RESUMO
Background: Cancer-associated fibroblasts (CAFs) are the key components of the immune barrier in liver cancer. Therefore, gaining a deeper understanding of the heterogeneity and intercellular communication of CAFs holds utmost importance in boosting immunotherapy effectiveness and improving clinical outcomes. Methods: A comprehensive analysis by combing single-cell, bulk, and spatial transcriptome profiling with multiplexed immunofluorescence was conducted to unravel the complexities of CAFs in liver cancer. Results: Through an integrated approach involving 235 liver cancer scRNA-seq samples encompassing over 1.2 million cells, we found that CAFs were particularly increased in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). FAP + fibroblasts were identified as the dominant subtype of CAFs, and which were mainly involved in extracellular matrix organization and angiogenesis. These CAFs were enriched in the tumor boundary of HCC, but diffusely scattered within ICC. The DAB2 + and SPP1 + tumor-associated macrophages (TAMs) reinforce the function of FAP + CAFs through signals such as TGF-ß, PDGF, and ADM. Notably, the interaction between DAB2 + TAMs and FAP + CAFs promoted the formation of immune barrier and correlated with poorer patient survival, non-response to immunotherapy in HCC. High FAP and DAB2 immunohistochemical scores predicted shorter survival and higher serum AFP concentration in a local clinical cohort of 90 HCC patients. Furthermore, this communication pattern might be applicable to other solid malignancies as well. Conclusions: The interaction between DAB2 + TAMs and FAP + CAFs appears crucial in shaping the immune barrier. Strategies aimed at disrupting this communication or inhibiting the functions of FAP + CAFs could potentially enhance immunotherapy effectiveness and improve clinical outcomes.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma Hepatocelular , Neoplasias Hepáticas , Microambiente Tumoral , Humanos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/imunologia , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/imunologia , Microambiente Tumoral/imunologia , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Colangiocarcinoma/terapia , Colangiocarcinoma/patologia , Colangiocarcinoma/imunologia , Colangiocarcinoma/metabolismo , Imunoterapia/métodos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Masculino , Feminino , EndopeptidasesRESUMO
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
Assuntos
Técnicas Biossensoriais , Endopeptidases , Staphylococcus aureus , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/virologia , Técnicas Biossensoriais/métodos , Endopeptidases/química , Endopeptidases/isolamento & purificação , Endopeptidases/genética , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Humanos , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/química , Fagos de Staphylococcus/isolamento & purificação , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Estafilocócicas/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Molecular imaging holds the potential for noninvasive and accurate grading of liver fibrosis. It is limited by the lack of biomarkers that strongly correlate with liver fibrosis grade. Here, we discover the grading potential of fibroblast activation protein alpha (FAPα) for liver fibrosis through transcriptional analysis and biological assays on clinical liver samples. The protein and mRNA expression of FAPα are linearly correlated with fibrosis grade (R2 = 0.89 and 0.91, respectively). A FAPα-responsive MRI molecular nanoprobe is prepared for quantitatively grading liver fibrosis. The nanoprobe is composed of superparamagnetic amorphous iron nanoparticles (AFeNPs) and paramagnetic gadoteric acid (Gd-DOTA) connected by FAPα-responsive peptide chains (ASGPAGPA). As liver fibrosis worsens, the increased FAPα cut off more ASGPAGPA, restoring a higher T1-MRI signal of Gd-DOTA. Otherwise, the signal remains quenched due to the distance-dependent magnetic resonance tuning (MRET) effect between AFeNPs and Gd-DOTA. The nanoprobe identifies F1, F2, F3, and F4 fibrosis, with area under the curve of 99.8%, 66.7%, 70.4%, and 96.3% in patients' samples, respectively. This strategy exhibits potential in utilizing molecular imaging for the early detection and grading of liver fibrosis in the clinic.
Assuntos
Endopeptidases , Cirrose Hepática , Imageamento por Ressonância Magnética , Proteínas de Membrana , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Gelatinases/metabolismo , Compostos Organometálicos/química , Masculino , Fígado/diagnóstico por imagem , Fígado/patologia , Fígado/metabolismo , Feminino , Compostos Heterocíclicos/química , Pessoa de Meia-Idade , Animais , Meios de Contraste/químicaRESUMO
Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5' Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation.
Assuntos
Cromatina , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/metabolismo , DNA/metabolismo , Ubiquitinação , Endopeptidases/metabolismo , DNA Fúngico/metabolismo , DNA Fúngico/genética , Enzimas Desubiquitinantes/metabolismo , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Ubiquitina TiolesteraseRESUMO
Stimuli-responsive nanomaterials show promise in eradicating Staphylococcus aureus biofilm from implants. Peptidoglycan hydrolases (PGHs) are cationic antimicrobials that can be bioengineered to improve the targeting of persisters and drug-resistant bacteria. However, these molecules can be degraded before reaching the target and/or present limited efficacy against biofilm. Therefore, there is an urgent need to improve their potency. Herein, PGH-polyphosphate nanoparticles (PGH-PP NPs) are formed by ionotropic gelation between cationic PGHs and anionic polyphosphate, with the aim of protecting PHGs and delivering them at the target site triggered by alkaline phosphatase (AP) from S. aureus biofilm. Optimized conditions for obtaining M23-PP NPs and GH15-PP NPs are presented. Size, zeta potential, and transmission electron microscopy imaging confirm the nanoscale size. The system demonstrates outstanding performance, as evidenced by a dramatic reduction in PGHs' minimum inhibitory concentration and minimum bactericidal concentration, together with protection against proteolytic effects, storage stability, and cytotoxicity towards the Caco-2 and HeLa cell lines. Time-kill experiments show the great potential of these negatively charged delivery systems in overcoming the staphylococcal biofilm barrier. Efficacy under conditions inhibiting AP proves the enzyme-triggered delivery of PGHs. The enzyme-responsive PGH-PP NPs significantly enhance the effectiveness of PGHs against bacteria residing in biofilm, offering a promising strategy for eradicating S. aureus biofilm.
Assuntos
Antibacterianos , Biofilmes , Testes de Sensibilidade Microbiana , Nanopartículas , Staphylococcus aureus , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Biofilmes/efeitos dos fármacos , Nanopartículas/química , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Endopeptidases/metabolismo , Endopeptidases/farmacologia , Endopeptidases/química , Tamanho da Partícula , Polifosfatos/química , Polifosfatos/farmacologiaRESUMO
Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus. ZP3's endolysin LysZP was further studied for its bacteriolytic action, and the N-terminal transmembrane domain of LysZP is suggested to be a signal-arrest-release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage-Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen.
Assuntos
Bacteriófagos , Genoma Viral , Oryza , Doenças das Plantas , Xanthomonas , Xanthomonas/virologia , Xanthomonas/efeitos dos fármacos , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bacteriófagos/isolamento & purificação , Oryza/microbiologia , Oryza/virologia , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Endopeptidases/farmacologia , Endopeptidases/genética , Endopeptidases/química , Endopeptidases/metabolismo , Filogenia , Folhas de Planta/virologia , Folhas de Planta/microbiologia , China , Genômica/métodosRESUMO
Many cells in high glucose repress mitochondrial respiration, as observed in the Crabtree and Warburg effects. Our understanding of biochemical constraints for mitochondrial activation is limited. Using a Saccharomyces cerevisiae screen, we identified the conserved deubiquitinase Ubp3 (Usp10), as necessary for mitochondrial repression. Ubp3 mutants have increased mitochondrial activity despite abundant glucose, along with decreased glycolytic enzymes, and a rewired glucose metabolic network with increased trehalose production. Utilizing ∆ubp3 cells, along with orthogonal approaches, we establish that the high glycolytic flux in glucose continuously consumes free Pi. This restricts mitochondrial access to inorganic phosphate (Pi), and prevents mitochondrial activation. Contrastingly, rewired glucose metabolism with enhanced trehalose production and reduced GAPDH (as in ∆ubp3 cells) restores Pi. This collectively results in increased mitochondrial Pi and derepression, while restricting mitochondrial Pi transport prevents activation. We therefore suggest that glycolytic flux-dependent intracellular Pi budgeting is a key constraint for mitochondrial repression.
Assuntos
Glucose , Mitocôndrias , Fosfatos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Glucose/metabolismo , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fosfatos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Glicólise , Trealose/metabolismo , EndopeptidasesRESUMO
Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.
Assuntos
Vírus da Febre Aftosa , Vírus da Febre Aftosa/genética , Animais , Linhagem Celular , Genoma Viral/genética , Replicação Viral , Febre Aftosa/virologia , Cricetinae , Plasmídeos/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Deleção de Genes , EndopeptidasesAssuntos
Ubiquitina Tiolesterase , Humanos , Ubiquitina Tiolesterase/genética , Endopeptidases/genética , Reação em Cadeia da Polimerase/métodos , Adenoma Hipofisário Secretor de ACT/genética , Adenoma Hipofisário Secretor de ACT/patologia , Adenoma Hipofisário Secretor de ACT/diagnóstico , Feminino , Adenoma/genética , Adenoma/diagnóstico , Adenoma/patologia , Adulto , Masculino , Pessoa de Meia-Idade , Complexos Endossomais de Distribuição Requeridos para TransporteRESUMO
Fibroblast activation protein (FAP) is abundantly expressed in the stroma of most human solid tumors. Clinical-stage radiolabeled FAP ligands are increasingly used as tools for the detection of various cancer lesions. To unleash the full therapeutic potential of FAP-targeting agents, ligands need to remain at the tumor site for several days after administration. We recently described the discovery of OncoFAP, a high-affinity small organic ligand of FAP with a rapid accumulation in tumors and low uptake in healthy tissues in cancer patients. Trimerization of OncoFAP provided a derivative (named TriOncoFAP, or OncoFAP-23) with improved FAP affinity. In this work, we evaluated the tissue biodistribution profile and the therapeutic performance of OncoFAP-23 in tumor-bearing mice. Methods: OncoFAP-23 was radiolabeled with the theranostic radionuclide 177Lu. Preclinical experiments were conducted on mice bearing SK-RC-52.hFAP (BALB/c nude mice) or CT-26.hFAP (BALB/c mice) tumors. 177Lu-OncoFAP and 177Lu-FAP-2286 were included in the biodistribution study as controls. Toxicologic evaluation was performed on Wistar rats and CD1 mice by injecting high doses of OncoFAP-23 or its cold-labeled counterpart, respectively. Results: 177Lu-OncoFAP-23 emerged for its best-in-class biodistribution profile, high and prolonged tumor uptake (i.e., â¼16 percentage injected dose/g at 96 h), and low accumulation in healthy organs, which correlates well with its potent single-agent anticancer activity at low levels of administered radioactivity. Combination treatment with the tumor-targeted interleukin 2 (L19-IL2, a clinical-stage immunocytokine) further expands the therapeutic window of 177Lu-OncoFAP-23 by potentiating its in vivo antitumor activity. Proteomics studies revealed a potent tumor-directed immune response on treatment with the combination. OncoFAP-23 and natLu-OncoFAP-23 exhibited a favorable toxicologic profile, without showing any side effects or signs of toxicity. Conclusion: OncoFAP-23 presents enhanced tumor uptake and tumor retention and low accumulation in healthy organs, findings that correspond to a strongly improved in vivo antitumor efficacy. The data presented in this work support the clinical development of 177Lu-OncoFAP-23 for the treatment of FAP-positive solid tumors.
Assuntos
Endopeptidases , Gelatinases , Lutécio , Proteínas de Membrana , Radioisótopos , Compostos Radiofarmacêuticos , Animais , Camundongos , Compostos Radiofarmacêuticos/uso terapêutico , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/química , Lutécio/uso terapêutico , Ratos , Humanos , Distribuição Tecidual , Radioisótopos/uso terapêutico , Radioisótopos/química , Linhagem Celular Tumoral , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Neoplasias/radioterapia , Neoplasias/diagnóstico por imagem , Feminino , Camundongos Endogâmicos BALB CRESUMO
Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Methods: Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for 177Lu-TRT. Key pretargeting parameters (sdAb-injected mass and lag time) were optimized in healthy mice and U87MG (FAP+) xenografts. A TRT study in a pancreatic ductal adenocarcinoma (PDAC) patient-derived xenograft (PDX) model was performed as a pilot study for sdAb-based pretargeting applications. Results: Modification of 4AH29 with trans-cyclooctene (TCO) moieties did not modify the sdAb pharmacokinetic profile. A 200-µg injected mass of 4AH29-TCO and an 8-h lag time for the injection of [177Lu]Lu-DOTA-PEG7-tetrazine resulted in the highest kidney therapeutic index (2.0 ± 0.4), which was 5-fold higher than that of [177Lu]Lu-DOTA-4AH29 (0.4 ± 0.1). FAP expression in the tumor microenvironment was validated in a PDAC PDX model with both immunohistochemistry and PET/CT imaging. Mice treated with the pretargeting high-activity approach (4AH29-TCO + [177Lu]Lu-DOTA-PEG7-tetrazine; 3 × 88 MBq, 1 injection per week for 3 wk) demonstrated prolonged survival compared with the vehicle control and conventionally treated ([177Lu]Lu-DOTA-4AH29; 3 × 37 MBq, 1 injection per week for 3 wk) mice. Mesangial expansion was reported in 7 of 10 mice in the conventional cohort, suggesting treatment-related kidney morphologic changes, but was not observed in the pretargeting cohort. Conclusion: This study validates pretargeting to mitigate sdAbs' kidney retention with no observation of morphologic changes on therapy regimen at early time points. Clinical translation of click-chemistry-based pre-TRT is warranted on the basis of its ability to alleviate toxicities related to biovectors' intrinsic pharmacokinetic profiles. The absence of representative animal models with extensive stroma and high FAP expression on cancer-associated fibroblasts led to a low mean tumor-absorbed dose even with high injected activity and consequently to modest survival benefit in this PDAC PDX.
Assuntos
Compostos Radiofarmacêuticos , Anticorpos de Domínio Único , Animais , Camundongos , Compostos Radiofarmacêuticos/uso terapêutico , Compostos Radiofarmacêuticos/farmacocinética , Humanos , Linhagem Celular Tumoral , Anticorpos de Domínio Único/uso terapêutico , Distribuição Tecidual , Feminino , Endopeptidases , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/diagnóstico por imagem , Carcinoma Ductal Pancreático/radioterapia , Carcinoma Ductal Pancreático/diagnóstico por imagem , Rim/diagnóstico por imagem , Proteínas de MembranaRESUMO
The byproduct from wheat starch production contains approximately 70% gluten (WG) and is an inexpensive but demanding protein raw material for the food industry. This study attempted to determine the optimal hydrolysis conditions for such raw material to obtain peptides combining beneficial functional characteristics with health-promoting activity. The proteases Bromelain, Alcalase, Flavourzyme, and a protease from A. saitoi were used for hydrolysis. It was shown that the tested proteases differ both in terms of the effective hydrolysis conditions of gluten and the profile of the released hydrolysates. Bromelain was particularly effective in converting gluten into peptides, combining beneficial health and functional properties. It achieved maximum activity (189 U/g) against WG at pH 6 and 60 °C, and the best-balanced peptides in terms of desired properties were released at a dose of 2.5 U/g. These peptides were free from most allergenic epitopes, effectively inhibited ACE, and, at 0.34 g, were equivalent to the approved dose of BHT. Their emulsifying activity was higher than that of gluten, and the foaming formation and stabilization potential exceeded that of ovalbumin by 10% and 19%, respectively. It seems that Bromelain-released WG hydrolysates are a promising candidate for a safe fat stabilizer and egg white substitute.
Assuntos
Bromelaínas , Glutens , Triticum , Glutens/química , Hidrólise , Triticum/química , Bromelaínas/química , Hidrolisados de Proteína/química , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/química , Subtilisinas/metabolismo , Subtilisinas/química , Peptídeos/química , EndopeptidasesRESUMO
Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety.
Assuntos
Patos , Endopeptidases , Fagos de Salmonella , Salmonella typhimurium , Esgotos , Animais , Fagos de Salmonella/fisiologia , Esgotos/virologia , Esgotos/microbiologia , Endopeptidases/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/virologia , Salmonella enteritidis/efeitos dos fármacos , Salmonella enteritidis/virologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/prevenção & controle , Salmonelose Animal/microbiologia , Salmonelose Animal/prevenção & controle , Fezes/microbiologia , Fezes/virologiaRESUMO
Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications.
Assuntos
Bacillus subtilis , Endopeptidases , Proteínas Recombinantes de Fusão , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Endotoxinas/genética , Endotoxinas/metabolismo , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/metabolismo , Lisina-tRNA Ligase/química , Expressão GênicaRESUMO
Fibrous dysplasia (FD) is a mosaic skeletal disorder involving the development of benign, expansile fibro-osseous lesions during childhood that cause deformity, fractures, pain, and disability. There are no well-established treatments for FD. Fibroblast activation protein (FAPα) is a serine protease expressed in pathological fibrotic tissues that has promising clinical applications as a biomarker and local pro-drug activator in several pathological conditions. In this study, we explored the expression of FAP in FD tissue and cells through published genetic expression datasets and measured circulating FAPα in plasma samples from patients with FD and healthy donors. We found that FAP genetic expression was increased in FD tissue and cells, and present at higher concentrations in plasma from patients with FD compared to healthy donors. Moreover, FAPα levels were correlated with skeletal disease burden in patients with FD. These findings support further investigation of FAPα as a potential imaging and/or biomarker of FD, as well as a pro-drug activator specific to FD tissue.
Assuntos
Endopeptidases , Displasia Fibrosa Óssea , Gelatinases , Proteínas de Membrana , Serina Endopeptidases , Humanos , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Feminino , Masculino , Endopeptidases/metabolismo , Endopeptidases/genética , Gelatinases/metabolismo , Gelatinases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Displasia Fibrosa Óssea/metabolismo , Displasia Fibrosa Óssea/genética , Displasia Fibrosa Óssea/patologia , Adulto , Adolescente , Criança , Biomarcadores/metabolismo , Biomarcadores/sangue , Osteoblastos/metabolismo , Osteoblastos/patologia , Pessoa de Meia-IdadeRESUMO
Disulfiram (DSF), an FDA-approved drug for treating alcoholism, has been verified with Cu2+-dependent anticancer activity by forming Cu(DTC)2, the complex of one of its metabolites diethyldithiocarbamate (DTC) and Cu2+. Nevertheless, the antitumor effect is limited by insufficient Cu(DTC)2 formation in suit and off-target system toxicity. Herein, we developed a fibroblast activation protein α (FAPα) activatable nanoagent (HfD-HID-Cu) for co-delivery of DTC polymeric prodrug and exogenous Cu2+ to achieve enhanced cancer-specific therapy and activatable in situ fluorescence imaging meanwhile. HfD-HID-Cu was simply constructed through the co-assembly of the DTC polymeric prodrug (HA-fap-DTC) and the copper-loaded IR808-conjugated polymer (HA-IR-DPA-Cu), which could serve as the "OFF-to-ON" switch for chemotherapy and fluorescence. With the high expression of FAPα in tumor tissues, HA-fap-DTC could be activated specifically to release DTC, while maintaining inactive in normal tissues. The liberated DTC within tumor tissues could contend for Cu2+ from HA-IR-DPA-Cu, resulting in the formation of highly cytotoxic Cu(DTC)2in situ for chemotherapy, concomitant with the fluorescence recovery of cyanine dye for tumor imaging. This work provides an effective strategy for co-delivery of DTC prodrug and Cu2+ for tumor theranostic with improved selectivity and minimal side effects. STATEMENT OF SIGNIFICANCE: DSF-based antitumor therapy is highly dependent on Cu2+. However, the non-synchronous distribution of DSF/DTC and Cu2+ in tumor tissues attenuates the antitumor efficacy. The insufficient Cu(DTC)2 formation in suit and off-target distribution greatly limit the anti-tumor application. This study provides a nanoagent for co-delivery of DTC polymeric prodrug and Cu2+ by simple co-assembly to achieve their synchronous tumor distribution. It can be selectively activated by FAPα, forming cytotoxic Cu(DTC)2in suit for tumor-specific chemotherapy and reducing the systemic toxicity. In addition to chemotherapy, the nanoagent can emit fluorescence under the sequential triggering of FAPα and released DTC for tumor imaging. Overall, this study renders a promising strategy for improved Cu(DTC)2-based antitumor therapy and imaging.
Assuntos
Cobre , Ditiocarb , Endopeptidases , Pró-Fármacos , Cobre/química , Cobre/farmacologia , Ditiocarb/química , Ditiocarb/farmacologia , Animais , Humanos , Linhagem Celular Tumoral , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Gelatinases/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Camundongos Endogâmicos BALB C , Nanopartículas/químicaRESUMO
Endolysins (lysins), a novel class of antibacterial agents derived from bacteriophages, efficiently lyse bacteria by degrading the peptidoglycan layer within the bacterial wall. Colistin, a classic peptide antibiotic with the ability to permeabilize the outer membrane, has recently shown great promise in synergizing with lysins against gram-negative bacteria. However, the exact mechanisms responsible for their synergy remain unclear. Here, we first demonstrated the synergistic bacterial killing of various lysin and colistin combinations. With a model lysin, LysAB2, we then confirmed that there is a threshold concentration of colistin causing sufficient permeabilization of the outer membrane for lysin to access the peptidoglycan layer and subsequently exert its lytic ability. The threshold colistin concentrations were found to range 0.2-0.8 µM for the tested bacteria, with the exact value largely depending on the density of lipopolysaccharides on the outer membrane. Beyond the threshold colistin level, LysAB2 could synergize with colistin at a concentration as low as 0.31 µM. Next, we proved for the first time that lysin-induced degradation of the peptidoglycan layer facilitated the disruption of cytoplasmic membrane by colistin, elevated the level of reactive oxygen species in bacterial cells, and boosted the killing effect of colistin. Additionally, the colistin-lysin combination could effectively eliminate established biofilms due to the biofilm dispersal ability of lysin. The in-vivo efficacy was preliminary confirmed in a Galleria mellonella infection model for combination with colistin doses (≥ 1.8 µg/larvae), which could reach beyond the threshold concentration, and a fixed LysAB2 dose (10 µg/larvae). In summary, our study provided the first experimental evidence unravelling the mechanisms behind the synergy of colistin and lysins. All these findings provided important insights in guiding the dosing strategy for applying this combination in future development.
Assuntos
Antibacterianos , Colistina , Farmacorresistência Bacteriana Múltipla , Endopeptidases , Bactérias Gram-Negativas , Colistina/farmacologia , Endopeptidases/farmacologia , Sinergismo Farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/farmacologia , Humanos , Linhagem CelularRESUMO
Fibroblast activation protein inhibitor (FAPI) positron emission tomography/computed tomography (PET/CT) is a multimodal imaging technique that combines PET and CT, utilizing FAP inhibitors as radiotracers. Fibroblast activation protein, a serine protease highly expressed in many epithelial tumor-associated fibroblasts, plays a crucial role in tumor stroma formation and remodeling. Through the detection of FAP expression, FAPI PET/CT facilitates the diagnosis and staging of both benign and malignant pulmonary tumors. In contrast to traditional fluorine-18-fluorodeoxyglucose (18F-FDG) PET/CT focusing on glucose metabolism, FAPI PET/CT offers benefits such as enhanced specificity, reduced background noise, accelerated imaging speed, and decreased radiation exposure. This review provides an overview of the progress in applying FAPI PET/CT and 18F-FDG PET/CT in pulmonary malignancies and discusses current challenges and future prospects.
Assuntos
Fluordesoxiglucose F18 , Neoplasias Pulmonares , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias Pulmonares/diagnóstico por imagem , Gelatinases/metabolismo , Endopeptidases , Proteínas de MembranaRESUMO
Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism.