Elevated c-di-GMP Levels and Expression of the Type III Secretion System Promote Corneal Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is generally believed to establish biofilm-associated infections under the regulation of the secondary messenger c-di-GMP. To evaluate P. aeruginosa biofilm physiology during ocular infections, comparative transcriptomic analysis was performed on wild-type P. aeruginosa PAO1, a ΔwspF mutant strain (high c-di-GMP levels), and a plac-yhjH-containing strain (low c-di-GMP levels) from mouse corneal infection, as well as in vitro biofilm and planktonic cultures. The c-di-GMP content in P. aeruginosa during corneal infection was monitored using a fluorescent c-di-GMP reporter strain. Biofilm-related genes were induced in in vivo PAO1 compared to in vitro planktonic bacteria. Several diguanylate cyclases and phosphodiesterases were commonly regulated in in vivo PAO1 and in vitro biofilm compared to in vitro planktonic bacteria. Several exopolysaccharide genes and motility genes were induced and downregulated, respectively, in in vivo PAO1 and the in vivo ΔwspF mutant compared to the in vivo plac-yhjH-containing strain. Elevation of c-di-GMP levels in P. aeruginosa began as early as 2 h postinfection. The ΔwspF mutant was less susceptible to host clearance than the plac-yhjH-containing strain and could suppress host immune responses. The type III secretion system (T3SS) was induced in in vivo PAO1 compared to in vitro biofilm bacteria. A ΔwspF mutant with a defective T3SS was more susceptible to host clearance than a ΔwspF mutant with a functional T3SS. Our study suggests that elevated intracellular c-di-GMP levels and T3SS activity in P. aeruginosa are necessary for establishment of infection and modulation of host immune responses in mouse cornea.

Host Defense Peptides at the Ocular Surface: Roles in Health and Major Diseases, and Therapeutic Potentials.

Sight is arguably the most important sense in human. Being constantly exposed to the environmental stress, irritants and pathogens, the ocular surface - a specialized functional and anatomical unit composed of tear film, conjunctival and corneal epithelium, lacrimal glands, meibomian glands, and nasolacrimal drainage apparatus - serves as a crucial front-line defense of the eye. Host defense peptides (HDPs), also known as antimicrobial peptides, are evolutionarily conserved molecular components of innate immunity that are found in all classes of life. Since the first discovery of lysozyme in 1922, a wide range of HDPs have been identified at the ocular surface. In addition to their antimicrobial activity, HDPs are increasingly recognized for their wide array of biological functions, including anti-biofilm, immunomodulation, wound healing, and anti-cancer properties. In this review, we provide an updated review on: (1) spectrum and expression of HDPs at the ocular surface; (2) participation of HDPs in ocular surface diseases/conditions such as infectious keratitis, conjunctivitis, dry eye disease, keratoconus, allergic eye disease, rosacea keratitis, and post-ocular surgery; (3) HDPs that are currently in the development pipeline for treatment of ocular diseases and infections; and (4) future potential of HDP-based clinical pharmacotherapy for ocular diseases.

Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights From Experimental and Molecular Dynamics Simulations Studies.

Background/Aim: Host defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and the secondary structure and underlying mechanism of action of CaD23. Methods: Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard and time-kill assays. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FIC<0.5), additive (FIC between 0.5-1.0), indifferent (FIC between >1.0 and ≤4), or antagonistic (FIC>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes. Circular dichroism (CD) spectroscopy was also performed to examine the secondary structures of CaD23. Results: CaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI = 0.60-0.69) and MRSA43300 (FICI = 0.56-0.60) but an indifferent effect against P. aeruginosa (FIC = 1.03-1.15). CaD23 (at 25 µg/ml; 2xMIC) completely killed S. aureus within 30 min. When used at sub-MIC concentration (3.1 µg/ml; 0.25xMIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23. The secondary structures of CaD23 observed in MD simulations were validated by CD spectroscopy. Conclusion: CaD23 is a novel alpha-helical, membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulations serves as a powerful tool in revealing the peptide secondary structure, dissecting the mechanism of action, and guiding the design and optimisation of HDPs.

Hybrid derivative of cathelicidin and human beta defensin-2 against Gram-positive bacteria: A novel approach for the treatment of bacterial keratitis.

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5-25.0 µg/ml (5.2-10.4 µM)] and S. epidermidis [MIC = 12.5 µg/ml (5.2 µM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 µg/ml (10.4-20.8 µM)]. CaD23 (at 25 µg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 µg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

Dissecting the Molecular Mechanism of Colistin Resistance in mcr-1 Bacteria.

Colistin or polymyxin B is the last resort antibiotic to treat infections of multidrug-resistant Gram-negative bacteria by disrupting their outer membranes. The recent emergence of Gram-negative bacteria that demonstrate colistin resistance, particularly plasmid-mediated mobile colistin resistance (mcr), poses a big challenge to the treatment of multidrug resistance infections. Using molecular dynamics simulations, we explore the mechanism of colistin resistance in a model lipid A bilayer mimicking the Gram-negative mcr-1 bacterial outer membrane. The simulation results reveal that the outer membrane of normal Gram-negative bacteria is stabilized by salt bridges between positively charged divalent ions and negatively charged phosphate groups of the membranes. In the presence of positively charged polymyxin B, these salt bridges are disrupted, and calcium is released into the aqueous phase, resulting in membrane disruption. In contrast, the lipid A in the outer membrane of mcr-1 bacteria has a novel modification, this being a covalently attached phosphoethanolamine group. This group enables the formation of a large number of hydrogen bonds between the amine and phosphate groups, resulting in an electrostatic net on the membrane. This extensive noncovalent electrostatic cross-linking between the lipid molecules collectively enhances the membrane stability and results in resistance to the action of cationic peptides such as polymyxin B. The simulation results shed new atomistic insights for understanding the mechanistic basis of colistin resistance and provide clues for the design of new membrane disruptors and permeabilizers to treat mcr-1 infections.

Strategies in Translating the Therapeutic Potentials of Host Defense Peptides.

The golden era of antibiotics, heralded by the discovery of penicillin, has long been challenged by the emergence of antimicrobial resistance (AMR). Host defense peptides (HDPs), previously known as antimicrobial peptides, are emerging as a group of promising antimicrobial candidates for combatting AMR due to their rapid and unique antimicrobial action. Decades of research have advanced our understanding of the relationship between the physicochemical properties of HDPs and their underlying antimicrobial and non-antimicrobial functions, including immunomodulatory, anti-biofilm, and wound healing properties. However, the mission of translating novel HDP-derived molecules from bench to bedside has yet to be fully accomplished, primarily attributed to their intricate structure-activity relationship, toxicity, instability in host and microbial environment, lack of correlation between in vitro and in vivo efficacies, and dwindling interest from large pharmaceutical companies. Based on our previous experience and the expanding knowledge gleaned from the literature, this review aims to summarize the novel strategies that have been employed to enhance the antimicrobial efficacy, proteolytic stability, and cell selectivity, which are all crucial factors for bench-to-bedside translation of HDP-based treatment. Strategies such as residues substitution with natural and/or unnatural amino acids, hybridization, L-to-D heterochiral isomerization, C- and N-terminal modification, cyclization, incorporation with nanoparticles, and "smart design" using artificial intelligence technology, will be discussed. We also provide an overview of HDP-based treatment that are currently in the development pipeline.

Wound healing properties of magnesium mineralized antimicrobial nanofibre dressings containing chondroitin sulphate - a comparison between blend and core-shell nanofibres.

The development of antimicrobial nanofibre dressings that can protect the injured tissues from commensal pathogens while promoting tissue regeneration finds enormous potential in plastic and reconstructive surgery practices. To achieve this goal, we investigated the effect of chondroitin sulphate on the morphology, mechanical properties, wettability and biocompatibility of polydopamine crosslinked electrospun gelatin nanofibres containing mineralized magnesium. To extend the durability of dressings, we prepared composite dressings containing polycaprolactone (PCL) and gelatin as blend or core-shell nanofibres. Nanofibre blends presented greater tensile strength and stretchability, while core-shell nanofibres displayed superior photoluminescent properties. In a porcine model of cutaneous burn injury, both the blend and core-shell nanofibre dressings displayed improved re-epithelialization, wound closure and clinical outcome in comparison to untreated burns. Histology of the biopsied tissues indicated smooth regeneration and collagen organization of the burns treated with core-shell nanostructures than untreated burns. This study compared the physico-chemical and biological properties of composite nanofibres that are capable of accelerating burn wound healing and possess antimicrobial properties, highlighting their potential as wound dressings and skin substitutes.

Increased Protein S-Glutathionylation in Leber's Hereditary Optic Neuropathy (LHON).

Leber's hereditary optic neuropathy (LHON, MIM#535000) is the most common form of inherited optic neuropathies and mitochondrial DNA-related diseases. The pathogenicity of mutations in genes encoding components of mitochondrial Complex I is well established, but the underlying pathomechanisms of the disease are still unclear. Hypothesizing that oxidative stress related to Complex I deficiency may increase protein S-glutathionylation, we investigated the proteome-wide S-glutathionylation profiles in LHON (n = 11) and control (n = 7) fibroblasts, using the GluICAT platform that we recently developed. Glutathionylation was also studied in healthy fibroblasts (n = 6) after experimental Complex I inhibition. The significantly increased reactive oxygen species (ROS) production in the LHON group by Complex I was shown experimentally. Among the 540 proteins which were globally identified as glutathionylated, 79 showed a significantly increased glutathionylation (p < 0.05) in LHON and 94 in Complex I-inhibited fibroblasts. Approximately 42% (33/79) of the altered proteins were shared by the two groups, suggesting that Complex I deficiency was the main cause of increased glutathionylation. Among the 79 affected proteins in LHON fibroblasts, 23% (18/79) were involved in energetic metabolism, 31% (24/79) exhibited catalytic activity, 73% (58/79) showed various non-mitochondrial localizations, and 38% (30/79) affected the cell protein quality control. Integrated proteo-metabolomic analysis using our previous metabolomic study of LHON fibroblasts also revealed similar alterations of protein metabolism and, in particular, of aminoacyl-tRNA synthetases. S-glutathionylation is mainly known to be responsible for protein loss of function, and molecular dynamics simulations and 3D structure predictions confirmed such deleterious impacts on adenine nucleotide translocator 2 (ANT2), by weakening its affinity to ATP/ADP. Our study reveals a broad impact throughout the cell of Complex I-related LHON pathogenesis, involving a generalized protein stress response, and provides a therapeutic rationale for targeting S-glutathionylation by antioxidative strategies.

Mechanism of polyamine induced colistin resistance through electrostatic networks on bacterial outer membranes.

Naturally occurring linear polyamines are known to enable bacteria to be resistant to cationic membrane active peptides. To understand this protective mechanism, molecular dynamics simulations are employed to probe their effect on a model bacterial outer membrane. Being protonated at physiological pH, the amine groups of the polyamine engage in favorable electrostatic interactions with the negatively charged phosphate groups of the membrane. Additionally, the amine groups form large number of hydrogen bonds with the phosphate groups. At high concentrations, these hydrogen bonds and the electrostatic network can non-covalently crosslink the lipid A molecules, resulting in stabilization of the outer membrane against membrane active antibiotics such as colistin and polymyxin B. Moreover, large polyamine molecules (e.g., spermidine) have a stronger stabilization effect than small polyamine molecules (e.g., ethylene diamine). The atomistic insights provide useful guidance for the design of next generation membrane active amine-rich antibiotics, especially to tackle the growing threat of multi-drug resistance of Gram negative bacteria.

Rabbit Fungal Keratitis Model of Fusarium solani Tested Against Three Commercially Available Antifungal Drugs.

OBJECTIVES: The purpose of this study was to develop a reproducible preclinical Fusarium solani keratitis model, which would allow comparative testing of currently available antifungals (NATACYN [Alcon, Fort Worth, TX], voriconazole 1%, and amphotericin B 0.1%) as well as efficacy testing of new antifungals for translation into clinical practice in the future. METHODS: The rabbit F. solani keratitis model was developed in New Zealand white rabbits using local and systemic immunosuppression. Infection was introduced by intrastromal injection of F. solani spores into one of the immunosuppressed rabbit eyes while the contralateral eye was a control. Progress of the infection was assessed by the clinical features, histopathology, and viable fungal counts. In this study, the efficacy of currently available antifungals (NATACYN [Alcon], voriconazole 1%, and amphotericin B 0.1%) was compared. Rabbits were randomly divided (n=4 in each group), and the respective antifungal was instilled topically 5 times/day for 7 days. Treatment effects were analyzed by evaluating the anterior segment with the help of slit-lamp, histopathological findings and viable fungal culture at the end of the experiment. RESULTS: We report the development of a reproducible and progressive rabbit F. solani keratitis model as shown by the substantial viable fungal counts (3 log CFU), the presence of large patchy lesions and substantial hypopyon in the 12-day model correlated with specific histopathological analysis for fungus (extended F. solani hyphae from midcorneal stroma into the anterior chamber and traverse Descemet membrane with anterior chamber suppurative plaque). Voriconazole 1% and NATACYN revealed significant reduction of the fungal wound area (P=0.02 and 0.021), respectively, while amphotericin B 0.1% exhibited P value of 0.083 compared with their infected nontreated controls. Voriconazole 1% and amphotericin B 0.1% showed significant viable fungal count differences (P=0.004 and 0.01), respectively, whereas P value of NATACYN was 0.337 compared with control infected corneas. CONCLUSION: The reported rabbit fungal keratitis model can be used for screening new antifungals and evaluating currently available antifungals to facilitate better clinical outcomes. Voriconazole 1% showed the best efficacy among the three tested currently available antifungals by showing the significant differences in both wound size and viable fungal count comparisons in our F. solani rabbit keratitis model.

Rational Substitution of ε-Lysine for α-Lysine Enhances the Cell and Membrane Selectivity of Pore-Forming Melittin.

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept in vivo experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by ε-lysylation.

Multifunctional Antimicrobial Nanofiber Dressings Containing ε-Polylysine for the Eradication of Bacterial Bioburden and Promotion of Wound Healing in Critically Colonized Wounds.

Bacterial colonization of acute and chronic wounds is often associated with delayed wound healing and prolonged hospitalization. The rise of multi-drug resistant bacteria and the poor biocompatibility of topical antimicrobials warrant safe and effective antimicrobials. Antimicrobial agents that target microbial membranes without interfering with the mammalian cell proliferation and migration hold great promise in the treatment of traumatic wounds. This article reports the utility of superhydrophilic electrospun gelatin nanofiber dressings (NFDs) containing a broad-spectrum antimicrobial polymer, ε-polylysine (εPL), crosslinked by polydopamine (pDA) for treating second-degree burns. In a porcine model of partial thickness burns, NFDs promoted wound closure and reduced hypertrophic scarring compared to untreated burns. Analysis of NFDs in contact with the burns indicated that the dressings trap early colonizers and elicit bactericidal activity, thus creating a sterile wound bed for fibroblasts migration and re-epithelialization. In support of these observations, in porcine models of Pseudomonas aeruginosa and Staphylococcus aureus colonized partial thickness burns, NFDs decreased bacterial bioburden and promoted wound closure and re-epithelialization. NFDs displayed superior clinical outcome than standard-of-care silver dressings. The excellent biocompatibility and antimicrobial efficacy of the newly developed dressings in pre-clinical models demonstrate its potential for clinical use to manage infected wounds without compromising tissue regeneration.

Biological Mechanisms of Atropine Control of Myopia.

Myopia is a global problem that is increasing at an epidemic rate in the world. Although the refractive error can be corrected easily, myopes, particularly those with high myopia, are susceptible to potentially blinding eye diseases later in life. Despite a plethora of myopia research, the molecular/cellular mechanisms underlying the development of myopia are not well understood, preventing the search for the most effective pharmacological control. Consequently, several approaches to slowing down myopia progression in the actively growing eyes of children have been underway. So far, atropine, an anticholinergic blocking agent, has been most effective and is used by clinicians in off-label ways for myopia control. Although the exact mechanisms of its action remain elusive and debatable, atropine encompasses a complex interplay with receptors on different ocular tissues at multiple levels and, hence, can be categorized as a shotgun approach to myopia treatment. This review will provide a brief overview of the biological mechanisms implicated in mediating the effects of atropine in myopia control.

A serum metabolomics study of patients with nAMD in response to anti-VEGF therapy.

Intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) is the current standard of treatment for choroidal neovascularization (CNV) secondary to neovascular age-related macular degeneration (nAMD), but there are no diagnostic tools to predict response of these therapies. We hypothesize that differences in baseline metabolic profiles of patients with nAMD may influence responsiveness to anti-VEGF therapy, and thus provide prognosticating information for these patients. A prospective study was performed on 100 patients with nAMD treated with anti-VEGF therapy. We classified patients into two groups: responders (n = 54) and non-responders (n = 46). The expression levels of glycerophosphocholine,LysoPC (18:2) and PS (18:0/20:4) were higher in non-responders and these findings were verified in the validation cohort, implicating that reductions in these three metabolites can be used as predictors for responsiveness to anti-VEGF therapy during the initial loading phase for patients with nAMD. Our study also provided new insights into the pathophysiological changes and molecular mechanism of anti- VEGF therapy for nAMD patients.

Mussel-Inspired Durable Antimicrobial Contact Lenses: The Role of Covalent and Noncovalent Attachment of Antimicrobials.

Contact lens is a major risk factor for microbial keratitis among contact lens wearers. Chemical strategies that can prevent microbial adhesion and biofilm formation are required to improve a wearer's hygiene and safety. Taking advantage of the material-independent properties of a polydopamine (pDA) coating, we investigated the role of covalent/noncovalent interactions of the antimicrobials and pDA in conferring long-term antimicrobial activities. The developed antimicrobial contact lenses not only retain their antibacterial efficiency against different bacterial strains for 2 weeks but also inhibit microbial adhesion and biofilm formation on the lens surfaces. The designed antimicrobial coatings were found to be safe for ocular cell lines. Moreover, the antimicrobial coatings did not affect the functional and surface properties of coated contact lenses. This methodology can be used to protect the contact lenses from microbial contamination for prolonged periods and has the potential to be extended for designing antimicrobial coatings for other medical devices as well.

Surface Immobilization of Nano-Silver on Polymeric Medical Devices to Prevent Bacterial Biofilm Formation.

: Bacterial biofilm on medical devices is difficult to eradicate. Many have capitalized the anti-infective capability of silver ions (Ag+) by incorporating nano-silver (nAg) in a biodegradable coating, which is then laid on polymeric medical devices. However, such coating can be subjected to premature dissolution, particularly in harsh diseased tissue microenvironment, leading to rapid nAg clearance. It stands to reason that impregnating nAg directly onto the device, at the surface, is a more ideal solution. We tested this concept for a corneal prosthesis by immobilizing nAg and nano-hydroxyapatite (nHAp) on poly(methyl methacrylate), and tested its biocompatibility with human stromal cells and antimicrobial performance against biofilm-forming pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Three different dual-functionalized substrates-high Ag (referred to as 75:25 HAp:Ag); intermediate Ag (95:5 HAp:Ag); and low Ag (99:1 HAp:Ag) were studied. The 75:25 HAp:Ag was effective in inhibiting biofilm formation, but was cytotoxic. The 95:5 HAp:Ag showed the best selectivity among the three substrates; it prevented biofilm formation of both pathogens and had excellent biocompatibility. The coating was also effective in eliminating non-adherent bacteria in the culture media. However, a 28-day incubation in artificial tear fluid revealed a ~40% reduction in Ag+ release, compared to freshly-coated substrates. The reduction affected the inhibition of S. aureus growth, but not the P. aeruginosa. Our findings suggest that Ag+ released from surface-immobilized nAg diminishes over time and becomes less effective in suppressing biofilm formation of Gram-positive bacteria, such as S. aureus. This advocates the coating, more as a protection against perioperative and early postoperative infections, and less as a long-term preventive solution.

Protective Action of Linear Polyethylenimine against Staphylococcus aureus Colonization and Exaggerated Inflammation in Vitro and in Vivo.

Increased evolution of multidrug resistant pathogens necessitates the development of multifunctional antimicrobials. There is a perceived need for developing new antimicrobials that can interfere with acute inflammation after bacterial infections. Here, we investigated the therapeutic potential of linear polyethylenimine (LPEI) in vitro and in vivo. The minimum inhibitory concentration of LPEI ranged from 8 to 32 µg/mL and elicited rapid bactericidal activity against clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). The polymer was biocompatible for human cultured ocular and dermal cells. Prophylactic addition of LPEI inhibited the bacterial colonization of human primary dermal fibroblasts (hDFs). In a scratch wound cell migration assay, LPEI attenuated the migration inhibitory effects of bacterial secretions. The polymer neutralized the cytokine release by hDFs exposed to bacterial secretions, possibly by blocking their accessibility to host cell receptors. Topical instillation of LPEI (1 mg/mL) was noncytotoxic and did not affect the re-epithelialization of injured porcine cornea. In a prophylactic in vivo model of S. aureus keratitis, LPEI was superior to gatifloxacin in terms of reducing stimulation of cytokines, corneal edema, and overall severity of the infection. These observations demonstrate therapeutic potential of LPEI for antimicrobial prophylaxis.

Age-associated changes in human tear proteome.

BACKGROUND: Prevalence of many eye and ocular surface diseases increases with age. While the clinical characteristics and pathophysiologic mechanisms of these conditions are often either known or extensively studied, the effects of normal aging on tear film and ocular surface have not been as widely researched. METHODS: In order to examine the effects of aging on tear fluid proteomics, tear fluid samples were collected preoperatively from 115 subjects undergoing strabismus or refractive surgery using glass microcapillary tubes. In addition to their refractive error or strabismus, the subjects did not have any other current, known eye diseases. The non-pooled samples were analysed using NanoLC-TripleTOF implementing a sequential window acquisition of all theoretical fragment ion spectra mass spectrometry, resulting in quantified data of 849 proteins. RESULTS: According to correlation results, 17 tear proteins correlated significantly with increased age and many of these proteins were connected to inflammation, immune response and cell death. According to enrichment analysis, growth and survival of cells decreased while immune response and inflammation increased with aging. We also discovered several well-known, activated and inhibited upstream regulators, e.g. NF-κB, which has been previously connected to aging in numerous previous studies. CONCLUSIONS: Overall, the results show the common age-dependent alterations in tear fluid protein profile, which demonstrate similar age-associated alterations of biological functions previously shown in other tissue and sample types.

Mediators of Corneal Haze Following Implantation of Presbyopic Corneal Inlays.

Purpose: To identify protein mediators of corneal haze following presbyopic corneal inlay surgery. Methods: Tears were collected from eyes with corneal haze following surgery with a shape-changing corneal inlay. Samples were subjected to quantitative proteomic analysis using iTRAQ and proteins significantly increased or decreased (1.3-fold or more) in haze eyes relative to fellow eyes were identified. Expression ratios were compared to postoperative eyes without corneal haze to identify proteins selectively increased or decreased in corneal haze eyes. Results: Inlay-associated haze occurred in 35% of eyes (6 of 17). Of 1443 unique tear proteins identified, eight proteins were selectively reduced in tears from postoperative haze eyes and one protein selectively increased. Proteins reduced in haze eyes included complement 4a (level relative to nonhaze eyes 0.464, P = 0.037), complement factor H (0.589, P = 0.048), immunoglobulin kappa variable 2-29 (0.128, P = 0.006), immunoglobulin kappa variable 2D-28 (0.612, P = 0.025), immunoglobulin lambda variable 7-46 (0.482, P = 0.007), S100 calcium binding protein A4 (0.614, P = 0.048), Shootin-1 (0.614, P = 0.048), and tissue inhibitor of metalloproteinase-1 (0.736, P = 0.023). The Xaa-Pro aminopeptidase 1 was increased in haze eyes relative to nonhaze eyes (1.517, P = 0.023). Conclusions: Corneal haze following corneal inlay surgery is associated with reduction in levels of known inflammatory and immune mediators. These findings represent a starting point for elucidation of pathways involved in corneal haze following synthetic inlay implantation and may enable development of targeted therapies that modulate the haze response.

Mathematical models of amino acid panel for assisting diagnosis of children acute leukemia.

BACKGROUND: The altered concentrations of amino acids were found in the bone marrow or blood of leukemia patients. Metabolomics technology combining mathematical model of biomarkers could be used for assisting the diagnosis of pediatric acute leukemia (AL). METHODS: The concentrations of 17 amino acids was measured by targeted liquid chromatograph-tandem mass spectrometry in periphery blood collected using dried blood spots. After evaluation, the mathematical models were further evaluated by prospective clinical validation cohort for AL diagnosis. RESULTS: The concentrations of 13 in 17 amino acids were statistically different between the periphery blood dried serum dots measured by targeted LC-MS/MS. The receiver operating characteristic analysis for the models of amino acid panel showed that the area under curve for AL diagnosis were 0.848, 0.834 and 0.856 by SVM, RF and XGBoost. The Kappa values in further prospectively evaluated clinical cohort were 0.697, 0.703 and 0.789 (p > 0.05) respectively, and the accuracies for the models were 84.86%, 85.20% and 89.46% respectively with further clinical validation. CONCLUSIONS: The established mathematical model is a faster, cheaper and more convenient way than conventional methods, and no significant difference on the effect of diagnosis comparing with conventional methods. The mathematical model can be clinically useful for assisting pediatric AL diagnosis.