RESUMEN
Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.
Asunto(s)
Leucemia Mielomonocítica Crónica , Síndromes Mielodisplásicos , Humanos , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Leucemia Mielomonocítica Crónica/genética , Leucemia Mielomonocítica Crónica/metabolismo , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Células Madre Hematopoyéticas/metabolismo , Monocitos , Células ClonalesRESUMEN
Peptidomimetics, such as oligo-N-alkylglycines (peptoids), are attractive alternatives to traditional cationic cell-penetrating peptides (such as R9) due to their robust proteolytic stability and reduced cellular toxicity. Here, monomeric N-alkylglycines, incorporating amino-functionalized hexyl or triethylene glycol (TEG) side chains, were synthesized via a three-step continuous-flow reaction sequence, giving the monomers N-Fmoc-(6-Boc-aminohexyl)glycine and N-Fmoc-((2-(2-Boc-aminoethoxy)ethoxy)ethyl)glycine in 49% and 41% overall yields, respectively. These were converted into oligomers (5, 7, and 9-mers) using an Fmoc-based solid-phase protocol and evaluated as cellular transporters. Hybrid oligomers, constructed of alternating units of the aminohexyl and amino-TEG monomers, were non-cytotoxic and exhibited remarkable cellular uptake activity compared to the analogous fully TEG or lysine-like compounds.
Asunto(s)
Péptidos de Penetración Celular/química , Glicina/química , Lisina/química , Microondas , Peptoides/química , Peptoides/farmacocinética , Polietilenglicoles/química , Transporte Biológico , Péptidos de Penetración Celular/farmacocinética , Células Cultivadas , Diseño de Fármacos , Células HEK293 , Células HeLa , Humanos , Peptidomiméticos/química , Distribución TisularRESUMEN
DPANN archaea are a diverse group of microorganisms characterised by small cells and reduced genomes. To date, all cultivated DPANN archaea are ectosymbionts that require direct cell contact with an archaeal host species for growth and survival. However, these interactions and their impact on the host species are poorly understood. Here, we show that a DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. During these interactions, the nanohaloarchaeon appears to enter, or be engulfed by, the host cell. Our results provide experimental evidence for a predatory-like lifestyle of an archaeon, suggesting that at least some DPANN archaea may have roles in controlling host populations and their ecology.
Asunto(s)
Halorubrum , Simbiosis , Halorubrum/genética , Halorubrum/fisiología , Archaea/genética , Archaea/fisiología , Nanoarchaeota/genética , Nanoarchaeota/fisiología , Genoma Arqueal , FilogeniaRESUMEN
Polymeric styrene microspheres have a great potential at the interface of chemistry and biology. The progress of the synthetic development of multifunctional microspheres and their use as delivery agents of different biomolecules into cells is discussed. Their multifunctional properties open a wide range of applications from intracellular real-time sensors, to the use of microspheres as catalysts performing exogenous chemistry within cells.
Asunto(s)
Materiales Biocompatibles , Nanopartículas , Polímeros/administración & dosificación , Polímeros/químicaRESUMEN
Screening of a 65,536-member one-bead-one-compound (OBOC) combinatorial library of glycopeptide dendrimers of structure ((betaGal)(n)(+1)X(8)X(7)X(6)X(5))(2)DapX(4)X(3)X(2)X(1)(beta-Gal)(m) (betaGal=beta-galactosyl-thiopropionic acid, X(8-1)=variable amino acids, Dap=l-2,3-diaminopropionic acid, n, m=0, or 1 if X(8)=Lys resp. X(1)=Lys) for binding of Jurkat cells to the library beads in cell culture, resynthesis and testing lead to the identification of dendrimer J1 (betaGal-Gly-Arg-His-Ala)(2)Dap-Thr-Arg-His-Asp-CysNH(2) and related analogues as delivery vehicles. Cell targeting is evidenced by FACS with fluorescein conjugates such as J1F. The colchicine conjugate J1C is cytotoxic with LD(50)=1.5 microM. The beta-galactoside groups are necessary for activity, as evidenced by the absence of cell-binding and cytotoxicity in the non-galactosylated, acetylated analogue AcJ1F and AcJ1C, respectively. The pentagalactosylated dendrimer J4 betaGal(4)(Lys-Arg-His-Leu)(2)Dap-Thr-Tyr-His-Lys(betaGal)-Cys) selectively labels Jurkat cell as the fluorescein derivative J4F, but its colchicine conjugate J4C lacks cytotoxicity. Tubulin binding assays show that the colchicine dendrimer conjugates do not bind to tubulin, implying intracellular degradation of the dendrimers releasing the active drug.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Colchicina/análogos & derivados , Dendrímeros/química , Glicopéptidos/química , Línea Celular Tumoral , Células Cultivadas , Colchicina/química , Colchicina/farmacología , Dendrímeros/farmacología , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Glicopéptidos/farmacología , Humanos , Células Jurkat , Modelos Biológicos , Estructura MolecularRESUMEN
[structure: see text] A 15 625-membered peptide dendrimer combinatorial library was acylated with an alpha-C-fucosyl residue at its four N-termini and screened for binding to fucose-specific lectins. Dendrimer FD2 (Fuc-alpha-CH2CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2 was identified as a potent ligand against Ulex europaeus lectin UEA-I (IC50 = 11 microM) and Pseudomonas aeruginosa lectin PA-IIL (IC50 = 0.14 microM).
Asunto(s)
Bases de Datos de Proteínas , Dendrímeros/química , Dendrímeros/metabolismo , Glicopéptidos/química , Glicopéptidos/metabolismo , Lectinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Fucosa/química , Ligandos , Estructura Molecular , Unión ProteicaRESUMEN
Nano materials are commonly functionalized to boost their physicochemical properties. However, there is little known about the impact of these modifications on cellular systems. Herein, we synthesized eight types of polymeric nanoparticles (NPs) bearing different functional groups, and investigated their effects on interactions with cellular membranes. As models for particle membrane interactions, hemolysis assays using human red blood cells and culture with A549 cells were utilized. Under protein-free conditions, the NPs showed a wide distribution of zeta potentials (ζPs) which showed a good correlation with their hemolytic potential. However, in the presence of serum or lung lining fluid, the ζPs of all NPs coalesced towards a single common negative value and showed neither hemolytic activity nor cytotoxicity to A549 cells. Lipase and protease treatment of the coronated particles did not restore their reactivity. These result simply proves that particle functionalization influences the stability of the particle corona which, if intact, prevents hemolytic activity and membrane disrupture.
Asunto(s)
Fenómenos Electromagnéticos , Hemólisis/efectos de los fármacos , Nanopartículas/química , Polímeros/química , Propiedades de Superficie , Línea Celular Tumoral , Eritrocitos/citología , Humanos , Interleucina-8/análisis , Interleucina-8/metabolismo , Nanopartículas/toxicidad , Polímeros/toxicidad , Proteínas/química , Tensoactivos/químicaRESUMEN
Nanotechnology is finding ever increasing application in the life science arena where nanoparticles can be used to deliver cargoes in cells. However, a clear understanding of the relationship between the chemical properties of the particle and its uptake efficiency is lacking. Herein, the effects on particle cellular uptake following modification with a variety of spacers, all bearing a positive charge, but differing in length, and the influence on formation of the protein corona are investigated. Although no significant differences in the composition of the protein corona are detected, the spacer length influences the cellular uptake of the nanoparticles. These findings will allow the target-orientated functionalisation of particles to increase the specificity of cellular uptake.
Asunto(s)
Endocitosis , Nanopartículas/química , Polímeros/metabolismo , Fluorescencia , Células HeLa , Humanos , Tamaño de la Partícula , Electricidad EstáticaRESUMEN
We have developed miniaturized heterogeneous Pd(0)-catalysts (Pd(0)-microspheres) with the ability to enter cells, stay harmlessly within the cytosol and mediate efficient bioorthogonal organometallic chemistries (e.g., allylcarbamate cleavage and Suzuki-Miyaura cross-coupling). This approach is a major addition to the toolbox available for performing chemical reactions within cells. Here we describe a full protocol for the synthesis of the Pd(0)-microspheres from readily available starting materials (by the synthesis of size-controlled amino-functionalized polystyrene microspheres), as well as for their characterization (electron microscopy and palladium quantitation) and functional validation ('in solution' and 'in cytoplasm' conversions). From the beginning of the synthesis to functional evaluation of the catalytic device requires 5 d of work.
Asunto(s)
Bioquímica/métodos , Citosol/química , Nanopartículas del Metal/química , Microesferas , Paladio/química , Poliestirenos/síntesis química , Células HeLa , Humanos , Microscopía Electrónica , Poliestirenos/químicaRESUMEN
Many important intracellular biochemical reactions are modulated by transition metals, typically in the form of metalloproteins. The ability to carry out selective transformations inside a cell would allow researchers to manipulate or interrogate innumerable biological processes. Here, we show that palladium nanoparticles trapped within polystyrene microspheres can enter cells and mediate a variety of Pd(0)-catalysed reactions, such as allylcarbamate cleavage and Suzuki-Miyaura cross-coupling. The work provides the basis for the customization of heterogeneous unnatural catalysts as tools to carry out artificial chemistries within cells. Such in cellulo synthesis has potential for a plethora of applications ranging from cellular labelling to synthesis of modulators or inhibitors of cell function.
Asunto(s)
Nanopartículas del Metal/química , Paladio/química , Carbamatos/química , Catálisis , Células HeLa , Humanos , Poliestirenos/químicaRESUMEN
The fucose-specific lectin LecB is implicated in tissue binding and biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa, which causes severe respiratory tract infections mainly in immunocompromised patients or cancer patients undergoing chemotherapy. With a view to developing multivalent LecB inhibitors as novel antibacterial agents, a combinatorial library containing 15 625 tetravalent C-fucosyl peptide dendrimers with the basic structure (CFuc-X(6)X(5)X(4))(4)(LysX(3)X(2)X(1))(2)LysIleHisNH(2) (CFuc=alpha-L-fucosyl acetic acid, X(1-6)=amino acids, Lys=lysine branching) was screened for lectin binding using on-bead binding assays. Ten tetravalent and three octavalent dendrimers derived from the identified sequences were prepared by solid-phase peptide synthesis (SPPS), cleaved from the resin, and purified by preparative HPLC. Relative affinities of these soluble ligands to LecB were determined by an enzyme-linked lectin assay (ELLA). Strong binding was observed for tetravalent and octavalent ligands, with up to 440-fold enhancement in potency over fucose for the octavalent cationic dendrimer 2G3 (CFuc-LysPro)(8)(LysLeuPhe)(4)(LysLysIle)(2)LysHisIleNH(2)). Mono- and divalent controls showed affinities similar to fucose, highlighting the importance of multivalency for binding. Docking studies showed that the C-fucosyl group of the dendrimers can adopt the same binding mode as fucose itself, with the peptide arms protruding from the binding pocket and establishing specific contacts with the lectin.
Asunto(s)
Dendrímeros/síntesis química , Glicopéptidos/química , Lectinas/química , Pseudomonas aeruginosa/química , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Dendrímeros/química , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , Rodaminas/químicaRESUMEN
The human pathogenic bacterium Pseudomonas aeruginosa produces a fucose-specific lectin, LecB, implicated in tissue attachment and the formation of biofilms. To investigate if LecB inhibition disrupts these processes, high-affinity ligands were obtained by screening two 15,536-member combinatorial libraries of multivalent fucosyl-peptide dendrimers. The most potent LecB-ligands identified were dendrimers FD2 (C-Fuc-LysProLeu)(4)(LysPheLysIle)(2)LysHisIleNH(2) (IC(50) = 0.14 microM by ELLA) and PA8 (OFuc-LysAlaAsp)(4)(LysSerGlyAla)(2)LysHisIleNH(2) (IC(50) = 0.11 microM by ELLA). Dendrimer FD2 led to complete inhibition of P. aeruginosa biofilm formation (IC(50) approximately 10 microM) and induced complete dispersion of established biofilms in the wild-type strain and in several clinical P. aeruginosa isolates. These experiments suggest that LecB inhibition by high-affinity multivalent ligands could represent a therapeutic approach against P. aeruginosa infections by inhibition of biofilm formation and dispersion of established biofilms.