) induced a strong light emission on the polyfluorene derivative-modified ITO glass electrode. The label-free immunosensor was successfully applied in the detection of a cancer biomarker: ovarian protein antigen (CA125) with high sensitivity, specificity, and stability. The results indicated that the ECL signal decreased regularly with the CA125 concentration increasing from 0.50 to 500 U mL−1, and the detection limit was 0.087 U mL−1. Moreover, the polyfluorene derivative-based ECL immunosensor can be accurately employed to detect the CA125 concentration in 10 human serum samples, suggesting the promising application of the as-proposed ECL immunosensor in the field of clinical screening of cancer biomarkers.

Abstract 3298 Introduction: Thrombotic thrombocytopenic purpura (TTP) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and microvascular thrombosis. TTP is usually due to acquired, autoimmune deficiency of ADAMTS13, a metalloprotease that cleaves von Willebrand factor (VWF) and inhibits the growth of platelet thrombi. Most patients respond to treatment with plasma exchange, but inhibitory autoantibodies and persistent ADAMTS13 deficiency are associated with an increased risk of refractory or relapsing disease. Therefore, ADAMTS13 activity and inhibitor assays can be useful for diagnosis, prognosis, and monitoring the response to therapy. ADAMTS13 assays currently use the fluorogenic substrate FRETS-VWF73, which absorbs/emits at 340 nm/430 nm. These spectral properties make FRETS-VWF73 subject to interference from plasma proteins, hemoglobin and bilirubin. To avoid this problem, plasma is diluted at least 1:20, which reduces assay sensitivity to 5% of normal ADAMTS13 levels and prevents the detection of some clinically relevant inhibitors. We have addressed these limitations by developing FRETS-rVWF71, a recombinant fluorogenic substrate with chromophores that absorb/emit in the near infrared. Methods: The substrate polypeptide corresponds to VWF residues Gln1599-Arg1668, with mutation N1610C to introduce a reactive thiol and K1617R to remove an amino group that competes with the N-terminus for chemical modification. This peptide was expressed in E. coli as a thioredoxin-(His6)-fusion protein, purified by Ni2+-NTA chromatography, and digested with TEV protease to remove the thioredoxin. After modification at Cys1610 with DyLight 633-maleimide (abs 638 nm, em 658 nm) and at the N-terminus with IRDye QC-1 N-hydroxysuccinimidyl ester (abs 500–800 nm), the substrate FRETS-rVWF71 was purified by C18-HPLC. Assays were performed with 1 μM FRETS-rVWF71 under physiological buffer conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM CaCl2) to facilitate the assay of samples containing up to 95% plasma (Figure, panel A). Inhibitor assays were performed by preincubating equal volumes of pooled normal plasma and serially diluted patient plasma, followed by addition of an equal volume of buffer containing FRETS-rVWF71. Product generation was monitored in a fluorescence microplate reader with 635 nm excitation and 660 nm emission filters. Results: Serum and matched samples of plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity that was stable indefinitely at −20°C. Bilirubin (>20 mg/dL) did not inhibit ADAMTS13 activity. As reported, hemoglobin was a weak inhibitor (EC50 approximately 1g/dL). Neither bilirubin nor hemoglobin interfered with product detection. Healthy donors (Li+-heparin plasma, n = 96) had a mean ADAMTS13 activity of 107.1 ± 18% (SD). Intra-assay and inter-assay coefficients of variation (CV) were Conclusions: The use of chromophores that absorb/emit in the near infrared avoids interference from blood proteins, bilirubin or hemoglobin. The combination of brighter chromophores and compatibility with undiluted plasma makes ADAMTS13 activity assays with FRETS-rVWF71 substantially more sensitive than with FRETS-VWF73. Higher sensitivity allows discrimination between very low levels of activity that may influence the risk of relapse in congenital or acquired TTP. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses to therapy and should help to determine why some patients with acquired TTP relapse and others do not. Disclosures: No relevant conflicts of interest to declare.

Abstract 2211 The von Willebrand factor (VWF) cleaving protease, ADAMTS13, and the aggrecanase, ADAMTS5, have a modular structure that includes metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), cysteine-rich (C), and spacer (S) domains. Both enzymes utilize some combination of DTCS domains to bind substrates and facilitate proteolysis, thereby maintaining the homeostatic balance between the synthesis and degradation of their substrates. We constructed chimeric metalloproteinases and substrates to examine the activity of ADAMTS13 and ADAMTS5 towards their respective physiological cleavage sites at VWF Tyr1605-Met1606 and aggrecan interglobular domain (IGD) Glu373-Ala374 (as shown in table). Cleavage rates were determined by ELISA, and C-terminal products were sequenced to confirm specificity. ADAMTS13 truncated after the S domain (MDTCS13) cleaved recombinant VWF Asp1596-Arg1668 (VWF73) much more rapidly than did constructs MDT13/CS5 and MD13/TCS5, in which distal ADAMTS13 domains were replaced by those of ADAMTS5. Similarly, replacement of the C-terminal 52 residues of VWF73 by aggrecan IGD residues Glu394-Gly458 (VWF/IGD) markedly impaired cleavage by MDTCS13, MDT13/CS5, and MD13/TCS5.Therefore, optimal cleavage of VWF73 depends on interactions between ADAMTS13 TCS domains and C-terminal sites of VWF73. In contrast, MDTCS5 was inactive toward VWF73 or VWF/IGD, whereas MD5/TCS13 efficiently cleaved VWF73 (at Glu1615-Ile1616) but not VWF/IGD. Thus, the TCS domains of ADAMTS13 interact with the Lys1617-Arg1668 segment of VWF73. In the context of ADAMTS13, this interaction accelerates the physiological cleavage of VWF. In the context of the chimeric MD5/TCS13, this interaction allows the ADAMTS5 active site to recognize and cleave a new site in VWF73, which is otherwise resistant to ADAMTS5. As expected, MDTCS5 readily cleaved recombinant IGD residues Thr331-Gly458 (IGD). Replacing the C-terminal 65 residues of IGD with VWF Lys1617-Arg1668 (IGD/VWF) minimally altered the rate of cleavage by MDTCS5, and replacement of the ADAMTS5 TCS domains by those of ADAMTS13 (MD5/TCS13) had little effect on the cleavage of either IGD or IGD/VWF. MDTCS5 and MD5/TCS13 also cleaved the same Glu373-Ala374 site in purified aggrecan with similar efficiency (not shown in table), indicating that recognition of the major cleavage site in the IGD domain does not depend strongly on specific TCS domains. In contrast, the other major site of aggrecan proteolysis by MDTCS5, at Glu1480-Ala1481, was completely resistant to MD5/TCS13, indicating that the TCS domains of ADAMTS13 cannot substitute for those of ADAMTS5. These results show that non-catalytic domains, particularly TCS domains, are principal modifiers of physiological substrate recognition and cleavage by ADAMTS5 and ADAMTS13, and these domains may have a similar function in other members of the ADAMTS family. Values (mean ± SD) for kcat/Km (×105 M-1s-1) were calculated from the initial rate of substrate cleavage determined by ELISA of products. Cleavage of aggrecan at the Glu1480-Ala1481 bond was analyzed by Western blotting with a site-specific antibody. N/A (no activity) indicates no significant cleavage identified by Western blotting or ELISA. Disclosures: No relevant conflicts of interest to declare.

ADAMTS13 reduces the size of plasma von Willebrand factor (VWF) multimers by cleaving the Y1605-M1606 bond within the central A2 domain of the VWF subunit. The C-terminus of VWF domain A2 is known to bind an exosite in the Spacer domain of ADAMTS13. However, ADAMTS13 cleaves smaller substrates that lack this C-terminal binding segment of the A2 domain, which suggests that additional sites in domain A2 interact with additional exosites on ADAMTS13. To identify these recognition sites, we constructed a series of recombinant substrates and analyzed their cleavage by ADAMTS13 variants (Table 1). Products were characterized by Western blotting and with a quantitative MALDI-MS assay. The progressive deletion of C-terminal amino acids of VWF domain A2 progressively reduced the rate of cleavage by ADAMTS13. In addition, the removal of 9 amino acids at the N-terminus of VWF73 (VWF64n) abolished cleavage. Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the first TSP1 domain (construct MDT) cleaved VWF64 ∼9-fold slower than did ADAMTS13 truncated after the Cys-rich domain (construct MDTC). Removal of 18 amino acids from the C-terminus of VWF64 (VWF46) also decreased the rate of cleavage by MDTC ∼9-fold, suggesting that the Cys-rich domain interacts with the segment I1642-R1659. Further C-terminal deletions of the substrate (VWF35, VWF28) had little effect on the rate of cleavage by enzymes MDTC or MDT. Comparing substrates VWF73 and VWF73d3, internal deletion of the region missing from VWF35 and VWF28 (Q1624 to R1641) had only a small effect ( Specificity Constants (kcat/Km) for Substate Cleavage by ADAMTS13 Variants Substrate VWF Sequence MDT MDTC ADAMTS13 Values (×105M−1s−1) are based on at least two independent experiments. No entry: not done. VWF73 D1596-R1668 1.0 7.5 VWF64 D1596-R1659 0.1 0.9 0.5 VWF46 D1596-R1641 0.04 0.1 VWF35 D1596-V1630 0.05 0.1 VWF28 D1596-I1623 0.02 VWF64n Y1605-R1668 Inactive Inactive Inactive VWF73d1 D1596-R1641+E1660-R1668 0.12 0.21 0.24 VWF73d2 D1596-R1641+D1653-R1668 0.16 0.36 0.84 VWF73d3 D1596-I1623+I1642-R1668 0.11 1.5 2.5 VWF73d4 D1596-T1608+Q1624-R1668 Inactive Inactive Inactive

ADAMTS13 is a plasma metalloproteinase that limits platelet-rich thrombi by cleaving von Willebrand factor (VWF) at the Tyr1605-Met1606 bond in the A2 domain. A minimal substrate that consists of GST linked to VWF residues Asp1596-Arg1668 with a C-terminal 6×His tag (GST-VWF73) is cleaved rapidly by plasma ADAMTS13. Further removal of Glu1660-Arg1668, which comprises a predicted C-terminal α-helix (GST-VWF64) markedly reduced the rate of cleavage, suggesting this helix comprises an exosite for substrate recognition. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of GST-VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. After removal of the GST moiety by proteolysis, the kinetic constants for cleavage of VWF73 and VWF64 were determined for several recombinant ADAMTS13 variants using a quantitative MALDI-MS assay (Table). Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the spacer domain (construct MDTCS) cleaved VWF73 ~20-fold faster than did a similar enzyme without the spacer domain (construct MDTC). In contrast, both MDTCS and MDTC cleaved VWF64 slowly at a rate similar to the cleavage of VWF73 by MDTC. Most of the variation in cleavage rates was explained by differences in Km, suggesting that the spacer domain recognizes an exosite at the C-terminus of VWF73 that is missing from VWF64. Cleavage of VWF73 yields a C-terminal product (cVWF63). Purified cVWF63 (7.5 μM) inhibited MDTCS activity toward VWF73 or VWF64 (1.5 μM) by »90%, but did not inhibit MDTC, suggesting that the exosite of VWF73 or cVWF63 interacts directly with the spacer domain. Moreover, cVWF63 inhibited the cleavage of multimeric VWF by full-length ADAMTS13 and MDTCS up to 80%, but did not inhibit MDTC. The selective effects of deleting the ADAMTS13 spacer domain and Glu1660-Arg1668 of the VWF domain A2 suggest that the C-terminal exosite of the VWF A2 domain accelerates substrate cleavage by binding specifically to the ADAMTS13 spacer domain. Table Kinetics studies of VWF73 and VWF64 cleavage by ADAMTS13 and its truncations VWF73 VWF64 Enzyme K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) FL-ADAMTS13 1.7±0.4 1.3±0.1 7.5±2.0 37.7±12.8 1.9±0.4 0.5±0.3 MDTCS 0.8±0.2 1.7±0.1 20.5±6.6 5.5±1.4 0.6±0.1 1.0±0.3 MDTC 16.0±4.5 1.8±0.3 1.1±0.5 17.9±6.3 1.6±0.3 0.9±0.5