bluuebunny/crossref_metadata_2025 huggingface dataset:
Conservation, alteration, and redistribution of mammalian striatal interneurons
Authors: Corrigan Emily K , DeBerardine Michael , Poddar Aunoy , Turrero Garcia Miguel , Schmitz Matthew T , Harwell Corey , Paredes Mercedes , Krienen Fenna M , Pollen Alex Aaron
Published: July 2024

Mammalian brains vary in size, structure, and function, but the extent to which evolutionarily novel cell types contribute to this variation remains unresolved. Recent studies suggest there is a primate-specific population of striatal inhibitory interneurons, the TAC3 interneurons5. However, there has not yet been a detailed analysis of the spatial and phylogenetic distribution of this population. Here, we profile single cell gene expression in the developing pig (an ungulate) and ferret (a carnivore), representing 94 million years divergence from primates, and assign newborn inhibitory neurons to initial classes first specified during development. We find that the initial class of TAC3 interneurons represents an ancestral striatal population that is also deployed towards the cortex in pig and ferret. In adult mouse, we uncover a rare population expressing Tac2, the ortholog of TAC3, in ventromedial striatum, prompting a reexamination of developing mouse striatal interneuron initial classes by targeted enrichment of their precursors. We conclude that the TAC3 interneuron initial class is conserved across Boreoeutherian mammals, with the mouse population representing Th striatal interneurons, a subset of which expresses Tac2. This study suggests that initial classes of telencephalic inhibitory neurons are largely conserved and that during evolution, neuronal types in the mammalian brain change through redistribution and fate refinement, rather than by derivation of novel precursors early in development.
Oxidation of Four Monoterpenoid Indole Alkaloid Classes by Three Cytochrome P450 Monooxygenases fromTabernaemontana litoralis
Authors: Mai Zhan , Kim Kyunghee , Richardson Matthew Bailey , Deschênes Daniel André Ramey , Garza-Garcia Jorge Jonathan Oswaldo , Shahsavarani Mohammadamin , Perley Jacob Owen , Njoku Destiny Ichechi , Deslongchamps Ghislain , De Luca Vincenzo , Qu Yang
Published: July 2024

Cytochrome P450 monooxygenases (CYPs) are well known for their ability to catalyze diverse oxidation reactions, playing a significant role in the biosynthesis of various natural products. In the realm of monoterpenoid indole alkaloids (MIAs), one of the largest groups of alkaloids in nature, CYPs are integral to reactions such as hydroxylation, epoxidation, ring opening, ring rearrangement, and aromatization, contributing to the extensive diversification of these compounds. In this study, we investigate the transcriptome, metabolome, and MIA biosynthesis inTabernaemontana litoralis(milky way tree), a prolific producer of rare pseudoaspidosperma-type MIAs. Alongside known pseudoaspidosperma biosynthetic genes, we identify and characterize three new CYPs that facilitate regio- and stereospecific oxidation of four MIA skeletons: iboga, aspidosperma, pseudoaspidosperma, and quebrachamine. Notably, the tabersonine 14,15-β-epoxidase catalyzes the formation of pachysiphine, the stereoisomer of 14,15-α-epoxytabersonine (lochnericine) found inCatharanthus roseus(Madagascar periwinkle) roots. The pseudovincadifformine 18-hydroxylase is the first CYP identified to modify a pseudoaspidosperma skeleton. Additionally, we demonstrate that the enzyme responsible for C10-hydroxylation of the iboga MIA coronaridine also catalyzes the same reaction on voaphylline, which bears a quebrachamine skeleton. With the discovery of a new MIA, 11-hydroxypseudovincadifformine, this study provides a comprehensive understanding of MIA biosynthesis and diversification inT. litoralis, highlighting its potential for further exploration.
Genetic diversity analysis of main agronomic traits and ISSR markers in 35 ornamental rape germplasm resources
Authors: Xia Mang , Chen Meizhu , Dong Xiaoxiao , Chen Jingdong , Cheng Miao , Wan Heping , Dong Yuanhuo , Zeng Changli , Dai Xigang
Published: July 2024

Rape (Brassica napusL.) is a major oil crop in our country, valued for its oil and ornamental uses. This study analyzed 35 ornamental rape germplasm resources from different origins to examine differences in agronomic traits and molecular markers. Nine agronomic traits were assessed in the field for variability, correlation, principal component analysis, and cluster analysis. Genetic diversity was analyzed using microsatellite (ISSR) markers and the unweighted pair group method with arithmetic mean (UPGMA). Our findings revealed a notable average coefficient of variation of 22.59% across the nine agronomic traits, with flower color exhibiting the highest variability and corolla width the least. The observed range of variation spanned from 9.24% to 83.38%, the correlation among these traits was generally low, with a mere 13.9% demonstrating significant correlations. The four principal components accounted for an impressive 84.62% of the cumulative contribution rate, while the genetic similarity, as gauged by eight ISSR primers, varied from 0.675 to 0.980. Most strikingly, we observed that plants from the same geographical region displayed molecular-level differences, underscoring the rich genetic diversity inherent in the 35 ornamental rape resources under study. Employing UPGMA cluster analysis on the primary agronomic traits and ISSR molecular markers, the 35 ornamental rape resources were categorized into seven and four distinct groups, respectively. Although the clustering outcomes from these two methodologies did not align perfectly, they served to complement each other. Collectively, these insights offer a theoretical framework for the innovation of ornamental rape germplasm resources and the cultivation of novel varieties.
Appendage regeneration requires IMPDH2 and creates a sensitized environment for enzyme filament formation
Authors: McCartney Morgan E. , Wheeler Gavin M. , O’Neill Audrey G. , Patel Jeet H. , Litt Zoey R. , Calise S. John , Kollman Justin M. , Wills Andrea E.
Published: July 2024

Regeneration of lost tissue requires biosynthesis of metabolites needed for cell proliferation and growth. Among these are the critical purine nucleotides ATP and GTP. The abundance and balance of these purines is regulated by inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyzes the committing step of GTP synthesis. IMPDH2 assembles into filaments that resist allosteric inhibition under conditions of high GTP demand. Here we asked whether IMPDH2 is required in the highly proliferative context of regeneration, and whether its assembly into filaments takes place in regenerating tissue. We find that inhibition of IMPDH2 leads to impaired tail regeneration and reduced cell proliferation in the tadpoleXenopus tropicalis. We find that both endogenous and fluorescent fusions of IMPDH2 robustly assemble into filaments throughout the tadpole tail, and that the regenerating tail creates a sensitized condition for filament formation. These findings clarify the role of purine biosynthesis in regeneration and reveal that IMPDH2 enzyme filament formation is a biologically relevant mechanism of regulation in vertebrate regeneration.

Graphical Abstract
Methods for detecting “missing” dimensions in genetic covariance matrices
Authors: Dochtermann Ned A.
Published: July 2024

Blows and Hoffmann (2005) and others have suggested that low levels of genetic variation in some dimensions of an additive genetic variance-covariance matrix (G) will be detectable as eigenvalues approaching zero. These dimensions may be indicative of trade-offs (Blows and Hoffmann 2005) or, potentially, of “holes” in fitness landscapes (Dochtermann et al. 2023).

However, because the estimation ofGtypically constrains variances to be positive, and matrices to therefore be positive definite, “approaching zero” is challenging to statistically define. It is, therefore, not currently clear how to statistically identify dimensions with effectively zero variances. Being able to identify dimensions ofGwith variances approaching zero would improve our ability to understand trade-offs which, typically and inappropriately, focus on the signs of bivariate correlations (Houle 1991). Viable approaches would also allow better understanding of the structure ofGand the evolutionary processes that have shapedGacross taxa.

Mezey and Houle (2005) and Kirkpatrick and Lofsvold (1992) used matrix rank to determine if there were dimensions lacking variation. Rank is the number of eigenvalues for a matrix that are greater than zero. If aG’s rank is less than its dimensionality, this would be evidence that there are dimensions without variation. However, most contemporary analysis procedures force estimates ofGto be positive semi-definite. Consequently, estimatedGs will not have eigenvalues of 0 or negative. Therefore, the estimated eigenvalues on their own will not allow the identification of absolute constraints (sensu(Houle 2001)) or more generally dimensions lacking variation. One alternative would be to compare the eigenvalues of observed matrices to those of random matrices and, if there were dimensions with less variation than expected by chance, this comparison could be used to identify dimensions in which evolution is constrained.

If trade-offs or landscape holes result in dimensions ofGthat have minimal variance, we would expect that the associated eigenvalues exhibit less variation than expected by chance. Here, I tested whether the distribution of eigenvalues from randomly generated correlated matrices can be used for such testing.
Topological mixing and irreversibility in animal chromosome evolution
Authors: Schultz Darrin T. , Blümel Arno , Destanović Dalila , Sarigol Fatih , Simakov Oleg
Published: July 2024

Abstract/Summary Paragraph

Animal chromosomes can persist with recognizable homology over hundreds of millions of years, in spite of homology-obfuscating processes such as chromosomal fusion and translocation. The frequency and pace of these major genome structural changes are unknown, and it remains unclear whether or how they impact long-term genome evolution. Here, we compare whole chromosomal sequences of 3,631 genomes from 2,291 species spanning all major animal clades and show that animal karyotypes evolve primarily via karyotype contraction, associated with increased rates of chromosomal fusion-with-mixing and dispersion that largely obey chromosomal algebra1, or karyotype expansion, which breaks up ancestral linkage groups and forms new chromosomal elements via non-algebraic changes. We show that chromosomal changes can be associated with major extinction events. Using a multi-scale encoding of pan-animal genome homology and a manifold representation of genomic changes, we find that genome evolution is not only driven by changes at the chromosomal level, but that subchromosomal mixing and irreversibility define clade-specific evolution. Using this ‘evolutionary genome topology’ approach, we calculate extrema of irreversible genomic configurations and identify species that occupy intermediate manifold positions, providing evidence for distinct macro-evolutionary trajectories. We propose that investigation of mixed state accumulation around important gene loci (such as Hox) will be crucial in capturing and further study of clade-specific regulatory innovations.
Nuclear Argonaute protein NRDE-3 switches small RNA partners during embryogenesis to mediate temporal-specific gene regulatory activity
Authors: Chen Shihui , Phillips Carolyn M.
Published: July 2024

RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. We also observe that many other RNAi factors form foci in embryonic cells distinct from “SIMR granules”, including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Prior work has shown that NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
A modular system to label endogenous presynaptic proteins using split fluorophores inC. elegans
Authors: Kurashina Mizuki , Snow Andrew W. , Mizumoto Kota
Published: July 2024

Visualizing the subcellular localization of presynaptic proteins with fluorescent proteins is a powerful tool to dissect the genetic and molecular mechanisms underlying synapse formation and patterning in live animals. Here, we utilize split green and red fluorescent proteins to visualize the localization of endogenously expressed presynaptic proteins at a single neuron resolution inCaenorhabditis elegans.By using CRISPR/Cas9 genome editing, we generated a collection ofC. elegansstrains in which endogenously expressed presynaptic proteins (RAB-3/Rab3, CLA-1/Piccolo, SYD-2/Liprin-α, UNC-10/RIM and ELKS-1/ELKS) are tagged with tandem repeats of GFP11and/or wrmScarlet11. We show that the expression of wrmScarlet1-10and GFP1-10under neuron-specific promoters can robustly label presynaptic proteins in different neuron types. We believe that combinations of knock-in strains and wrmScarlet1-10and GFP1-10plasmids are a versatile modular system to examine the localization of endogenous presynaptic proteins in any neuron type.
Comparative metabolomics of wild and cultivatedTaxus cuspidata: insights into metabolic diversity and environmental adaptability
Authors: Wang Dandan , Zhang Yanwen
Published: July 2024

Taxus cuspidatais a well-known gymnosperm with great ornamental and medicinal value. The study aims to reveal the metabolic differences between wild and cultivated species ofT. cuspidata, and to analyze the genetic and ecological factors behind these differences. This study conducted a comparative metabolomics analysis of wild and cultivatedT. cuspidatabased on LC-MS/MS technology. The results showed: (1) A total of 7030 metabolites were identified, primarily including flavonoids, organic acids, phenolic acids, amino acids and their derivatives, lipids, and alkaloids, among others; (2) 2381 differential metabolites were confirmed: 949 higher in cultivated species and 1432 higher in wild species, which suggesting wild species have more advantageous metabolites. (3) Through KEGG annotation, 20 significant metabolic pathways were identified, with sugar metabolism, photosynthesis, and sulfur metabolism were significantly higher in wild species. In contrast, pathways related to most amino acids and their derivatives, flavonoids, terpenoids, polyphenols, alkaloids, and plant hormones were significantly higher in cultivated species. In conclusion, the wild species are inferior to cultivated species in terms of drought resistance, growth rate, and medicinal value for cancer treatment. However, the advantage of wild species lies in their significantly higher diversity of upregulated secondary metabolites due to their rich genetic resources, which exhibit greater metabolic diversity and environmental adaptability, which enhances their survival capability. Furthermore, the wild species exhibit significantly higher efficiency in photosynthesis and sugar metabolism compared to cultivated species, enabling them to better withstand low-temperature stress. This study has provided the direction for research into the mechanisms of stress resistance inT. cuspidataand has also established a theoretical basis for formulating scientifically and reasonable conservation strategies for wild species.
Integrative, high-resolution analysis of single cells across experimental conditions with PARAFAC2
Authors: Ramirez Andrew , Orcutt-Jahns Brian T. , Pascoe Sean , Abraham Armaan , Remigio Breanna , Thomas Nathaniel , Meyer Aaron S.
Published: July 2024

Effective tools for exploration and analysis are needed to extract insights from large-scale single-cell measurement data. However, current techniques for handling single-cell studies performed across experimental conditions (e.g., samples, perturbations, or patients) require restrictive assumptions, lack flexibility, or do not adequately deconvolute condition-to-condition variation from cell-to-cell variation. Here, we report that the tensor decomposition method PARAFAC2 (Pf2) enables the dimensionality reduction of single-cell data across conditions. We demonstrate these benefits across two distinct contexts of single-cell RNA-sequencing (scRNA-seq) experiments of peripheral immune cells: pharmacologic drug perturbations and systemic lupus erythematosus (SLE) patient samples. By isolating relevant gene modules across cells and conditions, Pf2 enables straightforward associations of gene variation patterns across specific patients or perturbations while connecting each coordinated change to certain cells without pre-defining cell types. The theoretical grounding of Pf2 suggests a unified framework for many modeling tasks associated with single-cell data. Thus, Pf2 provides an intuitive universal dimensionality reduction approach for multi-sample single-cell studies across diverse biological contexts.

Highlights

PARAFAC2 enables tensor-based analysis of single-cell experiments across conditions.

PARAFAC2 separates condition-specific effects from cell-to-cell variation.

PARAFAC2 provides intuitive isolation of patterns into condition-, cell-, and gene-specific patterns.
Glucocorticoids desensitize hypothalamic CRH neurons to norepinephrine and somatic stress activation via rapid nitrosylation-dependent regulation of α1 adrenoreceptor trafficking
Authors: Weiss Grant L , Harrison Laura M. , Jiang Zhiying , Nielsen Alyssa M. , Feygin Maximillian S. , Nguyen Sandy , Tirrell Parker S. , Tasker Jeffrey
Published: July 2024

Noradrenergic afferents to hypothalamic corticotropin releasing hormone (CRH) neurons provide a major excitatory drive for somatic stress activation of the hypothalamic-pituitary-adrenal (HPA) axis. We showed that glucocorticoids rapidly desensitize CRH neurons to norepinephrine and suppress inflammation-induced HPA activation via a glucocorticoid receptor- and endocytosis-dependent mechanism. Here, we show that α1 adrenoreceptor (ARα1) trafficking is regulated by convergent glucocorticoid and nitric oxide synthase signaling mechanisms. Live-cell imaging of ARα1b-eGFP-expressing hypothalamic cells revealed rapid corticosterone-stimulated redistribution of internalized ARα1 from rapid recycling endosomes to late endosomes and lysosomes via a nitrosylation-regulated mechanism. Proximity assay demonstrated interaction of glucocorticoid receptors with ARα1b and β-arrestin, and showed corticosterone blockade of norepinephrine-stimulated ARα1b/β-arrestin interaction, which may prevent ARα1b from entering the rapid recycling endosomal pathway. These findings demonstrate a rapid glucocorticoid regulation of G protein-coupled receptor trafficking and provide a molecular mechanism for rapid glucocorticoid desensitization of noradrenergic signaling in CRH neurons.
White matter connections of human ventral temporal cortex are organized by cytoarchitecture, eccentricity, and category-selectivity from birth
Authors: Kubota Emily , Yan Xiaoqian , Tung Sarah , Fascendini Bella , Tyagi Christina , Duhameau Sophie , Ortiz Danya , Grotheer Mareike , Natu Vaidehi S. , Keil Boris , Grill-Spector Kalanit
Published: July 2024

Category-selective regions in ventral temporal cortex (VTC) have a consistent anatomical organization, which is hypothesized to be scaffolded by white matter connections. However, it is unknown how white matter connections are organized from birth. Here, we scanned newborn to 6-month-old infants and adults to determine the organization of the white matter connections of VTC. We find that white matter connections are organized by cytoarchitecture, eccentricity, and category from birth. Connectivity profiles of functional regions in the same cytoarchitectonic area are similar from birth and develop in parallel, with decreases in endpoint connectivity to lateral occipital, and parietal, and somatosensory cortex, and increases to lateral prefrontal cortex. Additionally, connections between VTC and early visual cortex are organized topographically by eccentricity bands and predict eccentricity biases in VTC. These data show that there are both innate organizing principles of white matter connections of VTC, and the capacity for white matter connections to change over development.
Time-resolved tracking of cellulose biosynthesis and microfibril network assembly during cell wall regeneration in live Arabidopsis protoplasts
Authors: Huh Hyun , Jayachandran Dharanidaran , Sun Junhong , Irfan Mohammad , Lam Eric , Chundawat Shishir P. S. , Lee Sang-Hyuk
Published: July 2024

Plant cell walls are composed of polysaccharides among which cellulose is the most abundant component. Cellulose is processively synthesized as bundles of linear β-1,4-glucan homopolymer chains via the coordinated action of multiple enzymes in cellulose synthase complexes (CSCs) embedded within the plasma cell membrane. Plant cell walls are composed of multiple layers of cellulose fibrils that form highly intertwined extracellular matrix networks. However, it is not yet clear as to how cellulose fibrils synthesized by multiple CSCs are assembled into the intricate cellulose network deposited on plant cell surfaces. Herein, we have established an in vivo time-resolved imaging platform for visualizing cellulose during its biosynthesis and assembly into a complex fibrillar network on the surface of Arabidopsis thaliana mesophyll protoplasts as the primary cell wall regenerates. We performed total internal reflection fluorescence microscopy (TIRFM) with fluorophore-conjugated tandem carbohydrate binding modules (tdCBMs) that were engineered to specifically bind to nascent cellulose fibrils. Together with a well-controlled environment, it was possible to monitor in vivo cellulose fibril synthesis dynamics in a time-resolved manner for nearly one day of continuous cell wall regeneration on protoplast cell surfaces. Our observations provide the basis for a novel model of cellulose fibril network development in protoplasts driven by complex interplay of multi-scale dynamics that include: rapid diffusion and coalescence of short nascently synthesized cellulose fibrils; processive elongation of single fibrils; and cellulose fibrillar network rearrangement during cell wall maturation. This platform is valuable for exploring mechanistic aspects of cell wall synthesis while visualizing cellulose microfibrils assembly.
Age-related myelin deficits in the auditory brain stem contribute to cocktail-party deficits
Authors: Poleg Shani , Li Ben-Zheng , Sergison Matthew , Ridenour Matthew , Hughes Ethan G. , Tollin Daniel , Klug Achim
Published: July 2024

Age-related hearing loss consists of both peripheral and central components and is an increasing global health concern. While peripheral hearing loss is well understood, central hearing loss— age-related changes in the central auditory pathways resulting in a listener’s inability to process sound correctly —remains poorly understood. In this study, we focus on the pathway from the cochlear nucleus to the medial nucleus of the trapezoid body (MNTB), which depends on heavily myelinated axons for microsecond-level temporal precision required for sound localization.

Using a combination of auditory brainstem response recordings (ABR), advanced light and electron microscopy, and behavioral testing with prepulse inhibition of the acoustic startle response (PPI) we identified a correlation between oligodendrocyte loss, abnormal myelination in MNTB afferents, altered ABR wave III morphology indicating MNTB dysfunction, and deficits in spatial hearing behaviors in aging Mongolian gerbils.

These findings provide a mechanistic explanation of how demyelination contributes to age-related dysfunction in the auditory brainstem’s sound localization pathway.
Amyloid peptide – synthetic polymer blends with enhanced mechanical and biological properties
Authors: Wang Xianjun , Mondal Malay , Jankoski Penelope E. , Kemp Lisa K. , Clemons Tristan D. , Rangachari Vijayaraghavan , Morgan Sarah E.
Published: July 2024

Interest in utilizing amyloids to develop biomaterials is increasing due to their potential for biocompatibility, unique assembling morphology, mechanical stability, and biophysical properties. However, challenges include the complexity of peptide chemistry and the practical techniques required for processing amyloids into bulk materials. In this work, two decapeptides with fibrillar and globular morphologies were selected, blended with poly(ethylene oxide), and fabricated into composite mats via electrospinning. Notable enhancements in mechanical properties were observed, attributed to the uniform distribution of the decapeptide assemblies within the PEO matrix. Morphological differences, such as the production of thinner nanofibers, are attributed to the increased conductivity from the zwitterionic nature of the decapeptides. Blend rheology and post-processing analysis revealed how processing might affect the amyloid aggregation and secondary structure of the peptides. Both decapeptides demonstrated good biocompatibility and strong antioxidant activity, indicating their potential for safe and effective use as biomaterials. By evaluating these interdependencies, this research lays the foundation for understanding the structure-property-processing relationships of peptide-polymer blends and highlights the strong potential for developing applications in biotechnology.
Formation of stress granules and non-canonical survival responses in arsenite-exposed cells
Authors: Hirano Seishiro , Udagawa Osamu , Kanno Sanae
Published: July 2024

The concentration-dependent decrease in viable cells is a well-documented phenomenon in cytotoxicity assays for most toxic substances. We report that arsenite (As3+), a widely recognized oxidative toxicant, exhibited lower cytotoxic effects at 300 µM compared to 100 µM As3+in CHO-K1 and Jurkat cells. Formation of stress granules (SGs), which appear in the cytoplasm shortly after exposure to hypertonicity, heat shock, and high concentrations of As3+is considered as a pro-survival cellular event. We hypothesized that unusual cytotoxicity profile of As3+could be attributed to SG formation. In both CHO-K1 and Jurkat cells stably expressing GFP-tagged G3BP1, SGs were more rapidly and distinctly induced by 300 µM As3+than 100 µM As3+. Other toxic metals and a metalloid such as Cd2+, Cu2+, Ag+, and Se4+did not clearly induce SG formation and instead reduced the viability in a concentration-dependent manner. Exposure to As3+led to phosphorylation of eIF2α, a key regulator of polysome stability and a hallmark of SG formation. Depletion of intracellular glutathione (GSH) increased the susceptibility of cells to As3+, highlighting its role in cellular defense mechanisms. Exposure to As3+activated small ubiquitin-like modifier (SUMO) which is implicated in phase separation. However, neither depletion of GSH nor overexpression of SUMO contributed As3+-induced SG formation. Consistently, THP-1 and HL60 cells exposed to As3+also exhibited non-canonical cytotoxic features, albeit at higher concentrations (1 mM). These findings underscore the need for further mechanistic investigations into As3+-induced SG formation, given that As3+is a promising anti-cancer agent, and resistance of tumor cells to As3+is a critical issue.
CaLMPhosKAN: Prediction of General Phosphorylation Sites in Proteins via Fusion of Codon-Aware Embeddings with Amino Acid-Aware Embeddings and Wavelet-based Kolmogorov–Arnold Network
Authors: Pratyush Pawel , Carrier Callen , Pokharel Suresh , Ismail Hamid D. , Chaudhari Meenal , KC Dukka B.
Published: July 2024

The mapping from codon to amino acid is surjective due to the high degeneracy of the codon alphabet, suggesting that codon space might harbor higher information content. Embeddings from the codon language model have recently demonstrated success in various downstream tasks. However, predictive models for phosphorylation sites, arguably the most studied Post-Translational Modification (PTM), and PTM sites in general, have predominantly relied on amino acid-level representations. This work introduces a novel approach for prediction of phosphorylation sites by incorporating codon-level information through embeddings from a recently developed codon language model trained exclusively on protein-coding DNA sequences. Protein sequences are first meticulously mapped to reliable coding sequences and encoded using this encoder to generate codon-aware embeddings. These embeddings are then integrated with amino acid-aware embeddings obtained from a protein language model through an early fusion strategy. Subsequently, a window-level representation of the site of interest is formed from the fused embeddings within a defined window frame. A ConvBiGRU network extracts features capturing spatiotemporal correlations between proximal residues within the window, followed by a Kolmogorov-Arnold Network (KAN) based on the Derivative of Gaussian (DoG) wavelet transform function to produce the prediction inference for the site. We dub the overall model integrating these elements as CaLMPhosKAN. On independent testing with Serine-Threonine (combined) and Tyrosine test sets, CaLMPhosKAN outperforms existing approaches. Furthermore, we demonstrate the model’s effectiveness in predicting sites within intrinsically disordered regions of proteins. Overall, CaLMPhosKAN emerges as a robust predictor of general phosphosites in proteins. CaLMPhosKAN will be released publicly soon.
The origin of septin ring size control in budding yeast
Authors: Kukhtevich Igor , Persson Sebastian , Padovani Francesco , Schneider Robert , Cvijovic Marija , Schmoller Kurt M.
Published: July 2024

The size of organelles and cellular structures needs to be tightly controlled and adjusted to the overall cell size to ensure cell function. A prominent example of a self-assembly process forming a structure whose size scales with cell size is Cdc42-driven cell polarization and subsequent septin ring formation inSaccharomyces cerevisiae. Despite extensive research, the mechanisms that determine Cdc42 cluster and septin ring size are still unclear. Combining computational modeling, live-cell imaging and genetic manipulations, we show here that positive feedback in the polarization pathway, together with the amount of polarity proteins increasing with cell size, can account for the increase of the Cdc42 cluster area with cell size, and as a consequence also the scaling of the septin ring diameter. We demonstrate that inbni1Δcells, which have a larger septin ring compared to wild-type cells but a similarly sized Cdc42-GTP cluster, disruption of F-actin cable assembly and polarization toward the bud site results in diffuse exocytosis, causing septin ring enlargement. Furthermore, we find that in cells with impaired negative feedback in the polarization pathway, the Cdc42-GTP cluster area expands compared to wild-type cells, while the septin ring diameter remains mostly unchanged. This decoupling can be partially explained by a high recruitment rate of septin via polarity factors. Taken together, we provide insights into the origin of septin ring size control, in particular the scaling with cell volume, by integrating new experimental findings and mechanistic models of budding yeast polarization.
Distributed Collaboration for Data, Analysis Pipelines, and Results in Single-Cell Omics
Authors: Hutton Alexandre , Ai Lizhuo , Meyer Jesse G.
Published: July 2024

Single-cell omics data analysis pipelines are complicated to design and difficult to share or reproduce. We describe a web platform that enables no-code analysis pipeline design, simple computing via the Open Science Grid, and sharing of entire data analysis pipelines, their input data, and interactive results. We expect this platform to increase the accessibility and reproducibility of single-cell omics.
Evolution of the RNA alternative decayciselement into a high-affinity target for the immunomodulatory protein Roquin
Authors: Tants Jan-Niklas , Friedrich Katharina , Neumann Jasmina , Schlundt Andreas
Published: July 2024

RNAciselements play pivotal roles in regulatory processes, e.g. in transcriptional and translational regulation. Two stem-loopedciselements, the constitutive and alternative decay elements (CDE and ADE, respectively) are shape-specifically recognized in mRNA 3’ untranslated regions (UTRs) by the immune-regulatory protein Roquin. Roquin initiates mRNA decay and contributes to balanced transcript levels required for immune homeostasis. While the interaction of Roquin with several CDEs is described, our knowledge about ADE complex formation is limited to the mRNA ofOx40, a gene encoding for a T-cell co-receptor. TheOx403’UTR comprises both a CDE and ADE, each sufficient for Roquin-mediated control. Opposed to highly conserved and abundant CDE structures, ADEs are rarer, but predicted to exhibit a greater structural heterogeneity. This raises the question how and when two structurally distinctciselements evolved as equal target motifs for Roquin. Using an interdisciplinary approach we here monitor the evolution of sequence and structure features of theOx40ADE across species. We designed RNA variants to probe en-detail determinants steering Roquin-RNA complex formation. Specifically, those reveal the contribution of a second RNA-binding interface of Roquin for recognition of the ADE basal stem region. In sum, our study sheds light on how the conserved Roquin protein selected ADE-specific structural features to evolve a second high-affinity mRNA targetcis-element relevant for adaptive immune regulation. As our findings also allow expanding the RNA target spectrum of Roquin, the approach can serve a paradigm for understanding RNA-protein specificity through back-tracing the evolution of the RNA element.
Pharyngeal neuronal mechanisms governing sour taste perception inDrosophila melanogaster
Authors: Shrestha Bhanu , Sang Jiun , Rimal Suman , Lee Youngseok
Published: July 2024

Sour taste, which is elicited by low pH, may serve to help animals distinguish appetitive from potentially harmful food sources. In all species studied to date, the attractiveness of oral acids is contingent on concentration. Many carboxylic acids are attractive at ecologically relevant concentrations but become aversive beyond some maximal concentration. Recent work found thatDrosophilaionotropic receptors IR25a and IR76b expressed by sweet-responsive gustatory receptor neurons (GRNs) in the labellum, a peripheral gustatory organ, mediate appetitive feeding behaviors toward dilute carboxylic acids. Here, we disclose the existence of pharyngeal sensors inD. melanogasterthat detect ingested carboxylic acids and are also involved in the appetitive responses to carboxylic acids. These pharyngeal sensors rely on IR51b, IR94a, and IR94h, together with IR25a and IR76b, to drive responses to carboxylic acids. We then demonstrate that optogenetic activation of eitherIr94a+orIr94h+GRNs promotes an appetitive feeding response, confirming their contributions to appetitive feeding behavior. Our discovery of internal pharyngeal sour taste receptors opens up new avenues for investigating the internal sensation of tastants in insects.
Extracellular matrix integrity regulates GABAergic plasticity in the hippocampus
Authors: Jabłońska Jadwiga , Wiera Grzegorz , Mozrzymas Jerzy W.
Published: July 2024

The brain’s extracellular matrix (ECM) is crucial for neural circuit functionality, synaptic plasticity, and learning. While the role of the ECM in excitatory synapses has been extensively studied, its influence on inhibitory synapses, particularly on GABAergic long-term plasticity, remains poorly understood. This study aims to elucidate the effects of ECM components on inhibitory synaptic transmission and plasticity in the hippocampal CA1 region. We focus on the roles of chondroitin sulfate proteoglycans (CSPGs) and hyaluronic acid in modulating inhibitory postsynaptic currents (IPSCs) at two distinct inhibitory synapses formed by somatostatin (SST)-positive and parvalbumin (PV)-positive interneurons onto pyramidal cells (PCs). Using optogenetic stimulation in brain slices, we observed that acute degradation of ECM constituents by hyaluronidase or chondroitinase-ABC did not affect basal inhibitory synaptic transmission. However, short-term plasticity, particularly burst-induced depression, was enhanced at PV→PC synapses following enzymatic treatments. Long-term plasticity experiments demonstrated that CSPGs are essential for NMDA-induced iLTP at SST→PC synapses, whereas the digestion of hyaluronic acid by hyaluronidase impaired iLTP at PV→PC synapses. This indicates a synapse-specific role of CSPGs and hyaluronic acid in regulating GABAergic plasticity. Additionally, we report the presence of cryptic GABAergic plasticity at PV→PC synapses induced by prolonged NMDA application, which became evident after CSPG digestion and was absent under control conditions. Our results underscore the differential impact of ECM degradation on inhibitory synaptic plasticity, highlighting the synapse-specific interplay between ECM components and specific GABAergic synapses. This offers new perspectives in studies on learning and critical period timing.
Efficient generation of CAR NK cells from human umbilical cord blood CD34+stem and progenitors for democratizing affordable immunotherapy
Authors: Li Jianhuan , Wang Yao , Zheng Xiujuan , Lin Yunqing , Weng Qitong , Liu Xiaofei , Geng Yang , Wu Hongling , Liu Lijuan , Peng Huan , Wu Bingyan , Huang Dehao , Xia Chengxiang , Wang Tongjie , Zhang Mengyun , Du Xin , Zeng Hui , Dong Fang , Zhang Yingchi , Zhu Xiaofan , Hu Fangxiao , Wang Jinyong
Published: July 2024

Chimeric antigen receptor (CAR) natural killer cells (CAR NK) cells, leveraging safety and not requiring HLA match in adoptive infusion, have emerged as promising alternative cells to CAR-T cells for immunotherapies. High and multiple doses of CAR NK cell infusions are essential to maintain therapeutic efficacy in clinical trials. This requires efficient methods for generating large-scale CAR NK cells and significantly reducing CAR engineering costs. In this study, we develop a three-step strategy to generate highly high yields of induced NK (iNK) and CAR iNK cells from human umbilical cord blood CD34+hematopoietic stem and progenitor cells (CD34+HSPCs). Starting from a single umbilical cord blood CD34+HSPC, our reliable method efficiently produces 14-83 million mature iNK cells or 7-32 million CAR iNK cells with high expression levels of CD16 and zero T cell contaminations. Introducing CAR expression elements at the HSPC level reduces the quantities of CAR pseudoviruses to 1 / 140.000 - 1 / 600,000 compared to engineering CARs in mature NK cells. The iNK and CAR iNK cells, including fresh cells and thawed cells from cryopreserved conditions, demonstrate remarkable tumoricidal activities against various human cancer cells and significantly prolong the survival of human tumor-bearing animals. The high yields of CAR NK cells and negligible costs of CAR engineering of our method support the broad applications of CAR NK cells for treating cancer patients.
Proteomics Landscape of Triple-Negative Apocrine Breast Carcinoma Reveals Molecular Mechanisms of Tumorigenesis and Characteristics of Chemo-insensitivity
Authors: Zhu Yiying , Long Mengping , Shi Wenhao , He Tianlong , Xie Fangzhou , Qian Annan , Liu Yuqiao , Hu Taobo , Tang Shaojun
Published: July 2024

Triple-negative apocrine breast carcinoma (TNAC) is a rare type of triple-negative breast cancer (TNBC) characterized by apocrine morphology. Due to its low clinical incidence, TNAC has not been well studied at the molecular level in terms of cellular mechanisms of tumorigenesis and treatment response. In this study, we collected formalin-fixed paraffin-embedded samples from 31 patients diagnosed with invasive TNAC. Mass spectrometry analysis and whole-exome sequencing were performed to systematically construct the proteome and mutation landscape of the TNAC. Comparing ductal carcinoma in situ (DCIS), invasive, and adjacent normal tissues, we observed increased cadherin binding and ligase activity during tumorigenesis, heightened extracellular matrix signaling at tumor initiation, and elevated GTPase activity during tumor progression. Besides, we discovered genes such as C3 and COL18A1 enriched in somatic mutations are also significantly dysregulated in invasive TNAC. Additionally, by assessing biopsies before neo-adjacent chemotherapy, we provided molecular evidence supporting clinical observations of chemotherapy insensitivity in TNAC patients. We further identified a protein panel (CAPN1, CORO1B, HK1, RAP1B, and VPS4B) differentiating TNAC from non-apocrine TNBC, which could potentially serve as diagnostic markers. Taken together, this study represents the first large-scale proteomics analysis of TNAC and may provide guidance on decisions regarding the chemotherapy treatment of TNAC patients.
Structures of H2A.Z-associated human chromatin remodelers SRCAP and TIP60 reveal divergent mechanisms of chromatin engagement
Authors: Park Giho , Patel Avinash B , Wu Carl , Louder Robert K
Published: July 2024

H2A.Z is a conserved histone variant that is localized to specific genomic regions where it plays important roles in transcription, DNA repair, and replication. Central to the biochemistry of human H2A.Z are the SRCAP and TIP60 chromatin remodelers, homologs of yeast SWR1 which catalyzes ATP-dependent H2A.Z exchange. Here, we use cryo-electron microscopy to resolve six structural states of the native SRCAP complex, uncovering conformational intermediates interpreted as a stepwise path to full nucleosome engagement. We also resolve the structure of the native TIP60 complex which consists of a structured core from which flexibly tethered chromatin binding domains emerge. Despite the shared subunit composition, the core of TIP60 displays divergent architectures from SRCAP that structurally disfavor nucleosome engagement, suggesting a distinct biochemical function.
Disruption of mitochondrial folate metabolism leads to mitochondrial DNA leakage and activation of apoptotic or inflammatory pathways
Authors: Hwang Sinwoo , Baker Cameron , Field Martha S.
Published: July 2024

Folate-mediated one-carbon metabolism (FOCM) is required for the biosynthesis of purines, thymidylate (dTMP), and methionine. Maintenance of adequate cellular dTMP levels is essential to preserve the integrity of the nuclear and mitochondrial genomes. Inadequate dTMP production leads to uracil misincorporation into mitochondrial DNA (mtDNA) and impaired mitochondrial function. However, the mechanisms whereby uracil in mtDNA impairs mitochondrial function are uncharacterized. The release of mtDNA into the cytosol acts as a damage-associated molecular pattern (DAMP), resulting in inflammation and cell death. Previously, we reported that disrupting mitochondrial dTMP synthesis through serine hydroxymethyltransferase 2 (Shmt2) heterozygosity increases uracil in mtDNA and impairs mitochondrial function in mice. This study aimed to investigate whether impairment of mitochondrial FOCM throughShmt2disruption leads to the release of mtDNA into the cytosol.Shmt2+/-MEF cells showed a > 2-fold increase in cytosolic mtDNA leakage compared toShmt2+/+cells (p<0.05). There was no significant difference in total mtDNA content betweenShmt2+/+andShmt2+/-MEF cells. MEFs with decreasedShmt2expression activated apoptosis by the ratio of cleaved caspase-3 to caspase-3. In addition, SHMT2 knock-out (SHMT2 KO) haploid chronic myeloid leukemia (HAP1) cells also exhibited increased cytosolic mtDNA content compared to the wild-type (WT) HAP1 cells. HAP1 lackingShmt2expression activated the cGAS/STING pathway, but suppressed apoptosis, compared to WT HAP1 cells. This study demonstrates that decreasedShmt2expression leads to cytosolic mtDNA leakage and that the downstream effects of mtDNA leakage vary by cell type.
diaPASEF analysis for HLA-I peptides enables quantification of common cancer neoantigens
Authors: Oliinyk Denys , Gurung Hem , Zhou Zhenru , Leskoske Kristin , Rose Christopher M. , Klaeger Susan
Published: July 2024

Human leukocyte antigen class I (HLA-I) molecules present short peptide sequences from endogenous or foreign proteins to cytotoxic T cells. The low abundance of HLA-I peptides poses significant technical challenges for their identification and accurate quantification. While mass spectrometry (MS) is currently a method of choice for direct system-wide identification of cellular immunopeptidome, there is still a need for enhanced sensitivity in detecting and quantifying tumor specific epitopes. As gas phase separation in data-dependent MS data acquisition (DDA) increased HLA-I peptide detection by up to 50%, here, we aimed to evaluate the performance of data-independent acquisition (DIA) in combination with ion mobility (diaPASEF) for high-sensitivity identification of HLA presented peptides. Our streamlined diaPASEF workflow enabled identification of 11,412 unique peptides from 12.5 million A375 cells and 3,426 8-11mers from as low as 500,000 cells with high reproducibility. By taking advantage of HLA binder-specificin-silicopredicted spectral libraries, we were able to further increase the number of identified HLA-I peptides. We applied SILAC-DIA to a mixture of labeled HLA-I peptides, calculated heavy-to-light ratios for 7,742 peptides across 5 conditions and demonstrated that diaPASEF achieves high quantitative accuracy up to 4-fold dilution. Finally, we identified and quantified shared neoantigens in a monoallelic C1R cell line model. By spiking in heavy synthetic peptides, we verified the identification of the peptide sequences and calculated relative abundances for 13 neoantigens. Taken together, diaPASEF analysis workflows for HLA-I peptides can increase the peptidome coverage for lower sample amounts. The sensitivity and quantitative precision provided by DIA can enable the detection and quantification of less abundant peptide species such as neoantigens across samples from the same background.