Proceedings of the National Academy of Sciences of the United States of America最新文献

Gene delivery has emerged as a groundbreaking technique for altering gene expression, offering new possibilities in treating a vast array of diseases. We report a layer-by-layer elastin-like polypeptide nucleic acid nanoparticle (LENN) system for mRNA delivery as an attractive alternative to viral vectors and lipid nanoparticle (LNP) systems. This study focuses on determining the physical characteristics of LENN bearing mRNA cargo and assessing their biological performance in T24 bladder tumor cells. Our data show that mRNA encoding luciferase forms stable 30 to 130 nm LENN particles via batch mixing to efficiently encapsulate the mRNA strands, are resistant to heparin challenge, and are capable of storage at -20 °C for 3 d as lyophilized powders while retaining full biological activity after rehydration. We also demonstrate that LENN targeted to the epidermal growth factor receptor (EGFR) can efficiently deliver the mRNA cargo to the cytosol of EGFR+ T24 human bladder cancer cells via clathrin-mediated endocytosis where it is translationally active. Lipid profiling analyses show the significant role that upregulated phospholipid biosynthesis plays in nanoparticle internalization and endosomal escape compared to untargeted LENN, indicating the importance of the clathrin pathway in contributing to the delivery efficiency of LENN. Endocytosis inhibition experiments further support the involvement of the clathrin pathway. These findings highlight the compelling features of LENN with respect to their size, in vitro and in vivo targetability, mRNA encapsulation efficiency, complex stability, gene expression, and "green" manufacturability, offering an attractive alternative to existing methods for gene delivery.

Ductal carcinoma in situ (DCIS) is a precursor mammary lesion characterized by abnormal epithelial cells in mammary ducts that remain confined to the luminal space. Not all DCIS becomes invasive, and no strategy currently exists in patients to stratify indolent DCIS from DCIS at risk of progression. Several studies of human DCIS and breast cancer suggest that TP53 mutations occur early in DCIS. However, TP53 mutation alone is insufficient for DCIS formation or transformation to invasive disease. Using an autochthonous somatic mouse model of Trp53^R245W induced breast cancer (equivalent to the TP53^R248W hotspot mutation in humans), we identified DCIS lesions. Through exome sequencing and low-pass whole-genome sequencing, we identified additional genomic changes shared between DCIS and invasive tumors. This comparison nominated seven murine candidate genes, with eight human orthologs. We assessed the cooperativity of these genes with mutant TP53 in human breast cells using acinar morphogenesis and migration assays. Overexpression of TMEM267, which encodes a transmembrane protein overexpressed in 22% of TP53 missense mutant breast cancer cases, in cells with mutant TP53 caused a significant increase in the filled duct, DCIS-like phenotype. We nominate TMEM267 as a cooperating event with mutant TP53 in DCIS progression.

Immune cell populations are dysregulated in COVID-19 for currently unknown reasons: Plasmacytoid dendritic cell (pDC) populations are reduced, thus hampering antiviral responses. CD8⁺ T cell populations are reduced, the level of which has emerged as an index of disease severity. Recent work has shown that the proteome of SARS-CoV-2 is a rich reservoir of antimicrobial peptide-like sequence motifs (xenoAMPs) which can chaperone and organize dsRNA for amplified Toll-Like Receptor 3 (TLR3)-mediated inflammation in vitro and in vivo. Here, we demonstrate that proteolytic digestion of the SARS-CoV-2 spike protein by host trypsin-like serine proteases directly produces xenoAMPs. Synchrotron Small Angle X-ray Scattering, mass spectrometry, and a theoretical analysis based on continuum membrane elasticity show that proteolytically generated xenoAMPs from SARS-CoV-2 proteins in vitro and machine learning-predicted high-scoring xenoAMPs all induce negative Gaussian curvature (NGC) necessary for pore formation in membranes. We find that xenoAMPs alone as well as xenoAMPs synergistically with endogenous AMP LL-37 can induce NGC in membranes. A computational analysis of immune cells with morphologically complex shapes (e.g., pDC, CD8⁺, and CD4⁺ T cells) suggests that surfaces with high local NGC can concentrate AMP-like sequences and promote selective membrane disruption. Consistent with this hypothesis, experiments with freshly isolated human peripheral blood mononuclear cells confirm that viable pDCs, DCs, and T cells are significantly depleted after xenoAMP exposure, in contrast to monocytes and neutrophils, the immune cell subsets with spheroidal morphology. Structural data from Omicron variant xenoAMP homologs indicate reduced pore formation, consistent with clinical observations of reduced T cell cytopenia in Omicron variant infections.

Post-acute sequelae of COVID-19 (PASC) encompasses persistent neurological disease, including olfactory and cognitive dysfunction. The basis for this dysfunction is poorly understood. Here, we report neurological dysfunction for at least 120 d postinfection in mice infected with a virulent nonneurotropic mouse-adapted SARS-CoV-2. Long after recovery from nasal infection, we observed diminished tyrosine hydroxylase expression in olfactory bulb glomeruli and in substantia nigra. Similar changes were observed in brains of COVID-19 deceased patients. Vulnerability of dopaminergic neurons in these brain areas was accompanied by increased proinflammatory cytokines, and neurobehavioral changes. RNAseq analysis unveiled persistent microglia activation, similar to human neurodegenerative diseases. Treatment with antivirals (nirmatrelvir and molnupiravir) at the time of infection minimally prevented neurological abnormalities, consistent with patient data. In contrast, antivirals plus corticosteroids resulted in nearly complete recovery of neurological function. Remarkably, initiation of combined therapy even three days after infection improved outcomes. Together these results demonstrate that neurological dysfunction in SARS-CoV-2 infected mice resembles human neurodegenerative disease and indicate that minimizing inflammation early after SARS-CoV-2 infection may be critical for decreasing neurological PASC. The requirement for decreasing inflammation soon after infection may also explain why antiviral therapy has had inconsistent effects in patients.

Aging is associated with the decline of many bodily functions including motor coordination. Aging-related impairment in motor coordination can result in falls, which reduce independence, health span, and quality of life in the elderly. To study the neural mechanisms that underlie this decline, we studied aged mice and observed a progressive decline in motor coordination on multiple motor coordination assays. The cerebellum is critically involved in motor coordination and balance, and cerebellar Purkinje cells play an important role in modulating motor output and coordinated movements. Purkinje cells fire high-frequency and high-regularity action potentials in healthy young adult mice. We wondered whether this firing remained stable across lifespan in aging mice. We performed juxtacellular recordings from Purkinje cells in acute cerebellar slices and observed a reduction in the rate of firing in aged animals without changes in firing regularity. To understand whether reduced Purkinje cell firing rate caused impaired motor performance in aged mice, we used chemogenetics to modulate Purkinje cell firing. Reducing Purkinje cell firing rates in young mice impaired motor performance, while elevating Purkinje cell firing rates in aged mice improved motor performance. Our results suggest that Purkinje cell firing rate impacts motor coordination and that the aging-related reduction of Purkinje cell firing rate that we observed contributes to impaired motor coordination and could contribute to declining health span and quality of life in the elderly.