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Phillips, M.

Publications and source records attributed to Phillips, M..

At least 19 recordsLinked to original sources

FEATMAP: Targeted Correction of Acquisition Signatures Harmonizes Medical Foundation Model Embeddings and Enables Robust Task Generalization

AO_SCPLOWBSTRACTC_SCPLOWMedical foundation models compress biomedical data into embeddings that support diverse downstream clinical tasks. However, successful model deployment is hampered by performance degradation on external data. It is recognized that embeddings capture acquisition signatures, such as hardware and technical differences, in addition to biology. Effective harmonization must remove the acquisition signature while preserving biological signals, a trade-off that current methods fail to balance adequately. Input-level normalization fails to eliminate acquisition signatures from embeddings, whereas embedding-level methods adjust features in an untargeted manner. We present FEATMAP, a harmonization approach that models acquisition signatures as geometric distortions between manifolds of similarly arranged embeddings. Using paired data that isolate the effect of acquisition signatures, FEATMAP fits a single global affine transformation per foundation model to correct acquisition signatures directly in the embedding space. This targeted, reusable correction aims to preserve biological and demographic variation while harmonizing across acquisition signatures. Across scanner and foundation-model harmonization in digital pathology and field-strength harmonization in brain MRI, FEATMAP improves cross-condition embedding similarity, reduces performance gaps without retraining, and suggests potential for the alignment of disparate embedding spaces.

bioinformatics↗

Highly diverged non-vertebrate Ki-67 proteins with conserved biophysical and functional properties

The Ki-67 protein is a widely used marker of mammalian cell proliferation that plays critical roles in heterochromatin organisation and mitotic perichromosomal layer formation. Thus, its apparent restriction to vertebrates is puzzling. However, sequences with limited homology to subdomains of Ki-67 are present in genomes from some non-vertebrate species. Here, using structural modelling, biophysical and functional analysis, we identify Ki-67-like proteins across diverse non-vertebrate eukaryotes, including tunicates, sponges, molluscs, insects and fungi. Despite extremely low sequence homology, the disordered domains of Ki-67 homologues have generally conserved biophysical and molecular properties. This is further reflected by comparison of cellular localisation, dynamics and effects on chromatin organisation of human, Ciona and Drosophila Ki-67 homologues. These results reinforce the emerging paradigm that functions may be encoded by general biophysical features in highly diverged intrinsically disordered domains. Our approach provides a blueprint for comparative biology of disordered proteins and a foundation for future studies exploring functions of Ki-67 homologues across eukaryotes.

Cell Biology↗

Chorismate and isochorismate turnover reveal hidden dynamics of aromatic metabolism in Arabidopsis

The shikimate pathway provides precursors for phenolic metabolites in plant primary and secondary metabolism. Carbon flux measurements in intact Arabidopsis leaves using a novel isotopologue MS/MS methodology revealed unexpected dynamics between chorismate and isochorismate pools. 13CO2 labeling kinetics point to chloroplast-derived shikimate as a major bifurcation point, with approximately one third diverted away from the chloroplast. In contrast, the small pool of chorismate was almost exclusively chloroplast-localized and turned over rapidly, reaching more than 70% labeling within minutes. Isochorismate, a precursor to salicylic acid (SA) in the Brassicales, was present at 50-fold molar excess over chorismate but labeled much more slowly and appeared primarily extra-chloroplastic. Total isochorismate declined by 90% in the Arabidopsis enhanced disease susceptibility mutant, which lacks the isochorismate exporter. Non-aqueous fractionation further supported a primarily chloroplast-localized chorismate pool but extra-plastidic isochorismate. Populus trichocarpa and Nicotiana benthamiana leaves contained chorismate but only trace isochorismate, consistent with their use of the benzoyl-CoA route to SA. Carbon commitment calculations indicated that one third of the total chorismate pool in Arabidopsis leaves is diverted to isochorismate. Global leaf calculations based on elemental analysis-isotope ratio mass spectrometry and targeted isotope recovery indicate that only 0.03% of total assimilated carbon (5.27 pmol{middle dot}mg-1 D.W.{middle dot}min-1) enters the shikimate pathway in photosynthetically active mesophyll. For comparison [~]0.05% (7.75 pmol{middle dot}mg-1 D.W.{middle dot}min-1) enters the 2-C-methyl-D-erythritol 4-phosphate pathway, which provides precursors for photosynthetic pigments and electron carriers. The compartmentalization and turnover dynamics of shikimate, chorismate, and isochorismate suggest continuous demand for aromatic precursors in mesophyll tissue is comparable to demand for MEP-pathway derived pigments.

plant biology↗

Scalable longitudinal imaging and transcriptomics of cells in dynamic enclosures

Dynamic transitions between cell states underlie both normal physiology and disease. However, most single-cell technologies capture only static snapshots. To address this gap, we developed a platform that integrates light-guided hydrogel polymerization with computer vision to generate on-demand compartments around live cells, enabling longitudinal imaging of cellular behavior paired with whole-transcriptome profiling of the same cells at scale. These data link dynamic phenotypes with molecular programs, enabling deeper characterization of cellular states. This approach revealed an adaptive, drug-resistant state in lung cancer cells characterized by potassium channel upregulation and p53-dependent quiescence. In models of adipogenesis and microglial phagocytosis, joint analysis of imaging and transcriptomic data identified key drivers of cellular function that were missed by transcriptomic clustering alone. These results establish the value of paired functional and transcriptomic analysis to resolve molecular drivers of complex cellular behaviors.

systems biology↗

Inhibition of p65 NF-κB enhances production of galactose-deficient IgA1 through suppression of C1GALT1 and SP1 in plasmablast-like cell subpopulations

IgA nephropathy (IgAN) is a common primary glomerulonephritis characterized by glomerular immune-complex deposits with (co)dominant IgA. These deposits are enriched for IgA1 glycoforms with some O-glycans deficient in galactose (Gd-IgA1). Circulating Gd-IgA1 is bound by IgG autoantibodies to form immune complexes, some of which deposit in glomeruli. Genomic and immunologic studies indicate involvement of pro-inflammatory signaling pathways in the production of Gd-IgA1 in IgAN. Genomic studies identified multiple genetic loci associated with IgAN and suggested a convergence on the NF-{kappa}B pathway, including RELA, the gene encoding the NF-{kappa}B subunit p65. However, the mechanisms by which NF-{kappa}B pathways may affect O-glycosylation in IgA1-producing cells are unknown. Using EBV-immortalized B cells derived from peripheral-blood mononuclear cells of IgAN patients and healthy controls that have constitutively activated NF-{kappa}B, we report that inhibition of NF-{kappa}B/p65 by a selective IKK{beta} inhibitor TPCA-1 reduced phosphorylation of NF-{kappa}B/p65 at S536 and decreased production of IgA1 and, conversely, increased Gd-IgA1 production. This was likely related to reduced expression of C1GALT1 gene that encodes the enzyme responsible for galactosylation of IgA1 O-glycans. Flow-cytometry imaging revealed changes in nuclear translocation and co-localization of the NF-{kappa}B/p65 with co-transcriptional factor SP1, a transcriptional activator of C1GALT1, suggesting that NF-{kappa}B pathway affects IgA1 O-glycosylation via SP1 transcriptional control of C1GALT1 expression. Furthermore, prolonged IKK{beta} inhibition altered B cell subpopulations, enhancing generation of cells with a plasmablast-like phenotype, characterized by high SSC MFI and CD138 expression. Together, these findings provide functional evidence for involvement of NF-{kappa}B/p65 and its transcriptional partners in IgA1 O-glycosylation. HighlightsO_LIIKK{beta} inhibition reduced C1GALT1 expression and thereby increased galactose-deficient IgA1 (Gd-IgA1) production in immortalized human B cells. C_LIO_LISP1+ subpopulations, a transcriptional activator of C1GALT1, declined after sustained NF-{kappa}B inhibition. C_LIO_LINF-{kappa}B inhibition shifted a subpopulation of B cells into a plasmablast-like phenotype. C_LIO_LIThis study links NF-{kappa}B signaling with the GWAS-identified RELA susceptibility locus and IgA1 O-glycosylation. C_LI

immunology↗

Neuronal silencing through depolarization block underlies odor discrimination

The olfactory system enables animals to distinguish between attractive and aversive odor sources through the perception of odorant molecules. A fundamental challenge is maintaining stable odor identity perception despite fluctuating stimulus intensities during navigation. Using the numerically simple olfactory system of the Drosophila larva, we combined a quantitative odor discrimination assay, electrophysiology, computational modelling, and ectopic expression of odorant receptors to study the encoding of odor identity. We found that depolarization block, which silences olfactory sensory neurons (OSNs) at odor concentrations 3-4 log units above the detection threshold, enables odor discrimination by creating distinct patterns of OSN activity. In a minimal system with two functional OSNs, larvae discriminate between odors when depolarization block creates differential OSN silencing but fail to distinguish odors producing qualitatively similar activity patterns in both OSNs. This principle extends to odor mixtures and the fully functional olfactory system, where the high affinity of the OR42b odorant receptor for one of the test odors enables discrimination regardless of which OSN expresses OR42b. Our results indicate that depolarization block serves as an integral feature of olfactory coding alongside combinatorial OSN activation. Selective silencing of high-affinity OSNs creates distinct neural representations and enables discrimination between odors that would otherwise produce similar activity patterns. This mechanism appears evolutionarily conserved across sensory systems, from olfactory sensory neurons to photoreceptors, suggesting that selective neuronal silencing serves as a mechanism for maintaining distinct sensory representations across stimulus intensities.

neuroscience↗

Transient optical clearing using absorbing molecules for ex-vivo and in-vivo imaging

Dynamic imaging is a foundational tool in biology and medicine but is limited by light scattering in live tissues caused by refractive index mismatches, which reduces penetration depth and resolution in biological tissues. Our previous work introduced a novel approach to achieve an optical transparency in live animals by utilizing strongly absorbing molecules. When these absorbing molecules, such as Tartrazine, an FDA approved dye commonly used in foods, dissolve in water, they modify the refractive index of the aqueous medium through Kramers-Kronig relations. This modulation allows reduction of the refractive index mismatch between separate components in tissue with distinct refractive indices, thus mitigating scattering. In this article, we report detailed methodology of our tissue clearing technique using a Tartrazine-based aqueous solution, applied to both ex-vivo and in-vivo samples. This approach reversibly renders live mice bodies transparent in the visible spectrum, enabling clearer and deeper imaging of internal processes and structures. We then utilized an advanced image processing algorithm to improve the contrast and segmentation of organs of interests. We also show high transparency in ex-vivo samples, such as chicken breast, when treated with Tartrazine based solution. This method offers a versatile and accessible approach to enhancing imaging capabilities across various modalities and paves the way for advancement in our understanding of complex biological systems. SUMMARYOptical clearing techniques, such as absorbing molecules solutions, reduce light scattering temporarily to enhance biological imaging and offer biocompatible, reversible in-vivo imaging strategies for deeply embedded organs. This protocol presents detailed experimental procedures as well as advanced image processing to achieve high-quality, dynamic imaging for real-time dynamics in living animals.

bioengineering↗

The CECR2 bromodomain displays distinct binding modes to select for acetylated histone proteins versus non-histone ligands.

The cat eye syndrome chromosome region candidate 2 (CECR2) protein is an epigenetic regulator involved in chromatin remodeling and transcriptional control. The CECR2 bromodomain (CECR2-BRD) plays a pivotal role in directing the activity of CECR2 through its capacity to recognize and bind acetylated lysine residues on histone proteins. This study elucidates the binding specificity and structural mechanisms of CECR2-BRD interactions with both histone and non-histone ligands, employing techniques such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and a high-throughput peptide assay. The CECR2-BRD selectively binds acetylated histone H3 and H4 ligands, exhibiting a preference for multi-acetylated over mono-acetylated targets. The highest affinity was observed for tetra-acetylated histone H4. Neighboring post-translational modifications, including methylation and phosphorylation, modulate acetyllysine recognition, with significant effects observed for histone H3 ligands. Additionally, this study explored the interaction of the CECR2-BRD with the acetylated RelA subunit of NF-{kappa}B, a pivotal transcription factor in inflammatory signaling. Dysregulated NF-{kappa}B signaling is implicated in numerous pathologies, including cancer progression, with acetylation of RelA at lysine 310 (K310ac) being critical for its transcriptional activity. Recent evidence linking the CECR2-BRD to RelA suggests it plays a role in inflammatory and metastatic pathways, underscoring the need to understand the molecular basis of this interaction. We found the CECR2-BRD binds to acetylated RelA with micromolar affinity, and uses a distinctive binding mode to recognize this non-histone ligand. These results provide new insight on the role of CECR2 in regulating NF-{kappa}B-mediated inflammatory pathways. Functional mutagenesis of critical residues, such as Asn514 and Asp464, highlight their roles in ligand specificity and binding dynamics. Notably, the CECR2-BRD remained monomeric in solution and exhibited differential conformational responses upon ligand binding, suggesting adaptive recognition mechanisms. Furthermore, the CECR2-BRD exclusively interacts with nucleosome substrates containing multi-acetylated histones, emphasizing its role in transcriptional activation within euchromatic regions. These findings position the CECR2-BRD as a key chromatin reader and a promising therapeutic target for modulating transcriptional and inflammatory processes, particularly through the development of selective bromodomain inhibitors. HIGHLIGHTSO_LIThe CECR2 bromodomain recognizes a range of combinatorial PTMs on the histone H3 and H4 N-terminal tails. C_LIO_LIThe CECR2 bromodomain binds to an acetylated RelA ligand with micromolar affinity. C_LIO_LINMR perturbation studies delineate the distinct binding modes driving CECR2-BRD recognition of histone versus non-histone ligands. C_LIO_LISite-directed mutagenesis reveals the specificity determinants of CECR2-BRD ligand binding. C_LIO_LIThe bromodomain of CECR2 exhibits a strong interaction with multi-acetylated nucleosomes. C_LI

biochemistry↗

In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

Chimeric antigen receptor (CAR) T cells are highly effective in hematologic malignancies. However, loss of CAR T cells can contribute to relapse in a significant number of patients. These limitations could potentially be overcome by targeted gene editing to increase CAR T cell persistence. Here, we performed in vivo loss-of-function CRISPR screens in BCMA-targeting CAR T cells to investigate genes that influence CAR T cell persistence, function and efficacy in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR-library edited T cells in vitro and then at early and late timepoints in vivo to track the performance of gene modified CAR T cells from manufacturing to survival in tumors. The screens revealed several context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, while loss of PTPN2, ZC3H12A, and RC3H1 conferred early selective growth advantages to CAR T cells in vivo. Strikingly, we identified cyclin-dependent kinase inhibitor 1B (CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late timepoints in vivo. CDKN1B ablation increased BCMA CAR T cell proliferation and effector function in response to antigen, significantly enhancing tumor clearance and overall survival. Thus, our findings reveal differing effects of gene-perturbation on CAR T cells over time and in different selective environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma, and underscore the importance of in vivo screening as a tool for identifying genes to enhance CAR T cell function and efficacy.

immunology↗

A generalizable normative deep autoencoder for brain morphological anomaly detection: application to the multi-site StratiBip dataset on bipolar disorder in an external validation framework.

The heterogeneity of psychiatric disorders makes researching disorder-specific neurobiological markers an ill-posed problem. Here, we face the need for disease stratification models by presenting a generalizable multivariate normative modelling framework for characterizing brain morphology, applied to bipolar disorder (BD). We employed deep autoencoders in an anomaly detection framework, combined with a confounder removal step integrating training and external validation. The model was trained with healthy control (HC) data from the human connectome project and applied to multi-site external data of HC and BD individuals. We found that brain deviating scores were greater, more heterogeneous, and with increased extreme values in the BD group, with volumes prominently from the basal ganglia, hippocampus and adjacent regions emerging as significantly deviating. Similarly, individual brain deviating maps based on modified z scores expressed higher abnormalities occurrences, but their overall spatial overlap was lower compared to HCs. Our generalizable framework enabled the identification of subject- and group-level brain normative-deviating patterns, a step forward towards the development of more effective and personalized clinical decision support systems and patient stratification in psychiatry.

neuroscience↗

Experience-dependent modulation of collective behavior in larval zebrafish

Complex group behavior can emerge from simple inter-individual interactions. Commonly, these interactions are considered static and hardwired and little is known about how experience and learning affect collective group behavior. Young larvae use well described visuomotor transformations to guide interindividual interactions and collective group structure. Here, we use naturalistic and virtual-reality (VR) experiments to impose persistent changes in population density and measure their effects on future visually evoked turning behavior and the resulting changes in group structure. We find that neighbor distances decrease after exposure to higher population densities, and increase after the experience of lower densities. These adaptations develop slowly and gradually, over tens of minutes and remain stable over many hours. Mechanistically, we find that larvae estimate their current group density by tracking the frequency of neighbor-evoked looming events on the retina and couple the strength of their future interactions to that estimate. A time-varying state-space model that modulates agents social interactions based on their previous visual-social experiences, accurately describes our behavioral observations and predicts novel aspects of behavior. These findings provide concrete evidence that inter-individual interactions are not static, but rather continuously evolve based on past experience and current environmental demands. The underlying neurobiological mechanisms of experience dependent modulation can now be explored in this small and transparent model organism.

animal behavior and cognition↗

Metabolic immaturity of newborns and breast milk bile acids are the central determinants of heightened neonatal vulnerability to norovirus diarrhea

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

microbiology↗

Deep super-resolution imaging of thick tissue using structured illumination with adaptive optics

Three-dimensional structured illumination microscopy (3D-SIM) doubles the resolution of fluorescence imaging in all directions and increases the image contrast. However, 3D-SIM has not been widely applied to imaging deep in thick tissues due to its sensitivity to sample-induced aberrations, making the method difficult to apply beyond 10 {micro}m in depth. Furthermore, 3D-SIM has not been available in an upright configuration, limiting its use for live imaging while manipulating the specimen, for example with electrophysiology. Here, we have overcome these barriers by developing a novel upright 3D-SIM system (termed Deep3DSIM) that incorporates adaptive optics for aberration correction and remote focusing, reducing artefacts, improving contrast, restoring resolution, and removing the need to move the specimen or the objective lens in volume imaging. These advantages are equally applicable to inverted 3D-SIM systems. We demonstrate high-quality 3D-SIM imaging in various samples, including an example of imaging 130 {micro}m into Drosophila brain.

cell biology↗

Clinically relevant orthotopic pancreatic cancer models for adoptive T cell therapy

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor. Prognosis is poor and survival is low in patients diagnosed with this disease; [~]12% at 5 years. Immunotherapy, including adoptive T cell transfer therapy, has not impacted outcomes in PDAC patients, due in part to the hostile tumor microenvironment (TME) which limits T cell trafficking and persistence. We posit that murine models serve as useful tools to study the fate of T cell therapy. Currently, genetically engineered mouse models (GEMM) for PDAC are considered a "gold-standard" as they recapitulate many aspects of human disease. However, these models have limitations, including marked tumor variability across individual mice and cost of colony maintenance. We characterized the immunologic features and trafficking patterns of adoptively transferred T cells in orthotopic PDAC models using two mouse cell lines, KPC-Luc and MT-5, isolated from KPC-GEMM mouse models (KrasLSL-G12D/+p53-/- and KrasLSL-G12D/+p53LSL-R172H/+, respectively). The MT-5 orthotopic model best recapitulates the cellular and stromal features of the TME in the PDAC GEMM. In contrast, far more host immune cells infiltrate KPC-Luc tumors, which have less stroma. Albeit CD4+ T cells were similarly detected in MT-5 tumors compared to KPC-GEMM in mice. Interestingly, we found that CAR T cells redirected to recognize mesothelin on these tumors that signal via CD3{sigma} and 41BB (Meso-41BB{sigma}-CAR T cells) post antigen recognition infiltrated the tumors of mice bearing stroma-devoid KPC-Luc orthotopic tumors, but not MT-5 tumors. Our data establish for the first time a reproducible and realistic clinical system useful for modeling stroma-rich and stroma-devoid PDAC tumors. These models shall serve in-depth study of how to overcome barriers that limit anti-tumor activity of adoptively transferred T cells.

cancer biology↗

Regulation of ATAD2B bromodomain binding activity by the histone code

The ATPase family AAA+ domain containing 2 (ATAD2) protein, and its paralog ATAD2B, have a C-terminal bromodomain that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2 and ATAD2B bromodomains to select acetylated lysine among multiple histone post-translational modifications. Isothermal titration calorimetry experiments revealed that the ATAD2 and ATAD2B bromodomains selectively recognize distinct patterns of acetylated lysine residues on the N-terminal tails of histone proteins. Adjacent methylation or phosphorylation marks were found to either enhance or weaken the recognition of acetylated lysine by the ATAD2/B bromodomains. Complementary structural studies provide mechanistic insights into how residues within the bromodomain binding pocket coordinate the acetyllysine group in the context of adjacent post- translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands, either as onco mutations or as histone variants, impact the recognition of an adjacent acetylated lysine residue. In summary, our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2 and ATAD2B bromodomains, resulting in distinct binding modes of the two bromodomains. KEY POINTSO_LIMultiple independent ATAD2 gene duplication events are evident during metazoan evolution, indicating expansion of functionality in the ATAD2 gene family and suggesting distinct functions for ATAD2 and ATAD2B. C_LIO_LIHigh-resolution structures of the ATAD2 and ATAD2B bromodomains in complex with their histone ligands demonstrate how multiple post-translational modifications are coordinated. C_LIO_LIRecognition of different subsets acetylated histone ligands by the ATAD2 and ATAD2B bromodomains is driven by unique features within the binding pockets of these paralogous proteins. C_LIO_LIOnco-histone mutations and histone variants that change the amino acid sequence of the histone tails modulate the ATAD2 and ATAD2B bromodomain activity. C_LIO_LIThis study demonstrates how the combinatorial activity of multiple post- translational modifications forms a histone code and influences the recognition of acetylated lysine by bromodomain-containing proteins. C_LI

biochemistry↗

Structural insights into acetylated histone ligand recognition by the BDP1 bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to up-regulate and down-regulate certain genes required for invasion and replication. The P. falciparum genome encodes six bromodomaincontaining proteins, of which Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and the 8 human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

biophysics↗

Combining metabolomics and experimental evolution reveals key mechanisms underlying longevity differences in laboratory evolved Drosophila melanogaster populations

Experimental evolution with Drosophila melanogaster has been used extensively for decades to study aging and longevity. In recent years, the addition of DNA and RNA sequencing to this framework has allowed researchers to leverage the statistical power inherent to experimental evolution study the genetic basis of longevity itself. Here we incorporated metabolomic data into to this framework to generate even deeper insights into the physiological and genetic mechanisms underlying longevity differences in three groups of experimentally evolved D. melanogaster populations with different aging and longevity patterns. Our metabolomic analysis found that aging alters mitochondrial metabolism through increased consumption of NAD+ and increased usage of the TCA cycle. Combining our genomic and metabolomic data produced a list of biologically relevant candidate genes. Among these candidates, we found significant enrichment for genes and pathways associated with neurological development and function, and carbohydrate metabolism. While we do not explicitly find enrichment for aging canonical genes, neurological dysregulation and carbohydrate metabolism are both known to be associated with accelerated aging and reduced longevity. Taken together, our results provide plausible genetic mechanisms for what might be driving longevity differences in this experimental system. More broadly, our findings demonstrate the value of combining multiple types of omic data with experimental evolution when attempting to dissect mechanisms underlying complex and highly polygenic traits like aging.

evolutionary biology↗

Coordination of di-acetylated histone ligands by the ATAD2 bromodomain

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four -helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histones. In addition, ATAD2 is shown to be up regulated in multiple forms of cancer including breast, lung, gastric, endometrial, renal, and prostate. Furthermore, up-regulation of ATAD2 is strongly correlated with poor prognosis in many types of cancer, making the ATAD2 bromodomain an innovative target for cancer therapeutics. In this study, we describe the recognition of histone acetyllysine modifications by the ATAD2 bromodomain. Residue-specific information on the complex formed between the histone tail and the ATAD2 bromodomain, obtained through nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography, illustrates key residues lining the binding pocket, which are involved in coordination of di-acetylated histone tails. Analytical ultracentrifugation, NMR relaxation data, and isothermal titration calorimetry further confirm the monomeric state of the functionally active ATAD2 bromodomain in complex with di-acetylated histone ligands. Overall, we describe histone tail recognition by ATAD2 BRD and illustrate that one acetyllysine group is primarily engaged by the conserved asparagine (N1064), the "RVF" shelf residues, and the flexible ZA loop. Coordination of a second acetyllysine group also occurs within the same binding pocket, but is essentially governed by unique hydrophobic and electrostatic interactions making the di-acetyllysine histone coordination more specific than previously presumed.

biochemistry↗