Application of a derivative of human defensin 5 to treat ionizing radiation-induced enterogenic infection.

Enterogenic infection is a common complication for patients with radiation injury and requires efficient therapeutics in the clinic. Herein, we evaluated the promising drug candidate T7E21RHD5, which is a peptide derived from intestinal Paneth cell-secreted human defensin 5. Oral administration of this peptide alleviated the diarrhea symptoms of mice that received total abdominal irradiation (TAI, γ-ray, 12 Gy) and improved survival. Pathologic analysis revealed that T7E21RHD5 elicited an obvious mitigation of ionizing radiation (IR)-induced epithelial damage and ameliorated the reduction in the levels of claudin, zonula occluden 1 and occludin, three tight junction proteins in the ileum. Additionally, T7E21RHD5 regulated the gut microbiota in TAI mice by remodeling ß diversity, manifested as a reversal of the inverted proportion of Bacteroidota to Firmicutes caused by IR. T7E21RHD5 treatment also decreased the abundance of pathogenic Escherichia-Shigella but significantly increased the levels of Alloprevotella and Prevotellaceae_NK3B31, two short-chain fatty acid-producing bacterial genera in the gut. Accordingly, the translocation of enterobacteria and lipopolysaccharide to the blood, as well as the infectious inflammatory responses in the intestine after TAI, was all suppressed by T7E21RHD5 administration. Hence, this versatile antimicrobial peptide possesses promising application prospects in the treatment of IR-induced enterogenic infection.

Clustered Cobalt Nanodots Initiate Ferroptosis by Upregulating Heme Oxygenase 1 for Radiotherapy Sensitization.

High cobalt (Co) levels in tumors are associated with good clinical prognosis. An anticancer regimen that increases intratumoral Co through targeted nanomaterial delivery is proposed in this study. Bovine serum albumin and cobalt dichloride are applied to prepare cobaltous oxide nanodots using a facile biomineralization strategy. After iRGD peptide conjugation, the nanodots are loaded into dendritic mesoporous silica nanoparticles, generating a biocompatible product iCoDMSN. This nanocomposite accumulates in tumors after intravenous injection by deep tissue penetration and can be used for photoacoustic imaging. Proteomics research and molecular biology experiments reveal that iCoDMSN is a potent ferroptosis inducer in cancer cells. Mechanistically, iCoDMSNs upregulate heme oxygenase 1 (HMOX1), which increases transferrin receptors and reduces solute carrier family 40 member 1 (SLC40A1), resulting in Fe2+ accumulation and ferroptosis initiation. Furthermore, upregulated nuclear factor erythroid 2-related factor 2 (NRF2), arising from the reduction in Kelch-like ECH-associated protein 1 (KEAP1) expression, is responsible for HMOX1 enhancement after iCoDMSN treatment. Owing to intensified ferroptosis, iCoDMSN acts as an efficient radiotherapy enhancer to eliminate cancer cells in vitro and in vivo. This study demonstrates a versatile Co-based nanomaterial that primes ferroptosis by expanding the labile iron pool in cancer cells, providing a promising tumor radiotherapy sensitizer.

Irradiation accelerates SARS-CoV-2 infection by enhancing sphingolipid metabolism.

Cancer patients who receive radiotherapy have a high risk of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, but the concrete reason remains unclear. Herein, we investigated the influence of irradiation on the vulnerability of cancer cells to SARS-CoV-2 using S pseudovirions and probed the underlying mechanism via RNA-seq and other molecular biology techniques. Owing to the enhancement of sphingolipid metabolism, irradiation accelerated pseudovirion infection. Mechanistically, irradiation induced the expression of acid sphingomyelinase (ASM), which catalyses the hydrolysis of sphingomyelin to ceramide, contributing to lipid raft formation and promoting SARS-CoV-2 invasion. Inhibition of lipid raft formation with methyl-ß-cyclodextrin (MßCD) or the tyrosine kinase inhibitor genistein and ASM suppression through small interfering RNA or amitriptyline (AMT) treatment abolished the enhancing effect of irradiation on viral infection. Animal experiments supported the finding that irradiation promoted SARS-CoV-2 S pseudovirion infection in A549 cell tumour-bearing BALB/c nude mice, whereas AMT treatment dramatically decreased viral infection. This study discloses the role of sphingolipid metabolism in irradiation-induced SARS-CoV-2 infection, thus providing a potential target for clinical intervention to protect patients receiving radiotherapy from COVID-19.

γ-Core Guided Antibiotic Design Based on Human Enteric Defensin 5.

An increase in the number of infections caused by resistant bacteria worldwide necessitates the development of alternatives to antibiotics. Human defensin (HD) 5 is an innate immune peptide with broad-spectrum antibacterial activity, but its complicated structure makes its preparation difficult. Herein, we truncated the HD5 structure by extracting the highly conserved γ-core motif. A structure-activity study showed that this motif was ineffective in killing bacteria in the absence of specific spatial conformation. Notably, after the introduction of two intramolecular disulfide bonds, its antibacterial activity was markedly improved. Glu and Ser residues were then replaced with Arg to create the derivative RC18, which exhibited stronger potency than HD5, particularly against methicillin-resistant S. aureus (MRSA). Mechanistically, RC18 bound to lipid A and lipoteichoic acid at higher affinities than HD5. Furthermore, RC18 was more efficient than HD5 in penetrating the bacterial membranes. Molecular dynamics simulation revealed that five Arg residues, Arg1, Arg7, Arg9, Arg15, and Arg18, mediated most of the polar interactions of RC18 with the phospholipid head groups during membrane penetration. In vivo experiments indicated that RC18 decreased MRSA colonization and dramatically improved the survival of infected mice, thus demonstrating that RC18 is a promising drug candidate to treat MRSA infections.

An angiotensin-converting enzyme-2-derived heptapeptide GK-7 for SARS-CoV-2 spike blockade.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global concern and necessitates efficient drug antagonists. Angiotensin-converting enzyme-2 (ACE2) is the main receptor of SARS-CoV-2 spike 1 (S1), which mediates viral invasion into host cells. Herein, we designed and prepared short peptide inhibitors containing 4-6 critical residues of ACE2 that contribute to the interaction with SARS-CoV-2 S1. Among the candidates, a peptide termed GK-7 (GKGDFRI), which was designed by extracting residues ranging from Gly353 to Ile359 in the ligand-binding domain of ACE2, exhibited the highest binding affinity (25.1 nM) with the SARS-CoV-2 spike receptor-binding domain (RBD). GK-7 bound to the RBD and decreased SARS-CoV-2 S1 attachment to A549 human alveolar epithelial cells. Owing to spike blockade, GK-7 inhibited SARS-CoV-2 spike pseudovirion infection in a dose-dependent manner, with a half-maximal inhibitory concentration of 2.96 µg/mL. Inspiringly, pulmonary delivery of GK-7 by intranasal administration did not result in toxicity in mice. This study revealed an easy-to-produce peptide inhibitor for SARS-CoV-2 spike blockade, thus providing a promising candidate for COVID-19 treatment.

Human Cathelicidin Inhibits SARS-CoV-2 Infection: Killing Two Birds with One Stone.

SARS-CoV-2 infection begins with the association of its spike 1 (S1) protein with host angiotensin-converting enzyme-2 (ACE2). Targeting the interaction between S1 and ACE2 is a practical strategy against SARS-CoV-2 infection. Herein, we show encouraging results indicating that human cathelicidin LL37 can simultaneously block viral S1 and cloak ACE2. LL37 binds to the receptor-binding domain (RBD) of S1 with high affinity (11.2 nM) and decreases subsequent recruitment of ACE2. Owing to the RBD blockade, LL37 inhibits SARS-CoV-2 S pseudovirion infection, with a half-maximal inhibitory concentration of 4.74 µg/mL. Interestingly, LL37 also binds to ACE2 with an affinity of 25.5 nM and cloaks the ligand-binding domain (LBD), thereby decreasing S1 adherence and protecting cells against pseudovirion infection in vitro. Intranasal administration of LL37 to C57 mice infected with adenovirus expressing human ACE2 either before or after pseudovirion invasion decreased lung infection. The study identified a versatile antimicrobial peptide in humans as an inhibitor of SARS-CoV-2 attachment using dual mechanisms, thus providing a potential candidate for coronavirus disease 2019 (COVID-19) prevention and treatment.

Membrane Nanoparticles Derived from ACE2-Rich Cells Block SARS-CoV-2 Infection.

The ongoing COVID-19 pandemic worldwide necessitates the development of therapeutics against SARS-CoV-2. ACE2 is the main receptor of SARS-CoV-2 S1 and mediates viral entry into host cells. Herein, membrane nanoparticles (NPs) prepared from ACE2-rich cells were discovered to have potent capacity to block SARS-CoV-2 infection. The membranes of human embryonic kidney-239T cells highly expressing ACE2 were applied to prepare NPs using an extrusion method. The nanomaterials, termed ACE2-NPs, contained 265.1 ng mg-1 ACE2 on the surface and acted as baits to trap S1 in a dose-dependent manner, resulting in reduced recruitment of the viral ligand to HK-2 human renal tubular epithelial cells. Aside from affecting receptor recongnition, S1 translocated to the cytoplasm and induced apoptosis by reducing optic atrophy 1 expression and increasing cytochrome c release, which was also inhibited by ACE2-NPs. Further investigations revealed that ACE2-NPs efficiently suppressed SARS-CoV-2 S pseudovirions entry into host cells and blocked viral infection in vitro and in vivo. This study characterizes easy-to-produce memrbane nanoantagonists of SARS-CoV-2 that enrich the existing antiviral arsenal and provide possibilities for COVID-19 treatment.

Platelet-membrane-camouflaged bismuth sulfide nanorods for synergistic radio-photothermal therapy against cancer.

Bismuth-containing nanoparticles (BNPs) are potential enhancers for tumor radiotherapy. Improving the bioavailability and developing synergistic therapeutic regimens benefit the drug transformation of BNPs. In the present study, we prepare a mesoporous silica-coated bismuth nanorod (BMSNR) camouflaged by a platelet membrane (PM). This biomimetic material is termed BMSNR@PM. The PM camouflage enhances the immune escape of the BMSNRs by lowering endocytosis by macrophages in the reticuloendothelial system. Additionally, the PM camouflage strengthens the material tumor-targeting capacity and leads to better radiotherapeutic efficacy compared with bare BMSNRs. Owing to the photothermal effect, BMSNR@PMs alters the cell cycle of 4T1 cancer cells post-treatment with 808 nm near-infrared irradiation (NIR). The proportions of S phase and G2/M phase cells decrease and increase, respectively, which explains the synergistic effect of NIR on BMSNR@PM-based radiotherapy. BMSNR@PMs efficiently eradicates cancer cells by the combined action of photothermal therapy (PTT) and radiotherapy in vivo and markedly improves the survival of 4T1-tumor-bearing mice. The synergistic therapeutic effect is superior to the outcomes of PTT and radiotherapy performed alone. Our study demonstrates a versatile bismuth-containing nanoplatform with tumor-targeting, immune escape, and radiosensitizing functionalities using an autologous cell membrane biomimetic concept that may promote the development of radiotherapy enhancers.

Succinylated casein-coated peptide-mesoporous silica nanoparticles as an antibiotic against intestinal bacterial infection.

Increasing drug resistance necessitates the discovery of novel bactericides. Human defensin (HD) peptides can eliminate resistant bacteria and are promising candidates for next-generation antibiotics. T7E21R-HD5 is a potent bactericide designed by site mutations at enteric HD5. To facilitate the development of T7E21R-HD5 into an intestinal antibiotic, we employed a mesoporous silica nanoparticle (MSN) as the peptide carrier. Despite its ineffectiveness at killing bacteria, the MSN intensified the outer membrane penetration and inner membrane permeabilization abilities of T7E21R-HD5 and thus enhanced its antibacterial action against multidrug resistant (MDR) E. coli, which broadened the role of MSNs in drug delivery. For the reduction in T7E21R-HD5 losses in the stomach, we further modified MSN@T7E21R-HD5 with succinylated casein (SCN), a milk protein that can be specifically degraded by intestinal protease. SCN coating decreased T7E21R-HD5 release from the MSNs, especially in a highly acidic environment. The controlled release of MSN@T7E21R-HD5 from SCN encapsulation was confirmed in the presence of trypsin. MSN@T7E21R-HD5@SCN was nontoxic to host cells, and it was capable of inactivating MDR E. coli in vivo and alleviating intestinal inflammation by suppressing the production of inflammatory factors TNF-α, IL-1ß, and MMP-9. This study provides a peptide-based nanobiotic with efficacy to combat intestinal infection, especially against drug-resistant bacteria. The biocompatible and readily prepared MSN/SCN delivery system may benefit further intestinal antibiotic design and promote the drug transformation of additional enterogenic functional molecules.

A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.

The increasing incidence of multidrug-resistant Acinetobacter baumannii (MDRAb) infections worldwide has necessitated the development of novel antibiotics. Human defensin 5 (HD5) is an endogenous peptide with a complex architecture and antibacterial activity against MDRAb In the present study, we attempted to simplify the structure of HD5 by removing disulfide bonds. We found that the Cys2-4 bond was most indispensable for HD5 to inactivate MDRAb, although the antibacterial activity of the derivative was significantly attenuated. We then replaced the noncationic and nonhydrophobic residues with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb Further investigations revealed that HD5d5 efficiently bound to outer membrane lipid A and penetrated membranes, leading to bacterial collapse and peptide translocation. Compared to HD5, more HD5d5 molecules were located in the cytoplasm of MDRAb, and HD5d5 was more efficient at reducing the activities of superoxide dismutase and catalase, causing the accumulation of reactive oxygen species that are detrimental to microbes. In addition, HD5 failed to suppress the pathogenic outer membrane protein A of Acinetobacter baumannii (AbOmpA) at concentrations up to 50 µg/ml, whereas HD5d5 strongly bound to AbOmpA and exhibited a dramatic toxin-neutralizing ability, thus expanding the repertoire of drugs that is available to treat MDRAb infections.

Dopamine induces platelet production from megakaryocytes via oxidative stress-mediated signaling pathways.

Dopamine (DA), a catecholamine neurotransmitter, is known to for its diverse roles on hematopoiesis, yet its function in thrombopoiesis remains poorly understood. This study shows that DA stimulation can directly induce platelet production from megakaryocytes (MKs) in the final stages of thrombopoiesis via a reactive oxygen species (ROS)-dependent pathway. The mechanism was suggested by the results that DA treatment could significantly elevate the ROS levels in MKs, and time-dependently activate oxidative stress-mediated signaling, including p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and caspase-3 signaling pathways, while the antioxidants N-acetylcysteine and L-glutathione could effectively inhibit the activation of these signaling pathways, as well as the ROS increase and platelet production triggered by DA. Therefore, our data revealed that the direct role and mechanism of DA in thrombopoiesis, which provides new insights into the function recognition of DA in hematopoiesis.

Development of a sandwich enzyme-linked immunosorbent assay for dTMP-GH fusion protein by rational immunogen selection.

dTMP-GH is a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide (dTMP) fused to human growth hormone (hGH) prepared previously by our team. It shows significant bioactivity in promoting thrombocytopoiesis, but detection of intact dTMP-GH in plasma is still a challenge due to the presence of endogenous hGH. In this study, a rabbit polyclonal antibody with high affinity to dTMP was obtained with a BSA-conjugated immunogen composed of 20 amino acids sequence spanning two TMP and the linker. A monoclonal antibody termed as 3B2 was screened out by using immunizing mice with whole dTMP-GH, which was proved to simultaneously interact with rhGH, TMP-GH, and dTMP-GH, respectively. In this study, we developed a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies (one polyclonal and one HRP-conjugated monoclonal) to quantify dTMP-GH. The polyclonal antibody and HRP-conjugated monoclonal antibody 3B2 were applied as the capture antibody and detection antibody, respectively. A good correlation between ELISA and bicinchoninic acid (BCA) assay in the quantification of diluted dTMP-GH was observed (r2 = 0.996). Meanwhile, the standard curve of this ELISA method was found in a linear relationship between 0.2 and 10 ng/mL in the presence of rabbit plasma. In vivo experiments demonstrate that the newly developed method is effective to detect dTMP-GH in rabbits, which paves the way for further pharmacokinetic evaluation.

Sympathetic stimulation facilitates thrombopoiesis by promoting megakaryocyte adhesion, migration, and proplatelet formation.

The effect of sympathetic stimulation on thrombopoiesis is not well understood. Here, we demonstrate that both continual noise and exhaustive exercise elevate peripheral platelet levels in normal and splenectomized mice, but not in dopamine ß-hydroxylase-deficient (Dbh(-/-)) mice that lack norepinephrine (NE) and epinephrine (EPI). Further investigation demonstrates that sympathetic stimulation via NE or EPI injection markedly promotes platelet recovery in mice with thrombocytopenia induced by 6.0 Gy of total-body irradiation and in mice that received bone marrow transplants after 10.0 Gy of lethal irradiation. Unfavorably, sympathetic stress-stimulated thrombopoiesis may also contribute to the pathogenesis of atherosclerosis by increasing both the amount and activity of platelets in apolipoprotein E-deficient (ApoE(-/-)) mice. In vitro studies reveal that both NE and EPI promote megakaryocyte adhesion, migration, and proplatelet formation (PPF) in addition to the expansion of CD34(+) cells, thereby facilitating platelet production. It is found that α2-adrenoceptor-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) activation is involved in NE- and EPI-induced megakaryocyte adhesion and migration, and PPF is regulated by ERK1/2 activation-mediated RhoA GTPase signaling. Our data deeply characterize the role of sympathetic stimulation in the regulation of thrombopoiesis and reevaluate its physiopathological implications.