Shouping Liu

Identification of Tear Fluid Biomarkers in Dry Eye Syndrome Using iTRAQ Quantitative Proteomics

The proteins found in tears have an important role in the maintenance of the ocular surface and changes in the quality and quantity of tear components reflect changes in the health of the ocular surface. In this study, we have used quantitative proteomics, iTRAQ technology coupled with 2D-nanoLC-nano-ESI-MS/MS and with a statistical model to uncover proteins that are significantly and reliably changed in the tears of dry eye patients in an effort to reveal potential biomarker candidates. Fifty-six patients with dry eye and 40 healthy subjects were recruited for this study. In total, 93 tear proteins were identified with a ProtScore >or=2 (>or=99% confidence). Associated with dry eye were 6 up-regulated proteins, alpha-enolase, alpha-1-acid glycoprotein 1, S100 A8 (calgranulin A), S100 A9 (calgranulin B), S100 A4 and S100 A11 (calgizzarin) and 4 down-regulated proteins, prolactin-inducible protein (PIP), lipocalin-1, lactoferrin and lysozyme. Receiver operating curves (ROC) were evaluated for individual biomarker candidates and a biomarker panel. With the use of a 4-protein biomarker panel, the diagnostic accuracy for dry eye was 96% (sensitivity, 91.0%; specificity, 90.0%). Two biomarker candidates (alpha-enolase and S100 A4) generated from iTRAQ experiments were successfully verified using an ELISA assay. The levels of these 10 tear proteins reflect aqueous secretion deficiency by lacrimal gland, inflammatory status of the ocular surface. The clinical classification of the severity of the dry eye condition was successfully correlated to the proteomics by using three proteins that are associated with inflammation, alpha1-acid glycoprotein 1, S100 A8 and S100 A9. The nine tear protein biomarker candidates (except alpha1-acid glycoprotein 1) were also verified using an independent age-matched patient sample set. This study demonstrated that iTRAQ technology combined with 2D-nanoLC-nanoESI-MS/MS quantitative proteomics is a powerful tool for biomarker discovery.

Characterization and thermal degradation of polyimide and polyamide liquid crystalline polymers

Abstract The wholly aromatic thermotropic liquid crystalline polymers, polyimide [1,2,4,5-benzentetracarboxylic dianhydride (PMDA) and 1,3-bis [4-(4′-aminophenoxy) cumyl] benzene (BACB)] and polyamide [terephthaloyl chloride (TPC) and BACB] were characterized by FTIR-ATR, and DSC measurements. Three endothermic peak temperatures observed (274, 284, 307°C) for the polyamide are lower than those observed (277, 297, 337°C) for the polyimide liquid crystalline polymers. The major difference between the polyamide and polyimide is that their infrared peaks are at 1644 and 1722 cm −1 , respectively. The thermal stability, apparent activation energy and evolved gas analysis of these LCPs was done using thermo-gravimetric analysis (TGA) coupled with Fourier transform infrared (FTIR) spectroscopy. Decomposition results show that the polyamide is much less thermally stable than the polyimide. The evolved gases are found to be H 2 O, CO, CO 2 and various hydrocarbon fragments. A substantial amount of CO 2 detected during the decomposition is due to degradation of the carbonyl functional groups from the polyimide liquid crystalline polymers. Activation energies for the initial thermal degradation of this polyimide in nitrogen and air are 236 and 201 kJ/mol, and those for polyamide are 207 and 219 kJ/mol, respectively. A jump in the activation energy is observed around 40% weight loss, beyond which it decreases in the case of polyimide. However, an unusual observation was made during the degradation of polyamide. The apparent activation energy values are found to be higher under air environment than in the nitrogen environment.

Rapid bactericidal action of alpha-mangostin against MRSA as an outcome of membrane targeting.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created the need for better therapeutic options. In this study, five natural xanthones were extracted and purified from the fruit hull of Garcinia mangostana and their antimicrobial properties were investigated. α-Mangostin was identified as the most potent among them against Gram-positive pathogens (MIC=0.78-1.56 μg/mL) which included two MRSA isolates. α-Mangostin also exhibited rapid in vitro bactericidal activity (3-log reduction within 5 min). In a multistep (20 passage) resistance selection study using a MRSA isolated from the eye, no resistance against α-mangostin in the strains tested was observed. Biophysical studies using fluorescence probes for membrane potential and permeability, calcein encapsulated large unilamellar vesicles and scanning electron microscopy showed that α-mangostin rapidly disrupted the integrity of the cytoplasmic membrane leading to loss of intracellular components in a concentration-dependent manner. Molecular dynamic simulations revealed that isoprenyl groups were important to reduce the free energy for the burial of the hydrophobic phenyl ring of α-mangostin into the lipid bilayer of the membrane resulting in membrane breakdown and increased permeability. Thus, we suggest that direct interactions of α-mangostin with the bacterial membrane are responsible for the rapid concentration-dependent membrane disruption and bactericidal action.

Structure-Dependent Charge Density as a Determinant of Antimicrobial Activity of Peptide Analogues of Defensin

Defensins are small (3-5 kDa) cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. Despite intensive research, bactericidal and cytotoxic mechanisms of defensins are still largely unknown. Moreover, we recently demonstrated that small peptides derived from defensins are even more potent bactericidal agents with less toxicity toward host cells. In this paper, structures of three C-terminal (R36-K45) analogues of human beta-defensin-3 were studied by 1H NMR spectroscopy and extensive molecular dynamics simulations. Because of indications that these peptides might target the inner bacterial membrane, they were reconstituted in dodecylphosphocholine or dodecylphosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] mixed micelles, and lipid bicelles mimicking the phospholipid-constituted bilayer membrane of mammalian and bacterial cells. The results show that the binding affinity and partitioning into the lipid phase and the ability to dimerize and accrete well-defined structures upon interactions with lipid membranes contribute to compactization of positive charges within peptide oligomers. The peptide charge density, mediated by corresponding three-dimensional structures, was found to directly correlate with the antimicrobial activity. These novel observations may provide a new rationale for the design of improved antimicrobial agents.

Linear Analogues of Human β-Defensin 3: Concepts for Design of Antimicrobial Peptides with Reduced Cytotoxicity to Mammalian Cells

A series of engineered linear analogues [coded as F6, W6, Y6, A6, S6 and C(Acm)6] were modeled, designed, synthesized and structurally characterized by mass spectra, circular dichroism, hydrophobicity analysis and molecular modeling. We have screened antimicrobial activity, hemolysis to rabbit erythrocytes, and cytotoxicity to human conjunctival epithelial cells. No significant hemolytic effect was observed for hBD3 or from five of the six analogues [F6, Y6, A6, S6 and C(Acm)6] over the range of 3–100 μg mL−1. The six linear analogues have reduced cytotoxicity to human conjunctival epithelial cells over the range of 6–100 μg mL−1 compared to hBD3. By tuning the overall hydrophobicity of linear hBD3 analogues, reduced cytotoxicity and hemolysis were obtained while preserving the antimicrobial properties. The decreased cytotoxicity of the linear analogues is suggested to be structurally related to the removal of disulfide bridges, and the flexible structure of the linear forms, which seem to be associated with loss of secondary structure. These results suggest a new approach for guiding the design of new linear analogues of defensin peptides with strong antibiotic properties and reduced cytotoxicity to mammalian cells.

Amino acid modified xanthone derivatives: novel, highly promising membrane-active antimicrobials for multidrug-resistant Gram-positive bacterial infections.

Antibiotic resistance is a critical global health care crisis requiring urgent action to develop more effective antibiotics. Utilizing the hydrophobic scaffold of xanthone, we identified three components that mimicked the action of an antimicrobial cationic peptide to produce membrane-targeting antimicrobials. Compounds 5c and 6, which contain a hydrophobic xanthone core, lipophilic chains, and cationic amino acids, displayed very promising antimicrobial activity against multidrug-resistant Gram-positive bacteria, including MRSA and VRE, rapid time-kill, avoidance of antibiotic resistance, and low toxicity. The bacterial membrane selectivity of these molecules was comparable to that of several membrane-targeting antibiotics in clinical trials. 5c and 6 were effective in a mouse model of corneal infection by S. aureus and MRSA. Evidence is presented indicating that 5c and 6 target the negatively charged bacterial membrane via a combination of electrostatic and hydrophobic interactions. These results suggest that 5c and 6 have significant promise for combating life-threatening infections.

Bio-inspired in situ crosslinking and mineralization of electrospun collagen scaffolds for bone tissue engineering.

Bone disorders are the most common cause of severe long term pain and physical disability, and affect millions of people around the world. In the present study, we report bio-inspired preparation of bone-like composite structures by electrospinning of collagen containing catecholamines and Ca(2+). The presence of divalent cation induces simultaneous partial oxidative polymerization of catecholamines and crosslinking of collagen nanofibers, thus producing mats that are mechanically robust and confer photoluminescence properties. Subsequent mineralization of the mats by ammonium carbonate leads to complete oxidative polymerization of catecholamines and precipitation of amorphous CaCO3. The collagen composite scaffolds display outstanding mechanical properties with Youngs modulus approaching the limits of cancellous bone. Biological studies demonstrate that human fetal osteoblasts seeded on to the composite scaffolds display enhanced cell adhesion, penetration, proliferation, differentiation and osteogenic expression of osteocalcin, osteopontin and bone matrix protein when compared to pristine collagen or tissue culture plates. Among the two catecholamines, mats containing norepinephrine displayed superior mechanical, photoluminescence and biological properties than mats loaded with dopamine. These smart multifunctional scaffolds could potentially be utilized to repair and regenerate bone defects and injuries.

Membrane Active Antimicrobial Peptides: Translating Mechanistic Insights to Design

Antimicrobial peptides (AMPs) are promising next generation antibiotics that hold great potential for combating bacterial resistance. AMPs can be both bacteriostatic and bactericidal, induce rapid killing and display a lower propensity to develop resistance than do conventional antibiotics. Despite significant progress in the past 30 years, no peptide antibiotic has reached the clinic yet. Poor understanding of the action mechanisms and lack of rational design principles have been the two major obstacles that have slowed progress. Technological developments are now enabling multidisciplinary approaches including molecular dynamics simulations combined with biophysics and microbiology toward providing valuable insights into the interactions of AMPs with membranes at atomic level. This has led to increasingly robust models of the mechanisms of action of AMPs and has begun to contribute meaningfully toward the discovery of new AMPs. This review discusses the detailed action mechanisms that have been put forward, with detailed atomistic insights into how the AMPs interact with bacterial membranes. The review further discusses how this knowledge is exploited toward developing design principles for novel AMPs. Finally, the current status, associated challenges, and future directions for the development of AMP therapeutics are discussed.

Progressive Structuring of a Branched Antimicrobial Peptide on the Path to the Inner Membrane Target

Background: A cationic branched peptide was designed with antimicrobial activities against Gram-negative bacteria. Results: B2088 penetrated the outer membrane through inducing phase transitions of LPS and caused inner membrane depolarization by lipid redistribution. Conclusion: Our findings support the interfacial activity model and extend it to more complex interfaces. Significance: We provide a functional structural motif for developing new antimicrobials. In recent years, interest has grown in the antimicrobial properties of certain natural and non-natural peptides. The strategy of inserting a covalent branch point in a peptide can improve its antimicrobial properties while retaining host biocompatibility. However, little is known regarding possible structural transitions as the peptide moves on the access path to the presumed target, the inner membrane. Establishing the nature of the interactions with the complex bacterial outer and inner membranes is important for effective peptide design. Structure-activity relationships of an amphiphilic, branched antimicrobial peptide (B2088) are examined using environment-sensitive fluorescent probes, electron microscopy, molecular dynamics simulations, and high resolution NMR in solution and in condensed states. The peptide is reconstituted in bacterial outer membrane lipopolysaccharide extract as well as in a variety of lipid media mimicking the inner membrane of Gram-negative pathogens. Progressive structure accretion is observed for the peptide in water, LPS, and lipid environments. Despite inducing rapid aggregation of bacteria-derived lipopolysaccharides, the peptide remains highly mobile in the aggregated lattice. At the inner membranes, the peptide undergoes further structural compaction mediated by interactions with negatively charged lipids, probably causing redistribution of membrane lipids, which in turn results in increased membrane permeability and bacterial lysis. These findings suggest that peptides possessing both enhanced mobility in the bacterial outer membrane and spatial structure facilitating its interactions with the membrane-water interface may provide excellent structural motifs to develop new antimicrobials that can overcome antibiotic-resistant Gram-negative pathogens.

Molecular simulations suggest how a branched antimicrobial peptide perturbs a bacterial membrane and enhances permeability

A covalently, branched antimicrobial peptide (BAMP) B2088 demonstrating enhanced antimicrobial effects and without additional toxicity when compared to its linear counterpart, has been developed. Atomistic molecular dynamics simulations have been used to investigate the mode of interaction of B2088 with model bacterial and mammalian membranes. These simulations suggest that both long-range electrostatic interactions and short-range hydrogen bonding play important roles in steering B2088 toward the negatively charged membranes. The reason why B2088 is selective towards the bacterial membrane is postulated to be the greater density of negative charges on the bacterial membrane which enables rapid accumulation of B2088 on the bacterial membrane to a high surface concentration, stabilizing it through excess hydrogen bond formation. The majority of hydrogen bonds are seen between the side chains of the basic residues (Arg or Lys) with the PO4 groups of lipids. In particular, formation of the bidentate hydrogen bonds between the guanidinium group of Arg and PO4 groups are found to be more favorable, both geometrically and energetically. Moreover, the planar gaunidinium group and its hydrophobic character enable the Arg side chains to solvate into the hydrophobic membrane. Structural perturbation of the bacterial membrane is found to be concentration dependent and is significant at higher concentrations of B2088, resulting in a large number of water translocations across the bacterial membrane. These simulations enhance our understanding of the action mechanism of a covalently branched antimicrobial peptide with model membranes and provide practical guidance for the design of new antimicrobial peptides.