Single-molecule, hybridization-based strategies for short nucleic acids detection and recognition with nanopores.

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.

Teaching an old dog new tricks: A lipid membrane-based electric immunosensor for real-time probing of the spike S1 protein subunit from SARS-CoV-2.

Fast, cheap, and easy to implement point-of-care testing for various pathogens constituted a game changer in past years due to its potential for early disease diagnosis. Herein, we report on the proof-of-concept of a simple method enabling in vitro detection of a structural spike protein subunit from the SARS-CoV-2 (S1 ) in aqueous samples. At the core of this discovery lies the well-known paradigm of monitoring the capacitive current across a reconstituted zwitterionic lipid membrane subjected to a periodic transmembrane potential, followed by the real-time spectral analysis enabling the extraction of the second harmonic of the capacitive current. Subsequent changes in the amplitude of this harmonic recorded during lipid membrane-S1 interactions were correlated with alterations induced in the inner membrane potential profile by the S1 protein subunit adsorption, and were shown to be augmented by ionic strength, the presence of a specific monoclonal antibody designed against the S1 subunit and the angiotensin-converting enzyme 2 (ACE2) protein receptor, and uninhibited by the presence of other human serum proteins.

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.

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.

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.

Comparative Antimicrobial Activity of Hp404 Peptide and Its Analogs against Acinetobacter baumannii.

An amphipathic α-helical peptide, Hp1404, was isolated from the venomous gland of the scorpion Heterometrus petersii. Hp1404 exhibits antimicrobial activity against methicillin-resistant Staphylococcus aureus but is cytotoxic. In this study, we designed antimicrobial peptides by substituting amino acids at the 14 C-terminal residues of Hp1404 to reduce toxicity and improve antibacterial activity. The analog peptides, which had an amphipathic α-helical structure, were active against gram-positive and gram-negative bacteria, particularly multidrug-resistant Acinetobacter baumannii, and showed lower cytotoxicity than Hp1404. N-phenyl-1-naphthylamine uptake and DisC3-5 assays demonstrated that the peptides kill bacteria by effectively permeating the outer and cytoplasmic membranes. Additionally, the analog peptides inhibited biofilm formation largely than Hp1404 at low concentrations. These results suggest that the analog peptides of Hp1404 can be used as therapeutic agents against A. baumannii infection.

Intra-Articular Administration of Cramp into Mouse Knee Joint Exacerbates Experimental Osteoarthritis Progression.

Osteoarthritis (OA) is the most common type of arthritis and is associated with wear and tear, aging, and inflammation. Previous studies revealed that several antimicrobial peptides are up-regulated in the knee synovium of patients with OA or rheumatoid arthritis. Here, we investigated the functional effects of cathelicidin-related antimicrobial peptide (Cramp) on OA pathogenesis. We found that Cramp is highly induced by IL-1ß via the NF-κB signaling pathway in mouse primary chondrocytes. Elevated Cramp was also detected in the cartilage and synovium of mice suffering from OA cartilage destruction. The treatment of chondrocytes with Cramp stimulated the expression of catabolic factors, and the knockdown of Cramp by small interfering RNA reduced chondrocyte catabolism mediated by IL-1ß. Moreover, intra-articular injection of Cramp into mouse knee joints at a low dose accelerated traumatic OA progression. At high doses, Cramp affected meniscal ossification and tears, leading to cartilage degeneration. These findings demonstrate that Cramp is associated with OA pathophysiology.

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.

Single-Crystal Poly[4-(4,4-dihexadecyl-4H-cyclopenta[1,2-b:5,4-b']dithiophen-2-yl)- alt-[1,2,5]thiadiazolo[3,4- c]pyridine] Nanowires with Ultrahigh Mobility.

We fabricated single-crystal poly[4-(4,4-dihexadecyl-4H-cyclopenta[1,2-b:5,4-b']-dithiophen-2-yl)- alt-[1,2,5]thiadiazolo-[3,4- c]pyridine] (PCDTPT) nanowires with ultrahigh mobility using a liquid-bridge-mediated nanotransfer molding method. The structural analysis of the single-crystal PCDTPT nanowires reveals that PCDTPT crystals have a triclinic structure, and the nanowires grow parallel to PCDTPT backbone chains, which provide important insights into its intrinsic charge transport. The single-crystal PCDTPT nanowire exhibits a superior charge carrier mobility of 72.94 ± 18.02 cm2 V-1 s-1 (maximum mobility up to 92.64 cm2 V-1 s-1), which is a record high value among conjugated polymers to date. In the single-crystal PCDTPT nanowire, the backbone chains in the linear structure along the nanowire growth axis lead to strong backbone delocalization, resulting in highly conductive polymer backbones and a drastic increase in charge carrier mobility. In addition, the single-crystal PCDTPT nanowire shows good environmental stability under air conditions compared to small-molecule organic semiconductors.

All d-Lysine Analogues of the Antimicrobial Peptide HPA3NT3-A2 Increased Serum Stability and without Drug Resistance.

Novel antibiotic drugs are urgently needed because of the increase in drug-resistant bacteria. The use of antimicrobial peptides has been suggested to replace antibiotics as they have strong antimicrobial activity and can be extracted from living organisms such as insects, marine organisms, and mammals. HPA3NT3-A2 ([Ala1,8] HPA3NT3) is an antimicrobial peptide that is an analogue of the HP (2-20) peptide derived from Helicobacter pylori ribosomal protein L1. Although this peptide was shown to have strong antimicrobial activity against drug-resistant bacteria, it also showed lower toxicity against sheep red blood cells (RBCs) and HaCaT cells compared to HPA3NT3. The l-Lys residues of HPA3NT3-A2 was substituted with d-Lys residues (HPA3NT3-A2D; [d-Lys2,5,6,9,10,15] HPA3NT3-A2) to prevent the cleavage of peptide bonds by proteolytic enzymes under physiological conditions. This peptide showed an increased half-life and maintained its antimicrobial activity in the serum against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (pathogen). Furthermore, the antimicrobial activity of HPA3NT3-A2D was not significantly affected in the presence of mono- or divalent ions (Na+, Mg2+, Ca2+). Finally, l- or d-HPA3NT3-A2 peptides exhibited the strongest antimicrobial activity against antibiotic-resistant bacteria and failed to induce resistance in Staphylococcus aureus after 12 passages.

Nanopore-Assisted, Sequence-Specific Detection, and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs.

We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's ß-barrel entry permits discrimination between sequences that differ by one base pair.

Antibacterial and anti-biofilm activity, and mechanism of action of pleurocidin against drug resistant Staphylococcus aureus.

The abuse of antibiotics has resulted in the emergence of multi-drug-resistant bacteria. Staphylococcus aureus is a frequent cause of infections, and antibiotic-resistant S. aureus has become a serious problem. Antimicrobial peptides play an important role in innate immunity and are attracting increasing attention as alternative antibiotics. In a previous study, pleurocidin, derived from winter flounder, was identified as a 25-amino acid antimicrobial peptide with no cytotoxicity toward mammalian cells and low hemolytic activity. In the present study, pleurocidin was observed to exhibit antimicrobial activity against gram-positive and gram-negative bacteria, especially against drug resistant S. aureus. Pleurocidin retained its antibacterial activity against drug resistant S. aureus in the presence of a physiological salt concentration. Membrane depolarization assays and propidium iodide uptake indicated that pleurocidin kills bacteria by damaging the integrity of the bacterial membrane. DNA binding assays revealed that pleurocidin binds to DNA. Thus, pleurocidin targets not only the bacterial membrane, but also their DNA. S. aureus biofilms have become a serious problem because of increased resistance to antibiotics. Therefore, we investigated the effect of pleurocidin on biofilm inhibition and eradication using crystal violet staining and microscopic observation. Pleurocidin inhibited and eradicated biofilms at low concentrations. Taken together, the results suggested that pleurocidin is a promising candidate therapeutic agent to treat drug-resistant bacteria and biofilm-related infections.

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.

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.

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.

Antibacterial activity and mechanism of action of analogues derived from the antimicrobial peptide mBjAMP1 isolated from Branchiostoma japonicum.

Objectives: The worldwide increase in antibiotic-resistant bacteria is a growing threat to public health. Antimicrobial peptides (AMPs) are potentially effective alternatives to conventional antibiotics. We therefore tested analogues of the AMP mBjAMP1 from Branchiostoma japonicum, which we produced by adding and/or replacing amino acids to increase antimicrobial activity against Gram-negative bacteria. Methods: We compared the antimicrobial activities of mBjAMP1 analogues against Gram-negative bacteria reference strains and 52 strains of Klebsiella pneumoniae isolated from patients. Antibiofilm activity and cytotoxicity were evaluated, and the mechanisms of action were then studied. Results: Analogue peptides exhibited greater antimicrobial and antibiofilm activities than mBjAMP1. In particular, the analogue IARR-Anal10 displayed not only the greatest antimicrobial and antibiofilm activities, but also no toxicity against human red blood cells or other mammalian cells. IARR-Anal10 had little or no effect on bacterial outer membrane permeability, membrane polarization or membrane integrity. Instead, it appears IARR-Anal10 binds bacterial DNA, as evidenced in DNA gel retardation assays. Thus, IARR-Anal10 likely kills bacteria through an intracellular mechanism. We also confirmed that IARR-Anal10 suppresses the virulence of K. pneumoniae to a degree similar to tigecycline, used to treat carbapenem-resistant Enterobacteriaceae infections. Notably, IARR-Anal10 did not induce development of resistance by K. pneumoniae, though both meropenem and tigecycline did so within a short time. Conclusions: These results suggest that IARR-Anal10 is a promising agent for treating infections caused by bacteria resistant to tigecycline and meropenem.

If Squeezed, a Camel Passes Through the Eye of a Needle: Voltage-Mediated Stretching of Dendrimers Facilitates Passage Through a Nanopore.

Herein, we report uni-molecular observations of electric potential- and electrolyte-dependent elasticity of poly(amidoamine) (PAMAM)-G1.5 dendrimers containing sodium carboxylate surface groups, using the electric field-assisted migration through the α-hemolysin nanopore (α-HL). Although at moderate transmembrane potentials the dendrimer (~ 2.5 nm in diameter) is sterically excluded from translocation across the constriction region of the nanopore (~ 1.5 nm in diameter), we found a threshold for its translocation that depends on both the electrolyte pH and ionic strength. We posit that the decreased repulsive intramolecular interactions among dendrimer's branches at low when compared to neutral pH, caused mainly by the protonation of surface groups on the dendrimer, determine a larger propensity of the dendrimer to collapse and deform. This in turns enables the dendrimer to adopt more favorably conformations that facilitate its optimal squeezing through the α-HL's constriction region at low pH, despite the fact that the estimated net force acting on it becomes approximately one order of magnitude lower than at neutral pH. Experiments performed in a low ionic strength buffer, which decreases Coulombic screening, enhance the intramolecular forces on the dendrimer and renders the dendrimer stiffer than in high ionic strength buffer, confirming the dendrimer elastic properties-dependent threshold for deformation inside the nanopore.

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.

Anti-endotoxin mechanism of the KW4 peptide in inflammation in RAW 264.7 cells induced by LTA and drug-resistant Staphylococcus aureus 1630.

Drug-resistant microorganism infections cause serious disease and can lead to mortality and morbidity. In particular, Staphylococcus aureus induces pyrogenic and toxigenic infections, and drug-resistance occurs rapidly. Multidrug-resistant S. aureus, such as methicillin-resistant S. aureus and methicillin-sensitive S. aureus, can also cause immunodeficiency and immune deficiency syndrome from lipoteichoic acid. However, antimicrobial peptides, such as KW4, have strong antimicrobial activity, low cytotoxicity, and high neutralization activity against endotoxin substances from Gram-negative bacteria. The objective of this study was to use a synthetic KW4 antimicrobial peptide to evaluate the inhibition of drug-resistance development, antimicrobial activity, and neutralizing activity in S. aureus Gram-positive bacteria. The KW4 peptide showed strong antimicrobial activity against drug-resistant S. aureus strains and significantly increased the anti-neutralizing activity of lipoteichoic acid in S. aureus 1630 drug-resistant bacteria. In addition, S. aureus ATCC 29213 did not develop resistance to KW4 as with other antibiotic drugs. These results suggest that the KW4 peptide is an effective antibiotic and anti-neutralizing agent against multidrug-resistant S. aureus strains.

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.