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Two fundamentally different approaches are routinely used for protein engineering: user-defined mutagenesis and random mutagenesis, each with its own strengths and weaknesses. Here, we invent a unique mutagenesis protocol, which combines the advantages of user-defined mutagenesis and random mutagenesis. The new method, termed the reverse Kunkel method, allows the user to create random mutations at multiple specified regions in a one-pot reaction. We demonstrated the reverse Kunkel method by mimicking the somatic hypermutation in antibodies that introduces random mutations concentrated in complementarity-determining regions. Coupling with the phage display and yeast display selections, we successfully generated dramatically improved antibodies against a model protein and a neurotransmitter peptide in terms of affinity and immunostaining performance. The reverse Kunkel method is especially suitable for engineering proteins whose activities are determined by multiple variable regions, such as antibodies and adeno-associated virus capsids, or whose functional domains are composed of several discontinuous sequences, such as Cas9 and Cas12a.

Cancer cells have dramatically increased demands for energy as well as biosynthetic precursors to fuel their restless growth. Enhanced glutaminolysis is a hallmark of cancer metabolism which fulfills these needs. Two glutamine transporters, SLC1A5 and SLC38A2, have been previously reported to promote glutaminolysis in cancer with controversial perspectives. In this study, we harnessed the proximity labeling reaction to map the protein interactome using mass spectrometry-based proteomics and discovered a potential protein-protein interaction between SLC1A5 and SLC38A2. The SLC1A5/SLC38A2 interaction was further confirmed by bimolecular fluorescence complementation assay. We further investigated the metabolic influence of SLC1A5 and SLC38A2 overexpression in human cells, respectively, and found that only SLC38A2, but not SLC1A5, resulted in a cancer-like metabolic profile, where the intracellular concentrations of essential amino acids and lactate were significantly increased as quantified by nuclear magnetic resonance spectroscopy. Finally, we analyzed the 5-year survival rates in a large pan-cancer cohort and found that the SLC1A5hi /SLC38A2lo group did not relate to a poor survival rate, whereas the SLC1A5lo /SLC38A2hi group significantly aggravated the lethality. Intriguingly, the SLC1A5hi /SLC38A2hi group resulted in an even worse prognosis, suggesting a cooperative effect between SLC1A5 and SCL38A2. Our data suggest that SLC38A2 plays a dominant role in reprogramming the cancer-like metabolism and promoting the cancer progression, whereas SLC1A5 may augment this effect when co-overexpressed with SLC38A2. We propose a model to explain the relationship between SLC1A5, SLC38A2 and SCL7A5, and discuss their impact on glutaminolysis and mTOR signaling.

APEX2, an engineered ascorbate peroxidase for high activity, is a powerful tool for proximity labeling applications. Owing to its lack of disulfides and the calcium‐independent activity, APEX2 can be applied intracellularly for targeted electron microscopy imaging or interactome mapping when fusing to a protein of interest. However, APEX2 fusion is often deleterious to the protein expression, which seriously hampers its wide utility. This problem is especially compelling when APEX2 is fused to structurally delicate proteins, such as multi‐pass membrane proteins. In this study, we found that a cysteine‐free single mutant C32S of APEX2 dramatically improved the expression of fusion proteins in mammalian cells without compromising the enzyme activity. We fused APEX2 and APEX2(C32S) to four multi‐transmembrane solute carriers (SLCs), SLC1A5, SLC6A5, SLC6A14, and SLC7A1, and compared their expressions in stable HEK293T cell lines. Except the SLC6A5 fusions expressing at decent levels for both APEX2 (70%) and APEX2(C32S) (73%), other three SLC proteins showed significantly better expression when fusing to APEX2(C32S) (69 ± 13%) than APEX2 (29 ± 15%). Immunofluorescence and western blot experiments showed correct plasma membrane localization and strong proximity labeling efficiency in all four SLC‐APEX2(C32S) cells. Enzyme kinetic experiments revealed that APEX2 and APEX2(C32S) have comparable activities in terms of oxidizing guaiacol. Overall, we believe APEX2(C32S) is a superior fusion tag to APEX2 for proximity labeling applications, especially when mismatched disulfide bonding or poor expression is a concern.

Series of composite thin films consisted of graphene nanodots (GNDs) and water-based binder (i.e., polyvinylpyrrolidone and polyvinyl alcohol) are designed and fabricated for nano-engineering devices with enhanced thermal and electrical conductivities. A thermal pyrolysis of citric acid and urea is adopted to synthesize crystalline GNDs under IR irradiation. The as-prepared GNDs are uniformly coated over three types of substrates including Cu foil, cotton cloth and filter paper. The GND thin films emit tunable fluorescence upon thermal treatment of GNDs at 400 °C in helium atmosphere. The thermally treated GND-based thin film exhibits excellent thermal as well as electrical conductivity compared to bare GNDs and reduced graphene oxide sheets. The enhanced conductivity is due to the reduced oxidation level induced by the thermal treatment on GNDs samples which subsequently decreases the photon scattering. With increasing weight loading, GNDs can serve not only as connective point but also as stuff, offering a well-developed conductive path for the heat dissipation. Accordingly, the design of GND thin film is promising for enhanced thermal management for electronic and photonic applications since it enables engineering the fluorescence emission with substantially increased thermal and electrical conductivities.

A silicone-based hydrogel was synthesized from poly(dimethylsiloxane) dialkanol (PDMS), isophorone diisocyanate (IPDI), 2-hydroxyethyl methacrylate (HEMA) and poly(ethylene glycol) methacrylate (PEGMA). The hydrophilicity of the resulting block copolymer was adjustable by manipulating the ratio of PDMS and PEGMA. The results showed that higher PEGMA content led to a lower water contact angle, higher water content, lower elastic modulus and higher glucose permeability. At a PEGMA content of 20%, the protein adsorption decreased to 23% and 18% for lysozyme and human serum albumin (HSA), respectively, of those of the control (PDMS-PU). This indicated that the PDMS-PU–PEGMA hydrogels exhibited an ability to resist protein adsorption. The oxygen permeability ( Dk ) was 92 barrers for the hydrogel with 20% PEGMA. Furthermore, these hydrogels were non-cytotoxic according to an in vitro L929 fibroblast assay. Overall, the results demonstrated that the PDMS-PU–PEGMA hydrogels exhibited not only relatively high oxygen permeability and relative optical transparency, but also hydrophilicity and anti-protein adsorption; therefore, they would be applicable as a contact lens material. Furthermore, this study demonstrated a new approach to controlling the performance of silicone hydrogels.

Montgomery modular multiplication is widely used in public-key cryptosystems. This work shows how to relax the data dependency in conventional word-based algorithms to maximize the possibility of reusing the current words of variables. With the greatly relaxed data dependency, we then proposed a novel scheduling scheme to alleviate the number of memory access in the developed scalable architecture. Analytical results show that the memory bandwidth requirement of the proposed scalable architecture is almost 1/(w - 1) times that of conventional scalable architectures, where w denotes word size. The proposed one also retains a latency of exactly one cycle between the operations of the same words in two consecutive iterations of the Montgomery modular multiplication algorithm when employing enough processing elements. Compared to the design in the related work, experimental results demonstrate that the proposed one achieves an almost 54 percent reduction in power consumption with no degradation in throughput. The reduced number of memory access not only leads to lower power consumption, but also facilitates the design of scalable architectures for any precision of operands.

Modular multiplication is a crucial operation in public key cryptosystems like RSA and elliptic curve cryptography (ECC). This paper presents a new word-based Montgomery modular multiplication algorithm which can be used to achieve a low-latency scalable architecture for efficient hardware implementations. We show how to relax the data dependency in conventional word-based algorithms so that a latency of exactly one cycle can be obtained regardless of the chosen word size w (w > 1). With the presented operand reduction scheme, the proposed scalable architecture can operate at high speeds and suitable data paths can be chosen for specific applications. Complexity analysis shows that the proposed architecture has the lowest latency and area complexity compared to related scalable architectures. Experimental results demonstrate that our design has area, speed, and flexibility advantages over related schemes.

With rapid increases in communication and network applications, cryptography has become a crucial issue to ensure the security of transmitted data. In this paper, we propose a microcode-based architecture with a novel reconfigurable datapath which can perform either prime field GF(p) operations or binary extension field GF(2m) operations for arbitrary prime numbers, irreducible polynomials, and precision. Using these field arithmetic units, users are capable of programming cryptographic algorithms in microcode sequences for full compliance with a majority of public-key cryptographic algorithms such as Rivest-Shamir-Adleman (RSA) and elliptic curve cryptosystems. An algorithmic optimization or refinement can thus be made at a higher level based on the reconfigurable datapath. Experimental results show that the developed processor has full cryptography algorithm flexibility, high hardware utilization, and high performance.

The novel poly(dimethyl siloxane-urethane)/Pluronic hydrogel was fabricated to develop a new ophthalmically compatible material. In this study, the soft segment consists of poly(dimethylsiloxane) dialkanol having hydroxyethyl propoxy end groups and hard segment consists of isophorone diisocyanate (IPDI). The poly(ethylene glycol) methacrylate (PEGMA) was added as the chain-extender to form UV-curable silicone marcomer. Finally, the semi-interpenetrating network (semi-IPN) hydrogel was achieved by reacting with silicone marcomer and Pluronic F127 triblock copolymer under UV-photopolymerization (mSi–PU/F127). It was found that the increase in Pluronic F127 content led to decreased water contact angle and increased the water content of silicone hydrogels. Elastic modulus also decreased with Pluronic F127 content, while surface roughness did not significantly differ from silicone controls. The Pluronic F127 content reached 4%, the apparent protein adsorption amount decreased about 60% in comparison with that of mSi–PU control. It indicated that the mSi–PU/F127 hydrogel membrane had an excellent ability to resist protein adsorption. Additionally, the oxygen permeability ( Dk ) would decrease 24%, as compared with mSi–PU control. Furthermore, these hydrogel membranes were regarded as non-cytotoxic through in vitro L929 fibroblasts proliferation assay. Overall results demonstrated that the mSi–PU/F127 semi-IPN hydrogel provided silicone hydrogel materials not only having relatively high oxygen permeability and a relatively low modulus, but also enhancing hydrophilicity and anti-protein adsorption.

Modular exponentiation with a large modulus, which is usually accomplished by repeated modular multiplications, has been widely used in public key cryptosystems for secured data communications. To speed up the computation, the Montgomery modular multiplication algorithm is used to relax the process of quotient determination, and the carry-save addition (CSA) is employed to reduce the critical path delay. In this paper, based on the inherent data dependency between the modular multiplication and square operations in the H-algorithm of modular exponentiation, we present a new modular exponentiation architecture with a unified modular multiplication/square module and show how to reduce the number of input operands for the CSA tree by mathematical manipulation. The developed architecture has the following advantages. 1) There is no need to convert the carry-save form of an operand into its binary representation at the end of each modular multiplication. In this way, except the final step to get the result of modular exponentiation, the time-consuming carry propagation can then be eliminated. 2) The number of input operands for the CSA tree is reduced in a very efficient way. 3) The hardware saving is achieved with very limited impact on the original critical path delay when designed with two distinct modular multiplication and square components. Experimental results show that our modular exponentiation design obtains the least hardware complexity compared with the existing work and outperforms them in terms of area-time (AT) complexity as well.

Novel silver/poly(vinyl alcohol) (PVA) nanocomposite films were obtained by an in situ reduction method, in which the silver nitrate, sodium poly(γ-glutamic acid) (PGA) and PVA acted as precursor, stabilizer and polyol reducant, respectively. The surface-enhanced Raman scattering (SERS)-activity of as-prepared nanocomposite films was investigated using benzoic acid (BA) as probed molecule. The results showed that the surface plasmon resonance (SPR) absorption band on silver films was more symmetric with stronger intensity than silver colloidal solution, indicating that the Ag nanoparticle distribution on the film substrate was uniform. The fact was further confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM) and field-emission scanning electron microscope (FE-SEM) measurements. It was found that PGA-stabilized silver/PVA nanocomposite film revealed the presence of well-dispersed and spherical silver nanoparticles with average diameter of 90 nm, while the particle sizes were enlarged as the PGA concentration increased. The new substrate presented high SERS enhancement and the enhanced factor was estimated to be 106 for the detection of benzoic acid.

The improvement of hydrophilicity and hemocompatibility of thermoplastic polyurethane (TPU) film was developed using surface modification of polyelectrolyte multilayers (PEMs) deposition. The polysaccharide PEMs included chitosan (CS, as a positive-charged agent) and dextran sulfate (DS, as a negative-charged and an antiadhesive agent) that were successfully prepared on the aminolyzed TPU film in a layer-by-layer (LBL) self-assembly manner. X-ray photoelectron spectroscopy (XPS), field-emission scanning electronic microscopy (FE-SEM), and atomic force microscopy (AFM) data will verify the progressive buildup of the PEMs film. The obtained results showed that the contact angle and Zeta-potential reached the steady value after four bilayers of coating, hence proving that the full coverage of coating with PEM layers was achieved. It could be found that the PEMs-deposited TPU films with DS as the outmost layer could resist the platelet adhesion and human plasma fibrinogen (HPF) adsorption, thereby prolonging effectively the blood coagulation times. Besides, the results of growth inhibition index (GI) of L929 fibroblast proliferation suggested that the as-fabricated TPU films were noncytotoxic. Overall results demonstrated that such an easy, valid, shape-independent, and noncytotoxic processing should be potential for the ion of TPU substrate in the application of hemodialysis or cardiovascular devices.

Surface-enhanced Raman scattering (SERS)-active substrates with high enhancement were prepared by an in situ reduction method. Novel silver/poly(vinyl alcohol) (PVA) nanocomposite films were obtained, in which the silver nitrate, poly(γ-glutamic acid) (PGA), and PVA acted as precursor, stabilizer, and polyol reducant, respectively. The UV-visible spectra of the as-fabricated films showed that the surface plasmon resonance (SPR) absorption band was narrow and of a stronger intensity, which indicates that the Ag nanoparticle Size distribution on the substrate was highly uniform. This finding was further confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM), and field-emission scanning electron microscope (FE-SEM) measurements. It was found that a PGA-stabilized PVA nanocomposite film revealed the presence of well-dispersed spherical silver nanoparticles with an average diameter of 90 nm. The new substate presents high SERS enhancement and the enhanced factor is estimated to be 10 6 for the detection of benzoic acid.

Binary blend membranes of biodegradable poly(l-lactide) (PLLA) with poly(tetramethylene adipate-co-terephthalate) (PTAT) copolymer were prepared by solution casting via air evaporation. The miscibility of PLLA/PTAT blends was studied by dynamic mechanical analysis (DMA) and thermal mechanical analysis (TMA) in a tensile mode. Differential scanning calorimetry (DSC) measurement was carried out. The surface microstructure and tensile properties of the blend membranes were examined using atomic force microscopy (AFM) and tensile tester. It was concluded that PLLA/PTAT blends should be partially miscible for all ranges of compositions. Higher roughness and porosity were observed for the blend containing 50% PTAT, suggesting more phase separation occurred. The DSC analysis showed that the fusion enthalpy and crystallinity (Xc) of the PLLA-rich phase decreased with increasing PTAT content. Solidification process strongly suggested that the crystallization rate was accelerated by blending with 25% PTAT content, which served as the nucleation agent. Furthermore, the crystallization rate coefficient (CRC) depended on the blending miscibility and cooling rate in the non-isothermal crystallization process. Besides, PTAT addition could be proved to enhance the thermal stability and elongation of resulting blend membranes, even superior to those properties of poly(lactic acid-co-glycolic acid) (PLGA).

Plasma proteins were covalently immobilized onto polyacrylonitrile (PAN) membrane to evaluate the hemocompatibility and anaphylatoxin formation. This is used as a model to study the effect of protein-adsorption on the blood-contacting response of hemodializing membranes. The proteins used were either platelet-adhesion-promoting collagen (COL) or platelet-adhesion-inhibiting human serum albumin (HSA). The microstructure and characterization of the protein-immobilizing PAN membranes were evaluated by Coomassie dye assay, atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle measurement. PAN-HSA membrane improved not only hemocompatibility including less platelet adhesion, longer blood coagulation times, and higher thrombin inactivity level, but also induced lower complement activation. On the other hand, PAN-COL membrane exhibited blood incompatibility, although induced less increase of C3, C4 antigens of serum. Overall results of this study demonstrated that the immobilization of HSA onto the surface of PAN membrane would be beneficial to improve the hemocompatibility and to reduce the anaphylatoxin formation during hemodialysis.

Polyacrylonitrile (PAN) was blended with polyvinylidine fluoride (PVDF) at various ratios and made into membranes. The hemocompatibility of the resulting membranes was evaluated based on human plasma proteins adsorption, platelet adhesion, thrombus formation, and blood coagulation time. The PAN/PVDF blends exhibited partial miscibility according to the inward shifting of their two glass transition temperatures. The microstructures of blend membranes examined using atomic force microscopy (AFM) indicated that the roughness increased with the PVDF content, and the phase separation was too severe to form a membrane when the PVDF content was more than 30%. The water contact angle of PAN/PVDF blend membranes increased with the PVDF content. By blending with 20wt% apolar PVDF the adsorption of blood proteins could be reduced, and hence the platelet adhesion and thrombus formation was also reduced. However, when the PVDF content was 30wt%, severe thrombogenicity was observed due probably to the more porous structure of blend membrane. These results demonstrated that the hemocompatibility would be improved for PAN/PVDF blend membranes with appropriate hydrophilicity and roughness. Copyright # 2005 John Wiley & Sons, Ltd.

We identify a concordant model for the intergalactic medium (IGM) at redshift z=1.9 that uses popular values for cosmological and astrophysical parameters and accounts for all baryons with an uncertainty of 6%. We have measured the amount of absorption, DA, in the Ly-alpha forest at redshift 1.9 in spectra of 77 QSO from the Kast spectrograph. We calibrated the continuum fits with realistic artificial spectra, and we found that averaged over all 77 QSOs the mean continuum level is within 1-2% of the correct value. Absorption from all lines in the Ly-alpha forest at z=1.9 removes DA=15.1 +/- 0.7% of the flux between 1070 and 1170 (rest) Angstroms. This is the first measurement using many QSOs at this z, and the first calibrated measurement at any redshift. Metal lines absorb 2.3 +/- 0.5%, and LLS absorb 1.0 +/- 0.4% leaving 11.8 +/- 1.0% from the lower density bulk of the IGM. Averaging over Delta z=0.1 or 154 Mpc, the dispersion is 6.1 +/- 0.3% including LLS and metal lines, or 3.9 (+0.5, -0.7)% for the lower density IGM alone, consistent with the usual description of large scale structure. LLS and metal lines are major contributors to the variation in the mean flux, and they make the flux field significantly non-Gaussian. We find that a hydrodynamic simulation on a 1024 cubed grid in a 75.7 Mpc box reproduces the observed DA from the low density IGM with parameters values H_o=71 km/s/Mpc, Omega_Lambda=0.73, Omega_m=0.27, Omega_b=0.044, sigma_8=0.9 and a UV background that has an ionization rate that is 1.08 +/- 0.08 times the prediction by Madau, Haardt & Rees (1999).

Stable water quality is necessary for the growth of aqua biological to survive conditions, and how to achieve a stable water quality suitable for biological growth to copy the environment and can energy-saving and exhibitions of both goals, it is the purpose of "Museum of Marine Biology" and must established means. This study used the TRIZ Innovation Principles to make solving water quality problems in the NMMBA (National Museum of Marine & Biology) Open ocean exhibition tank - for many years' pH value was too low and unstable can't improved issues, And maintained at 7.8 to 8.4 in pH values as well as the savings of electricity consumption has reached 1,296,000 kWh, which is equivalent to reduce 894,240kg of annual carbon emissions.

Scope Traditional medicinal herbs are increasingly used as alternative therapies in patients with inflammatory diseases. Here we evaluated the effect of Wedelia chinensis, a medicinal herb commonly used in Asia, on the prevention of dextran sulfate sodium (DSS)-induced acute colitis in mice. General safety and the effect of different extraction methods on the bioactivity of W. chinensis were also explored. Methods and Results C57BL/6 mice were administrated hot water extract of fresh W. chinensis (WCHF) orally for one week followed by drinking water containing 2% DSS for nine days. WCHF significantly attenuated the symptoms of colitis including diarrhea, rectal bleeding and loss of body weight; it also reduced the shortening of colon length and histopathological damage caused by colonic inflammation. Among four W. chinensis extracts prepared using different extraction techniques, WCHF showed the highest anti-colitis efficacy. Analyses of specific T-cell regulatory cytokines (TNF-α, IL-4, IFN-γ, IL-17, TGF-β, IL-12) revealed that WCHF treatment can suppress the Th1 and Th17, but not Th2, responses in colon tissues and dendritic cells of DSS-induced colitis mice. A 28-day subacute toxicity study showed that daily oral administration of WCHF (100, 500, 1000 mg/kg body weight) was not toxic to mice. Conclusion Together, our findings suggest that specific extracts of W. chinensis have nutritional potential for future development into nutraceuticals or dietary supplements for treatment of inflammatory bowel disease.

This work presents a look-up table-based (LUT-based) algorithm for scanline-based rendering of OpenVG. The proposed method can deal with arbitrary number of scissoring rectangles. The rasterization and scissoring in the proposed architecture can be performed concurrently to reduce rendering time. The scanline-size buffers used as scissoring LUTs result in low area overhead. Moreover, a linked list structure of scissoring rectangles is proposed in order that only the scissoring rectangles interacted with the processing scanline are accessed to increase bus efficiency and reduce power consumption. Implementation results based on TSMC 0.13-µm CMOS technology show that the proposed rasterization design with LUT-based scissoring can operate at 200 MHz with 77K gate counts. The proposed design can render 16.8 tiger images with 392×483 resolution per second assuming ideal bus latency. Compared to existing works, the proposed design achieves a smaller area and more functionality for higher display resolution with comparable throughput.

As the de facto standard for data mining models, the Predictive Model Markup Language (PMML) provides tremendous benefits for business, IT, and the data mining industry in general, since it allows for predictive models to be easily moved between applications. Due to the cross-platform and vendor-independent nature of such an open-standard, auto-generated PMML code is often represented in different versions of PMML. A tool may export PMML 2.1 and another import PMML 4.0. This problem raises the issue of conversion. For true interoperability, PMML needs to be easily converted from one version to another.In this paper, we describe the capabilities associated with the "PMML Converter". This application represents a great step in the PMML path towards true interoperability in data mining. Besides converting older versions of PMML to its latest, the PMML converter checks PMML files for syntax issues and, if issues are encountered, automatically corrects them.This paper also describes the capabilities associated with an interactive PMML-based application, the "Transformations Generator". Auto-generated PMML code can omit important data pre-processing steps which are an integral part of a predictive solution. The Transformations Generator aims to bridge this gap by providing a graphical interface for the development and expression of data pre-processing steps in PMML.

This paper describes PMML extensions for the modular open source data analytics platform KNIME adding pre-processing support and the ability to edit existing PMML code. It is also shown how the PMML model representation in KNIME can be used within meta learning schemes such as boosting and bagging.

In this paper, we reformulated the conventional iterative division algorithm by substituting the pre-defined variable and then updating its initial value accordingly. The reformulated division algorithm allows a restructuring of the divider architecture to further improve its operating speed without increasing latency and area cost. Using the proposed fast algorithm, we developed a high-speed bit-serial GF(2m) divider. Analytical results show that the cost of the initial value update and variable transformation in the reformulated algorithm is almost negligible in the hardware implementation. Our divider reduces the critical path delay. Compared with related divider designs, the proposed design has time and area advantages.