An injectable, chitosan-based hydrogel prepared by Schiff base reaction for anti-bacterial and sustained release applications.

Injectable hydrogels, providing sustained release as implanted materials, have received tremendous attention. In this study, chitosan-based hydrogels were prepared via Schiff base reaction of the aldehyde groups on Poly(NIPAM-co-FBEMA) and the amine groups on chitosan. Owing to the dynamic covalent linkage, the SC/PNF hydrogels exhibit pH-responsive, reversible sol-gel transition, injectable, and self-healing capacity. The mechanical strength of SC/PNF hydrogels can be operated simply by switching the composition or solid content of Poly(NIPAM-co-FBEMA) copolymers. Rheological analyses, including frequency sweeps, strain sweep scanning, and dynamic time sweeps, were employed to demonstrate the relationship between storage modulus (G'), loss modulus (G″), and composition of the SC/PNF hydrogels. In vitro release behaviors reveal that vancomycin-loaded SC/PNF hydrogel could contribute to both the initial burst release (over 1000 ppm within 4 h) and the sustained release (3000 ppm for at least 30 days). Pristine SC/PNF hydrogel holds good biocompatibility toward L929 cells and S. aureus that it degrades as incubated with S. aureus. However, vancomycin-wrapped SC/PNF hydrogel possesses a rapid bacterial-killing effect with a clear inhibition zone. In short, the SC/PNF hydrogels deliver not only sustainable release ability but also tunable physical properties, which are expected to be an outstanding candidate for non-invasive, anti-infection applications.

Factors Governing the Different Functions of Zn2+-Sites with Identical Ligands in Proteins.

In Zn-proteins, structural Zn-sites are mostly Cys-rich lined by two or more Cys residues, whereas catalytic Zn-sites usually contain His or Asp/Glu residues and a water molecule. Here, we reveal many examples outside this trend with Zn2+ bound to ligands commonly found in both structural and catalytic Zn-sites, namely, Zn-CC(C/H)x (x = D, E, or H2O) sites. We show that these atypical Zn-sites are found in all known life forms (i.e., eukaryotes, bacteria, archaea, and viruses) and can serve structural roles in some proteins but catalytic roles in others. By calculating the physical properties of these atypical Zn-binding sites, we elucidate why Zn-CC(C/H)x sites of the same composition can serve structural and catalytic roles in proteins. Furthermore, we found new sequence/structural motifs characteristic of catalytic Zn-CCHw sites and provide guidelines to predict the structural/catalytic role of atypical Zn-CC(C/H)x sites of unknown function. We discuss how our results could help to design inhibitors targeting catalytic Zn-CC(C/H) H2O sites.

Using an Old Drug to Target a New Drug Site: Application of Disulfiram to Target the Zn-Site in HCV NS5A Protein.

In viral proteins, labile Zn-sites, where Zn(2+) is crucial for maintaining the native protein structure but the Zn-bound cysteines are reactive, are promising drug targets. Here, we aim to (i) identify labile Zn-sites in viral proteins using guidelines established from our previous work and (ii) assess if clinically safe Zn-ejecting agents could eject Zn(2+) from the predicted target site and thus inhibit viral replication. As proof-of-concept, we identified a labile Zn-site in the hepatitis C virus (HCV) NS5A protein and showed that the antialcoholism drug, disulfiram, could inhibit HCV replication to a similar extent as the clinically used antiviral agent, ribavirin. The discovery of a novel viral target and a new role for disulfiram in inhibiting HCV replication will enhance the therapeutic armamentarium against HCV. The strategy presented can also be applied to identify labile sites in other bacterial or viral proteins that can be targeted by disulfiram or other clinically safe Zn-ejectors.

Modeling Zn²âº release from metallothionein.

Mammalian metallothioneins (MTs) comprise a Zn3Cys9 cluster in the ß domain and a Zn4Cys11 cluster in the α domain. They play a crucial role in storing and donating Zn(2+) ions to target metalloproteins and have been implicated in several diseases, thus understanding how MTs release Zn(2+) is of widespread interest. In this work, we present a strategy to compute the free energy for releasing Zn(2+) from MTs using a combination of classical molecular dynamics (MD) simulations, quantum-mechanics/molecular-mechanics (QM/MM) minimizations, and continuum dielectric calculations. The methodology is shown to reproduce the experimental observations that (1) the Zn-binding sites do not have equal Zn(2+) affinity and (2) the isolated ß domain is thermodynamically less stable and releases Zn(2+) faster with oxidizing agents than the isolated α domain. It was used to compute the free energies for Zn(2+) release from the metal cluster in the absence and presence of the protein matrix (protein architecture and coupled protein-water interactions) to yield the respective disulfide-bonded product. The results show the importance of the protein matrix as well as protein dynamics and coupled conformational changes in accounting for the differential Zn(2+)-releasing propensity of the two domains with oxidizing agents.

An azido-BODIPY probe for glycosylation: initiation of strong fluorescence upon triazole formation.

We have designed a low fluorescent azido-BODIPY-based probe AzBOCEt (Az10) that undergoes copper(I)-catalyzed 1,3-dipolar cycloadditions with alkynes to yield strongly fluorescent triazole derivatives. The fluorescent quantum yield of a triazole product T10 is enhanced by 52-fold as compared to AzBOCEt upon excitation at a wavelength above 500 nm. Quantum mechanical calculations indicate that the increase in fluorescence upon triazole formation is due to the lowering of the HOMO energy level of the aryl moiety to reduce the process of acceptor photoinduced electron transfer. AzBOCEt is shown to label alkyne-functionalized proteins in vitro and glycoproteins in cells with excellent selectivity, and enables cell imaging and visualization of glycoconjugates in alkynyl-saccharide-treated cells at extremely low concentration (0.1 µM). Furthermore, the alkyne-tagged glycoproteins from cell lysates can be directly detected with AzBOCEt in gel electrophoresis.

Identification of labile Zn sites in drug-target proteins.

Labile Zn fingers (Zfs) in proteins contain Zn-bound thiolates that can react with electrophilic agents, causing Zn(2+) ejection and protein unfolding. Such labile Zfs have been shown to be Cys4 or Cys3His cores whose Zn-bound Cys have no hydrogen bonds. Our aim here is to identify labile Zfs in proteins that are promising drug targets using these features. To prove the strategy used, we showed that five proteins with predicted labile Zfs reacted with Zn-ejecting agents, whereas five proteins with no or inert Zfs did not. The comprehensive set of labile Zfs provides new drug targets and guidelines to redesign Zn-ejecting compounds with improved specificity.

Improvement in running economy after 8 weeks of whole-body vibration training.

The purpose of this study was to investigate the effects of 8-week whole-body vibration (WBV) training on running economy (RE) and power performance. Twenty-four male collegiate athletes were recruited and randomly assigned to experimental (WBV) and placebo (PL) groups. The WBV subjects performed semisquat vibration training (30 Hz, ±1-2 mm, 3 times per week), whereas PL subjects performed identical training without vibration. The isometric maximum voluntary contraction tests were used to evaluate maximal isometric force (F(max)) and rate of force development (RFD) of lower extremities, before and after the intervention, and RE was measured on a level treadmill at 3 velocities (2.68, 3.13, and 3.58 m·s(-1)). The F(max) of the lower leg (plantar flexion, from 80.8 ± 24.5 to 99.0 ± 33.9 N·m, p < 0.05, η(2) = 0.567; dorsiflexion, from 38.1 ± 6.5 to 43.0 ± 7.7 N·m, p < 0.05), and the RFD of 0-200 milliseconds during plantar flexion (from 186.0 ± 69.2 to 264.6 ± 87.2 N·m·s(-1), p < 0.05, η(2) = 0.184) were significantly increased in the WBV group after training. The averaged RE values for the 3 running velocities were significantly improved after WBV training (pretraining vs. posttraining, 4.31 ± 0.33 vs. 4.65 ± 0.34 m·ml(-1)·kg(-1), p = 0.001, η(2) = 0.654); however, no significant differences were found in the PL group (pretraining vs. posttraining, 4.18 ± 0.26 vs. 4.26 ± 0.44 m·ml(-1)·kg(-1), p = 0.476). The WBV training significantly improved RE at selected speeds (∼5.0-8.5%, p < 0.05). These results indicated that short-term WBV training could be an effective stimulus to enhance RE and lower extremity power performance in competitive athletes.

Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold.

Factors controlling the reactivity of zinc finger cores.

Although the Zn(2+) cation in Zn·Cys(4), Zn·Cys(3)His, Zn·Cys(2)His(2), and Zn(2)Cys(6) cores of zinc finger (Zf) proteins typically plays a structural role, the Zn-bound thiolates in some Zf cores are reactive. Such labile Zf cores can serve as drug targets for retroviral or cancer therapies. Previous studies showed that the reactivity of a Zn-bound thiolate toward electrophiles is significantly reduced if it forms S---NH hydrogen bonds with the backbone amide. However, we found several well-known inactive Zf cores containing Cys ligands with no H-bonding interactions. Here, we show that H bonds from the peptide backbone or bonds from a second Zn cation to Zn-bound S atoms suppress the reactivity not only of these S atoms, but also of Zn-bound S* atoms with no interactions. Indeed, two or more indirect NH---S hydrogen bonds raise the free energy barrier for methylation of a Zn-bound S* in a Cys(4) core more than a direct NH---S* hydrogen bond. These findings help to elucidate why several well-known Zf cores have Cys ligands with no H bonds, but are unreactive. They also help to provide guidelines for distinguishing labile Cys-rich Zn sites from structural ones, which in turn help to identify novel potential Zf drug targets.

Conserved structural motif for recognizing nicotinamide adenine dinucleotide in poly(ADP-ribose) polymerases and ADP-ribosylating toxins: implications for structure-based drug design.

The ADP-ribosylating toxins (ADPRTs) and poly(ADP-ribose) polymerases (PARPs) are two important drug-target protein families. Although the Y-X(10)-Y motif for the diphtheria toxin group and the STS motif for the other ADPRTs have been found to recognize the NAD(+) substrate, it is not known (i) if these two different motifs share any structural similarity, (ii) the key forces/residues contributing to NAD(+) binding, and (iii) if they recognize the same or different NAD(+) conformations. Here, we show that even though the different toxin groups and PARPs share insignificant sequence identity, they share a similar 3D structure shaped like a scorpion (the "scorpion" motif) whose first three and last residues interact mainly with the NAD(+) nicotinamide ring via van der Waals forces. This locally conserved structure binds the nicotinamide mononucleotide moiety in a structurally conserved ringlike conformation. The biological implications/applications of locally conserved structures for toxins/PARPs and the nicotinamide mononucleotide are discussed.

Zero-block mode decision algorithm for H.264/AVC.

In the previous paper , we proposed a zero-block intermode decision algorithm for H.264 video coding based upon the number of zero-blocks of 4 x 4 DCT coefficients between the current macroblock and the co-located macroblock. The proposed algorithm can achieve significant improvement in computation, but the computation performance is limited for high bit-rate coding. To improve computation efficiency, in this paper, we suggest an enhanced zero-block decision algorithm, which uses an early zero-block detection method to compute the number of zero-blocks instead of direct DCT and quantization (DCT/Q) calculation and incorporates two adequate decision methods into semi-stationary and nonstationary regions of a video sequence. In addition, the zero-block decision algorithm is also applied to the intramode prediction in the P frame. The enhanced zero-block decision algorithm brings out a reduction of average 27% of total encoding time compared to the zero-block decision algorithm.

Physical basis of structural and catalytic Zn-binding sites in proteins.

Zn(2+), an element that is essential to all life forms, can play a catalytic or a solely structural role. Previous works have shown that Zn(2+) binds preferentially to water molecules and His in catalytic sites, but to Cys(-) instructural sites, but the molecular basis for the observed ligand preference is unclear. Here, we show that the different Zn(2+) roles are also reflected in the different bond distances to Zn(2+) in structural and catalytic sites. We reveal the physical basis for the observed differences between structural and catalytic Zn sites: In most catalytic sites, water is found bound to Zn(2+) as it transfers the least charge to Zn(2+) and is less bulky compared to the protein ligands, enabling Zn(2+) to serve as a Lewis acid in catalysis. In most structural sites, however, >/= 2 Cys(-) are found bound to Zn(2+), as Cys(-) transfers the most charge to Zn(2+) and reduces the Zn charge to such an extent that Zn(2+) can no longer act as a Lewis acid; furthermore, steric repulsion among the bulky Cys(S(-)) prevents Zn(2+) from accommodating another ligand. Based on the observed ligand preference and Zn-ligand distance differences between structural and catalytic Zn sites, we present a simple method for distinguishing the two types of sites and for verifying the catalytic role of Zn(2+). Finally, we discuss how the physical bases revealed aid in designing potential drug molecules that target Zn proteins.

Differential effects of the Zn-His-Bkb vs Zn-His-[Asp/Glu] triad on Zn-core stability and reactivity.

The most common partner of the Zn-bound His is the Asp/Glu carboxylate side chain in catalytic Zn sites and the backbone (Bkb) carbonyl group in structural Zn sites. To elucidate the factors governing the selection of the second-shell partner of the Zn-bound His in structural/catalytic Zn sites, systematic studies using density functional theory and continuum dielectric calculations were performed to determine the relative contributions of the second-shell Bkb carbonyl and the Asp/Glu carboxylate to the Zn-core stability and reactivity. The results show that the contributions of the second-shell Bkb carbonyl and Asp/Glu carboxylate to the Zn-core stability depend mainly on the solvent accessibility of the Zn-site and the composition of the Zn-core. They reveal the advantage of a second-shell Bkb carbonyl in anionic Zn cavities: it stabilizes anionic, buried Zn-cores more than the corresponding negatively charged Asp/Glu carboxylate, thus explaining the absence of the Zn-His-Asp/Glu triad in structural [Zn(Cys)3(His)]- cores. They also reveal the advantage of a second-shell Asp/Glu carboxylate in catalytic Zn-cores: relative to a Bkb carbonyl group, it increases (i) the HOMO energy of the cationic/neutral zinc core, (ii) the reactivity of the attacking Zn-bound OH-, (iii) electron transfer to the substrate, and (iv) the stability of the metal complex upon electron transfer. Furthermore, a second-shell Asp/Glu carboxylate could facilitate product release in the common cationic catalytic cores, by acting as a proton acceptor of the Zn-bound His creating an Asp...His- dyad that stabilizes the zinc dication more than the respective Bkb...His0 dyad.