RESUMO
Phase segregation of multicomponent lipid bilayers leads to, under phase-coexistence conditions, domain formation, featuring delimitation by essentially one-dimensional borders. (Micro-)phase segregation of bilayers is proposed to influence the physiological behaviour of cell membranes and provides the driving force for lipid-raft formation. Experiments show a maximum in the electrical-conductivity of membranes at the phase-transition point, which has been conjectured to arise from border-nucleated transmembrane-conducting defects or pores. However, recent electroporation experiments on phase-segregated bilayers demonstrate electro-pore detection in the liquid disordered phase (Ld), wherein they diffuse over macroscopic periods without absorption into the liquid ordered phase (Lo). Here, we scrutinise transmembrane-pore formation via molecular dynamics simulations on a multicomponent phase-segregated bilayer. We find that pores created in Lo domains always migrate spontaneously to the Ld phase, via 'recruitment' of unsaturated lipids to the pore's rim to transport the pore to the fluid phase under a large stress-field driving force. Once in Ld domains, pores migrate towards their centre, never returning or pinning to Lo. These findings are explained by thermodynamics. By comparing the free-energy cost for creating pores in the bulk of Ld and Lo membranes, and in the phase-segregated system, we show that it is always more energetically tractable to create pores in Ld domains, independent of the pore size.
RESUMO
T-cell large granular lymphocytic leukemia and chronic lymphoproliferative disorder of natural killer cells are intriguing entities between benign and malignant lymphoproliferation. The molecular pathogenesis has partly been uncovered by the recent discovery of somatic activating STAT3 and STAT5b mutations. Here we show that 43% (75/174) of patients with T-cell large granular lymphocytic leukemia and 18% (7/39) with chronic lymphoproliferative disorder of natural killer cells harbor STAT3 mutations when analyzed by quantitative deep amplicon sequencing. Surprisingly, 17% of the STAT3-mutated patients carried multiple STAT3 mutations, which were located in different lymphocyte clones. The size of the mutated clone correlated well with the degree of clonal expansion of the T-cell repertoire analyzed by T-cell receptor beta chain deep sequencing. The analysis of sequential samples suggested that current immunosuppressive therapy is not able to reduce the level of the mutated clone in most cases, thus warranting the search for novel targeted therapies. Our findings imply that the clonal landscape of large granular lymphocytic leukemia is more complex than considered before, and a substantial number of patients have multiple lymphocyte subclones harboring different STAT3 mutations, thus mimicking the situation in acute leukemia.
Assuntos
Artrite Reumatoide/genética , Biomarcadores/análise , Evolução Clonal/genética , Leucemia Linfocítica Granular Grande/genética , Mutação/genética , Fator de Transcrição STAT3/genética , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Seguimentos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Leucemia Linfocítica Granular Grande/tratamento farmacológico , Leucemia Linfocítica Granular Grande/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Adulto JovemRESUMO
Cell-penetrating peptides (CPPs) enable the transport of nanoparticles through cell membranes. Using molecular simulations, we conduct an in-depth investigation into the thermodynamic forces governing the passive translocation of CPP-coated nanoparticles across lipid bilayers, contrasting their behavior with that of bare particles to dissect the contribution of the peptides. Our analysis unveils a distinctive two-stage translocation mechanism, where the adsorption energy of the particles overcomes the cost of forming a hydrophilic transmembrane pore. Proper evaluation of the translocation mechanisms is only possible when using two reaction coordinates, in particular, one that explicitly includes the density of the lipids on the binding site of the particle. An analysis of adsorption and activation free energies in terms of a simple kinetic model provides a clearer understanding of the CPP effect. Experimental validation using nonendocytic cells confirms the superior membrane permeation of CPP-coated particles. Our findings have implications for the rational design of more efficient cell-permeating particles.
Assuntos
Peptídeos Penetradores de Células , Nanopartículas , Peptídeos Penetradores de Células/química , Bicamadas Lipídicas/química , Membrana Celular/química , TermodinâmicaRESUMO
The t(5;11)(q35;p15.4) is a clinically significant marker of poor prognosis in acute myeloid leukemia (AML), which is difficult to detect due to sub-telomeric localization of the breakpoints. To facilitate the detection of this rearrangement, we studied NUP98-NSD1 transcript variants in patients with the t(5;11) using paired-end RNA sequencing and standard molecular biology techniques. We discovered three NUP98-NSD1 transcripts with two fusion junctions (NUP98 exon 11-12/NSD1 exon 6), alternative 5' donor site in NUP98 exon 7, and NSD1 exon 7 skipping. Two of the transcripts were in-frame and occurred in all t(5;11) samples (N = 5). The exonic splicing events were present in all samples (N = 23) regardless of the NUP98-NSD1 suggesting that these novel splice events are unassociated with t(5;11). In conclusion, we provide evidence of two different NUP98-NSD1 fusion transcripts in adult AML, which result in functional proteins and represent suitable molecular entities for monitoring t(5;11) AML patients.