RESUMO
The bacterial transposon Tn7 directs transposition into actively replicating DNA by a mechanism involving the transposon-encoded protein TnsE. Here we show that TnsE physically and functionally interacts with the processivity factor of the DNA replication machinery in vivo and in vitro. Our work establishes an in vitro TnsABC+E transposition reaction reconstituted from purified proteins and target DNA structures. Using the in vitro reaction we confirm that the processivity factor specifically reorders TnsE-mediated transposition events on target DNAs in a way that matches the bias with active DNA replication in vivo. The TnsE interaction with an essential and conserved component of the replication machinery, and a DNA structure reveals a mechanism by which Tn7, and probably other elements, selects target sites associated with DNA replication.
Assuntos
Replicação do DNA , Elementos de DNA Transponíveis , Escherichia coli/genética , Escherichia coli/metabolismo , Sequência de Aminoácidos , DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis. [68Ga]Pentixafor is a promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of 68Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of [18F]RPS-534 (7.2 ± 0.3 %ID/g) and [18F]RPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background, [18F]RPS-547, and especially [18F]RPS-534, are promising 18F-labeled candidates for imaging CXCR4 expression.
Assuntos
Complexos de Coordenação/administração & dosagem , Imagem Molecular , Peptídeos Cíclicos/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Receptores CXCR4/genética , Animais , Linhagem Celular Tumoral , Complexos de Coordenação/química , Radioisótopos de Flúor/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Peptídeos Cíclicos/química , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Receptores CXCR4/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Integrins are cell surface receptors that transduce signals bidirectionally across the plasma membrane. The key event of integrin signaling is the allosteric regulation between its ligand-binding site and the C-terminal helix (alpha7) of integrin's inserted (I) domain. A significant axial movement of the alpha7 helix is associated with the open, active conformation of integrins. We describe the crystal structure of an engineered high-affinity I domain from the integrin alpha(L)beta(2) (LFA-1) alpha subunit in complex with the N-terminal two domains of ICAM-5, an adhesion molecule expressed in telencephalic neurons. The finding that the alpha7 helix swings out and inserts into a neighboring I domain in an upside-down orientation in the crystals implies an intrinsically unusual mobility of this helix. This remarkable feature allows the alpha7 helix to trigger integrin's large-scale conformational changes with little energy penalty. It serves as a mechanistic example of how a weakly bound adhesion molecule works in signaling.
Assuntos
Antígeno CD11a/química , Antígeno CD11a/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Regulação Alostérica , Animais , Células CHO , Moléculas de Adesão Celular/química , Cricetinae , Cricetulus , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
PURPOSE: To demonstrate the phase and quantitative susceptibility mapping (QSM) patterns created by solid and shell spatial distributions of magnetic susceptibility in multiple sclerosis (MS) lesions. MATERIALS AND METHODS: Numerical simulations and experimental phantoms of solid- and shell-shaped magnetic susceptibility sources were used to generate magnitude, phase, and QSM images. Imaging of 20 consecutive MS patients was also reviewed for this Institutional Review Board (IRB)-approved MRI study to identify the appearance of solid and shell lesions on phase and QSM images. RESULTS: Solid and shell susceptibility sources were correctly reconstructed in QSM images, while the corresponding phase images depicted both geometries with shell-like patterns, making the underlying susceptibility distribution difficult to determine using phase alone. In MS patients, of the 60 largest lesions identified on T2 , 30 lesions were detected on both QSM and phase, of which 83% were solid and 17% were shells on QSM, and of which 30% were solid and 70% were shell on phase. Of the 21 shell-like lesions on phase, 76% appeared solid on QSM, 24% appeared shell on QSM. Of the five shell-like lesions on QSM, all were shell-like on phase. CONCLUSION: QSM accurately depicts both solid and shell patterns of magnetic susceptibility, while phase imaging fails to distinguish them.
Assuntos
Encéfalo/patologia , Imagem de Tensor de Difusão/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Esclerose Múltipla/patologia , Substância Branca/patologia , Adulto , Idoso , Algoritmos , Feminino , Humanos , Aumento da Imagem/métodos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Ionizing radiation has garnered considerable attention as a combination partner for immunotherapy due to its potential immunostimulatory effects. In contrast to the more commonly used external beam radiation, we explored the feasibility of combining chimeric antigen receptor (CAR) T cell therapy with targeted radionuclide therapy (TRT), which is achieved by delivering ß-emitting 177Lu-DOTATATE to tumor via tumor-infiltrating CAR T cells that express somatostatin receptor 2 (SSTR2). We hypothesized that the delivery of radiation to tumors could synergize with CAR T therapy, resulting in enhanced antitumor immunity and tumor response. To determine the optimal dosage and timing of 177Lu-DOTATATE treatment, we measured CAR T cell infiltration and expansion in tumors longitudinally through positron emission tomography (PET) using a SSTR2-specific positron-emitting radiotracer,18F-NOTA-Octreotide. In animals receiving CAR T cells and a low-dose (2.5 Gy) of TRT following the administration of 177Lu-DOTATATE, we observed a rapid regression of large subcutaneous tumors, which coincided with a dramatic increase in serum proinflammatory cytokines. Tumor burden was also reduced when a higher radiation dose (6 Gy) was delivered to the tumor. However, this higher dose led to cell death in both the tumor and CAR T cells. Our study suggests that there may exist an optimum range of TRT dosage that can enhance T cell activity and sensitize tumor cells to T cell killing, which may result in more durable tumor control compared to a higher radiation dose.
Assuntos
Neoplasias , Animais , Neoplasias/tratamento farmacológico , Octreotida/uso terapêutico , Linfócitos T , Imunoterapia , Radioisótopos/uso terapêuticoRESUMO
Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.
Assuntos
Técnicas Citológicas/métodos , Lasers , Transfecção/métodos , Animais , Células CHO , Membrana Celular/metabolismo , Sobrevivência Celular , Corantes/metabolismo , Cricetinae , Cricetulus , DNA/genética , DNA/metabolismo , Plasmídeos/genética , Fatores de TempoRESUMO
OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.
Assuntos
Clatrina/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clatrina/química , Vesículas Revestidas/metabolismo , Vesículas Revestidas/ultraestrutura , Endocitose , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositóis/metabolismo , Fosfolipídeos , Monoéster Fosfórico Hidrolases/genética , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Alinhamento de SequênciaRESUMO
P-TEFb complex, a heterodimer of the kinase CDK9 and Cyclin T, is a critical factor that stimulates the process of transcription elongation. Here, we explored a fast and large-scale screening method to induce a temperature-dependent conditional disruption of the CDK9/Cyclin T interaction and developed an assay to validate their mutant phenotypes in a biological context. First, we used the yeast two-hybrid system to screen Drosophila melanogaster Cyclin T mutants at a large scale for temperature or cold sensitive (TS or CS) CDK9 interaction phenotypes. The isolated P-TEFb TS mutants were then expressed in Drosophila cells and were investigated for their effects on Drosophila hsp70 transcriptional activity. Our results showed that these P-TEFb TS mutants had a reduced level of hsp70 transcription at restrictive temperatures. A model structure of the Cyclin T and CDK9 complex suggested that the key TS mutations were found within the α2- and α3-helices at the interface of the complex, which may disrupt the binding of Cyclin T to CDK9 directly or indirectly by affecting the conformation of Cyclin T. The yeast two-hybrid-based screening strategy described here for isolating TS or CS interaction phenotypes can be directly applicable to other complexes in higher organisms. The use of TS or CS mutants will enable a 'real-time and reversible perturbation' restricted to specific protein-protein interactions, providing a mechanistic insight into the biological process mediated by a target complex.
Assuntos
Proteínas de Drosophila/genética , Mutação , Fator B de Elongação Transcricional Positiva/genética , Técnicas do Sistema de Duplo-Híbrido , Sequência de Aminoácidos , Animais , Ciclina T/química , Ciclina T/genética , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Modelos Moleculares , Dados de Sequência Molecular , Fator B de Elongação Transcricional Positiva/metabolismo , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Análise de Sequência de DNA , Temperatura , Transcrição GênicaRESUMO
BACKGROUND: The BRAF(V600E) mutation is present in 62% of radioactive iodine-resistant thyroid tumors and is associated with downregulation of the sodium-iodide symporter (NIS) and thyroid stimulating hormone receptor (TSHr). We sought to evaluate the combined effect of BRAF inhibition and TSH supplementation on (131)I uptake of BRAF(V600E)-mutant human thyroid cancer cells. MATERIALS AND METHODS: WRO cells (a BRAF(V600E)-mutant follicular-derived papillary thyroid carcinoma cell line) were transfected with small interfering RNA targeting BRAF for 72 h in a physiological TSH environment. NIS and TSHr expression were then evaluated at three levels: gene expression, protein levels, and (131)I uptake. These three main outcomes were then reassessed in TSH-depleted media and media supplemented with supratherapeutic concentrations of TSH. RESULTS: NIS gene expression increased 5.5-fold 36 h after transfection (P = 0.01), and TSHr gene expression increased 2.8-fold at 24 h (P = 0.02). NIS and TSHr protein levels were similarly increased 48 and 24 h after transfection, respectively. Seventy-two hours after BRAF inhibition, (131)I uptake was unchanged in TSH-depleted media, increased by 7.5-fold (P < 0.01) in physiological TSH media, and increased by 9.1-fold (P < 0.01) in supratherapeutic TSH media. CONCLUSIONS: The combined strategy of BRAF inhibition and TSH supplementation results in greater (131)I uptake than when either technique is used alone. This represents a simple and feasible approach that may improve outcomes in patients with radioactive iodine-resistant thyroid carcinomas for which current treatment algorithms are ineffective.
Assuntos
Adenocarcinoma Folicular/metabolismo , Inativação Gênica , Iodo/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/metabolismo , Tireotropina/farmacologia , Adenocarcinoma Folicular/patologia , Linhagem Celular Tumoral , Inativação Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Radioisótopos do Iodo , Mutação/genética , RNA Interferente Pequeno/farmacologia , Receptores da Tireotropina/metabolismo , Simportadores/metabolismo , Neoplasias da Glândula Tireoide/patologia , TransfecçãoRESUMO
A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (alpha(M)beta(2)) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigen-antibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-1, complement C3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications.
Assuntos
Especificidade de Anticorpos , Epitopos/imunologia , Antígeno de Macrófago 1/imunologia , Biblioteca de Peptídeos , Saccharomyces cerevisiae/imunologia , Adesão Celular , Movimento Celular , Humanos , Ligantes , Ativação de Neutrófilo , Neutrófilos/citologia , Neutrófilos/imunologiaRESUMO
Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells.
RESUMO
The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Interleucina-12/genética , Molécula de Adesão da Célula Epitelial , Imunoterapia Adotiva , Neoplasias/genética , Neoplasias/terapia , Antígenos de Neoplasias/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
A subset of thyroid cancers, recurrent differentiated thyroid cancers and anaplastic thyroid cancer (ATC), are difficult to treat by thyroidectomy and systemic therapy. A common mutation in thyroid cancer, BRAFV600E, has targetable treatment options; however, the results have been disappointing in thyroid cancers compared with BRAFV600E melanoma, as thyroid cancers quickly become resistant to BRAFV600E inhibitor (BRAFi). Here, we studied the molecular pathway that is induced in BRAFV600E thyroid cancer cells and patient-derived tumor samples in response to BRAFi, vemurafenib, using RNA-sequencing and molecular analysis. Both inducible response to BRAFi and acquired BRAFi resistance in BRAFV600E thyroid cancer cells showed significant activation of the JAK/STAT pathway. Functional analyses revealed that the combination of BRAFi and inhibitors of JAK/STAT pathway controlled BRAFV600E thyroid cancer cell growth. The Cancer Genome Atlas data analysis demonstrated that potent activation of the JAK/STAT signaling was associated with shorter recurrence rate in patients with differentiated thyroid cancer. Analysis of tumor RNA expression in patients with poorly differentiated thyroid cancer and ATC also support that enhanced activity of JAK/STAT signaling pathway is correlated with worse prognosis. Our study demonstrates that JAK/STAT pathway is activated as BRAFV600E thyroid cancer cells develop resistance to BRAFi and that this pathway is a potential target for anticancer activity and to overcome drug resistance that commonly develops to treatment with BRAFi in thyroid cancer. IMPLICATIONS: Dual inhibition of BRAF and JAK/STAT signaling pathway is a potential therapeutic treatment for anticancer activity and to overcome drug resistance to BRAFi in thyroid cancer.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Janus Quinases/genética , Janus Quinases/metabolismo , Janus Quinases/uso terapêutico , Sulfonamidas/farmacologia , Transdução de Sinais , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição STAT/uso terapêutico , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologia , Mutação , RNA , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular TumoralRESUMO
Helix-helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils.
Assuntos
Proteína gp41 do Envelope de HIV/química , Saccharomyces cerevisiae/química , Técnicas do Sistema de Duplo-Híbrido , HIV/efeitos dos fármacos , Cinética , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/virologiaRESUMO
This study demonstrates the dependence of non-local susceptibility effects on object orientation in gradient echo MRI and the reduction of non-local effects by deconvolution using quantitative susceptibility mapping. Imaging experiments were performed on a 3T MRI system using a spoiled 3D multi-echo GRE sequence on phantoms of known susceptibilities, and on human brains of healthy subjects and patients with intracerebral hemorrhages. Magnetic field measurements were determined from multiple echo phase data. To determine the quantitative susceptibility mapping, these field measurements were deconvolved through a dipole inversion kernel under a constraint of consistency with the magnitude images. Phantom and human data demonstrated that the hypointense region in GRE magnitude image corresponding to a susceptibility source increased in volume with TE and varied with the source orientation. The induced magnetic field extended beyond the susceptibility source and varied with its orientation. In quantitative susceptibility mapping, these blooming artifacts, including their dependence on object orientation, were reduced, and the material susceptibilities were quantified.
Assuntos
Mapeamento Encefálico/métodos , Encéfalo/patologia , Hemorragia Cerebral/patologia , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Imageamento por Ressonância Magnética/métodos , Algoritmos , Artefatos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
This study describes the synthesis of magnetite/amphiphilic polymer composite nanoparticles that can be potentially used simultaneously for cancer diagnosis and therapy. The synthesis method was a one-shot process wherein magnetite nanoparticles were mixed with core-crosslinked amphiphilic polymer (CCAP) nanoparticles, prepared using a copolymer of a urethane acrylate nonionomer (UAN) and a urethane acrylate anionomer (UAA). The CCAP nanoparticles had a hydrophobic core and a hydrophilic exterior with both PEG segments and carboxylic acid groups, wherein the magnetite nanoparticles were coordinated and stabilized. According to DLS data, the ratio of UAN to UAA and the ratio of magnetite to polymer are keys to controlling the size and thus, the stability of the composite nanoparticles. The magnetic measurement indicated that the composite nanoparticles had superparamagnetic properties and high saturation magnetization. The preliminary magnetic resonance imaging showed that the particles produced an enhanced image even when their concentration was as low as 80 microg/ml.
Assuntos
Nanopartículas de Magnetita/uso terapêutico , Nanopartículas/uso terapêutico , Polímeros/química , Animais , Cristalização/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas de Magnetita/química , Teste de Materiais , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMO
The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells.
Assuntos
Imunotoxinas , Linfócitos T , Humanos , Animais , Camundongos , Biomarcadores , Monitorização Fisiológica , Diagnóstico por Imagem , Interleucina-12/genéticaRESUMO
The immunoglobulin (Ig) superfamily is one of the largest families in the vertebrate genome, found most frequently in cell surface molecules. Intercellular adhesion molecule-1 (ICAM-1) contains five extracellular Ig superfamily domains (D1-D5) of which the first domain, D1, is the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1) and human rhinovirus. Despite the modular nature of many Ig superfamily domains with respect to domain folding and ligand recognition, D1 does not fold on its own due to the loss of its interaction with the second domain. The goal of this study was to engineer ICAM-1 D1 by introducing mutations that would stabilize the Ig superfamily domain fold while retaining its ability to bind to LFA-1 and rhinovirus. First, with a directed evolution approach, we isolated mutations in D1 that showed binding to conformation-specific antibodies and the ligand binding domain of LFA-1 called the inserted, or I, domain. Then, with a rational design approach we introduced mutations that contributed to the stability of ICAM-1 D1 in solution. The mutations that restored native folding of D1 in isolation were those that would convert hydrogen bond networks in buried regions into hydrophobic contacts. Notably, for most mutations, identical or similar types of substitutions were found in ICAM-1 molecules of different species and other ICAM family members. The systematic approach demonstrated in this study to engineer a single Ig superfamily fold in ICAM-1 can be broadly applicable to the engineering of modular Ig superfamily domains in other cell surface molecules.
Assuntos
Evolução Molecular Direcionada , Molécula 1 de Adesão Intercelular/química , Engenharia de Proteínas , Dobramento de Proteína , Animais , Células COS , Chlorocebus aethiops , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiaeRESUMO
The enzymes immobilized through yeast surface display (YSD) can be used in in vitro metabolic pathway reconstruction as alternatives to the enzymes isolated or purified through conventional biochemistry methods. They can be easily prepared by growing and collecting yeast cells harboring display constructs. This may provide an economical method for enriching certain enzymes for biochemistry characterization and application. Herein, we took the advantage of one-pot cascade reactions catalyzed by YSD-immobilized enzymes in the mevalonate pathway to produce geraniol in vitro. YSD-immobilized enzymes of 10 cascade reactions for geraniol production, together with optimization of catalytic components, cofactor regeneration, and byproduct removal, achieved a final yield of 7.55 mg L-1 after seven cycles. This study demonstrated that it is feasible to reconstitute a complex multi-enzymatic system for the chemical biosynthesis in vitro by exploiting YSD-immobilized cascade enzymes.
Assuntos
Vias Biossintéticas/fisiologia , Saccharomyces cerevisiae/metabolismo , Monoterpenos Acíclicos/metabolismo , Catálise , Enzimas Imobilizadas/metabolismo , Redes e Vias Metabólicas/fisiologia , Ácido Mevalônico/metabolismo , Complexos Multienzimáticos/metabolismoRESUMO
Obesity is characterized by an excessive body mass, but is also closely associated with metabolic syndrome. And, so far, only limited pharmacological treatments are available for obesity management. Celastrol, a pentacyclic triterpenoid from a traditional Chinese medicine (Tripterygium wilfordii Hook.f.), has shown remarkable potency against obesity, inflammation and cancer, but its high toxicity, low natural abundance and tedious chemical synthesis hindered its translation into clinics. In the present work, a triterpenoid library was screened for compounds with both high natural abundance and structural similarity to celastrol; from this library, glycyrrhetinic acid (GA), a compound present in extremely high yields in Glycyrrhiza uralensis Fisch. ex DC., was selected as a possible scaffold for a celastrol mimic active against obesity. A simple chemical modification of GA resulted in GA-02, a derivative that suppressed 68% of food intake in diet-induced obesity mice and led to 26.4% weight loss in 2 weeks. GA-02 plays a role in obesity treatment by re-activating leptin signaling and reducing systemic and, more importantly, hypothalamic inflammation. GA-02 was readily bioavailable with unnoticeable in vitro and in vivo toxicities. The strategy of scaffold search and modification on the basis of bio-content and structural similarity has proved to be a green, economic, efficient and practical way of widening the medicinal applications of "imperfect" bioactive natural compounds.