Pioneering PGC-1α-boosted secretome: a novel approach to combating liver fibrosis.

Purpose: Liver fibrosis is a critical health issue with limited treatment options. This study investigates the potential of PGC-Sec, a secretome derived from peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-overexpressing adipose-derived stem cells (ASCs), as a novel therapeutic strategy for liver fibrosis. Methods: Upon achieving a cellular confluence of 70%-80%, ASCs were transfected with pcDNA-PGC-1α. PGC-Sec, obtained through concentration of conditioned media using ultrafiltration units with a 3-kDa cutoff, was assessed through in vitro assays and in vitro mouse models. Results: In vitro, PGC-Sec significantly reduced LX2 human hepatic stellate cell proliferation and mitigated mitochondrial oxidative stress compared to the control-secretome. In an in vivo mouse model, PGC-Sec treatment led to notable reductions in hepatic enzyme activity, serum proinflammatory cytokine concentrations, and fibrosis-related marker expression. Histological analysis demonstrated improved liver histology and reduced fibrosis severity in PGC-Sec-treated mice. Immunohistochemical staining confirmed enhanced expression of PGC-1α, optic atrophy 1 (a mitochondrial function marker), and peroxisome proliferator-activated receptor alpha (an antifibrogenic marker) in the PGC-Sec-treated group, along with reduced collagen type 1A expression (a profibrogenic marker). Conclusion: These findings highlight the therapeutic potential of PGC-Sec in combating liver fibrosis by enhancing mitochondrial biogenesis and function, and promoting antifibrotic processes. PGC-Sec holds promise as a novel treatment strategy for liver fibrosis.

Liver Transplantation at a Small Volume Center-The Role of Network System.

BACKGROUND: Liver transplantation (LT) is typically performed at specialized, high-volume centers. However, some smaller centers also offer liver transplantation services, but their outcomes and safety have been a subject of debate. To overcome these difficulties, we tried to build a Catholic Medical Center (CMC) network to share our experiences and overcome the lack of volume. In this study, we reviewed the overall outcome of patients undergoing LT at a small-volume procedure center, with a focus on patient and graft survival rates. METHODS: Between July 2014 and September 2021, 60 adults underwent LT at Bucheon Saint Mary's Hospital. The overall outcomes were analyzed in terms of perioperative outcomes, complications, and overall survival rate. In addition, the patients were divided into a benign end-stage liver disease (ESLD) group (n = 44) and a hepatocellular carcinoma (HCC) group (n = 16). The baseline characteristics, perioperative outcomes, complications, and overall survival rate were analyzed between the 2 groups. RESULTS: Of a total of 60 LT, living donor liver transplantation (LDLT) was 26, and deceased donor liver transplantation was 34. LDLT was 14 (31.8%) in the ESLD group and 12 (75.0%) in the HCC group. The overall 1-year, 3-year, and 5-year survival rates were 86.7%, 79.7%, and 77.7%, respectively. The survival difference was not statistically significant (P = .214) between the 2 groups. CONCLUSION: We suggest that with appropriate patient selection and adequate resources, LT can be safely performed at smaller centers with the assistance of the CMC network, thus expanding access to this life-saving procedure.

Proposal of a Network System to Solve the Problem of Small Volume in Liver Transplantation; Catholic Medical Center Network.

INTRODUCTION: Liver transplantation (LT) is a complex and demanding procedure associated with significant perioperative challenges and risks. Concerns have arisen regarding LT outcomes in low-volume centers. We implemented an integrated training and surgical team network to address these concerns within the Catholic Medical Center (CMC) network. This study presents a comprehensive review of our 9-year LT experience within the CMC medical network. METHOD: A retrospective study of LT procedures conducted between January 2013 and August 2021 in 6 CMC-affiliated hospitals was performed. One center was categorized as a high-volume center, conducting over 60 cases annually, and the remaining 5 were considered small-volume centers. The primary endpoints assessed were 1-year and 5-year survival rates. RESULTS: A total of 793 LTs were performed during the study period. The high-volume center performed 411 living donor LT (LDLT) cases and 127 deceased donor LT (DDLT) cases. Also, 146 LDLT cases and 109 DDLT cases were performed in 5 small-volume centers. One-year and 5-year patient survival for LDLT recipients was 88.3% and 78.8% in the high-volume center and 85.6% and 80.6% in the low-volume center. Five-year survival was not significantly different in small-volume centers (P = .903). For DDLT recipients, 1-year and 5-year patient survival was 80.3% and 70.6% in the high-volume center and 76.1% and 67.6% in the low-volume center. In DDLT cases, 5-year survival was not significantly different in small-volume centers (P = .445). CONCLUSION: In conclusion, comparable outcomes for liver transplantation can be obtained in a small-volume center with a high level of integrated training systems and networks.

Study on P-AlGaAs/Al/Au Ohmic Contact Characteristics for Improving Optoelectronic Response of Infrared Light-Emitting Device.

The Al/Au alloy was investigated to improve the ohmic characteristic and light efficiency of reflective infrared light-emitting diodes (IR-LEDs). The Al/Au alloy, which was fabricated by combining 10% aluminum and 90% gold, led to considerably improved conductivity on the top layer of p-AlGaAs of the reflective IR-LEDs. In the wafer bond process required for fabricating the reflective IR-LED, the Al/Au alloy, which has filled the hole patterns in Si3N4 film, was used for improving the reflectivity of the Ag reflector and was bonded directly to the top layer of p-AlGaAs on the epitaxial wafer. Based on current-voltage measurements, it was found that the Al/Au alloyed material has a distinct ohmic characteristic pertaining to the p-AlGaAs layer compared with those of the Au/Be alloy material. Therefore, the Al/Au alloy may constitute one of the favored approaches for overcoming the insulative reflective structures of reflective IR-LEDs. For a current density of 200 mA, a lower forward voltage (1.56 V) was observed from the wafer bond IR-LED chip made with the Al/Au alloy; this voltage was remarkably lower in value than that of the conventional chip made with the Au/Be metal (2.29 V). A higher output power (182 mW) was observed from the reflective IR-LEDs made with the Al/Au alloy, thus displaying an increase of 64% compared with those made with the Au/Be alloy (111 mW).

Steroid-Resistant Rejection in Liver Transplant: A Single-Center Study for Risk Factor and Second-Line Treatment.

BACKGROUND: Steroid-resistant rejection (SRR) in liver transplant occurs in about 10% of T cell-mediated rejection; prognosis of SRR is known to be worse than steroid-sensitive rejection (SSR). Only a few studies describe treatment methods or features for SRR, and there is no clear consensus yet. Therefore, the purpose of this study is to describe the difference between SSR and SRR and to compare the effect of the SRR treatment method performed our institution. METHODS: This study is a 10-year, retrospective cohort study at Seoul St Mary's Hospital; clinical data were collected from January 2008 to December 2017. Of 663 cases, 154 patients (23.3%) underwent steroid pulse therapy for rejection; we excluded 30 patients who did not undergo liver biopsy. A total of 124 patients (18.7%) with biopsy-proven rejection were analyzed for this study. RESULTS: Child-Turcotte-Pugh score, cold ischemia time, and cytomegalovirus (CMV) infection showed a statistically significant difference in 2 groups. Multivariate analysis was performed on risk factors of SRR at first rejection. CMV infection and total bilirubin at first rejection and numbers of rejection were significant results. Both overall survival and allograft survival rate of SSR are higher than SRR (P < .001). Of second-line treatment patients, 13 patients (54.2%) recovered, and 11 patients (45.8%) failed to recover. Survival was the highest in patients using antithymocyte globulin and in patients with liver retransplant. CONCLUSIONS: When the first rejection in liver transplant occurs, patients with high bilirubin level ​​and previous CMV infections are more likely to have SRR, so if they do not respond to steroid pulse therapy for the first time, either using antithymocyte globulin or liver retransplant preparation should be considered.

Analysis of Prognostic Factors for Patients Undergoing Renal Replacement Therapy With Acute Kidney Injury Prior to Living Donor Liver Transplantation.

BACKGROUND: Acute kidney injury (AKI) is a common complication in patients undergoing liver transplantation (LT) for end-stage liver disease (ESLD), and renal replacement therapy (RRT) is required in many cases. This study was performed to identify the prognostic factors for patients undergoing RRT owing to AKI before living donor liver transplantation (LDLT). MATERIALS AND METHODS: From January 2010 to December 2018, LDLT was performed in 464 adult patients in our center. We reviewed 33 patients who underwent RRT before LDLT among 464 consecutive cases. Patients who continued to RRT after LDLT or who underwent subsequent kidney transplantation were considered to have not recovered from renal impairment. RESULTS: Among 33 patients, there were 23 patients in the recovery group and 10 patients in the nonrecovery group. The preoperative duration of RRT was shorter in the recovery group, but it was not statistically significant. In the nonrecovery group, diabetes mellitus was found to have a higher prevalence and ischemic time was longer. Other perioperative factors were not significantly different between the 2 groups. After LDLT, the peak total bilirubin level was higher, and the intensive care unit stay was longer in the nonrecovery group. The overall survival rate was higher in the recovery group. CONCLUSIONS: Liver transplant recipients who maintain RRT after LDLT have poor outcome. It is necessary to know the risk factors and manage them well, perioperatively.

Noninvasive Evaluations to Estimate Graft Steatosis in Living Donor Liver Transplant for Donor Safety and Successful Outcome.

BACKGROUND: In living donor liver transplant, graft steatosis is very important for donor safety and recipient outcome. The purpose of this study was to evaluate the accuracy of noninvasive methods to estimate graft steatosis and establish preoperative selection criteria based on the results. METHODS: From January 2014 to September 2018, a total of 204 patients underwent donor hemihepatectomy (right lobe) in our center. Imaging studies, such as computed tomography (CT) and magnetic resonance spectroscopy (MRS) were routinely performed. Patients were divided into 4 groups by the macrovesicular steatosis based on the pathologic report (group 1: <5%, group 2: ≥5 and <10%, group 3: ≥10 and <20%, group 4: ≥20%). Hepatic and splenic attenuation values were measured on noncontrast CT scans by using circular region-of-interest cursors in the liver and spleen. RESULTS: Of the 204 donors, 112 (55.1%) were in group 1, 59 (28.5%) were in group 2, 21 (10.8%) were in group 3, and 12 (5.6%) were in group 4. There were no statistical differences in age and sex among 4 groups, but, body mass index, aspartate aminotransferase, alanine aminotransferase, and all imaging studies were significantly different among the 4 groups. Body mass image, alanine aminotransferase, and imaging studies showed a linear relationship with pathologic data. As a result of drawing receiving operating characteristic curves, excellent area under the curve value is shown at average of regions of interest in liver and MRS. CONCLUSIONS: Preoperative CT and MRS provide and accurate method to estimate graft steatosis. If the 2 modalities are properly combined, they can be helpful for donor selection.

Clinical Analysis of Factors Affecting Hospital Mortality After Liver Transplant in Patients With High Model for End-Stage Liver Disease Score.

BACKGROUND: This study was undertaken to identify poor prognostic factors in patients with high Model for End-Stage Liver Disease (MELD) scores. METHODS: From September 2001 to December 2017, living donor liver transplant and deceased donor liver transplant were performed in 851 (84.4%) and 157 patients (15.6%), respectively, in our center. Eighty-one patients (8.0%) with MELD scores ≥ 35 were classified as patients with high MELD scores. RESULTS: The overall survival rates in patients with high MELD scores were significantly worse than those in patients with low MELD scores (P = .005). However, no significant difference in survival was found between the 2 groups when in-hospital mortality was excluded. In-hospital mortality occurred in 18 patients (22.2%), and the main cause of death was sepsis (n = 14, 77.8%). On univariate analysis, the risk factors for in-hospital mortality were mean age (P = .028), mean MELD score (P = .045), intubation status (P < .001), culture positivity (P = .042), and encephalopathy grade 3 or 4 (P = .014). On multivariate analysis, age (P = .006), intubation status (P = .042), and culture positivity (P = .036) were significant. CONCLUSIONS: The main cause of in-hospital mortality was sepsis, and the risk factors for in-hospital mortality of patients with high MELD score were older age, preoperative intubation, and culture positivity. Special attention should be paid to the prevention and treatment of infection in the liver transplant of patient with high MELD scores.

Single-Center Experience with Hepatic Artery Reconstruction During Living Donor Liver Transplantation: Microscope Versus Surgical Loupe.

BACKGROUND Hepatic artery reconstruction during living donor liver transplantation is a critical step. To perform this risky procedure, a microscope has been used. However, it takes a long time to complete the procedure and it has a long and steep learning curve. Recently, some transplant surgeons have performed the procedure using a surgical loupe. We conducted this study to compare the outcomes after hepatic artery reconstruction using a microscope versus using a surgical loupe. MATERIAL AND METHODS We retrospectively reviewed the outcomes of 300 patients at our institution from April 2014 to July 2020. From April 2014 to September 2017 (era 1), hepatic artery reconstruction was performed using a microscope by an experienced plastic surgeon. From September 2017 to the end date (era 2), it was performed using a loupe (×5.0) by an experienced transplantation surgeon. RESULTS There was no difference in most perioperative outcomes between the 2 groups, including the major postoperative complications of hepatic artery complications (2/150 versus 2/150, P=1.000), postoperative bleeding (10/150 versus 5/150, P=0.185), and biliary leakage (18/150 versus 13/150, P=0.343). There was a statistically significant difference between the 2 groups in anastomosis time (42.4±11.8 versus 24.2±7.8, P<0.001) and the entire operation time (436.6±83.9 versus 415.3±68.5, P=0.035). CONCLUSIONS We suggest that when the surgeon is familiar with a loupe and vascular anastomosis, hepatic artery reconstruction using a surgical loupe is a safe and feasible method with a shorter operation time.

PEP27-2, a Potent Antimicrobial Cell-Penetrating Peptide, Reduces Skin Abscess Formation during Staphylococcus aureus Infections in Mouse When Used in Combination with Antibiotics.

PEP27, a 27-amino acid (aa) peptide secreted by Streptococcus pneumoniae, is an autolytic peptide that functions as a major virulence factor. To develop a clinically applicable antimicrobial peptide (AMP), we designed PEP27 analogs with Trp substitutions to enhance its antimicrobial activity compared to that of PEP27. Particularly, PEP27-2 showed strong antimicrobial activity against a wide variety of bacteria, including multidrug-resistant (MDR) bacteria. It was found that the antimicrobial activity of PEP27-2 was increased by substituting Trp for the aa at the middle position of PEP27. We found that PEP27-2 acts as an effective cell-penetrating peptide in bacterial and mammalian cells. Here, we proved that subcutaneous infection with MDR Staphylococcus aureus induced skin lesions such as skeletal muscle damage, deep inflammation, and necrosis of the overlaying dermis in mice. Combination treatment with antibiotics revealed synergistic effects, remarkably reducing abscess size and improving the bacteria removal rate from the infection site. Moreover, PEP27-2-antibiotic combination treatment reduced inflammation, lowering levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, inducible NO synthase (iNOS), and cyclooxygenase (COX-2) in skin abscess tissue. The results suggest that the PEP27-2 peptide is a promising therapeutic option for combating MDR bacterial strains by enhancing antibiotic penetration and protecting against MDR bacteria.

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma.

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Non-Receptor-Mediated Lipid Membrane Permeabilization by the SARS-CoV-2 Spike Protein S1 Subunit.

Due to the pressing need to generate specific drugs or vaccines for COVID-19 and management of its outbreak, detailed knowledge regarding the SARS-CoV-2 entry into host cells and timely, cheap, and easy-to-use detection methods are of critical importance for containing the SARS-CoV-2 epidemic. Through electrophysiology and fluorescence spectroscopy experiments, we show that even in the absence of the angiotensin-converting enzyme 2 receptor, the S1 subunit from SARS-CoV-2 spike protein binding to neutral phospholipid membranes leads to their mechanical destabilization and permeabilization. A similar cytotoxic effect of the protein was seen in human lung epithelial cells. A monoclonal antibody generated toward the S1 subunit alleviates to a considerable extent the destabilizing potential of the protein in such model membranes. Finally, we demonstrate the proof-of-concept capability of an α-hemolysin (α-HL) protein nanopore to detect in aqueous buffer and real time the region-binding domain of the S1 subunit from SARS-CoV-2 spike protein by monitoring its immunological interaction with a target antibody. Our results may offer new perspectives in understanding the pathogenesis of the SARS-CoV-2 infection, its treatment, and real-time detection.

Scorpion-Venom-Derived Antimicrobial Peptide Css54 Exerts Potent Antimicrobial Activity by Disrupting Bacterial Membrane of Zoonotic Bacteria.

Antibiotic resistance is an important issue affecting humans and livestock. Antimicrobial peptides are promising alternatives to antibiotics. In this study, the antimicrobial peptide Css54, isolated from the venom of C. suffuses, was found to exhibit antimicrobial activity against bacteria such as Listeria monocytogenes, Streptococcus suis, Campylobacter jejuni, and Salmonella typhimurium that cause zoonotic diseases. Moreover, the cytotoxicity and hemolytic activity of Css54 was lower than that of melittin isolated from bee venom. Circular dichroism assays showed that Css54 has an α-helix structure in an environment mimicking that of bacterial cell membranes. We examined the effect of Css54 on bacterial membranes using N-phenyl-1-naphthylamine, 3,3'-dipropylthiadicarbbocyanine iodides, SYTOX green, and propidium iodide. Our findings suggest that the Css54 peptide kills bacteria by disrupting the bacterial membrane. Moreover, Css54 exhibited antibiofilm activity against L. monocytogenes. Thus, Css54 may be useful as an alternative to antibiotics in humans and animal husbandry.

Author Correction: Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach.

Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic investigations, food safety monitoring or evolution of infectious diseases. We report here a method based on the α-hemolysin (α-HL) nanopore, working in conjunction to unmodified citrate anion-coated gold nanoparticles (AuNPs), to detect nanomolar concentrations of short single-stranded DNA sequences (ssDNA). The core idea was to use charge neutral peptide nucleic acids (PNA) as hybridization probe for complementary target ssDNAs, and monitor at the single-particle level the PNA-induced aggregation propensity AuNPs during PNA-DNA duplexes formation, by recording ionic current blockades signature of AuNP-α-HL interactions. This approach offers advantages including: (1) a simple to operate platform, producing clear-cut readout signals based on distinct size differences of PNA-induced AuNPs aggregates, in relation to the presence in solution of complementary ssDNAs to the PNA fragments (2) sensitive and selective detection of target ssDNAs (3) specific ssDNA detection in the presence of interference DNA, without sample labeling or signal amplification. The powerful synergy of protein nanopore-based nanoparticle detection and specific PNA-DNA hybridization introduces a new strategy for nucleic acids biosensing with short detection time and label-free operation.

Bee venom-derived antimicrobial peptide melectin has broad-spectrum potency, cell selectivity, and salt-resistant properties.

Antimicrobial peptides have attracted attention as alternatives to conventional antibiotics. Previously, a novel antimicrobial peptide, melectin, consisting of 18 amino acids was isolated from the venom of a bee, Melecta albifrons. Here, we investigated the antibacterial activity of melectin against drug-resistant bacteria. Melectin showed broad-spectrum antimicrobial activity but low cytotoxicity and no hemolytic activity. Melectin maintained its antimicrobial activity at physiological salt concentrations. Melectin is an α-helical structure that binds to the bacterial membrane via electrostatic interactions and kills bacteria in a short time by bacterial membrane targeting. Collectively, our results suggest that melectin has antibacterial activity and anti-inflammatory activity.

Mechanism of action of antimicrobial peptide P5 truncations against Pseudomonas aeruginosa and Staphylococcus aureus.

Rates of microbial drug resistance are increasing worldwide; therefore, antimicrobial peptides (AMPs) are considered promising alternative therapeutic agents to antibiotics. AMPs are essential components of the innate immune system and exhibit broad-spectrum antimicrobial activity. P5 is a Cecropin A-Magainin 2 hybrid analog peptide with antimicrobial activity against Gram-negative and Gram-positive bacteria. In the present study, truncated peptides were designed to reduction length, retainment their antimicrobial activity and low toxicity at high concentrations compared with that of the parent peptide P5. The truncated peptides P5-CT1 and P5-NT1 exhibited antibacterial activities against both Gram-negative and Gram-positive bacteria. In contrast, P5-CT2, P5-CT3, P5-NT2, and P5-NT3 showed higher antibacterial activities against gram-positive bacteria compared to Gram-negative bacteria at low concentration of peptides. The truncated peptides showed lower hemolytic activity and toxic effects against mammalian cells compared with those of the parent peptide P5. The levels of several truncated peptides were maintained in the presence of physiological concentrations of salts, indicating their high stability. The results of flow cytometry, propidium iodide uptake, n-phenyl-1-naphthylamine uptake, and 3,3'-dipropylthiadicarbocyanine iodide assays showed that these truncated peptides killed microbial cells by increasing membrane permeability, thereby causing membrane damage. The results suggested that truncated peptides of P5 have good potential for use as novel antimicrobial agents.

Antimicrobial and Immunomodulatory Properties and Applications of Marine-Derived Proteins and Peptides.

Marine organisms provide an abundant source of potential medicines. Many of the marine-derived biomaterials have been shown to act as different mechanisms in immune responses, and in each case they can significantly control the immune system to produce effective reactions. Marine-derived proteins, peptides, and protein hydrolysates exhibit various physiologic functions, such as antimicrobial, anticancer, antioxidant, antihypertensive, and anti-inflammatory activities. Recently, the immunomodulatory properties of several antimicrobial peptides have been demonstrated. Some of these peptides directly kill bacteria and exhibit a variety of immunomodulatory activities that improve the host innate immune response and effectively eliminate infection. The properties of immunomodulatory proteins and peptides correlate with their amino acid composition, sequence, and length. Proteins and peptides with immunomodulatory properties have been tested in vitro and in vivo, and some of them have undergone different clinical and preclinical trials. This review provides a comprehensive overview of marine immunomodulatory proteins, peptides, and protein hydrolysates as well as their production, mechanisms of action, and applications in human therapy.

Nonfunctionalized PNAs as Beacons for Nucleic Acid Detection in a Nanopore System.

In this work, single-channel current recordings were used to selectively detect individual ssDNA strands in the vestibule of the α-hemolysin (α-HL) protein nanopore. The sensing mechanism was based on the detection of the intrinsic topological change of target ssDNA molecules after the hybridization with complementary PNA fragments. The readily distinguishable current signatures of PNA-DNA duplexes reversible association with the α-HL's vestibule, in terms of blockade amplitudes and kinetic features, allows specific detection of nucleic acid hybridization.

Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-Resistant Pseudomonas aeruginosa Infection.

Pseudin-2, isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity. In vivo, wounds infected with multidrug-resistant (MDR) Pseudomonas aeruginosa healed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.