Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition.

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

SARS-CoV-2 IgG Spike antibody levels and avidity in natural infection or following vaccination with mRNA-1273 or BNT162b2 vaccines.

Certain aspects of the immunogenicity and effectiveness of the messenger ribonucleic acid (mRNA) vaccines (mRNA-1273 and BNT162b2) developed in response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are still uncharacterized. Serum or plasma samples from healthy donor recipients of either vaccine (BNT162b2 n = 53, mRNA-1273 n = 49; age 23-67), and individuals naturally infected with SARS-CoV-2 (n = 106; age 18-82) were collected 0-2 months post-infection or 1- and 4 months after second dose of vaccination. Anti-Spike antibody levels and avidity were measured via an enzyme-linked immunosorbent assay (ELISA). Overall, vaccination induced higher circulating anti-Spike protein immunoglobulin G (IgG) antibody levels and avidity compared to infection at similar time intervals. Both vaccines produced similar anti-Spike IgG concentrations at 1 month, while mRNA-1273 demonstrated significantly higher circulating antibody concentrations after 4 months. mRNA-1273 induced significantly higher avidity at month 1 compared to BNT162b2 across all age groups. However, the 23-34 age group was the only group to maintain statistical significance by 4 months. Male BNT162b2 recipients were approaching statistically significant lower anti-Spike IgG avidity compared to females by month 4. These findings demonstrate enhanced anti-Spike IgG levels and avidity following vaccination compared to natural infection. In addition, the mRNA-1273 vaccine induced higher antibody levels by 4 months compared to BNT162b2.

High-throughput translational profiling with riboPLATE-seq.

Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages anti-rRNA ribosome immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases.

Endothelial Notch signaling directly regulates the small GTPase RND1 to facilitate Notch suppression of endothelial migration.

To control sprouting angiogenesis, endothelial Notch signaling suppresses tip cell formation, migration, and proliferation while promoting barrier formation. Each of these responses may be regulated by distinct Notch-regulated effectors. Notch activity is highly dynamic in sprouting endothelial cells, while constitutive Notch signaling drives homeostatic endothelial polarization, indicating the need for both rapid and constitutive Notch targets. In contrast to previous screens that focus on genes regulated by constitutively active Notch, we characterized the dynamic response to Notch. We examined transcriptional changes from 1.5 to 6 h after Notch signal activation via ligand-specific or EGTA induction in cultured primary human endothelial cells and neonatal mouse brain. In each combination of endothelial type and Notch manipulation, transcriptomic analysis identified distinct but overlapping sets of rapidly regulated genes and revealed many novel Notch target genes. Among the novel Notch-regulated signaling pathways identified were effectors in GPCR signaling, notably, the constitutively active GTPase RND1. In endothelial cells, RND1 was shown to be a novel direct Notch transcriptional target and required for Notch control of sprouting angiogenesis, endothelial migration, and Ras activity. We conclude that RND1 is directly regulated by endothelial Notch signaling in a rapid fashion in order to suppress endothelial migration.

The Role of Methyl Groups in the Early Stage of Thermal Polymerization of Polycyclic Aromatic Hydrocarbons Revealed by Molecular Imaging.

The initial thermal reactions of aromatic hydrocarbons are relevant to many industrial applications. However, tracking the growing number of heavy polycyclic aromatic hydrocarbon (PAH) products is extremely challenging because many reactions are unfolding in parallel from a mixture of molecules. Herein, we studied the reactions of 2,7-dimethylpyrene (DMPY) to decipher the roles of methyl substituents during mild thermal treatment. We found that the presence of methyl substituents is key for reducing the thermal severity required to initiate chemical reactions in natural molecular mixtures. A complex mixture of thermal products including monomers, dimers, and trimers was characterized by NMR, mass spectrometry, and noncontact atomic force microscopy (nc-AFM). A wide range of structural transformations including methyl transfer and polymerization reactions were identified. A detailed mechanistic understanding on the roles of H radicals during the polymerization of polycyclic aromatic hydrocarbons was obtained.

Elucidating the Geometric Substitution of Petroporphyrins by Spectroscopic Analysis and Atomic Force Microscopy Molecular Imaging.

Determination of the molecular structures of petroporphyrins has been crucial to understand the diagenetic pathways and maturation of petroleum. However, these studies have been hampered by their structural complexity and the challenges associated with their isolation. In comparison to the skeletal macrocyclic structures, much less is known about the substitutions, which are more sensitive to the maturation and diagenesis pathways. While these isolated vanadyl petroporphyrins largely consist of etioporphyrin and deoxophylloerythroetioporphyrin as expected, surprisingly, we find evidence that one or a few ß hydrogens are present in petroporphyrins of low carbon numbers using a combination of ultraviolet-visible spectroscopy, Fourier transform ion cyclotron resonance mass spectrometry, and non-contact atomic force microscopy. Petroporphyrins with ß hydrogens were not anticipated on the basis of their biological precursors. The data support dealkylation under catagenesis but not transalkylation or random alkylation of the ß and meso positions, despite the fact that more complex porphyrin structures are formed.

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knockout Mice.

FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome.

Cardiac recovery via extended cell-free delivery of extracellular vesicles secreted by cardiomyocytes derived from induced pluripotent stem cells.

The ability of extracellular vesicles (EVs) to regulate a broad range of cellular processes has recently been exploited for the treatment of diseases. For example, EVs secreted by stem cells injected into infarcted hearts can induce recovery through the delivery of stem-cell-specific miRNAs. However, the retention of the EVs and the therapeutic effects are short-lived. Here, we show that an engineered hydrogel patch capable of slowly releasing EVs secreted from cardiomyocytes derived from induced pluripotent stem (iPS) cells reduced arrhythmic burden, promoted ejection-fraction recovery, decreased cardiomyocyte apoptosis 24 hours after infarction, and reduced infarct size and cell hypertrophy 4 weeks post-infarction when implanted onto infarcted rat hearts. We also show that the EVs are enriched with cardiac-specific miRNAs known to modulate cardiomyocyte-specific processes. The extended delivery of EVs secreted from iPS-cell-derived cardiomyocytes into the heart may help understand heart recovery and treat heart injury.