RESUMEN
Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.
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Proteínas de Unión al ADN , Proteína Homóloga de MRE11 , Reparación del ADN por Recombinación , Humanos , ADN , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Recombinación Homóloga , Proteína Homóloga de MRE11/metabolismo , Ácido Láctico/metabolismoRESUMEN
TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca2+ permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.
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Venenos de Escorpión/farmacología , Canal Catiónico TRPA1/metabolismo , Animales , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Escorpiones/metabolismoRESUMEN
Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.
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Proteínas de la Cápside/metabolismo , Péptidos de Penetración Celular/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/genética , Endosomas/metabolismo , Aparato de Golgi/virología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiología , Humanos , Mutagénesis , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/genética , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Acoplamiento Viral , Internalización del VirusRESUMEN
The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging XpdTTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored.
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Envejecimiento/patología , Antibióticos Antineoplásicos/efectos adversos , Péptidos de Penetración Celular/farmacología , Doxorrubicina/efectos adversos , Envejecimiento/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacología , Apoptosis , Proteínas de Ciclo Celular , Línea Celular , Supervivencia Celular , Senescencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Femenino , Fibroblastos/citología , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/metabolismo , Humanos , Cuerpos de Inclusión/efectos de los fármacos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Riñón/efectos de los fármacos , Riñón/fisiología , Hígado/efectos de los fármacos , Hígado/fisiología , Masculino , Ratones , Síndromes de Tricotiodistrofia/tratamiento farmacológico , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Blood-brain barrier penetrating peptides (BBBPs) are short peptide sequences that possess the ability to traverse the selective blood-brain interface, making them valuable drug candidates or carriers for various payloads. However, the in vivo or in vitro validation of BBBPs is resource-intensive and time-consuming, driving the need for accurate in silico prediction methods. Unfortunately, the scarcity of experimentally validated BBBPs hinders the efficacy of current machine-learning approaches in generating reliable predictions. In this paper, we present DeepB3P3, a novel framework for BBBPs prediction. Our contribution encompasses four key aspects. Firstly, we propose a novel deep learning model consisting of a transformer encoder layer, a convolutional network backbone, and a capsule network classification head. This integrated architecture effectively learns representative features from peptide sequences. Secondly, we introduce masked peptides as a powerful data augmentation technique to compensate for small training set sizes in BBBP prediction. Thirdly, we develop a novel threshold-tuning method to handle imbalanced data by approximating the optimal decision threshold using the training set. Lastly, DeepB3P3 provides an accurate estimation of the uncertainty level associated with each prediction. Through extensive experiments, we demonstrate that DeepB3P3 achieves state-of-the-art accuracy of up to 98.31% on a benchmarking dataset, solidifying its potential as a promising computational tool for the prediction and discovery of BBBPs.
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Barrera Hematoencefálica , Péptidos , Aprendizaje Automático , Secuencia de Aminoácidos , Biología Computacional/métodosRESUMEN
The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), can trigger dysregulated immune responses known as the cytokine release syndrome (CRS), leading to severe organ dysfunction and respiratory distress. Our study focuses on developing an improved cell-permeable nuclear import inhibitor (iCP-NI), capable of blocking the nuclear transport of inflammation-associated transcription factors, specifically nuclear factor kappa B (NF-κB). By fusing advanced macromolecule transduction domains and nuclear localization sequences from human NF-κB, iCP-NI selectively interacts with importin α5, effectively reducing the expression of proinflammatory cytokines. In mouse models mimic SARS-CoV-2-induced pneumonitis, iCP-NI treatment demonstrated a significant decrease in mortality rates by suppressing proinflammatory cytokine production and immune cell infiltration in the lungs. Similarly, in hamsters infected with SARS-CoV-2, iCP-NI effectively protected the lung from inflammatory damage by reducing tumor necrosis factor-α, interleukin-6 (IL-6), and IL-17 levels. These promising results highlight the potential of iCP-NI as a therapeutic approach for COVID-19-related lung complications and other inflammatory lung diseases.
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COVID-19 , Ratones , Animales , Humanos , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , SARS-CoV-2 , FN-kappa B/metabolismo , Inflamación , Citocinas/metabolismo , Péptidos/metabolismoRESUMEN
Insulin resistance (IR) is the key pathophysiological cause of type 2 diabetes, and inflammation has been implicated in it. The death domain (DD) of the adaptor protein, MyD88 plays a crucial role in the transduction of TLR4-associated inflammatory signal. Herein, we have identified a 10-residue peptide (M10), from the DD of MyD88 which seems to be involved in Myddosome formation. We hypothesized that M10 could inhibit MyD88-dependent TLR4-signaling and might have effects on inflammation-associated IR. Intriguingly, 10-mer M10 showed oligomeric nature and reversible self-assembly property indicating the peptide's ability to recognize its own amino acid sequence. M10 inhibited LPS-induced nuclear translocation of NF-κB in L6 myotubes and also reduced LPS-induced IL-6 and TNF-α production in peritoneal macrophages of BALB/c mice. Remarkably, M10 inhibited IL-6 and TNF-α secretion in diabetic, db/db mice. Notably, M10 abrogated IR in insulin-resistant L6 myotubes, which was associated with an increase in glucose uptake and a decrease in Ser307-phosphorylation of IRS1, TNF-α-induced JNK activation and nuclear translocation of NF-κB in these cells. Alternate day dosing with M10 (10 and 20â mg/kg) for 30 days in db/db mice significantly lowered blood glucose and improved glucose intolerance after loading, 3.0 g/kg glucose orally. Furthermore, M10 increased insulin and adiponectin secretion in db/db mice. M10-induced glucose uptake in L6 myotubes involved the activation of PI3K/AKT/GLUT4 pathways. A scrambled M10-analog was mostly inactive. Overall, the results show the identification of a 10-mer peptide from the DD of MyD88 with anti-inflammatory and anti-diabetic properties, suggesting that targeting of TLR4-inflammatory pathway, could lead to the discovery of molecules against IR and diabetes.
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Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucemia , Dominio de Muerte , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inflamación/tratamiento farmacológico , Insulina/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/toxicidad , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Péptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo required Batf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.
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Vacunas contra el Cáncer , Péptidos de Penetración Celular , Inmunogenicidad Vacunal , Ganglios Linfáticos , Animales , Presentación de Antígeno , Antígenos , Vacunas contra el Cáncer/inmunología , Péptidos de Penetración Celular/farmacología , Reactividad Cruzada , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Ratones , Linfocitos T/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: The blood-brain barrier serves as a critical interface between the bloodstream and brain tissue, mainly composed of pericytes, neurons, endothelial cells, and tightly connected basal membranes. It plays a pivotal role in safeguarding brain from harmful substances, thus protecting the integrity of the nervous system and preserving overall brain homeostasis. However, this remarkable selective transmission also poses a formidable challenge in the realm of central nervous system diseases treatment, hindering the delivery of large-molecule drugs into the brain. In response to this challenge, many researchers have devoted themselves to developing drug delivery systems capable of breaching the blood-brain barrier. Among these, blood-brain barrier penetrating peptides have emerged as promising candidates. These peptides had the advantages of high biosafety, ease of synthesis, and exceptional penetration efficiency, making them an effective drug delivery solution. While previous studies have developed a few prediction models for blood-brain barrier penetrating peptides, their performance has often been hampered by issue of limited positive data. RESULTS: In this study, we present Augur, a novel prediction model using borderline-SMOTE-based data augmentation and machine learning. we extract highly interpretable physicochemical properties of blood-brain barrier penetrating peptides while solving the issues of small sample size and imbalance of positive and negative samples. Experimental results demonstrate the superior prediction performance of Augur with an AUC value of 0.932 on the training set and 0.931 on the independent test set. CONCLUSIONS: This newly developed Augur model demonstrates superior performance in predicting blood-brain barrier penetrating peptides, offering valuable insights for drug development targeting neurological disorders. This breakthrough may enhance the efficiency of peptide-based drug discovery and pave the way for innovative treatment strategies for central nervous system diseases.
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Péptidos de Penetración Celular , Enfermedades del Sistema Nervioso Central , Humanos , Barrera Hematoencefálica/química , Células Endoteliales , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Péptidos de Penetración Celular/uso terapéutico , Encéfalo , Enfermedades del Sistema Nervioso Central/tratamiento farmacológicoRESUMEN
The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.
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Mucosa Nasal , Polifenoles , Taninos , Taninos/química , Polifenoles/química , Polifenoles/administración & dosificación , Mucosa Nasal/metabolismo , Mucosa Nasal/inmunología , Animales , Nanopartículas/química , Humanos , Péptidos de Penetración Celular/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Adhesivos/química , Depuración Mucociliar/efectos de los fármacos , Inmunoglobulina A , RatonesRESUMEN
Tumor acidity-driven nanomotors may offer robust propulsion for tumor-specific penetrating drug delivery. Herein, an acidity-actuated poly(amino acid) calcium phosphate (CaP) hybrid nanomotor (PCaPmotor) was designed, using a mPEG-PAsp-PPhe@THZ531 micelle (Poly@THZ) for CaP mineralization accompanied by αPD-L1 antibody encapsulation. Dissolution of the CaP layer in an acidic tumor environment gave off heat energy to propel the nanomotor to augment the cellular uptake and penetration into deeply seated cancer cells while facilitating αPD-L1 release. THZ531 delivered by the PCaPmotor inhibited CDK12 and its down-streamed phosphorylation of RNAP-II to increase the cancer immunogenicity events such as the DNA damage, cell apoptosis, immunogenic cell death, lysosomal function disturbance, and MHC-I upregulation. THZ531 and αPD-L1 cosupplied by PCaPmotor significantly increased the frequency of DCs maturation and intratumoral infiltration of CTLs, but the two free drugs did not. Consequently, the PCaP@THZ/αPD-L1 nanomotor resulted in synergistic anticancer immunotherapy in mice. This acid-actuated PCaPmotor represented a new paradigm for penetrating drug delivery.
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Fosfatos de Calcio , Sistemas de Liberación de Medicamentos , Inmunoterapia , Fosfatos de Calcio/química , Animales , Ratones , Humanos , Línea Celular Tumoral , Polímeros/química , Micelas , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/administración & dosificación , Concentración de Iones de Hidrógeno , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Antígeno B7-H1 , Nanopartículas/químicaRESUMEN
Trp is unique among the amino acids since it is involved in many different types of noncovalent interactions such as electrostatic and hydrophobic ones, but also in π-π, π-cation, π-anion and π-ion pair interactions. In membranotropic peptides and proteins, Trp locates preferentially at the water-membrane interface. In antimicrobial or cell-penetrating peptides (AMPs and CPPs respectively), Trp is well-known for its strong role in the capacity of these peptides to interact and affect the membrane organisation of both bacteria and animal cells at the level of the lipid bilayer. This essential amino acid can however be involved in other types of interactions, not only with lipids, but also with other membrane partners, that are crucial to understand the functional roles of membranotropic peptides. This review is focused on this latter less known role of Trp and describes in details, both in qualitative and quantitative ways: (i) the physico-chemical properties of Trp; (ii) its effect in CPP internalisation; (iii) its importance in AMP activity; (iv) its role in the interaction of AMPs with glycoconjugates or lipids in bacteria membranes and the consequences on the activity of the peptides; (v) its role in the interaction of CPPs with negatively charged polysaccharides or lipids of animal membranes and the consequences on the activity of the peptides. We intend to bring highlights of the physico-chemical properties of Trp and describe its extensive possibilities of interactions, not only at the well-known level of the lipid bilayer, but with other less considered cell membrane components, such as carbohydrates and the extracellular matrix. The focus on these interactions will allow the reader to reevaluate reported studies. Altogether, our review gathers dedicated studies to show how unique are Trp properties, which should be taken into account to design future membranotropic peptides with expected antimicrobial or cell-penetrating activity.
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Antiinfecciosos , Péptidos de Penetración Celular , Animales , Aminoácidos , Antibacterianos/química , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Antimicrobianos , Carbohidratos , Cationes , Membrana Dobles de Lípidos , Triptófano/química , Triptófano/metabolismo , AguaRESUMEN
Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.
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Péptidos de Penetración Celular , Liposomas , Nanopartículas , Pinocitosis , Proteínas tau , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacología , Liposomas/química , Humanos , Proteínas tau/metabolismo , Proteínas tau/química , Nanopartículas/química , Pinocitosis/efectos de los fármacos , Péptidos/química , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Lisosomas/metabolismo , Sistemas de Liberación de Medicamentos/métodosRESUMEN
Gram-negative bacteria (GNB) pose a major global public health challenge as they exhibit a remarkable level of resistance to antibiotics. One of the factors responsible for promoting resistance against a wide range of antibiotics is the outer membrane (OM) of Gram-negative bacteria. The OM acts as a barrier that prevents the entry of numerous antibiotics by reducing their influx (due to membrane impermeability) and enhancing their efflux (with the help of efflux pumps). Our study focuses on analyzing the effect of IMT-P8, a cell-penetrating peptide (CPP), to enhance the influx of various Gram-positive specific antibiotics in multi-drug resistant Gram-negative pathogens. In the mechanistic experiments, IMT-P8 permeabilizes the OM at the same concentrations at which it enhances the activity of various antibiotics against GNB. Cytoplasmic membrane permeabilization was also observed at these concentrations, indicating that IMT-P8 acts on both the outer and cytoplasmic membranes. IMT-P8 interferes with the intrinsic resistance mechanism of GNB and has the potential to make Gram-positive specific antibiotics effective against GNB. IMT-P8 extends the post-antibiotic effect and in combination with antibiotics shows anti-persister activity. The IMT-P8/fusidic acid combination is effective in eliminating intracellular pathogens. IMT-P8 with negligible toxicity displayed good efficacy in murine lung and thigh infection models. Based on these findings, IMT-P8 is a potential antibiotic adjuvant to treat Gram-negative bacterial infections that pose a health hazard.
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BACKGROUND: Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. METHODS: The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. RESULTS: Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2 mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis. CONCLUSIONS: Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.
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Péptidos de Penetración Celular , Ratones , Animales , Péptidos de Penetración Celular/genética , Dependovirus/genética , Transducción Genética , Serogrupo , Línea Celular , Vectores Genéticos/genéticaRESUMEN
The modified cell-penetrating peptide Pas2r12 can deliver antibodies (IgG, 150 kDa) and enhanced green fluorescent protein (EGFP1, 27 kDa) into the cytosol through caveolae-dependent endocytosis. In this study, we determined the effect of Caveolin-1 overexpression on the cytosolic delivery of EGFP by Pas2r12. Three types of Caveolin-1 overexpressing strains were isolated, including Cav1L (low), Cav1M (medium), and Cav1H (high), using HEK293 as the parent cell line. We found that the number of caveolae on the surface of the Caveolin-1-overexpressing strains was similar to that of HEK293. We examined the cytosolic delivery rate of EGFP by Pas2r12. In the Cav1L and Cav1M cells, there was little change compared with HEK293; however, in Cav1H, the rate was significantly decreased. Moreover, the amount of EGFP uptake into the cells (total intracellular EGFP) showed an increasing trend in Cav1H compared with HEK293. These results indicate that in Cav1H, the amount of EGFP uptake into the cells increases, whereas the cytosolic delivery rate of EGFP decreases. This suggests that high overexpression of Caveolin-1 inhibits the transition of EGFP from endosomes to the cytosol.
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OBJECTIVE: Intragastric administration of ninjin'yoeito (NYT), a traditional Japanese herbal medicine, reportedly prevents the decrease in baseline cerebral blood flow (CBF) in the cortex following gastric administration of water. We investigated the effect of NYT on baseline and dynamic changes in cerebral cortical arteriole diameter. METHODS: Urethane-anesthetized mice were intragastrically administered 1 g/kg NYT or distilled water (DW). The artery in the left parietal cortex was imaged using two-photon microscopy. The baseline diameter of penetrating arterioles was measured before and 50-60 min after administration. Dynamic CBF and arteriole diameter changes before, during, and after transient occlusion of the left common carotid artery were measured approximately 10 min after administration. RESULTS: DW decreased the baseline diameter of the penetrating arterioles, whereas NYT did not. During occlusion, the increase in penetrating arteriole diameter was comparable for DW and NYT; however, during reperfusion, the return to preocclusion diameter was slower for NYT than DW. Laser-speckle contrast imaging confirmed that CBF, although comparable during occlusion, was higher during reperfusion for NYT than DW. CONCLUSIONS: These results suggest that NYT attenuates vasoconstriction in penetrating arterioles after intragastric administration and during cerebral reperfusion, contributing to CBF regulation.
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Corneal neovascularization (CoNV) is a major cause of visual impairment worldwide. Currently, available treatment options have limited efficacy and are associated with adverse effects due to biological barriers and clearance mechanisms. To address this challenge, a novel topical delivery system is developed-Gel 2_1&Eylea-an aflibercept-loaded eye-drop hydrogel mediated with cell-penetrating peptide 1. Gel 2_1&Eylea demonstrates superior membrane permeability, increased stability, and prolonged drug retention time on the ocular surface, and thus may improve drug efficacy. In a rabbit CoNV model, Gel 2_1&Eylea significantly reduces the density of neovascularization with no adverse effects on normal corneoscleral limbal vessels, demonstrating high efficacy and biocompatibility. This work identifies a promising treatment for CoNV which has the potential to benefit other ocular neovascular diseases.
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Péptidos de Penetración Celular , Neovascularización de la Córnea , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión , Animales , Conejos , Neovascularización de la Córnea/tratamiento farmacológico , Hidrogeles , Soluciones Oftálmicas/uso terapéuticoRESUMEN
Inflammatory Bowel Disease (IBD) is a chronic inflammatory condition affecting the gastrointestinal tract (GIT). Glucagon-like peptide-2 (GLP-2) analogs possess high potential in the treatment of IBD by enhancing intestinal repair and attenuating inflammation. Due to the enzymatic degradation and poor intestinal absorption, GLP-2 analogs are administered parenterally, which leads to poor patient compliance. This work aims to develop IBD-targeted nanoparticles (NPs) for the oral delivery of the GLP-2 analog, Teduglutide (TED). Leveraging the overproduction of Reactive Oxygen Species (ROS) in the IBD environment, ROS-sensitive NPs are developed to target the intestinal epithelium, bypassing the mucus barrier. PEGylation of NPs facilitates mucus transposition, but subsequent PEG removal is crucial for cellular internalization. This de-PEGylation is possible by including a ROS-sensitive thioketal linker within the system. ROS-sensitive NPs are established, with the ability to fully de-PEGylate via ROS-mediated cleavage. Encapsulation of TED into NPs resulted in the absence of absorption in 3D in vitro models, potentially promoting a localized action, and avoiding adverse effects due to systemic absorption. Upon oral administration to colitis-induced mice, ROS-sensitive NPs are located in the colon, displaying healing capacity and reducing inflammation. Cleavable PEGylated NPs demonstrate effective potential in managing IBD symptoms and modulating the disease's progression.
RESUMEN
In recent years, targeted drug delivery has attracted a great interest for enhanced therapeutic efficiency, with diminished side effects, especially in cancer therapy. Cell penetrating peptides (CPPs) like HIV1-TAT peptides, appear to be the perfect vectors for translocating drugs or other cargoes across the plasma membrane, but their application is limited mostly due to insufficient specificity for intended targets. Although these molecules were successfully used, the mechanism by which the peptides enter the cell interior still needs to be clarified. The tripeptide motif RGD (arginine-glycine-aspartate), found in extracellular matrix proteins has high affinity for integrin receptors overexpressed in cancer and it is involved in different phases of disease progression, including proliferation, invasion and migration. Discovery of new peptides with high binding affinity for disease receptors and permeability of plasma membranes is desirable for both, development of targeted drug delivery systems and early detection and diagnosis. To complement the TAT peptide with specific targeting ability, we conjugated it with an integrin-binding RGD motif. Although the idea of RGD-CPPs conjugates is not entirely new,[1] here we describe the permeability abilities and specificity of integrin receptors of RGD-TAT peptides in model membranes. Our findings reveal that this novel RGD sequence based on TAT peptide maintains its ability to permeate lipid membranes and exhibits specificity for integrin receptors embedded in giant unilamellar vesicles. This promising outcome suggests that the RGD-TAT peptide has significant potential for applications in the field of targeted drug delivery systems.