Biology subjects

Adams, P. D.

Publications and source records attributed to Adams, P. D..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Automation of high-throughput arrayed mammalian cell line cultivation

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

cell biology↗

bioRxiv2025

A knowledge-guided approach to recovering important rare signals from high-dimensional single-cell data

Single-cell transcriptomic data are high-dimensional, with many genes profiled in each cell. Dimensionality reduction is routinely applied to improve interpretability, remove noise and redundancy, and enable visualization. Most existing methods aim at preserving the most prominent data properties, which can lead to omission of rare but important signals. Here we propose a novel framework that uses knowledge-derived genes of interest to guide dimensionality reduction, which can help cluster rare cells and separate highly similar cell sub-populations. We demonstrate the utility of our framework in identifying endocrine cell subtypes in the pancreatic islet, highly similar hematopoietic sub-populations, and rare senescent cells.

bioinformatics↗

bioRxiv2025

ONC206 demonstrates potent anti-tumorigenic activity and is a potential novel therapeutic strategy for high-risk medulloblastoma

BackgroundMedulloblastoma is the most common malignant pediatric brain tumor, and has an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency, ONC206, induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC206 is currently in Phase I clinical trials for pediatric patients with primary brain tumors. MethodsIn this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma and investigated its mechanism of action. ResultsWe found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumors, compared to very low expression in normal brain tissue. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine SHH-driven and Group 3 medulloblastoma cells treated with ONC206 with low IC-50s. After treatment with ONC206, we observed an induction of integrated stress response and mitochondrial damage. To test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 and Group 4 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating survival extension from 70 to 140 days. PDX-bearing mice also responded to ONC206, which led to a significant survival benefit. ConclusionOur results highlight ONC206 as a novel therapeutic option for patients with high-risk medulloblastoma and provide strong rationale for testing the efficacy of ONC206 in the treatment of these patients. Key points (2-3)ONC206 potently kills medulloblastoma cells by inducing integrated stress response and mitochondrial damage. ONC206 prolongs survival of medulloblastoma-bearing mice in both murine and patient-derived xenograft models. Importance of studyThere is an unmet need for better therapies for high-risk medulloblastoma patients. ONC201 has shown promising responses and recently received FDA approval for diffuse midline glioma. ONC206 is a chemical derivative with higher potency and better brain penetrance. In this study, we analyzed the therapeutic potential of ONC206 for high-risk medulloblastoma and found that the drug effectively killed mouse and human medulloblastoma cells with high nanomolar potency. We also saw that ONC206 very significantly prolonged survival of medulloblastoma-bearing mice, both in genetically engineered mouse models and patient-derived xenografts. Our study provides a strong rationale for testing the efficacy of ONC206 in the treatment of patients with medulloblastoma and has set the stage for a clinical trial with this agent in pediatric patients with recurrent malignant brain tumors, including medulloblastoma (NCT04732065).

cancer biology↗

bioRxiv2025

Nucleosome stability safeguards cell identity, stress resilience and healthy aging

Nucleosomes are the minimal repeating units of chromatin. Their dynamic assembly and disassembly underpins chromatin organization and genome regulation. However, it remains unclear how intrinsic nucleosome stability contributes to higher-level yet fundamental cellular and organismal properties--such as preservation of cell identity, lineage specification, stress resilience and ultimately healthy aging. To address this, we tested the impact of decreased intrinsic nucleosome stability across multiple cell, tissue and organismal models by introducing histone mutants that weaken histone-histone interactions. While nucleosome instability did not broadly alter global chromatin accessibility, DNA damage, cell proliferation or viability, it impaired lineage-specific gene expression programs, altered lineage specification and activated intrinsic inflammatory and stress pathways in a manner reminiscent of aging in mouse tissues and human cells. Consistently, nucleosome instability accelerated the onset of age-associated transcriptional alterations and functional decline in Caenorhabditis elegans and Drosophila melanogaster, and reduced cellular resilience to exogenous perturbations-- including environmental, epigenetic and mitotic stress--in human cells and Saccharomyces cerevisiae. These cross-species findings identify nucleosome stability as an evolutionarily conserved epigenetic safeguard that preserves cell identity and stress resilience and supports organismal function and healthy aging.

genomics↗

bioRxiv2025

CD155 regulates tumor growth and immune evasion in diffuse midline glioma.

Diffuse midline glioma (DMG) is a devastating pediatric brain tumor with an unmet need for novel therapies. Immune checkpoint inhibitors have failed to prolong survival for DMG patients. In this study, we analyzed the expression of immune checkpoint molecules in human and murine DMG cells, as well as primary brain tumor samples, and identified CD155 as the most highly expressed. When murine DMG cells were co-cultured with CD8+ T cells, silencing of CD155 led to a marked increase in T cell-mediated killing. Strikingly, CD155-deficient DMG cells failed to grow in immunocompetent mice, and depletion of CD8+ T cells allowed these tumors to grow. CD155 also exerted cell-autonomous effects on tumor cells: silencing of CD155 led to induction of apoptosis of DMG cells and to delayed tumor growth in immunodeficient mice. Transcriptomic analyses identified FOXM1 as a key target of CD155. Notably, FOXM1 silencing also led to reduced proliferation of DMG cells in vitro and in vivo. Finally, treatment of DMG-bearing mice with Thiostrepton, a FOXM1-targeting agent, delayed tumor growth and prolonged survival. These studies demonstrate that CD155 regulates immune evasion and tumor growth in DMG, and suggest that targeting CD155 could be a valuable two-pronged therapeutic strategy for this disease. Conflict-of-interest statementThe authors have declared that no conflict of interest exists.

cancer biology↗

bioRxiv2025

Validated ligand geometries for macromolecular refinement restraints and molecular mechanics force fields

In macromolecular structure refinement the low observation-to-parameter ratio and the lack of high-resolution data is countered by using a priori information in the form of restraints. Having accurate geometries of the chemical entities in the sample is paramount for generating accurate chemical restraints and, therefore, accurate macromolecular structures. In particular, it is desirable to have accurate restraints for known and novel ligand entities. Quantum Mechanics (QM) can minimise the energy of a ligand by adjusting its geometry, and these geometries can be used to generate restraints macromolecular refinement. We describe here a library of 37,000 small molecules extracted from the Chemical Components Dictionary in the Protein Data Bank and minimized by density functional QM. The library includes restraint files for use in crystallography or cryo-EM refinement, along with files suitable for molecular dynamics simulation. Because the geometries are validated, the restraints library provides users with both functional restraints and minimised geometries. This work also provides procedures for generating new and accurate restraints.

molecular biology↗

bioRxiv2025

Targeting CyclinD1-CDK6 to Mitigate Senescence-Driven Inflammation and Age-Associated Functional Decline

Cellular senescence contributes to aging and age-related diseases by driving chronic inflammation through the Senescence Associated Secretory Phenotype (SASP) and interferon-stimulated genes (ISGs). Cyclin D1 (CCND1), a key cell cycle regulator, is paradoxically upregulated in these non-proliferating cells. We show that CCND1 and its kinase partner CDK6 drive SASP and ISG expression in senescent cells by promoting DNA damage accumulation. This leads to the formation of cytoplasmic chromatin fragments (CCFs) that activate pro-inflammatory CGAS-STING signaling. The tumor suppressor p53 (TP53) and its target p21 (CDKN2A) antagonize this CCND1-CDK6-dependent DNA damage accumulation pathway to suppress the SASP. In aged mouse livers, senescent hepatocytes show increased Ccnd1 expression. Hepatocyte-specific Ccnd1 knockout or treatment with the Cdk4/6 inhibitor Palbociclib reduces DNA damage and ISGs in aged mouse liver. Notably, Palbociclib also suppresses frailty and improves physical performance of aged mice. These findings reveal a novel role for CCND1/CDK6 in regulating DNA damage and inflammation in senescence and aging, highlighting it as a promising therapeutic target.

cell biology↗

bioRxiv2025

Rapamycin Reverses the Hepatic Response to Diet-Induced Metabolic Stress That Is Amplified by Aging.

Aging is associated with increased susceptibility to metabolic stress and chronic liver disease, yet the interactions between age and metabolic stressors and the potential for ameliorating interventions remain incompletely understood. Here, we examined the hepatic response of young (7-month-old) and old (25-month-old) C57BL/6 male mice to a 9-week high-fat diet (HFD) and assessed whether rapamycin, a well-established pro-longevity intervention, could mitigate age-exacerbated effects. While both age groups developed metabolic-associated steatohepatitis (MASH), older mice displayed more severe hepatic steatosis, inflammation, and transcriptional dysregulation. Transcriptomic profiling of whole livers and purified hepatocytes revealed that aging amplifies HFD-induced inflammatory and metabolic gene expression changes, including activation of immune pathways and suppression of metabolic pathways. Notably, treatment of aging mice with rapamycin reversed the majority of HFD-driven transcriptional alterations, including upregulation of pro-inflammatory regulators such as Stat1, and dysregulation of metabolic gene networks. Rapamycin also reduced hepatosteatosis, total body weight, and a tumorigenic transcriptomic signature associated with hepatocellular carcinoma risk. These findings demonstrate that aging intensifies hepatic sensitivity to metabolic stress and identify rapamycin as a promising therapeutic to counteract age-related liver dysfunction and MAFLD progression.

cell biology↗

bioRxiv2025

Biochemical and structural characterization of glycosyltransferase family 61 proteins reveal a key determinant of sugar donor specificity

Xylan, the most abundant non-cellulosic polymer in plant cell walls, is structurally diverse, especially in grasses where it is heavily substituted with arabinofuranose and further modified by various residues. Common substitutions across species include glucuronic and 4-O-methyl-glucuronic acid. Arabinose and xylose sidechains are synthesized by glycosyltransferase family 61 (GT61) proteins, many of which remain uncharacterized in plants, with limited structural and mechanistic understanding. In this study, we identified two novel GT61 enzymes in Sorghum bicolor, functioning as xylan arabinosyltransferase (SbXAT) and xylan xylosyltransferase (SbXXT). We resolved the crystal structure of SbXAT, which exhibits a GT-B fold with two Rossmann-like domains linked by a cleft that accommodates the catalytic site. Structural comparison with a predicted SbXXT model revealed a substrate-binding residue critical for sugar donor specificity, validated through site-directed mutagenesis and enzymatic assays. These findings enhance understanding of xylan biosynthesis and provide a foundation for engineering glycosyltransferases and predicting their functions.

biochemistry↗

bioRxiv2025

Predictive genome-wide CRISPR-mediated gene downregulation for enhanced bioproduction

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, conventional approaches for identifying target genes for CRISPRi-mediated downregulation have largely relied on heuristic methods and trial and error, which are labor-intensive and time-consuming. Additionally, the ability to achieve multigene knockdowns is limited by the constraints of molecular cloning techniques required for building multiplexed CRISPRi systems. In this study, we describe two novel methodologies FluxRETAP (Flux-REaction TArget Prioritization, a Genome-Scale Modeling Technique) and VAMMPIRE (a Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation). FluxRETAP accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers, outperforming traditional heuristic selection. The use of VAMMPIRE enabled accurate and position-independent assembly of CRISPRi constructs containing up to five sgRNA arrays. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

synthetic biology↗

bioRxiv2025

Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida, a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putidas native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a [~]16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing [~]900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/643695v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@533605org.highwire.dtl.DTLVardef@ff98fforg.highwire.dtl.DTLVardef@9592fdorg.highwire.dtl.DTLVardef@1e52993_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Modest functional diversity decline and pronounced composition shifts of microbial communities in a uranium-contaminated aquifer

BackgroundMicrobial taxonomic diversity declines with increased environmental stress. Yet, few studies have explored whether phylogenetic and functional diversities track taxonomic diversity along the stress gradient. Here, we investigated bacterial communities within an aquifer in Oak Ridge, Tennessee, USA, which is characterized by a broad spectrum of stressors, including extremely high levels of nitrate, heavy metals like cadmium and chromium, radionuclides such as uranium, and extremely low pH (<3). ResultsBoth taxonomic and phylogenetic -diversities were reduced in the most impacted wells, while the decline in functional -diversity was modest and statistically insignificant, indicating a more robust buffering capacity to environmental stress. Differences in functional gene composition (i.e., functional {beta}-diversity) were pronounced in highly contaminated wells, while convergent functional gene composition was observed in uncontaminated wells. The relative abundances of most carbon degradation genes were decreased in contaminated wells, but genes associated with denitrification, adenylylsulfate reduction, and sulfite reduction were increased. Compared to taxonomic and phylogenetic compositions, environmental variables played a more significant role in shaping functional gene composition, suggesting that niche selection could be more closely related to microbial functionality than taxonomy. ConclusionsOverall, we demonstrated that despite a reduced taxonomic -diversity, microbial communities under stress maintained functionality underpinned by environmental selection.

microbiology↗

bioRxiv2024

Age-associated modulation of TREM1/2- expressing macrophages promotes melanoma progression and metastasis

Aging is a known risk factor for melanoma, yet mechanisms underlying melanoma progression and metastasis in older populations remain largely unexplored. Among the current knowledge gaps is how aging alters phenotypes of cells in the melanoma microenvironment. Here we demonstrate that age enriches the immunosuppressor tumor microenvironment, which is linked to phenotypes associated with melanoma metastasis. Among cellular populations enriched by aging were macrophages with a tolerogenic phenotype expressing TREM2 and dysfunctional CD8-positive cells with an exhausted phenotype, while macrophages with profibrotic phenotype expressing TREM1 were depleted. Notably, TREM1 inhibition decreased melanoma growth in young but not old mice, whereas TREM2 inhibition prevented lung metastasis in aged mice. These data identify novel targets associated with melanoma metastasis and may guide aged-dependent immunotherapies.

cancer biology↗

bioRxiv2024

A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxyacids

Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here, we present a novel approach utilising a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and NADPH-dependent terminal thioreductases (TRs), previously untapped in engineered PKSs. Reduction of the terminal aldehyde with specific alcohol dehydrogenases enables production of diols, oxidation enables production of hydroxy acids, and transamination with specific transaminases enables production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A (CoA)-specific acyltransferase (AT) in the extension module with methyl- or ethylmalonyl- CoA-specific ATs enables production of branched-chain diols and amino alcohols. In total, we demonstrated production of nine 1,3-diols (including the difficult-to-produce insect repellent and cosmetic ingredient 2-ethyl-1,3-hexanediol), six amino alcohols, and two carboxylic acids using our PKS platform in Streptomyces albus. Finally, tuning production of the PKS acyl-CoA substrates enabled production of high titers of specific diols and amino alcohols (1 g/L diol titer in shake flasks), demonstrating high tunability and efficiency of the platform.

bioengineering↗

bioRxiv2024

Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer

Age is a major risk factor for liver cancer, as is the case for most adult human cancers. However, the underlying mechanisms are not well defined. A better understanding of the role of aging in liver and other cancers can facilitate approaches for risk assessment, early detection and prevention. We hypothesize that age-driven changes render aged liver more sensitive to oncogenic stress and hence tumorigenesis. To investigate how the liver changes with age, we documented the immune profile, transcriptome and epigenome of healthy livers from both young and aged mice, revealing pronounced alterations with aging. Notably, in aged hepatocytes, we identified heightened interferon (IFN) signaling, as well as simultaneous tumor suppressor and oncogene signaling at both bulk and single cell level, suggestive of an aged liver that is poised for neoplasia. To challenge this seemingly poised state, we employed adeno-associated virus (AAV)-mediated expression of a c-Myc oncogene in young and aged mouse liver hepatocytes in vivo. Analysis of aged hepatocytes expressing c-Myc revealed further elevated expression of IFN Stimulated Genes (ISGs). This ISG upregulation was evident in multiple models of oncogenic stress and transformation in older mice and also observed in aged humans with Metabolic dysfunction-Associated Steatohepatitis (MASH). We determined that Stat1 is both necessary and sufficient for the age specific elevated ISG expression in old wild type mice. Remarkably, inhibiting Jak/Stat signaling alongside ectopic c-Myc expression led to high-grade hepatocyte dysplasia and tumor formation, selectively in aged mice. Together, these results suggest that an aged liver is in a state of "precarious balance", due to concurrent activation of oncogenic and tumor suppressor pathways, but protected against neoplastic progression by IFN-signaling. Age-dependent activation of IFN signaling has been observed in many tissues and recent studies have demonstrated its detrimental consequences on aging, raising the question as to why IFN-signaling is activated during aging. We propose that aged tissues are intrinsically at higher risk of cancer and age-dependent activation of IFN-signaling is an adaptive process to protect from tumorigenesis, but one that also has maladaptive consequences.

cancer biology↗

bioRxiv2024

AQuaRef: Machine learning accelerated quantum refinement of protein structures

Cryo-EM and X-ray crystallography provide crucial experimental data for obtaining atomic-detail models of biomacromolecules. Refining these models relies on library-based stereochemical restraints, which, in addition to being limited to known chemical entities, do not include meaningful noncovalent interactions relying solely on nonbonded repulsions. Quantum mechanical (QM) calculations could alleviate these issues but are too expensive for large molecules. We present a novel AI-enabled Quantum Refinement (AQuaRef) based on AIMNet2 neural network potential mimicking QM at substantially lower computational costs. By refining 41 cryo-EM and 30 X-ray structures, we show that this approach yields atomic models with superior geometric quality compared to standard techniques, while maintaining an equal or better fit to experimental data. Notably, AQuaRef aids in determining proton positions, as illustrated in the challenging case of short hydrogen bonds in the parkinsonism-associated human protein DJ-1 and its bacterial homolog YajL.

biochemistry↗

bioRxiv2024

Likelihood-based interactive local docking into cryo-EM maps in ChimeraX

The interpretation of cryo-EM maps often includes the docking of known or predicted structures of the components, which is particularly useful when the map resolution is worse than 4 [A]. Although it can be effective to search the entire map to find the best placement of a component, the process can be slow when the maps are large. However, frequently there is a well-founded hypothesis about where particular components are located. In such cases, a local search using a map subvolume will be much faster because the search volume is smaller, and more sensitive because optimizing the search volume for the rotation search step enhances signal-to-noise. A Fourier-space likelihood-based local search approach, based on the previously-published em_placement software, has been implemented in the new emplace_local program. Tests confirm that the local search approach enhances speed and sensitivity of the computations. An interactive graphical interface in the ChimeraX molecular graphics program provides a convenient way to set up and evaluate docking calculations, particularly in defining the part of the map into which the components should be placed. SynopsisLikelihood-based cryo-EM docking using our emplace_local software is faster and more sensitive than our related software, em_placement, when the approximate location of a component is known, and is available conveniently through a plugin to the ChimeraX visualization software.

biophysics↗

bioRxiv2024

PROTGOAT : Improved automated protein function predictions using Protein Language Models

Accurate prediction of protein function is crucial for understanding biological processes and various disease mechanisms. Current methods for protein function prediction relies primarily on sequence similarities and often misses out on important aspects of protein function. New developments in protein function prediction methods have recently shown exciting progress via the use of large transformer-based Protein Language Models (PLMs) that allow for the capture of nuanced relationships between amino acids in protein sequences which are crucial for understanding their function. This has enabled an unprecedented level of accuracy in predicting the functions of previously little understood proteins. We here developed an ensemble method called PROTGOAT based on embeddings extracted from multiple and diverse pre-trained PLMs and existing text information about the protein in published literature. PROTGOAT outperforms most current state-of-the-art methods, ranking fourth in the Critical Assessment of Functional Annotation (CAFA 5), a global competition benchmarking such developments among 1600 methods tested. The high performance of our method demonstrates how protein function prediction can be improved through the use of an ensemble of diverse PLMs. PROTGOAT is publicly available for academic use and can be accessed here: https://github.com/zongmingchua/cafa5

bioinformatics↗