Exploring the Therapeutic Potential of Scorpion-Derived Css54 Peptide Against Candida albicans.

Candida albicans (C. albicans) is one of the most common opportunistic fungi worldwide, which is associated with a high mortality rate. Despite treatment, C. albicans remains the leading cause of life-threatening invasive infections. Consequently, antimicrobial peptides (AMPs) are potential alternatives as antifungal agents with excellent antifungal activity. We previously reported that Css54, found in the venom of Centrurodies suffusus suffusus (C. s. suffusus) showed antibacterial activity against zoonotic bacteria. However, the antifungal activity of Css54 has not yet been elucidated. The objective of this study was to identify the antifungal activity of Css54 against C. albicans and analyze its mechanism. Css54 showed high antifungal activity against C. albicans. Css54 also inhibited biofilm formation in fluconazole-resistant fungi. The antifungal mechanism of action of Css54 was investigated using membrane-related assays, including the membrane depolarization assay and analysis of the membrane integrity of C. albicans after treatment with Css54. Css54 induced reactive oxygen species (ROS) production in C. albicans, which affected its antifungal activity. Our results indicate that Css54 causes membrane damage in C. albicans, highlighting its value as a potential therapeutic agent against C. albicans infection.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

Induction of Corneal Endothelial-like Cells from Mesenchymal Stem Cells of the Umbilical Cord.

Because of the limited differentiation capacity of human corneal endothelial cells (CECs), stem cells have emerged as a potential remedy for corneal endothelial dysfunction (CED). This study aimed to demonstrate the differentiation of human umbilical cord-derived mesenchymal stem cells (UC-MSCs) into CECs and to investigate the efficacy of MSC-induced CEC injection into the anterior chamber in a rabbit model of CED. Human UC-MSCs were differentiated into CECs using medium containing glycogen synthase kinase 3ß inhibitor and two types of Rho-associated protein kinase inhibitors. In the MSC-induced CECs, CEC-specific proteins were identified through immunohistochemistry and changes in CEC-specific gene expressions over time were confirmed through quantitative RT-PCR. When MSC-induced CECs were injected into a rabbit model of CED, corneal opacity and neovascularization were improved compared with the non-transplanted control or MSC injection group. We also confirmed that MSC-induced CECs were well engrafted as evidenced by human mitochondrial DNA in the central cornea of an animal model. Therefore, we demonstrated the differentiation of UC-MSCs into CECs in vitro and demonstrated the clinical efficacy of MSC-induced CEC injection, providing in vivo evidence that MSC-induced CECs have potential as a treatment option for CED.

Cathelicidin-related antimicrobial peptide mediates skeletal muscle degeneration caused by injury and Duchenne muscular dystrophy in mice.

BACKGROUND: Cathelicidin, an antimicrobial peptide, plays a key role in regulating bacterial killing and innate immunity; however, its role in skeletal muscle function is unknown. We investigated the potential role of cathelicidin in skeletal muscle pathology resulting from acute injury and Duchenne muscular dystrophy (DMD) in mice. METHODS: Expression changes and muscular localization of mouse cathelicidin-related antimicrobial peptide (Cramp) were examined in the skeletal muscle of normal mice treated with chemicals (cardiotoxin and BaCl2 ) or in dystrophic muscle of DMD mouse models (mdx, mdx/Utrn+/- and mdx/Utrn-/- ). Cramp penetration into myofibres and effects on muscle damage were studied by treating synthetic peptides to mouse skeletal muscles or C2C12 myotubes. Cramp knockout (KO) mice and mdx/Utrn/Cramp KO lines were used to determine whether Cramp mediates muscle degeneration. Muscle pathophysiology was assessed by histological methods, serum analysis, grip strength and lifespan. Molecular factors targeted by Cramp were identified by the pull-down assay and proteomic analysis. RESULTS: In response to acute muscle injury, Cramp was activated in muscle-infiltrating neutrophils and internalized into myofibres. Cramp treatments of mouse skeletal muscles or C2C12 myotubes resulted in muscle degeneration and myotube damage, respectively. Genetic ablation of Cramp reduced neutrophil infiltration and ameliorated muscle pathology, such as fibre size (P < 0.001; n = 6) and fibrofatty infiltration (P < 0.05). Genetic reduction of Cramp in mdx/Utrn+/- mice not only attenuated muscle damage (35%, P < 0.05; n = 9-10), myonecrosis (53%, P < 0.05), inflammation (37-65%, P < 0.01) and fibrosis (14%, P < 0.05) but also restored muscle fibre size (14%, P < 0.05) and muscle force (18%, P < 0.05). Reducing Cramp levels led to a 63% (male, P < 0.05; n = 10-14) and a 124% (female, P < 0.001; n = 20) increase in the lifespan of mdx/Utrn-/- mice. Proteomic and mechanistic studies revealed that Cramp cross-talks with Ca2+ signalling in skeletal muscle through sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase1 (SERCA1). Cramp binds and inactivates SERCA1, leading to the activation of Ca2+ -dependent calpain proteases that exacerbate DMD progression. CONCLUSIONS: These findings identify Cramp as an immune cell-derived regulator of skeletal muscle degeneration and provide a potential therapeutic target for DMD.

A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids.

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA. We discovered that the length-variable poly-Arg tail marked distinctly the molecular processes associated with the nanopore-mediated duplexes capture, trapping and unzipping. This enabled the detection of ssDNA targets via the signatures of (poly-Arg)-PNA-cDNA blockade events, rendered most efficient from the ß-barrel entrance of the nanopore, and scaled proportional in efficacy with a larger poly-Arg moiety. We illustrate the approach by sensing synthetic ssDNAs designed to emulate fragments from two regions of SARS-CoV-2 nucleocapsid phosphoprotein N-gene.

Therapeutic Potency of Induced Pluripotent Stem-Cell-Derived Corneal Endothelial-like Cells for Corneal Endothelial Dysfunction.

Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

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.

A phage protein-derived antipathogenic peptide that targets type IV pilus assembly.

Phage-inspired antibacterial discovery is a new approach that recruits phages in search for antibacterials with new molecular targets, in that phages are the biological entities well adapted to hijack host bacterial physiology in favor of their own thrive. We previously observed that phage-mediated twitching motility inhibition was effective to control the acute infections caused by Pseudomonas aeruginosa and that the motility inhibition was attributed to the delocalization of PilB, the type IV pilus (TFP) assembly ATPase by binding of the 136-amino acid (aa) phage protein, Tip. Here, we created a series of truncated and point-mutant Tip proteins to identify the critical residues in the Tip bioactivity: N-terminal 80-aa residues were dispensable for the Tip activity; we identified that Asp82, Leu84, and Arg85 are crucial in the Tip function. Furthermore, a synthetic 15-aa peptide (P1) that corresponds to Leu73 to Ala87 is shown to suffice for PilB delocalization, twitching inhibition, and virulence attenuation upon exogenous administration. The transgenic flies expressing the 15-aa peptide were resistant to P. aeruginosa infections as well. Taken together, this proof-of-concept study reveals a new antipathogenic peptide hit targeting bacterial motility and provides an insight into antibacterial discovery targeting TFP assembly.

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.

Inverse regulation of serum osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand levels in patients with leg lesional vascular calcification: An observational study.

We hypothesized that circulating osteoprotegerin (OPG) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) levels could be associated with vascular calcification, which is predominant in diabetes.The study included 71 Korean participants (36 with diabetes and 35 without diabetes), who were sub-grouped according to the results of the ankle-brachial index (ABI) and/or X-ray computed tomography scan (CT scan). Serum OPG and TRAIL levels were assayed using the respective enzyme-linked immunosorbent assay kits. Statistical significance was analyzed using Student's t test between the 2 groups or analysis of variance (ANOVA) among the 4 groups.Serum OPG was up-regulated in the participants with diabetes, with peripheral arterial disease (PAD), and/or with vascular calcification. TRAIL down-regulation was more strictly controlled than OPG up-regulation; it was significantly downregulated in the participants with PAD and vascular calcification, but not in the participants with diabetes. Serum OPG and TRAIL were regulated in the participants with femoral, popliteal, and peroneal artery calcification but not in the participants with aortic calcification.OPG up-regulation and TRAIL down-regulation were found to be associated with leg lesional vascular calcification; therefore, the average OPG/TRAIL ratio was significantly increased by 3.2-fold in the leg lesional vascular calcification group.

Nanoscale Probing of Informational Polymers with Nanopores. Applications to Amyloidogenic Fragments, Peptides, and DNA-PNA Hybrids.

The decades long advances in nanotechnology, biomolecular sciences, and protein engineering ushered the introduction of groundbreaking technologies devoted to understanding how matter behaves at single particle level. Arguably, one of the simplest in concept is the nanopore-based paradigm, with deep roots in what is originally known as the Coulter counter, resistive-pulse technique. Historically, a nanopore system comprising the oligomeric protein generated by Staphylococcus aureus toxin α-hemolysin (α-HL) was first applied to detecting polynucleotides, as revealed in 1996 by John J. Kasianowicz, Eric Brandin, Daniel Branton, and David W. Deamer, in the Proceedings of the National Academy of Sciences. Nowadays, a wide variety of other solid-state or protein-based nanopores have emerged as efficient tools for stochastic sensing of analytes as small as single metal ions, handling single molecules, or real-time, label-free probing of chemical reactions at single-molecule level. In this Account, we demonstrate the usefulness of the α-HL nanopore on probing metal-induced folding of peptides, and to investigating the reversible binding of various metals to physiologically relevant amyloid fragments. The widely recognized Achilles heel of the approach, is the relatively short dwell time of the analytes inside the nanopore. This hinders the collection of sufficient data required to infer statistically meaningful conclusions about the physical or chemical state of the studied analyte. To mitigate this, various approaches were successfully applied in particular experiments, including but not restricted to altering physical parameters of the aqueous solution, downsizing the nanopore geometry, the controlled tuning of the balance between the electrostatic and electro-osmotic forces, coating nanopores with a fluid lipid bilayer, employing a pressure-voltage biased pore. From our perspective, in this Account, we will present two strategies aimed at controlling the analyte passage across the α-HL. First, we will reveal how the electroosmotic flow can be harnessed to control residence time, direction, and the sequence of spatiotemporal dynamics of a single peptide along the nanopore. This also allows one to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or ß-barrel moiety. Second, we lay out the principles of an approach dubbed "nanopore tweezing", enabling simultaneous capture rate increase and escape rate decrease of a peptide from the α-HL, with the applied voltage. At its core, this method requires the creation of an electrical dipole on the peptide under study, via engineering positive and negative amino acid residues at the two ends of the peptide. Concise applications of this approach are being demonstrated, as in proof-of-concept experiments we probed the primary structure exploration of polypeptides, via discrimination between selected neutral amino acid residues. Another useful venue provided by the nanopores is represented by single-molecule force experiments on captured analytes inside the nanopore, which proved useful in exploring force-induced rupture of nucleic acids duplexes, hairpins, or various nucleic acids-ligand conjugates. We will show that when applied to oppositely charged, polypeptide-functionalized PNA-DNA duplexes, the nanopore tweezing introduces a new generation of force-spectroscopy nanopore-based platforms, facilitating unzipping of a captured duplex and enabling the duplex hybridization energy estimation.

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.

The immunomodulatory effect of antimicrobial peptide HPA3P restricts Brucella abortus 544 infection in BALB/c mice.

The discovery of antimicrobial peptides (AMPs) in recent years has been promising for the treatment of multidrug resistant pathogenic microbes. Brucellosis is still considered one of the most common zoonoses in the world. In this study, we evaluated the effect HPA3P peptide in the bacterial uptake and intracellular growth of Brucella abortus (B. abortus) 544 in murine macrophages RAW 264.7. HPA3P was further utilized in a mouse model for infection and treatment. This peptide did not show cytotoxicity or bactericidal effect to B. abortus. However, it inhibited bacterial internalization at 0, 15 and 30 min incubation at two different doses at 12 and 24 µM as well as reduced intracellular growth after 2, 24 and 48 h incubation. Mice treated with HPA3P demonstrated a significant 1.01-log reduction (P < 0.0001) and spleen weight reduction compared to the nanocarrier control (P < 0.01). Significant increases in key cytokines Interferon-γ (IFN-γ) and Tumor necrosis factor (TNF) at 3, 7 and 14 days post-infection were observed in HPA3P treated mice similar to the antibiotic control group with both compared to the nanocarrier control. Monocyte chemoattractant protein-1 (MCP-1) was also heightened at 14 days post-infection. Histopathological analysis also suggests reduced bacterial granuloma in the liver and spleens of HPA3P treated group compared with the nanocarrier control group. In this study, the modulation of crucial cytokines IFN-γ and TNF might have led to a considerable reduction in the proliferation of B. abortus in a mouse model of brucellosis. Further investigation might be required to maximize the efficacy of HPA3P treatment in murine brucellosis.

Single-Molecule, Real-Time Dissecting of Peptide Nucleic Acid-DNA Duplexes with a Protein Nanopore Tweezer.

Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.

The regulation of combined treatment-induced cell death with recombinant TRAIL and bortezomib through TRAIL signaling in TRAIL-resistant cells.

BACKGROUND: Multiple trials have attempted to demonstrate the effective induction of cell death in TRAIL-resistant cancer cells, including using a combined treatment of recombinant TRAIL and various proteasome inhibitors. These studies have yielded limited success, as the mechanism of cell death is currently unidentified. Understanding this mechanism's driving forces may facilitate the induction of cell death in TRAIL-resistant cancer cells. METHODS: Three kinds of recombinant soluble TRAIL proteins were treated into TRAIL-resistant cells and TRAIL-susceptible cells, with or without bortezomib, to compare their respective abilities to induce cell death. Recombinant TRAIL was treated with bortezomib to investigate whether this combination treatment could induce tumor regression in a mouse syngeneic tumor model. To understand the mechanism of combined treatment-induced cell death, cells were analyzed by flow cytometry and the effects of various cell death inhibitors on cell death rates were examined. RESULTS: ILz:rhTRAIL, a recombinant human TRAIL containing isoleucine zipper hexamerization domain, showed the highest cell death inducing ability both in single treatment and in combination treatment with bortezomib. In both TRAIL-resistant and TRAIL-susceptible cells treated with the combination treatment, an increase in cell death rates was dependent upon both the dose of TRAIL and its intrinsic properties. When a syngeneic mouse tumor model was treated with the combination of ILz:rhTRAIL and bortezomib, significant tumor regression was seen as a result of the effective induction of cancer cell death. The combination treatment-induced cell death was both inhibited by TRAIL blocking antibody and caspase-dependent. However, it was not inhibited by various ER stress inhibitors and autophagy inhibitors. CONCLUSIONS: The combination treatment with ILz:rhTRAIL and bortezomib was able to induce cell death in both TRAIL-susceptible and TRAIL-resistant cancer cells through the intracellular TRAIL signaling pathway. The efficiency of cell death was dependent on the properties of TRAIL under the environment provided by bortezomib. The combination treatment-induced cell death was not regulated by bortezomib-induced ER stress response or by autophagy.

Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of IκBζ.

OBJECTIVE: IκBζ, an atypical IκB family member, regulates gene expression in the nucleus as a transcriptional cofactor. Although IκBζ has been extensively studied in the immune system, its specific roles in osteoarthritis (OA) are currently unknown. The objective of this study was to investigate the potential role of IκBζ in chondrocyte catabolism and OA pathogenesis. We also determined the molecular mechanism underlying its relationship to the transcription factor NF-κB. METHODS: We determined expression levels of IκBζ in mouse chondrocytes treated with interleukin-1ß (IL-1ß), in human OA cartilage, and in mouse experimental OA cartilage. Adenovirus-mediated overexpression and small interfering RNA knockdown of IκBζ were performed to determine the impact of IκBζ on catabolic gene expression in vitro. Cartilage-specific IκBζ-transgenic and -knockout mice were generated and used for in vivo studies. Experimental and spontaneous OA were induced by surgical destabilization of the medial meniscus and by aging, respectively. Coimmunoprecipitation assay was used to examine the association between IκBζ and NF-κB subunits. RESULTS: IκBζ was highly up-regulated in chondrocytes in response to IL-1ß and in OA cartilage of human and mouse knee joints. Overexpression of IκBζ in chondrocytes promoted spontaneous OA development by activating chondrocyte catabolism. Genetic ablation of IκBζ in chondrocytes abolished catabolic gene induction by IL-1ß and protected against the development of experimental OA. IκBζ formed complexes with NF-κB members to regulate catabolic factor expression. CONCLUSION: These findings demonstrate a critical role for IκBζ in OA pathogenesis. Inhibition of IκBζ function might be an effective therapeutic approach for OA treatment.

New antimicrobial peptide kills drug-resistant pathogens without detectable resistance.

Clavaspirin peptide (CSP) is derived from the pharyngeal tissues of the tunicate Styela clava. The 23-amino acid peptide is histidine-rich and amidated at the N-terminus. CSP possesses low antimicrobial and high hemolytic activity at pH 7.4. Therefore, we designed 4 CSP analogs with substituted hydrophobic amino acids to reduce hydrophobic amino acid interactions. These modifications reduced the aggregation and cytotoxicity of the analogs at pH 7.4. The analogs also showed potent antimicrobial activity by accumulating on bacterial cell surfaces and inducing the lytic mechanism against gram-negative and gram-positive cells at pH 5.5 and 7.4. Moreover, exposure to the CSP-4 analog for up to 29 passages did not induce drug resistance in Staphylococcus aureus. Application of CSP-4 to inflamed skin of hairless mice infected with drug-resistant S. aureus (DRSA) significantly reduced skin infections without damaging dermal collagen or elastin. Topically applied CSP-4 penetrated 25-40 µm in the dermis within 30 min, reducing the levels of Toll-like receptor-2, nuclear factor kappa B (NF-κB), and the pro-inflammatory cytokines tumor necrosis factor- α (TNF-α) and interleukin-1ß (IL-1 ß). These results suggest that CSP-4 could be a promising topical antimicrobial agent for skin diseases caused by DRSA such as S. aureus CCARM 0027.

Antitumor activity of HPA3P through RIPK3-dependent regulated necrotic cell death in colon cancer.

The antimicrobial peptide HPA3 shows anticancer activity in gastric cancer and leukaemia. However, how HPA3 exerts its anticancer activity, as well as whether it also exhibits activity in other cancers, remains unknown. Therefore, the aim of this study was to evaluate the anticancer activity of HPA3 and its analogues in colon cancer and to elucidate the mechanisms responsible for this activity. HPA3P decreased cell viability, whereas HPA3 and HPA3P2 did not decrease cell viability in colon cancer cells compared with control cells. This reduction in cell viability occurred through necrosis, a conclusion supported by our observation of the release of cellular contents, our intracellular PI staining results, and our observation of the release of HMGB1. Moreover, RIPK3 inhibition blocks the reduction of cell viability by HPA3P. Consistent with this finding, we found that knocking down RIPK3 and MLKL, key necroptosis proteins, attenuates the reductions in cell viability induced by HPA3P. Furthermore, HPA3P can improve the anticancer activity of chemotherapeutic agents and exhibits anticancer activity in other cancer cells. These results suggest that HPA3P may have potential as an anticancer agent in the treatment of colon cancer.

Therapeutic Properties and Biological Benefits of Marine-Derived Anticancer Peptides.

Various organisms exist in the oceanic environment. These marine organisms provide an abundant source of potential medicines. Many marine peptides possess anticancer properties, some of which have been evaluated for treatment of human cancer in clinical trials. Marine anticancer peptides kill cancer cells through different mechanisms, such as apoptosis, disruption of the tubulin-microtubule balance, and inhibition of angiogenesis. Traditional chemotherapeutic agents have side effects and depress immune responses. Thus, the research and development of novel anticancer peptides with low toxicity to normal human cells and mechanisms of action capable of avoiding multi-drug resistance may provide a new method for anticancer treatment. This review provides useful information on the potential of marine anticancer peptides for human therapy.

Protein Nanopore-Based Discrimination between Selected Neutral Amino Acids from Polypeptides.

Nanopore probing of biological polymers has the potential to achieve single-molecule sequencing at low cost, high throughput, portability, and minimal sample preparation and apparatus. In this article, we explore the possibility of discrimination between neutral amino acid residues from the primary structure of 30 amino acids long, engineered peptides, through the analysis of single-molecule ionic current fluctuations accompanying their slowed-down translocation across the wild type α-hemolysin (α-HL) nanopore, and molecular dynamics simulations. We found that the transient presence inside the α-HL of alanine or tryptophan residues from the primary sequence of engineered peptides results in distinct features of the ionic current fluctuation pattern associated with the peptide reversibly blocking the nanopore. We propose that α-HL sensitivity to the molecular exclusion at the most constricted region mediates ionic current blockade events correlated with the volumes that are occluded by at least three alanine or tryptophan residues, and provides the specificity needed to discriminate between groups of neutral amino acids. Further, we find that the pattern of current fluctuations depends on the orientation of the threaded amino acid residues, suggestive of a conformational anisotropy of the ensemble of conformations of the peptide on the restricted nanopore region, related to its relative axial orientation inside the nanopore.