Effect of tryptophan position and lysine/arginine substitution in antimicrobial peptides on antifungal action.

Every year, the overprescription, misuse, and improper disposal of antibiotics have led to the rampant development of drug-resistant pathogens and, in turn, a significant increase in the number of patients who die of drug-resistant fungal infections. Recently, researchers have begun investigating the use of antimicrobial peptides (AMPs) as next-generation antifungal agents to inhibit the growth of drug-resistant fungi. The antifungal activity of alpha-helical peptides designed using the cationic amino acids containing lysine and arginine and the hydrophobic amino acids containing isoleucine and tryptophan were evaluated using 10 yeast and mold fungi. Among these peptides, WIK-14, which is composed of a 14-mer with tryptophan sequences at the amino terminus, showed the best antifungal activity via transient pore formation and ROS generation. In addition, the in vivo antifungal effects of WIK-14 were investigated in a mouse model infected with drug-resistant Candida albicans. The results demonstrate the potential of AMPs as antifungal agents.

Effects of structural changes on antibacterial activity and cytotoxicity due to proline substitutions in chimeric peptide HnMc.

Owing to the rapidly increasing emergence of multidrug-resistant pathogens, antimicrobial peptides (AMPs) are being explored as next-generation antibiotics. However, AMPs present in nature are highly toxic and exhibit low antibacterial activity. Simple modifications, such as amino acid substitution, can enhance antimicrobial activity and cell selectivity. Herein, we show that HnMc-W, substituted by the Phe1Trp analog of HnMc, a chimeric peptide, resulted in membranolytic antibacterial action and enhanced salt tolerance, whereas HnMc-WP1 with one Ser9Pro substitution resulted in a proline-kink helical structure that increased salt-tolerant antibacterial effects and reduced cytotoxicity. In addition, the HnMc-WP2 peptide, designed with a PXXP motif, had a flexible central hinge in its α-helical structure due to the introduction of two Pro and two Gln (X positions, by deletion of two Gln at positions 16 and 17) residues instead of Ser at position. HnMc-WP2 exhibited excellent antibacterial effects without cytotoxicity in vitro. Moreover, its potent antibacterial activity was demonstrated in a drug-resistant Pseudomonas aeruginosa-infected mouse model in vivo. Our findings provide valuable information for the design of peptides with high antibacterial activity and cell selectivity.

Potent Antifungal Functions of a Living Modified Organism Protein, CP4-EPSPS, against Pathogenic Fungal Cells.

Various proteins introduced into living modified organism (LMO) crops function in plant defense mechanisms against target insect pests or herbicides. This study analyzed the antifungal effects of an introduced LMO protein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 (CP4-EPSPS). Pure recombinant CP4-EPSPS protein, expressed in Escherichia coli, inhibited the growth of human and plant fungal pathogens (Candida albicans, C. tropicalis, C. krusei, Colletotrichum gloeosporioides, Fusarium solani, F. graminearum, and Trichoderma virens), at minimum inhibitory concentrations (MICs) that ranged from 62.5 to 250 µg/mL. It inhibited fungal spore germination as well as cell proliferation on C. gloeosporioides. Rhodamine-labeled CP4-EPSPS accumulated on the fungal cell wall and within intracellular cytosol. In addition, the protein induced uptake of SYTOX Green into cells, but not into intracellular mitochondrial reactive oxygen species (ROS), indicating that its antifungal action was due to inducing the permeability of the fungal cell wall. Its antifungal action showed cell surface damage, as observed from fungal cell morphology. This study provided information on the effects of the LMO protein, EPSPS, on fungal growth.

Oxidative Stress Amplifying Polyprodrug Micelles as Drug Carriers for Combination Anticancer Therapy.

Cancer cells are more vulnerable to reactive oxygen species (ROS)-mediated oxidative stress than normal cells due to disturbed redox balance. It can be postulated that ROS-generating drug carriers exert anticancer actions, leading to combination anticancer therapy with drug payloads. Here, we report a ROS-generating polyprodrug of cinnamaldehyde (CA) that not only serves as a drug carrier but also synergizes with drug payloads. The polyprodrug of CA (pCA) incorporates ROS-generating CA in the backbone of an amphiphilic polymer through an acid-cleavable acetal linkage. pCA could self-assemble with tumor-targeting lipopeptide (DSPE-PEG-RGD) and encapsulate doxorubicin (DOX) to form T-pCAD micelles. At acidic pH, T-pCAD micelles release both CA and DOX to exert synergistic anticancer actions. Animal studies using mouse xenograft models revealed that T-pCAD micelles accumulate in tumors preferentially and suppress the tumor growth significantly. Based on the oxidative stress amplification and acid-responsiveness, ROS-generating pCAD micelles hold tremendous potential as drug carriers for combination anticancer therapy.

Functional Characterization of an Arabidopsis Profilin Protein as a Molecular Chaperone under Heat Shock Stress.

Profilins (PFNs) are actin monomer-binding proteins that function as antimicrobial agents in plant phloem sap. Although the roles of Arabidopsis thaliana profilin protein isoforms (AtPFNs) in regulating actin polymerization have already been described, their biochemical and molecular functions remain to be elucidated. Interestingly, a previous study indicated that AtPFN2 with high molecular weight (HMW) complexes showed lower antifungal activity than AtPFN1 with low molecular weight (LMW). These were bacterially expressed and purified to characterize the unknown functions of AtPFNs with different structures. In this study, we found that AtPFN1 and AtPFN2 proteins have LMW and HMW structures, respectively, but only AtPFN2 has a potential function as a molecular chaperone, which has never been reported elsewhere. AtPFN2 has better protein stability than AtPFN1 due to its higher molecular weight under heat shock conditions. The function of AtPFN2 as a holdase chaperone predominated in the HMW complexes, whereas the chaperone function of AtPFN1 was not observed in the LMW forms. These results suggest that AtPFN2 plays a critical role in plant tolerance by increasing hydrophobicity due to external heat stress.

Comparison of Postoperative Tunnel Widening After Hamstring Anterior Cruciate Ligament Reconstructions Between Anatomic and Nonanatomic Femoral Tunnels.

PURPOSE: To evaluate the effect of the location of the femoral tunnel on 3-dimensional (3D) computed tomography (CT) upon the postoperative tunnel widening after anterior cruciate ligament (ACL) reconstructions. METHODS: Inclusion criteria were patients who underwent hamstring ACL reconstructions using an adjustable-loop cortical suspension device, underwent 3D CT at the day after surgery, and were followed for a minimum of 2 years after surgery. Exclusion criteria were patients with combined ligament injury and reinjury after reconstruction. Using 3D CT, the center of the femoral tunnel aperture was located on a standardized grid system. The center of the ACL footprint was defined from the literature. The femoral tunnel location was classified as anatomic if it located within 2 standard deviations of the center position. If it was outside the 2 standard deviations, the tunnel was classified as nonanatomic. The patients were divided into either anatomic or nonanatomic groups. Femoral tunnel angles on both sagittal and coronal planes were measured. Both femoral and tibial tunnels measured on anteroposterior and lateral radiographs at immediate postoperative day and at 2 years after surgery. Postoperative knee stability and patient-reported outcomes were evaluated. RESULTS: There were 37 patients in anatomical group and 52 patients in nonanatomical group among enrolled 87 patients. There were no differences in demographics between the 2 groups. There were no differences in the femoral tunnel angles and postoperative tunnel widening between the 2 groups. A higher position correlated to the femoral tunnel widening at 2 years postoperatively. Postoperative knee stability and patient-reported outcomes showed no statistically significant differences between the 2 groups. CONCLUSIONS: There was no significant difference in postoperative tunnel widening or clinical outcomes between anatomic and nonanatomic femoral tunnel location after hamstring ACL reconstructions. A higher position correlated to the femoral tunnel widening at 2 years postoperatively. LEVEL OF EVIDENCE: Level III, Retrospective comparative study.

Functional Mechanisms Underlying the Antimicrobial Activity of the Oryza sativa Trx-like Protein.

Plants are constantly subjected to a variety of environmental stresses and have evolved regulatory responses to overcome unfavorable conditions that might reduce or adversely change a plant's growth or development. Among these, the regulated production of reactive oxygen species (ROS) as a signaling molecule occurs during plant development and pathogen defense. This study demonstrates the possible antifungal activity of Oryza sativa Tetratricopeptide Domain-containing thioredoxin (OsTDX) protein against various fungal pathogens. The transcription of OsTDX was induced by various environmental stresses known to elicit the generation of ROS in plant cells. OsTDX protein showed potent antifungal activity, with minimum inhibitory concentrations (MICs) against yeast and filamentous fungi ranging between 1.56 and 6.25 and 50 and 100 µg/mL, respectively. The uptake of SYTOX-Green into fungal cells and efflux of calcein from artificial fungus-like liposomes suggest that its killing mechanism involves membrane permeabilization and damage. In addition, irregular blebs and holes apparent on the surfaces of OsTDX-treated fungal cells indicate the membranolytic action of this protein. Our results suggest that the OsTDX protein represents a potentially useful lead for the development of pathogen-resistant plants.

Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant Pseudomonas aeruginosa and Staphylococcus aureus Planktonic Cells and Biofilm.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

Postoperative seizure outcome-guided machine learning for interictal electrocorticography in neocortical epilepsy.

The objective of this study was to introduce a new machine learning guided by outcome of resective epilepsy surgery defined as the presence/absence of seizures to improve data mining for interictal pathological activities in neocortical epilepsy. Electrocorticographies for 39 patients with medically intractable neocortical epilepsy were analyzed. We separately analyzed 38 frequencies from 0.9 to 800 Hz including both high-frequency activities and low-frequency activities to select bands related to seizure outcome. An automatic detector using amplitude-duration-number thresholds was used. Interictal electrocorticography data sets of 8 min for each patient were selected. In the first training data set of 20 patients, the automatic detector was optimized to best differentiate the seizure-free group from not-seizure-free-group based on ranks of resection percentages of activities detected using a genetic algorithm. The optimization was validated in a different data set of 19 patients. There were 16 (41%) seizure-free patients. The mean follow-up duration was 21 ± 11 mo (range, 13-44 mo). After validation, frequencies significantly related to seizure outcome were 5.8, 8.4-25, 30, 36, 52, and 75 among low-frequency activities and 108 and 800 Hz among high-frequency activities. Resection for 5.8, 8.4-25, 108, and 800 Hz activities consistently improved seizure outcome. Resection effects of 17-36, 52, and 75 Hz activities on seizure outcome were variable according to thresholds. We developed and validated an automated detector for monitoring interictal pathological and inhibitory/physiological activities in neocortical epilepsy using a data-driven approach through outcome-guided machine learning. NEW & NOTEWORTHY Outcome-guided machine learning based on seizure outcome was used to improve detections for interictal electrocorticographic low- and high-frequency activities. This method resulted in better separation of seizure outcome groups than others reported in the literature. The automatic detector can be trained without human intervention and no prior information. It is based only on objective seizure outcome data without relying on an expert's manual annotations. Using the method, we could find and characterize pathological and inhibitory activities.

Cell-selectivity of tryptophan and tyrosine in amphiphilic α-helical antimicrobial peptides against drug-resistant bacteria.

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, 'XWZX' basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.

Hydrophilic Linear Peptide with Histidine and Lysine Residues as a Key Factor Affecting Antifungal Activity.

Increases in the numbers of immunocompromised patients and the emergence of drug-resistance fungal pathogens have led to the need for new, safe, efficacious antifungal agents. In this study, we designed a histidine-lysine-lysine (HKK) motif and synthesized six HKK peptides with repetitions of the motif. These peptides showed length-dependent antifungal activity against drug-susceptible and drug-resistant fungal pathogens via membranolytic or non-membranolytic action. None of the peptides were cytotoxic to rat erythrocytes or NIH3T3 mouse embryonic fibroblasts. Short-length peptides were directly translocated in fungal cytosol and reacted with mitochondria, resulting in apoptosis. Membrane-permeabilizing activity occurred in the presence of long peptides, and peptides were able to transfer to the cytosol and induce reactive oxygen species. Our results suggest that peptides composed only of cationic amino acids may be good candidates as antifungal agents.

Functional characterization of the Arabidopsis universal stress protein AtUSP with an antifungal activity.

An antifungal protein, AtUSP protein (At3g53990), was isolated from Arabidopsis thaliana leaves by ion and size chromatography and sequenced by N-terminal sequencing. The AtUSP gene amplified from an Arabidopsis leaf cDNA library was transformed to Escherichia coli to express the AtUSP protein. The recombinant protein inhibited the cell growth of various pathogenic fungal strains. The levels of the AtUSP transcripts were increased by various stresses, including pathogenic infection and salt stress. These results suggest that Arabidopsis AtUSP plays a critical role in the plant tolerance to diverse pathogenic infections. The potent antifungal action, which is a new function of AtUSP, was attributed to fungal reactive oxygen species (ROS) generation and mitochondrial potential alteration.

Gamma-knife subcaudate tractotomy for treatment-resistant depression and target characteristics: a case report and review.

Stereotactic subcaudate tractotomy has previously been suggested to be an effective treatment for depression. This is the first study to report the use of gamma-knife subcaudate tractotomy for treatment-resistant depression. A 49-year-old woman with major depressive disorder had been treated for 30 years, with nine suicide attempts during that time. The right and left target maximum diameter was 11 mm within 50 % isodose lines. The target was located more posteriorly and inferiorly than the subgenual cingulate target typically used for deep-brain stimulation. The maximum radiation dose was 130 Gy. During the 4 months after surgery, the patient improved gradually from 23 to 4 according to the Hamilton Rating Scale for Depression and antidepressant medication was discontinued. Target-sized focal lesions were identified and no edema was seen postoperatively. No aggravation or neurologic deficit occurred during the 2.5 years of follow-up. Gamma-knife subcaudate tractotomy for depression is a minimally invasive technique. Investigations of the effectiveness and safety profile in a larger group are warranted.

Functional characterization of alpha-synuclein protein with antimicrobial activity.

Alpha-synuclein (α-Syn), a small (14 kDa) protein associated with Parkinson's disease, is abundant in human neural tissues. α-Syn plays an important role in maintaining a supply of synaptic vesicles in presynaptic terminals; however, the mechanism by which it performs this function are not well understood. In addition, there is a correlation between α-Syn over-expression and upregulation of an innate immune response. Given the growing body of literature surrounding antimicrobial peptides (AMPs) in the brain, and the similarities between α-Syn and a previously characterized AMP, Amyloid-ß, we set out to investigate if α-Syn shares AMP-like properties. Here we demonstrate that α-Syn exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, we demonstrate a role for α-Syn in inhibiting various pathogenic fungal strains such as Aspergillus flavus, Aspergillus fumigatus and Rhizoctonia solani. We also analyzed localizations of recombinant α-Syn protein in E. coli and Candida albicans. These results suggest that in addition to α-Syn's role in neurotransmitter release, it appears to be a natural AMP.

Algicidal effect of hybrid peptides as potential inhibitors of harmful algal blooms.

OBJECTIVES: To biochemically characterize synthetic peptides to control harmful algal blooms (HABs) that cause red tides in marine water ecosystems. RESULTS: We present an analysis of several short synthetic peptides and their efficacy as algicidal agents. By altering the amino acid composition of the peptides we addressed the mode of algicidal action and determine the optimal balance of cationic and hydrophobic content for killing. In a controlled setting, these synthetic peptides disrupted both plasma and chloroplast membranes of several species known to result in HABs. This disruption was a direct result of the hydrophobic and cationic content of the peptide. Furthermore, by using an anti-HAB bioassay in scallops, we determined that these peptides were algicidal without being cytotoxic to other marine organisms. CONCLUSIONS: These synthetic peptides may prove promising for general marine ecosystem remediation where HABs have become widespread and resulted in serious economic loss.

Evaluation of the interaction between contact force and decision making on lower extremity biomechanics during a side-cutting maneuver.

INTRODUCTION: The purposes of this study were (1) to compare the effect between contact force [first and second ground reaction force (GRF)] and decision making (anticipation vs unanticipation) on lower extremity biomechanics during a side-cutting maneuver in young soccer players; and (2) to identify which condition is more vulnerable to biomechanical risk factors of the anterior cruciate ligament (ACL) injury. MATERIALS AND METHODS: Sixteen young, male middle school soccer players with right leg dominance participated in this study. Three-dimensional motion analysis featuring GRF and electromyography (EMG) of the right leg was used during the stance phase of the side-cutting maneuver. Kinematics, kinetics, and EMG data for each athlete were analyzed and averaged for three successful anticipated and unanticipated side-cutting maneuvers. RESULTS: GRF was smaller and muscle activities were lower in most muscle groups in the first peak than that of the second peak. More flexion and internal rotational angles of the hip joint were observed in the unanticipated first peak that that of the second peak. Lesser flexion angle and moment of the knee joint observed in the first peak than that of the second peak, and abduction moment was increased after the unanticipation. CONCLUSION: The GRF and muscle activities were smaller in the first peak than those in the second peak; however, first peak showed a closer association with biomechanical risk factors of the ACL injury. There were some interactions between contact force and decision making and unanticipation made the first peak more risky to the ACL injury.

Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 µM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent.

Antimicrobial action of water-soluble ß-chitosan against clinical multi-drug resistant bacteria.

Recently, the number of patients infected by drug-resistant pathogenic microbes has increased remarkably worldwide, and a number of studies have reported new antibiotics from natural sources. Among them, chitosan, with a high molecular weight and α-conformation, exhibits potent antimicrobial activity, but useful applications as an antibiotic are limited by its cytotoxicity and insolubility at physiological pH. In the present study, the antibacterial activity of low molecular weight water-soluble (LMWS) α-chitosan (α1k, α5k, and α10k with molecular masses of 1, 5, and 10 kDa, respectively) and ß-chitosan (ß1k, ß5k, and ß10k) was compared using a range of pathogenic bacteria containing drug-resistant bacteria isolated from patients at different pH. Interestingly, ß5k and ß10k exhibited potent antibacterial activity, even at pH 7.4, whereas only α10k was effective at pH 7.4. The active target of ß-chitosan is the bacterial membrane, where the leakage of calcein is induced in artificial PE/PG vesicles, bacterial mimetic membrane. Moreover, scanning electron microscopy showed that they caused significant morphological changes on the bacterial surfaces. An in vivo study utilizing a bacteria-infected mouse model found that LMWS ß-chitosan could be used as a candidate in anti-infective or wound healing therapeutic applications.

Antimicrobial HPA3NT3 peptide analogs: placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action.

In an earlier study, we determined that HP(2-20) (residues 2-20 of parental HP derived from the N-terminus of the Helicobacter pylori ribosomal protein L1) and its analog, HPA3NT3, had potent antimicrobial effects. However, HPA3NT3 also showed undesirable cytotoxicity against HaCaT cells. In the present study, we designed peptide analogs including HPA3NT3-F1A (-F1A), HPA3NT3-F8A (-F8A), HPA3NT3-F1AF8A (-F1AF8A), HPA3NT3-A1 (-A1) and HPA3NT3-A2 (-A2) in an effort to investigate the effects of amino acid substitutions in reducing their hydrophobicity or increasing their cationicity, and any resulting effects on their selectivity in their interactions with human cells and pathogens, as well as their mechanism of antimicrobial action. With the exception of HPA3NT3-A1, all of these peptides showed potent antimicrobial activity. Moreover, substitution of Ala for Phe at positions 1 and/or 8 of the HPA3NT3 peptides (-F1A, -F8A and -F1AF8A) dramatically reduced their cytotoxicity. Thus the cytotoxicity of HPA3NT3 appears to be related to its Phe residues (positions 1 and 8), which strongly interact with sphingomyelin in the mammalian cell membrane. HPA3NT3 exerted its bactericidal effects through membrane permeabilization mediated by pore formation. In contrast, fluorescent dye leakage and nucleic acid gel retardation assays showed that -A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. Notably, Staphylococcus aureus did not develop resistance to -A2 as it did with rifampin. These results suggest that the -A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.

Antifungal Action of Arabidopsis thaliana TCP21 via Induction of Oxidative Stress and Apoptosis.

The realm of antimicrobial proteins in plants is extensive but remains relatively uncharted. Understanding the mechanisms underlying the action of plant antifungal proteins (AFPs) holds promise for antifungal strategies. This study aimed to bridge this knowledge gap by comprehensively screening Arabidopsis thaliana species to identify novel AFPs. Using MALDI-TOF analysis, we identified a member of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) family of transcription factors as a novel AFP, A. thaliana TCP21 (AtTCP21; accession number NP_196450). Bacterially purified recombinant AtTCP21 inhibited the growth of various pathogenic fungal cells. AtTCP21 was more potent than melittin, a well-known AFP, in combating Colletotrichum gloeosporioides. Growth inhibition assays against various fungal pathogens and yeasts confirmed the pH-dependent antimicrobial activity of AtTCP21. Without inducing any membrane alterations, AtTCP21 penetrates the fungal cell wall and membrane, where it instigates a repressive milieu for fungal cell growth by generating intracellular reactive oxygen species and mitochondrial superoxides; resulting in morphological changes and apoptosis. Our findings demonstrate the redox-regulating effects of AtTCP21 and point to its potential as an antimicrobial agent.