RESUMO
Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of â¼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.
Assuntos
Echinococcus granulosus , Proteínas de Helminto , Echinococcus granulosus/enzimologia , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Animais , Cães , Proteínas de Helminto/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/química , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/química , Bovinos , Sequência de Aminoácidos , Tripsina/química , Tripsina/metabolismoRESUMO
OBJECTIVE: This study aimed to assess the efficacy and tolerability of stereotactic body radiation therapy (SBRT) for the treatment of liver metastases. METHODS: Patients with up to 5 liver metastases were enrolled in this prospective multicenter study and underwent SBRT. Efficacy outcomes included in-field local control (LC), progression-free survival (PFS), and overall survival (OS). Acute and late toxicities were evaluated using CTCAE v.4.0. RESULTS: A total of 52 patients with 105 liver metastases were treated between 2015 and 2018. The most common primary tumor was colorectal cancer (72% of cases). Liver metastases were synchronous with the primary tumor diagnosis in 24 patients (46.2%), and 21 patients (40.4%) presented with other extrahepatic oligometastases. All patients underwent intensity-modulated radiation therapy (IMRT)/volumetric-modulated arc therapy (VMAT) with image-guided radiation therapy (IGRT) and respiratory gating, and a minimum biologically effective dose (BED10Gy) of 100 Gy was delivered to all lesions. With a median follow-up of 23.1 months (range: 13.4-30.9 months) since liver SBRT, the median actuarial local progression-free survival (local-PFS) was not reached. The actuarial in-field LC rates were 84.9% and 78.4% at 24 and 48 months, respectively. The median actuarial liver-PFS and distant-PFS were 11 and 10.8 months, respectively. The actuarial median overall survival (OS) was 27.7 months from SBRT and 52.5 months from metastases diagnosis. Patients with lesion diameter ≤ 5 cm had significantly better median liver-PFS (p = 0.006) and OS (p = 0.018). No acute or late toxicities of grade ≥ 3 were observed. CONCLUSIONS: This prospective multicenter study confirms that liver SBRT is an effective alternative for the treatment of liver metastases, demonstrating high rates of local control and survival while maintaining a low toxicity profile.
Assuntos
Neoplasias Hepáticas , Radiocirurgia , Radioterapia de Intensidade Modulada , Humanos , Radiocirurgia/métodos , Radiocirurgia/efeitos adversos , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/mortalidade , Masculino , Estudos Prospectivos , Feminino , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto , Radioterapia de Intensidade Modulada/métodos , Intervalo Livre de Progressão , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Neoplasias Colorretais/mortalidade , Radioterapia Guiada por Imagem , Taxa de SobrevidaRESUMO
In recent years, the concern derived from the presence of emerging contaminants in the environment and the possible effects on the One Health trilogy has increased. This study determined the concentration of pharmaceutical contaminants of emerging concern and their relationship with the extracellular enzymatic activity of microbial communities from two rivers in western Cuba. Two sampling stations were analyzed; one in the Almendares River (urban) and the other in the San Juan River (rural), taking into account the pollution sources that arrive at these stations and previous physicochemical characterizations. Extracellular protease, acid phosphatase, alkaline phosphatase, lipase, and catalase activities in water and sediments were determined and correlated with contaminants of emerging concern determined by liquid chromatography with mass spectrometry. This study evidenced the presence of different pharmaceutical contaminants found in the categories of antihypertensives, stimulants, anti-inflammatories, and antibiotics in both rivers. Concentrations of contaminants of emerging concern were greater in the Almendares River compared to the San Juan River. In addition, through the canonical redundancy analysis, the influence of these contaminants on the extracellular enzymatic activities of microbial communities was documented, where in most cases they inhibit protease, phosphatase, and lipase activities and enhance catalase activity in response to oxidative stress. The present investigation constitutes the first report in Cuba of the presence of pharmaceutical contaminants of emerging concern and one of the few works that exist in the Latin American region.
Assuntos
Microbiota , Poluentes Químicos da Água , Rios/química , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , Cuba , Catalase , Peptídeo Hidrolases , Lipase , Preparações Farmacêuticas , Monitoramento Ambiental/métodosRESUMO
BACKGROUND: Preoperative radiation therapy following by limb-sparing or conservative surgery is a standard approach for limb and trunk STS. Data supporting hypofractionated radiotherapy schedules are scarce albeit biological sensitivity of STS to radiation would justify it. We sought to evaluate the impact of moderate hypofractionation on pathologic response and its influence on oncologic outcomes. MATERIAL AND METHODS: From October 2018 to January 2023, 18 patients with limb or trunk STS underwent preoperative radiotherapy at a median dose of 52.5 Gy (range 49.5-60 Gy) in 15 fractions of 3.5 Gy (3.3-4 Gy) with or without neoadjuvant chemotherapy. A favorable pathologic response (fPR) was considered as ≥ 90% tumor necrosis on specimen examination. RESULTS: All patients completed planned preoperative radiotherapy. Eleven patients (61.1%) achieved a fPR, and 7 patients (36.8%) a complete pathologic response with total disappearance of tumor cells. Nine patients (47%) developed grade 1-2 acute skin toxicity, and 7 patients (38.8%) had wound complications on follow-up. With a median follow-up of 14 months (range 1-40), no cases of local relapse were observed, and actuarial 3-year overall survival (OS) and distant metastases-free survival (DMFS) are 87% and 76.4%, respectively. In the univariate analysis, the presence of a favorable pathologic response (fPR) was associated with improved 3-year OS (100% vs. 56.03%, p = 0.058) and 3-year DMFS (86.91% vs. 31.46%, p = 0.002). Moreover, both complete or partial RECIST response and radiological stabilization of the tumor lesion showed a significant association with higher rates of 3-year distant metastasis-free survival (DMFS) (83% vs. 83% vs. 56%, p < 0.001) and 3-year overall survival (OS) (100% vs. 80% vs. 0, p = 0.002). CONCLUSIONS: Preoperative moderate hypofractionated radiation treatment for STS is feasible and well tolerated and associates encouraging rates of pathologic response that could have a favorable impact on final outcomes.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Humanos , Hipofracionamento da Dose de Radiação , Recidiva Local de Neoplasia/patologia , Extremidades/patologia , Sarcoma/patologia , Terapia Neoadjuvante , Neoplasias de Tecidos Moles/radioterapia , Neoplasias de Tecidos Moles/patologia , Resultado do Tratamento , Estudos RetrospectivosRESUMO
OBJECTIVE: To assess the clinical outcomes of patients with spine metastases treated with SBRT at our institution. MATERIALS AND METHODS: Patients with spine metastases treated with SBRT (1 fraction/18 Gy or 5 fractions/7 Gy) during the last 12 years have been analyzed. All patients were simulated supine in a vacuum cushion or with a shoulder mask. CT scans and MRI image registration were performed. Contouring was based on International Spine-Radiosurgery-Consortium-Consensus-Guidelines. Highly conformal-techniques (IMRT/VMAT) were used for treatment planning. Intra and interfraction (CBCT or X-Ray-ExacTrac) verification were mandatory. RESULTS: From February 2010 to January 2022, 129 patients with spinal metastases were treated with SBRT [1 fraction/18 Gy (75%) or 5 fractions/7 Gy] (25%). For patients with painful metastases (74/129:57%), 100% experienced an improvement in pain after SBRT. With a median follow-up of 14.2 months (average 22.9; range 0.5-140) 6 patients (4.6%) experienced local relapse. Local progression-free survival was different, considering metastases's location (p < 0.04). The 1, 2 and 3 years overall survival (OS) were 91.2%, 85.1% and 83.2%, respectively. Overall survival was significantly better for patients with spine metastases of breast and prostate cancers compared to other tumors (p < 0.05) and significantly worse when visceral metastases were present (p < 0.05), when patients were metastatic de novo (p < 0.05), and in those patients receiving single fraction SBRT (p: 0.01). CONCLUSIONS: According to our experience, SBRT for patients with spinal metastases was effective in terms of local control and useful to reach pain relief. Regarding the intent of the treatment, an adequate selection of patients is essential to propose this ablative approach.
Assuntos
Radiocirurgia , Neoplasias da Coluna Vertebral , Masculino , Humanos , Radiocirurgia/métodos , Neoplasias da Coluna Vertebral/radioterapia , Neoplasias da Coluna Vertebral/cirurgia , Neoplasias da Coluna Vertebral/patologia , Recidiva Local de Neoplasia/etiologia , Mama/patologia , Dor/etiologia , Estudos RetrospectivosRESUMO
DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: ⢠An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. ⢠The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. ⢠rDyP-AU10 showed high activity at low temperatures. ⢠rDyP-AU10 is potentially useful for biotechnological applications.
Assuntos
Corantes , Peroxidase , Peroxidase/metabolismo , Corantes/metabolismo , Escherichia coli/genética , Regiões Antárticas , Peróxido de Hidrogênio , Peroxidases/metabolismoRESUMO
Phosphorylation carries chemical information in biological systems. In two-component systems (TCSs), the sensor histidine kinase and the response regulator are connected through phosphoryl transfer reactions that may be uni- or bidirectional. Directionality enables the construction of complex regulatory networks that optimize signal propagation and ensure the forward flow of information. We combined x-ray crystallography, hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, and systems-integrative kinetic modeling approaches to study phosphoryl flow through the Bacillus subtilis thermosensing TCS DesK-DesR. The allosteric regulation of the histidine kinase DesK was critical to avoid back transfer of phosphoryl groups and futile phosphorylation-dephosphorylation cycles by isolating phosphatase, autokinase, and phosphotransferase activities. Interactions between the kinase's ATP-binding domain and the regulator's receiver domain placed the regulator in two distinct positions in the phosphotransferase and phosphatase complexes, thereby determining whether a key glutamine residue in DesK was properly situated to assist in the dephosphorylation reaction. Moreover, an energetically unfavorable phosphotransferase conformation when DesK was not phosphorylated minimized reverse phosphoryl transfer. DesR dimerization and a dissociative phosphoryl transfer reaction also enforced the direction of phosphoryl flow. Shorter or longer distances between the phosphoryl acceptor and donor residues shifted the phosphoryl transfer equilibrium by modulating the stabilizing effect of the Mg2+ cofactor. These mechanisms control the directionality of signal transmission and show how structure-encoded allostery stores and transmits information in signaling systems.
Assuntos
Bacillus subtilis , Transdução de Sinais , Histidina Quinase/metabolismo , Bacillus subtilis/genética , Fosforilação , Monoéster Fosfórico Hidrolases , Proteínas de Bactérias/metabolismoRESUMO
Persulfides (RSSH/RSS-) are species closely related to thiols (RSH/RS-) and hydrogen sulfide (H2S/HS-), and can be formed in biological systems in both low and high molecular weight cysteine-containing compounds. They are key intermediates in catabolic and biosynthetic processes, and have been proposed to participate in the transduction of hydrogen sulfide effects. Persulfides are acidic, more acidic than thiols, and the persulfide anions are expected to be the predominant species at neutral pH. The persulfide anion has high nucleophilicity, due in part to the alpha effect, i.e., the increased reactivity of a nucleophile when the neighboring atom has high electron density. In addition, persulfides have electrophilic character, a property that is absent in both thiols and hydrogen sulfide. In this article, the biochemistry of persulfides is described, and the possible ways in which the formation of a persulfide could impact on the properties of the biomolecule involved are discussed.
RESUMO
The retropepsin (PR) of the Bovine leukemia virus (BLV) plays, as in other retroviruses, a crucial role in the transition from the non-infective viral particle to the infective virion by processing the polyprotein Gag. PR is expressed as an immature precursor associated with Gag, after an occasional -1 ribosomal frameshifting event. Self-hydrolysis of PR at specific N- and C-terminal sites releases the monomer that dimerizes giving rise to the active protease. We designed a strategy to express BLV PR in E. coli as a fusion protein with maltose binding protein, with a six-histidine tag at its N-terminal end, and bearing a tobacco etch virus protease hydrolysis site. This allowed us to obtain soluble and mature recombinant PR in relatively good yields, with exactly the same amino acid composition as the native protein. As PR presents relative promiscuity for the hydrolysis sites we designed four fluorogenic peptide substrates based on Förster resonance energy transfer (FRET) in order to characterize the activity of the recombinant enzyme. These substrates opened the way to perform kinetic studies, allowing us to characterize the dimer-monomer equilibrium. Furthermore, we obtained kinetic evidence for the existence of a conformational change that enables the interaction with the substrate. These results constitute a starting point for the elucidation of the kinetic properties of BLV-PR, and may be relevant not only to improve the chemical warfare against this virus but also to better understand other viral PRs.
Assuntos
Ácido Aspártico Proteases , Vírus da Leucemia Bovina , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Protease de HIV/metabolismo , Cinética , Vírus da Leucemia Bovina/genética , Vírus da Leucemia Bovina/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Introducción: El proceso de adherencia es fundamental en el desarrollo de la mayoría de las infecciones ocasionadas por Escherichia coli. Para los patotipos de esta especie se describen tres patrones de adherencia diferentes: adherencia localizada, adherencia difusa y adherencia agregativa, los cuales se relacionan con los procesos patogénicos específicos que ocasiona en la clínica. Sin embargo, son pocos los estudios en relación con los fenotipos de adherencia in vitro de E. coli aisladas del ambiente. Objetivo: Determinar los fenotipos de adherencia de cepas de E. coli aisladas de ecosistemas dulceacuícolas de La Habana. Métodos: Se analizaron 108 cepas de E. coli aisladas de los ríos Almendares, Quibú y Luyanó de La Habana. Se determinó el patrón de adherencia mediante ensayos de adherencia en cultivo celular de la línea HEp-2 así como el serotipo de cada cepa. Resultados: El 25 por ciento de las cepas de E. coli aisladas fueron adherentes y el 75 por ciento fueron no adherentes. Veintidós cepas mostraron el típico patrón de adherencia difusa y cinco cepas mostraron una adherencia agregativa. Se encontraron cepas de los dos patrones de adherencia en los tres ríos evaluados. Las cepas presentaron 24 serotipos diferentes. Conclusiones: Se demostró que las cepas de E. coli ambientales circulantes en estos ecosistemas presentan características adherentes, cuya patogenicidad implica un riesgo potencial para la salud humana, especialmente en edades pediátricas(AU)
Introduction: Adherence is crucial to the development of most Escherichia coli infections. Three different adherence patterns have been described for pathotypes of this species: localized, diffuse and aggregative adherence, based on the specific clinical pathogenic processes they bring about. However, few studies have been conducted about in vitro adherence phenotypes of E. coli isolated from the environment. Objective: Determine the adherence phenotypes of E. coli strains isolated from freshwater ecosystems in Havana. Methods: An analysis was conducted of 108 E. coli strains isolated from the rivers Almendares, Quibú and Luyanó in Havana. Determination was made of the adherence pattern by adherence assays in HEp-2 cell line cultures, as well as of the serotype for each strain. Results: Of the E. coli strains isolated, 25 percent were adherent and 75 percent were not. Twenty-two strains displayed the typical diffusely adherent pattern and five displayed aggregative adherence. Strains exhibiting the two adherence patterns were found in the three rivers evaluated. The strains contained 24 different serotypes. Conclusions: The environmental E. coli strains circulating in these ecosystems were found to have adherent characteristics whose pathogenicity implies a potential risk to human health, particularly in childhood(AU)