Biology subjects

Boone, M.

Publications and source records attributed to Boone, M..

13 recordsLinked to original sources

bioRxiv2026

Intraventricular infusion to circumvent the blood-brain barrier to gemcitabine.

The poor prognosis of brain tumors, including IDH-wild-type glioblastoma (GB), as well as brain and leptomeningeal metastases, is partly related to the blood-brain barrier (BBB), which limits the delivery of hydrophilic anticancer drugs to the tumor site and surrounding brain parenchyma. Early studies using vital dyes demonstrated that intracranial injection could bypass the BBB in cats. We confirmed that, in guinea pigs, the vital dye Bleu Patente V diffused efficiently into the brain after a bolus intracranial injection, whereas the brain remained unstained after intravenous administration. Similarly, brain concentrations of the hydrophilic anticancer drug gemcitabine were significantly higher following intracranial injection than after intravenous administration. Consistent with these findings, Bleu Patente penetrated deeply into the cerebral cortex of sheep after a 24-hour intraventricular infusion. At the end of a 24-hour intraventricular infusion of 20 mg gemcitabine in sheep, mean gemcitabine concentrations reached 1,415 {micro}g/L in cerebrospinal fluid and 850 {micro}g/kg in brain tissue. These concentrations exceeded the IC90 values of gemcitabine for A172, U87-MG, and U118-MG human glioblastoma cell lines, as determined in vitro after 24 hours of incubation. We hypothesize that Bleu Patente dye and gemcitabine circumvent the blood-brain barrier (BBB) by utilizing the glymphatic system. Tolerance of a single 24-hour intraventricular infusion of gemcitabine at doses of 5, 10, and 20 mg was good. Taken together, these encouraging preclinical results support the resumption of Phase I clinical trials evaluating intraventricular infusion of gemcitabine in patients with refractory primary or secondary brain tumors.

pharmacology and toxicology↗

bioRxiv2026

Stereoselective methyl-swapping demonstrates target specificity of cognitive enhancer

The Integrated Stress Response (ISR) couples cellular stress sensing to translational control, playing a critical role in the homeostatic regulation of cell health. However, prolonged and unmitigated ISR activation becomes maladaptive and drives the progression of a wide range of pathologies, including cognitive decline. Pharmacological inhibition of the ISR with the small, drug-like molecule ISRIB has proven remarkably effective in reversing cognitive deficits and pathology in animal models, highlighting its potential for therapeutic intervention in humans. We engineered an allele-specific ISRIB analog (mISRIB) that selectively targets a mutant form of eIF2B, the molecular target of ISRIB, without affecting wild-type eIF2B. Notably, mISRIB treatment in mice homozygous for the eIF2B mutant allele enhances synaptic plasticity and long-term memory, confirming the on-target mechanism underlying ISRIBs cognitive benefits. Our results provide a framework for dissecting the ISRs contributions within complex cellular networks, such as those governing brain function, with precise temporal and spatial resolution.

biochemistry↗

bioRxiv2025

Allosteric Disordering of eIF2B Regulates the Integrated Stress Response

The ternary complex (TC), composed of translation initiation factor eIF2, GTP, and initiator methionyl tRNA, delivers the first amino acid to the ribosome to initiate protein synthesis. The activity of the decameric eukaryotic initiation factor 2B complex (eIF2B) initiates TC assembly by catalyzing GDP to GTP exchange on eIF2, thereby setting the TC levels in the cell. Stress-induced phosphorylation converts eIF2 from the substrate of the GDP/GTP exchange reaction into an inhibitor (eIF2-P) of eIF2B. This conversion reduces the cells TC levels and induces the widespread reprogramming of translation known as the Integrated Stress Response (ISR). Here, we chart an allosteric axis running through eIF2B, revealing the importance of a protrusive -helix in its {beta}-subunit, the latch-helix, that locks onto the -subunit to induce eIF2B activity. eIF2-P binding unhooks the latch-helix, opening eIF2B, which inhibits its GDP/GTP exchange activity. Distinct viral proteins have convergently evolved to bind to eIF2B and stabilize the latch-helix-bound active state. Using these insights, we generated ISR-ACTivating compounds, ISRACTs, that stabilize eIF2B in its inhibited, unlatched state. Our study thus highlights how state-transitions in eIF2B are regulated via long-range allostery.

biochemistry↗

bioRxiv2024

Multi-year drought strengthens positive and negative functional diversity effects on tree growth response

O_LIMixed-species forests are proposed as strategy to increase the resistance and resilience of forests to drought stress. However, evidence suggest that increasing tree species richness does not consistently enhance tree growth responses to drought. Moreover, tree diversity effects under unprecedented multiyear droughts remain uncertain, calling for a better understanding of the underlying processes. C_LIO_LIHere, we used a network of planted tree diversity experiments to investigate how drought-induced growth responses of individual trees are influenced by neighborhood tree diversity and the functional traits of the focal tree species. We analyzed tree cores (948 trees across 16 species) from nine experiments across Europe featuring gradients of tree species richness (1-6 species), which experienced severe droughts in recent years. Radial growth response to drought was quantified as tree-ring biomass increment using X-ray computed tomography. We applied hydraulic trait-based growth models to analyze single-year drought responses across all sites and site-specific responses during consecutive drought years for six sites as a function of neighborhood tree diversity. C_LIO_LIThe large variability in tree growth responses to a single-year drought was partially explained by the focal species hydraulic safety margin (representing species drought tolerance) and drought intensity, but independent of neighborhood species richness or functional trait diversity. However, tree diversity effects on growth responses strengthened during consecutive drought years and were site-specific, with contrasting direction (both positive and negative). This indicated opposing pathways of diversity effects under consecutive drought events, possibly resulting from competitive release or greater water consumption in diverse mixtures. C_LIO_LIWe conclude that tree diversity effects on growth responses to single-year droughts may differ considerably from responses to consecutive drought years. Our study highlights the need to consider trait-based approaches (specifically, hydraulic traits) and tree neighborhood scale processes to understand the multifaceted growth responses of tree mixtures under prolonged drought stress. C_LI

ecology↗

bioRxiv2024

Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility

Heritable Fragile Bone Disorders (FBDs) encompass a spectrum of conditions, from widespread multifactorial to rare monogenic diseases, all characterized by an elevated risk of fractures. The process of validating causative genes and elucidating their pathogenic mechanisms remains a daunting and resource-intensive task. In this study, we evaluated the feasibility of a semi-high throughput zebrafish screening platform for rapid validation and in vivo functional testing and validation of candidate disease-causing genes for a wide range of heritable FBDs. Six genes associated with severe recessive forms of Osteogenesis Imperfecta (OI) and four genes associated with bone mineral density (BMD), a key osteoporosis indicator, identified through genome-wide association studies (GWAS) were selected. The crispant screening approach, based on CRISPR/Cas9 technology, was used to phenotype directly in F0 mosaic founder zebrafish. Next-Generation Sequencing (NGS) analysis revealed a mean indel efficiency of 88% across ten different crispants, indicating a high proportion of knock-out alleles and thus resembling stable knock-out models. We applied multiple techniques to evaluate skeletal characteristics at 7, 14 and 90 days post-fertilization (dpf), including microscopy for osteoblast reporter visualization and mineralization by Alizarin Red S staining, and microCT for quantitative skeletal analysis. While larval crispants exhibited variable differences in osteoblast-positive and mineralized surface areas, adult-stage crispants displayed more pronounced and consistent skeletal phenotypes. Notably, all crispants developed malformed neural and haemal arches, with a majority presenting vertebral fractures and fusions, and some showing significant alterations in vertebral bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR analysis of osteoblast differentiation and bone formation markers at larval stages indicated differential expression of osteogenic markers bglap and col1a1a in a substantial portion of the crispants, hinting at their utility as biomarkers for FBD crispant screening. In summary, our findings demonstrate that crispant screening in zebrafish offers a viable and efficient strategy for the functional assessment of FBD genes. We advocate for a novel comprehensive approach that integrates various techniques and evaluates distinct skeletal and molecular profiles across different developmental and adult stages. This methodology has the potential to provide new insights into the role of these genes in skeletal biology.

genetics↗

bioRxiv2021

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Translational control is primarily exerted through a conformational switch in eIF2s nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2. Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2Bs {beta} subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed (Schoof et al. 2021) A/I-State model of allosteric ISR regulation.

molecular biology↗

bioRxiv2021

Viral Evasion of the Integrated Stress Response Through Antagonistic eIF2-P Mimicry

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 43 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2B mediated by five aromatic fingers. NSs binding preserves eIF2B activity by maintaining eIF2Bs conformation in its active A-State.

biochemistry↗

bioRxiv2020

eIF2B Conformation and Assembly State Regulate the Integrated Stress Response

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2s nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2Bs -subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing an allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into conjoined tetramers with greatly diminished activity. Thus, ISRIBs effects in disease models could arise from eIF2B decamer stabilization, allosteric modulation, or both.

cell biology↗

bioRxiv2020

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

biochemistry↗

bioRxiv2020

Co-expressed subunits of dual genetic origin define a conserved supercomplex mediating essential protein import into chloroplasts

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons — termed TOC and TIC — located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56 and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TicToc), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and the here newly identified TicToc components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TicToc complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TicToc supercomplex in maintaining chloroplast proteostasis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2020

Small molecule cognitive enhancer reverses age-related memory decline in mice.

With increased life expectancy age-associated cognitive decline becomes a growing concern, even in the absence of recognizable neurodegenerative disease. The integrated stress response (ISR) is activated during aging and contributes to age-related brain phenotypes. We demonstrate that treatment with the drug-like small-molecule ISR inhibitor ISRIB reverses ISR activation in the brain, as indicated by decreased levels of activating transcription factor 4 (ATF4) and phosphorylated eukaryotic translation initiation factor eIF2. Furthermore, ISRIB treatment reverses spatial memory deficits and ameliorates working memory in old mice. At the cellular level in the hippocampus, ISR inhibition i) rescues intrinsic neuronal electrophysiological properties, ii) restores spine density and iii) reduces immune profiles, specifically interferon and T cell-mediated responses. Thus, pharmacological interference with the ISR emerges as a promising intervention strategy for combating age-related cognitive decline in otherwise healthy individuals. ONE SENTENCE SUMMARYInhibition of the integrated stress response restores neuronal and immune dysfunction and alleviates memory deficits in aged mice.

neuroscience↗

bioRxiv2019

Evaluating the data quality of iNaturalist termite records

Citizen science (CS) contributes to the combined knowledge about species distributions, which is a critical foundation in the studies of invasive species, biological conservation, and response to climatic change. In this study, we assessed the value of CS for termites worldwide. First, we compared the abundance and species diversity of geo-tagged termite records in iNaturalist to that of the University of Florida termite collection (UFTC) and the Global Biodiversity Information Facility (GBIF). Second, we quantified how the combination of these data sources affected the number of genera that satisfy data requirements for ecological niche modeling. Third, we assessed the taxonomic correctness of iNaturalist termite records in the Americas at the genus and family level through expert review based on photo identification. Results showed that iNaturalist records were less abundant than those in UFTC and in GBIF, although they complemented the latter two in selected world regions. A combination of GBIF and UFTC led to a significant increase in the number of termite genera satisfying the abundance criterion for niche modeling compared to either of those two sources alone, whereas adding iNaturalist observations as a third source only had a moderate effect on the number of termite genera satisfying that criterion. Although research grade observations in iNaturalist require a community-supported and agreed upon ID below the family taxonomic rank, our results indicated that iNaturalist data do not exhibit a higher taxonomic classification accuracy when they are designated research grade. This means that non-research grade observations can be used to more completely map the presence of termite locations in certain geographic locations without significantly jeopardizing data quality. We concluded that CS termite observation records can, to some extent, complement expert termite collections in terms of geographic coverage and species diversity. Based on recent data contribution patterns in CS data, the role of CS termite contributions is expected to grow significantly in the near future.

zoology↗

bioRxiv2018

Beyond the ribosome: proteome-wide secretability studies using SECRiFY

While transcriptome- and proteome-wide technologies to assess processes in protein biogenesis are now widely available, we still lack global approaches to assay post-ribosomal biogenesis events, in particular those occurring in the eukaryotic secretory system. We here developed a method, SECRiFY, to simultaneously assess the secretability of >105 protein fragments by two yeast species, S. cerevisiae and P. pastoris, using custom fragment libraries, surface display and a sequencing-based readout. Screening human proteome fragments with a median size of 50 - 100 amino acids, we generated datasets that enable datamining into protein features underlying secretability, revealing a striking role for intrinsic disorder and chain flexibility. SECRiFY is the first methodology that generates sufficient amounts of annotated data for advanced machine learning methods to deduce secretability predictors. The finding that secretability is indeed a learnable feature of protein sequences is of significant impact in the broad area of recombinant protein expression and de novo protein design.

molecular biology↗