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The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types. The protein transfection efficiency of the HEPES method was not inferior to that of commercially available systems that are both more expensive and time consuming. Studies using endocytotic inhibitors and endosomal markers have revealed that cells internalize HEPES-protein mixtures through endocytosis. Results that HEPES-protein mixtures exhibited a low diffusion coefficient suggest that HEPES might neutralize the charges of proteins and, thus, facilitate their cellular internalization. Upon internalization, the cytosolic antibodies caused the degradation of targeted proteins in TRIM21-expressing cells. In summary, the HEPES method is efficient for protein transfection and has potential for myriad clinical applications. Keywords: HEPES, protein transfection, intracellularly targeting

A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We develope...

Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

Droplet impingement experiments at low Weber number ( W e = 7.61) were conducted by digitizing silhouettes of impacting water drops onto four graphite surfaces with different hydrophobicities. The relaxation of the wetting diameter, the droplet height and the dynamic contact angle were determined for characterizing the peculiar impact and spreading dynamics for the unalike surfaces, typified by four distinctive topographies carefully analyzed by scanning electron microscopy. After the initial inertially dominated phase, during which the drops spreading onto all substrates showed a similar behavior, the expected recoil phase was not observed for droplets impacting onto graphite surfaces characterized by 90° and 120° advancing contact angles. A few milliseconds after the impact, the drops exhibited a pinned configuration due to the peculiar morphology of these graphite substrates: randomly distributed cavities on a generally smooth surface, in fact, stopped the movement of the triple line. This behavior, however, was not detected for water droplets impinging onto graphite surfaces characterized by 160° advancing contact angles, which instead presented the usual retraction phase. Finally, the graphite surface characterized by 140° advancing contact angles showed a mixed behavior due to a transition of the drop configuration from Cassie-Baxter to Wenzel state.

The roles that the impact velocity ( V i ) and the drop size ( D 0 ) play for water drops impinging on parafilm surfaces were systematically investigated for systems of entrapped air bubbles. Entrapped bubbles were successfully observed by using a high-speed video camera system from a 45-degree top view. A half-closed region on the V i – D 0 plot was identified for the formation of the entrapped bubble. The upper and lower boundaries of V i tended to decrease as D 0 increased. The entrapped bubble generally took the shape of a hemispherical bubble after settling down on the parafilm surface. Bubble sizes became smaller when V i approached the upper and lower limits. A monotonically increasing correlation between Re and We at a fixed drop size has been reported in the literature. This monotonic relationship was shown to be dependent on the drop size. Moreover, two main different mechanisms for the formation of entrapped air bubbles were proposed for water on parafilm systems.

A 10-residue, glycosaminoglycan-binding peptide, GBP

The formation of an entrapped air bubble for a water droplet impinging on a parafilm surface was studied by using a high-speed video camera system. The images were taken sequentially immediately after the droplet impingement. The relaxation of impinging droplets from both the top and side views was monitored, and a formation mechanism of the entrapped air bubble was proposed in this work. An investigation of droplets with different sizes ( D 0 ) and impact velocities ( V i ) was performed, and it was found that the formation of the entrapped air bubble was observed only at (i) V i = 0.35–0.56 m/s for droplets with D 0 = 2.14 mm, (ii) D 0 = 1.93–2.14 mm for V i = 0.57 ± 0.02 m/s, and (iii) D 0 = 2.30–2.46 mm for V i = 0.52 m/s. The fluid flow inside the droplet during the formation of air bubble entrapment was examined by performing two sets of droplet impact experiments: (i) droplets with various V i at a fixed droplet size and (ii) droplets with various volumes at a fixed V i . Once the water droplet touched the parafilm surface, the drop spread, and the central cone of the impinging droplet descended. After a few milliseconds, the central cone disappeared, an air cavity formed, and the droplet started to retract. The retraction generated an inward, radial flow mainly to the top and bottom of the central air cavity. A crucial condition for the formation of an entrapped air bubble in this work is the inward flow over the top of the air cavity closes earlier than the upward movement of the fluid below the central cavity.

A study on the initial wetting velocity ( V w0 ) for a water droplet impinging on glass and parafilm surfaces was performed using a fast-speed video camera system. The silhouette images were recorded sequentially from the moment the droplet made contact with the solid surface. The profile of the droplet during spreading revealed the droplet-wetting diameter (especially in the first millisecond), then the wetting velocity was determined. The relaxation of the wetting velocity, V w ( t ), was investigated at five different impact velocities ( V i ). The value of V w0 was found to be a few meters per second; V w then decreased dramatically in a few ms to approximately 1/10 of V w0 . Moreover, the V w for water on parafilm became negative (i.e., retraction started) at approximately 2–3 ms (dimensionless time t * ∼ 1). A general phenomenon was observed: (i) a higher V i results in a larger V w0 ; (ii) at the first millisecond, the wetting velocity was dominated primarily by V i and was nearly independent of the surface hydrophobicity; (iii) for t > 2 ms, the V w depends mainly on the surface hydrophobicity; and (iv) for t = 1–3 ms, the V w for the droplet with higher V i became smaller for both solid substrates while the droplet continued spreading.

A notable universal relationship has been proposed in the literature for the evolution of dimensionless droplet height and wetting diameter during the initial spreading stage of droplet impingement. In this study, this universal relationship was investigated by employing three sets of measurements. Sequential images were recorded, and the whole droplet profile ensembles were plotted to facilitate this study. These sets of experiments were designed by changing impact velocity, surface hydrophobicity, or solution property. The experimental results illustrate that the importance of parameters causing the data variation is in the order of surface hydrophobicity > initial impact velocity > surfactant on wetting diameter, and surface hydrophobicity ≈ initial impact velocity > surfactant on droplet height. No universal relationship was observed for dimensionless droplet height and wetting diameter.

The dynamic wetting behaviors, especially the droplet morphology, of a water droplet impinging on five substrate surfaces were investigated. A water drop was released from 13.6 mm above a solid surface and impinged on substrates. The images (the silhouette and 45 degrees top view) were sequentially recorded from the moment that the droplet impacted the solid surface until it reached equilibrium. The entire profile of each of the water droplets during spreading was obtained from the digitized recorded images. The digitized droplets were then used to detail the spreading mechanism, including information on the relaxations of the wetting diameter, droplet height, contact angle, and spreading velocity. A comparison of the full droplet profiles allows us to clarify the independent motion of two related but independent components, the central region and rim, of an impinging droplet. An interesting plateau region in the droplet height relaxation curve was observed in the first cycle for all substrate surfaces. For hydrophobic surfaces (paraffin and Teflon), three particular growth modes in the droplet height relaxation curve were detected in every oscillation cycle during the early spreading stages. It only took three and four oscillation cycles for a water droplet on the glass and quartz substrates, respectively, to dissipate its energy and reach its equilibrium state. However, it took 72 and 28 oscillation cycles for a water droplet on the Teflon and paraffin substrates, respectively. Moreover, several other new phenomena were also observed.

The adsorption of C12E4 onto a fresh air–water interface was reported to be diffusion-controlled. Hsu et al. applied a video-enhanced pendant bubble tensiometer and obtained the dynamic surface tension (DST) for different C12E4 concentrations [C.T. Hsu, M.J. Shao, S.Y. Lin, Langmuir 16 (2000) 3187]. The entire DST profiles were compared with the theoretical DST from a classical spherical model (CSM). CSM assumes that the effect of pendant bubble shape and the existence of needle on DST were negligible. In this study, the actual shape of pendant bubble and the existence of a needle are considered on generating the theoretical DST profiles. The diffusion-controlled simulation profiles in this work predict the DST data well, but a slight different diffusivity may be resulted. Without considering the existence of needle and bubble shape, CSM underestimates a diffusivity 10–12%. An alternative way for correcting the effect of bubble shape in order to use the simple CSM is also given at the end of this work.

Ethylene plays versatile functions in regulating plant physiology. Although the high affinity ethylene receptor and its downstream regulators have been identified, the molecular recognition of the receptor interacting domains remains to be established. It has been speculated that the cytoplasmic signaling of the ethylene receptor is a two-component regulatory system involving the conserved receiver domain (RD). Here, we report the NMR chemical shift assignments for RD from Arabidopsis thaliana ethylene receptor ETR1. Nearly complete backbone and side-chain assignments were achieved at pH 6.0 and 25 °C. The assignments and backbone dynamics revealed the secondary structure and showed that ETR1-RD is a monomer in solution. These results will make it possible to monitor downstream binding partners and elucidates our understanding of phosphotransfer in the plant two-component regulatory system in the ethylene signaling pathway.

Fructus gardeniae has long been used by traditional Chinese medical practitioners for its anti-inflammatory, anti-oxidant, anti-tumor and anti-hyperlipidemic characteristics. Here we describe our finding that F. gardeniae greatly reduces anti-enterovirus 71 (EV71) activity, resulting in significant decreases in EV71 virus yields, EV71 infections, and internal ribosome entry site activity. We also found that geniposide, a primary F. gardeniae component, inhibited both EV71 replication and viral IRES activity. Our data suggest the presence of a mechanism that blocks viral protein translation. According to our findings, F. gardeniae and geniposide deserve a closer look as potential chemopreventive agents against EV71 infections.

A modified selected-plane method for contact angle (theta) measurement is proposed in this study that avoids the difficulty of finding the real contact point and image-distortion effects adjacent to the contact point. This method is particularly suitable for superhydrophobic surfaces. The sessile-drop method coupled with the tangent line is the most popular method to find the contact angle in literature, but it entails unavoidable errors in determining the air-solid base line due to the smoothness problem and substrate tilting. In addition, the tangent-line technique requires finding the actual contact point. The measurement error due to the base line problem becomes more profound for superhydrophobic surfaces. A larger theta deviation results from a more superhydrophobic surface with a fixed base line error. The proposed modified selected-plane method requires only four data points (droplet apex, droplet height, and two interfacial loci close to the air-solid interface), avoiding the problem of the sessile-drop-tangent method in finding the contact point and saving the trouble of the sessile-drop-fitting method for best fitting of the numerous edge points with the theoretical profile. A careful error analysis was performed, and a user-friendly program was provided in this work. This method resulted in an accurate theta measurement and a method that was much improved over the classical selected plane and the sessile-drop-tangent methods. The theta difference between this method and the sessile-drop-fitting method was found to be less than three degrees.

A pendant drop tensiometer, coupled with image digitization technology and a best-fitting algorithm, was built to accurately measure the surface tension of glass melts at high temperatures. More than one thousand edge-coordinate points were obtained for a pendant glass drop. These edge points were fitted with the theoretical drop profiles derived from the Young-Laplace equation to determine the surface tension of glass melt. The uncertainty of the surface tension measurements was investigated. The measurement uncertainty (σ) could be related to a newly defined factor of drop profile completeness (Fc): the larger the Fc is, the smaller σ is. Experimental data showed that the uncertainty of the surface tension measurement when using this pendant drop tensiometer could be ±3 mN∕m for glass melts.