Elucidating mysteries of phase-segregated membranes: mobile-lipid recruitment facilitates pores' passage to the fluid phase.

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.

The analysis of clonal diversity and therapy responses using STAT3 mutations as a molecular marker in large granular lymphocytic leukemia.

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.

Membrane Binding Strength vs Pore Formation Cost─What Drives the Membrane Permeation of Nanoparticles Coated with Cell-Penetrating Peptides?

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.

Predictive and dynamic signature for anti-angiogenics in combination with PD-1 inhibitor in soft-tissue sarcoma: correlative studies linked to the IMMUNOSARC trial.

PURPOSE: IMMUNOSARC trial combined an anti-angiogenic agent (sunitinib) with a PD-1 inhibitor (nivolumab) in advanced sarcomas. Here we present the first correlative studies of the STS cohort enrolled in this trial. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded (FFPE) and peripheral blood samples were collected at baseline and week 13. FFPE were used for transcriptomics and multiplex immunofluorescence, while peripheral blood samples were used for multiplexed immunoassays. Flow cytometry and Luminex assays were performed to validate translational findings in tumor-isolated cells and peripheral blood mononuclear cells derived from patients. RESULTS: The density of intratumoral CD8+ T cells, measured by multiplexed immuno-phenotyping, was significantly increased after treatment. This augment was accompanied by the dynamic significant increase in gene expression of CD86, CHI3L1, CXCL10, CXCL9, LAG3, and VCAM1, and the decrease in the expression levels of NR4A1. In peripheral blood, 12 proteins were significantly modulated by treatment at W13. A score integrating the dynamic expression of the 7 genes and the 12 soluble factors separated two groups with distinct progression-free survival (PFS): 4.1 months (95% CI 3.5-NR) vs 17 months (95% CI 12.0 - NR), p=0.014. This molecular score was predictive of PFS when applied to the normalized data determined in the baseline samples. CONCLUSIONS: Treatment with sunitinib and nivolumab inflamed the sarcoma microenvironment, increasing CD8+ T cell density and the expression of several genes/ proteins with relevance in the response to PD-1 inhibitors. A molecular signature identified two groups of patients with distinct PFS for the combination of anti-angiogenics plus PD-1 inhibitor.

Chimeric NUP98-NSD1 transcripts from the cryptic t(5;11)(q35.2;p15.4) in adult de novo acute myeloid leukemia.

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.