Biology subjects

Nilsson, L.

Publications and source records attributed to Nilsson, L..

7 recordsLinked to original sources

bioRxiv2026

Structural heterogeneity in mRNA-LNP subpopulations revealed by AF4-SAXS: implications for cargo loading and cell transfection

Lipid nanoparticles are the leading platform for the delivery of nucleic acid therapeutics, yet their structural complexity remains a significant barrier to achieve rational design and predictable function. Part of this complexity arises from the non-equilibrium assemblies that are difficult to identify using ensemble average techniques given the substantial heterogeneity in all properties. Aiming to overcome the limitations of traditional characterization methods, we combined asymmetric flow field-flow fractionation with in-line small-angle X-ray scattering and spectroscopic analyses, nanoflow cytometry, and cryo-EM to construct detailed structural models of mRNA-loaded nanoparticles formulated with different amounts of mRNA loading (N/P ratios of 3 and 6). This combination of techniques revealed that microfluidic formulation produces structurally diverse nanoparticle subpopulations differing in size, anisotropy, and cargo loading. Notably, these variations extend to the particle internal organization: spheroidal geometries display densely loaded mRNA cores, whereas bleb-like morphologies exhibit reduced mRNA content relative to the lipid amount within segregated domains at the core. NanoFCM further shows that the N/P ratio modulates cargo distribution across individual nanoparticles, with N/P=6 yielding a more uniform mRNA copy number per particle across subpopulations than N/P=3. These differences resulted in higher transfection efficacies for the N/P=6 formulation, highlighting core organization and loading homogeneity as key parameters for efficacious delivery. Together, these results establish a direct link between LNP architecture, internal organization, cargo distribution, and transfection efficiency, underscoring the importance of accounting for heterogeneity in the rational design of nucleic acid delivery systems.

biophysics↗

bioRxiv2023

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eyes uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients succumb to recurrent disease after two years. This study aimed to provide a path towards enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA sequencing of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR sequencing, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

cancer biology↗

bioRxiv2023

IL-17 activation undermines longevity and stress tolerance by inhibiting a protective transcriptional network

Aberrant cytokine secretion contributes to the pathogenesis of autoimmune diseases and age-related disorders, but the molecular mechanism underlying this is not entirely clear. Here, we elucidate how interleukin-17 (IL-17) overactivation shortens lifespan and damages defense mechanisms against stress in C. elegans. Our analysis reveals that NHR-49, the C. elegans ortholog of human PPAR and HNF4, is the central component in the transcriptional network undermined by increased IL-17 signaling. Both NHR-49 and its coactivator MDT-15 physically interact with the downstream components of IL-17 pathway, and their expression is significantly decreased when IL-17 signaling is enhanced. IL-17 overactivation also induces the expression and nucleus entry of the C. elegans ortholog of NF-{kappa}B inhibitor NFKI-1/I{kappa}B{zeta} to repress the activity of transcriptional coactivator MDT-15 and CBP-1. IL-17 signaling acts on neurons to modulate the activity of NFKI-1/I{kappa}B{zeta} and NHR-49. In addition, persistent IL-17 activation decreases the expression of HLH-30/TFEB, leading to the reduced transcription of lysosomal lipase genes in the distal tissues. All these jointly contribute to the increased sensitivity to oxidative stress of animals with enhanced IL-17 signaling. Collectively, our work illustrates a transcription system undermined by IL-17 overactivation in the animals without NF-{kappa}B, and provides mechanistic insight into the pathogenesis of abnormal IL-17 secretion.

cell biology↗

bioRxiv2021

A spindle and thread-mechanism unblocks translation of N-terminally disordered proteins

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.

biochemistry↗

bioRxiv2019

INTERACTION OF QUERCETIN AND EPIGALLOCATECHIN GALLATE (EGCG) AGGREGATES WITH PANCREATIC LIPASE UNDER SIMPLIFIED INTESTINAL CONDITIONS

Diets rich in flavonoids have been related with low obesity rates, which could be related with their potential to inhibit pancreatic lipase, the main enzyme of fat assimilation. Some flavonoids can aggregate in aqueous medium suggesting that the inhibition mechanism could occur on both molecular and colloidal levels. This study investigates the interaction of two flavonoid aggregates, quercetin and epigallocatechin gallate (EGCG), with pancreatic lipase under simplified intestinal conditions. The stability and the morphology of these flavonoid aggregates were studied in four different solutions: Control (water), salt, low lipase concentration and high lipase concentration. Particles were found by optical microscopy in almost all the solutions tested, except EGCG-control. The results show that the precipitation rate decreases for quercetin and increases for EGCG in salt solution and that lipase stabilize quercetin aggregates. In addition, both flavonoids were shown to precipitate together with pancreatic lipase resulting in a sequestering of the enzyme.

biochemistry↗

bioRxiv2019

INTERACTION BETWEEN MYRICETIN AGGREGATES AND LIPASE UNDER SIMPLIFIED INTESTINAL CONDITIONS

Myricetin, a flavonoid found in the plant kingdom, has previously been identified as a food molecule with beneficial effects against obesity. This property has been related with its potential to inhibit lipase, the enzyme responsible of fat digestion. In this study we investigate the interaction between myricetin and lipase under simplified intestinal conditions from a colloidal point of view. The results show that myricetin form aggregates in aqueous medium and under simplified intestinal condition, where it was found that lipase is in its monomeric form. Although lipase inhibition by myricetin at a molecular level has been reported previously, the results of this study suggest that myricetin aggregates inhibit lipase by a sequestering mechanism as well. The size of these aggregates was determined to be in the range of a few nm to >200 nm.

biochemistry↗

bioRxiv2017

Two distinct DNA sequences recognized by transcription factors represent enthalpy and entropy optima

Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima in the Gibbs free energy of binding ({Delta}G). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar {Delta}G. However, the interaction with the CAA sequence was driven by change in enthalpy ({Delta}H), whereas the TCG site was bound with similar affinity due to smaller loss of entropy ({Delta}S). The common presence of at least two local optima is general to all macromolecular interactions, as {Delta}G depends on two partially independent variables {Delta}H and {Delta}S according to the central equation of thermodynamics, {Delta}G = {Delta}H - T{Delta}S.

biochemistry↗