Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To determine whether a tag would affect de novo filament assembly, we used vimentin fused at the N-terminus with two different sized tags, AcGFP (239 residues, 27 kDa) and 3 × FLAG (22 residues; 2.4 kDa) to assemble into filaments in two vimentin-deficient epithelial cells, MCF-7 and A431. We showed that regardless of tag size, N-terminally tagged vimentin aggregated into globules with a significant proportion co-aligning with β-catenin at cell–cell junctions. However, the tagged vimentin aggregates could form filaments upon adding untagged vimentin at a ratio of 1:1 or when introduced into cells containing pre-existing filaments. The resultant filament network containing a mixture of tagged and untagged vimentin was less stable compared to that formed by only untagged vimentin. The data suggest that placing a tag at the N-terminus may create steric hinderance in case of a large tag (AcGFP) or electrostatic repulsion in case of highly charged tag (3 × FLAG) perhaps inducing a conformational change, which deleteriously affects the association between head and rod domains. Taken together our results shows that a free N-terminus is essential for filament assembly as N-terminally tagged vimentin is not only incapable of forming filaments, but it also destabilises when integrated into a pre-existing network.     

Blocking the PCNA/NKp44 Checkpoint to Stimulate NK Cell Responses to Multiple Myeloma

Simple SummaryMembrane-associated PCNA is expressed on the surface of human MM cell lines and primary MM cells. Mab 14-25-9 interacts with membrane-associated PCNA and blocks its binding to NK-expressed NKp44, thus activating NK function. We showed that mAb 14-25-9 can serve as an immune checkpoint blocker, enhancing the function of NK cells on target human MM cell lines and primary cells.AbstractMultiple Myeloma (MM) is a devastating malignancy that evades immune destruction using multiple mechanisms. The NKp44 receptor interacts with PCNA (Proliferating Cell Nuclear Antigen) and may inhibit NK cells’ functions. Here we studied in vitro the expression and function of PCNA on MM cells. First, we show that PCNA is present on the cell membrane of five out of six MM cell lines, using novel anti-PCNA mAb developed to recognize membrane-associated PCNA. Next, we stained primary bone marrow (BM) mononuclear cells from MM patients and showed significant staining of membrane-associated PCNA in the fraction of CD38⁺CD138⁺ BM cells that contain the MM cells. Importantly, blocking of the membrane PCNA on MM cells enhanced the activity of NK cells, including IFN-γ-secretion and degranulation. Our results highlight the possible blocking of the NKp44-PCNA immune checkpoint by the mAb 14-25-9 antibody to enhance NK cell responses against MM, providing a novel treatment option.     

A proposed approach for setup time reduction through integrating conventional SMED method with multiple criteria decision-making techniques

Short setup time is an essential element for the effective implementation of many lean pillars, i.e., JIT, and Kanban. Most of the current setup reduction methodologies are based on Shingo’s Single Minute Exchange of Dies (SMED) that suggests the conversion of internal setup operations to external operations. However, the conventional SMED approach – as proposed by Shingo – does not possess a systematic approach to accomplish this conversion. Thus, a new approach is proposed in order to aid the process engineers in implementing SMED. The proposed approach is based on the conventional SMED, but also it incorporates Multiple Criteria Decision-Making Techniques (MCDM) to the third implementation phase. The MCDM techniques used in this work are Analytical Hierarchal Process (AHP), Preference Selection Index (PSI) and Technique for Order Preference by Similarity to Ideal Solution (TOPSIS). The proposed approach provides a systematic procedure for selecting the best setup technique among the available alternatives, and takes also into consideration other factors that affect the decision-making process; including: cost, energy, facility layout, safety, life, quality and maintenance. A real example of PVC industry is used to exemplify the approach. The results demonstrate the capability of the proposed approach in setup time reduction, which in turn will improve machines’ utilization, and increase the productivity and flexibility of the whole facility.     

Production of in situ aluminum-titanium diboride master alloy formed by slag-metal reaction

Al-TiB₂ master alloys have received much attention in recent years owing to their potential as efficient grain refiners for aluminum foundry alloys. In this study, the process of production of master alloys was investigated to develop a low cost method, namely, slag-metal reaction. This method can be used to fabricate Al-TiB₂ master alloy in situ from the TiO₂-H₃BO₃-Na₃AlF₆ and Al system. Since the price of the raw materials is low and the technology is simple, the processing technique appears to reduce the cost of the master alloy. Because of exothermic reactions, not much energy is needed to melt materials. In this process, Titanium diboride particles were formed in situ through the reactions of TiO₂, H₃BO₃ and Na₃AlF₆. Results showed that when the aluminum melted, the condensed TiB₂ particles that formed in situ were spherical with an average diameter of 1 μm. Furthermore, these TiB₂ particles were distributed uniformly through the master alloy.     

Investigation of structural and optoelectronic properties of BaThO3

Structural and optoelectronic properties of BaThO₃ cubic perovskite are calculated using all electrons full potential linearized augmented plane wave (FP-LAPW) method. Wide and direct band gap, 5.7 eV, of the compound predicts that it can be effectively used in UV based optoelectronic devices. Different characteristic peaks in the wide UV range emerges mainly due to the transition of electrons between valance band state O-p and conduction band states Ba-d, Ba-f, Th-f and Th-d.     

64抽头LED线性亮度控制器用数字电位计控制LED亮度

采用LED但缺少微控制器或其它形式控制功能的应用可以得益于一种简单的电路,该电路能对LED的光强作手动控制.适合这种目的的器件有机械式(模拟)和电子式(数字)电位器.     

Synthesis,characterization, and supercapacitor studies of manganese (IV) oxide nanowires

One-dimensional manganese (IV) oxide (MnO₂) (～20 nm in average diameter) were synthesized by cathodic electrodeposition and heat treatment. The mechanism of electrodeposition and nanowire formation were discussed. The product was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR). Nanowires with varying lengths and diameters were found in TEM and SEM images of the sample. The results of N₂ adsorption–desorption analysis indicated that the BET surface area of the MnO₂ nanowires was 157 m² g^?1 and the pore size distributions were 2.5 and 4.5 nm. The electrochemical performances of the prepared MnO₂ as an electrode material for supercapacitors were evaluated by cyclic voltammetry and galvanostatic charge/discharge measurements in a solution of 0.5 M Na₂SO₄. The higher specific capacitance of 318 F g^?1 and good capacity retention of 86% were achieved after 1000 charge–discharge cycles had been observed for the MnO₂ nanowires electrode.     

A secure search protocol for lightweight and low-cost RFID systems

In radio frequency identification (RFID) systems, search protocols are used to find a specific item in a large number of tagged products. These protocols should be secure against RFID attacks such as traceability, impersonation, DoS and eavesdropping. Sundaresan et al. (IEEE Trans Dependable Secure Comput, 2015) presented a server-less search protocol based on 128-bits PRNG function and claimed that their method can address all vulnerabilities of previous protocols. In this paper, we prove that Sundaresan et al.’s protocol is vulnerable to traceability attack with the high probability. In addition, we present an improved protocol to solve the proposed problem and analyze its security level informally and formally based on AVISPA tool and BAN logic.     

A Zero-Pole Reposition Based, 0.95-mW, 68-dB,Linear-in-dB,Constant-Bandwidth Variable Gain Amplifier

A variable-gain amplifier with very low power consumption and wide tuning range is presented. The operational principle of this unique structure is discussed, its most important formulas are derived and its outstanding performance is verified by simulation in TSMC 0.18-μm N-well CMOS fabrication process. Owing to the novel zero-pole repositioning technique, the proposed circuit demonstrates very high frequency bandwidth of 79 MHz while drawing only 0.52 mA from 1.8 V power supply. The interesting results such as a very small core area of about 0.0025 mm² as well as a wide linear-in-dB and constant-bandwidth tuning range of 68.2 dB along with a very low power consumption of 0.95 mW are achieved utilizing standard CMOS technology. The stability of the proposed VGA is verified through transient sinusoidal response analysis. Full process, voltage and temperature (PVT) variation analysis of the circuit is also investigated through Monte Carlo and corner case analysis in order to approve the robustness of the structure. Monte Carlo simulations show standard deviation values of 4.6 dB and 78.3 MHz in gain and gain-bandwidth product, respectively. These results show that our zero-pole repositioning method would lend itself well for use in low-power and high-frequency applications, especially in high-speed automatic gain control amplifiers.