bluuebunny/crossref_metadata_2025 huggingface dataset:
Damsel: Analysis and visualisation of DamID sequencing in R
Authors: Page Caitlin G , Londsdale Andrew , Mitchell Katrina A , Schröder Jan , Harvey Kieran F. , Oshlack Alicia
Published: June 2024

DamID sequencing is a technique to map the genome-wide interaction of a protein with DNA. Damsel is the first Bioconductor package to provide an end to end analysis for DamID sequencing data within R. Damsel performs quantification and testing of significant binding sites along with exploratory and visual analysis. Damsel produces results consistent with previous analysis approaches.

Availability and implementation

The R package Damsel is available for install through the Bioconductor projecthttps://bioconductor.org/packages/release/bioc/html/Damsel.htmland the code is available on GitHubhttps://github.com/Oshlack/Damsel/

Contact

caitlin.page@petermac.org

Supplementary information

Available through the journal.
High-order Michaelis-Menten equations allow inference of hidden kinetic parameters in enzyme catalysis
Authors: Singh Divya , Robin Tal , Urbakh Michael , Reuveni Shlomi
Published: June 2024

Single-molecule measurements provide a platform for investigating the dynamical properties of enzymatic reactions. To this end, the single-molecule Michaelis-Menten equation was instrumental as it asserts that the first moment of the enzymatic turnover time depends linearly on the reciprocal of the substrate concentration. This, in turn, provides robust and convenient means to determine the maximal turnover rate and the Michaelis-Menten constant. Yet, the information provided by these parameters is incomplete and does not allow full characterization of enzyme kinetics at the single molecule level. Here we show that the missing kinetic information can be accessed via a set of high-order Michaelis-Menten equations that we derive. These equations capture universal linear relations between the reciprocal of the substrate concentration and distinguished combinations of turnover time moments, essentially generalizing the Michaelis-Menten equation to moments of any order. We demonstrate how key observables such as the lifetime of the enzyme-substrate complex, the rate of substrate-enzyme binding, and the probability of successful product formation, can all be inferred using these high-order Michaelis-Menten equations.
Optimizing drug synergy prediction through categorical embeddings in Deep Neural Networks
Authors: Lastre Manuel González , de Prado Salas Pablo González , Guantes Raúl
Published: June 2024

Cancer treatments often lose effectiveness as tumors develop resistance to single–agent therapies. Com-bination treatments can overcome this limitation, but the overwhelming combinatorial space of drug-dose interactions makes exhaustive experimental testing impractical. Data-driven methods, such as Machine and Deep Learning, have emerged as promising tools to predict synergistic drug combinations. In this work, we benchmark the use of categorical embeddings within Deep Neural Networks to enhance drug synergy predictions. These learned and transferable encodings capture similarities between the elements of each category, demonstrating particular utility in scarce data scenarios.
Addressing the dynamic nature of reference data: a new nt database for robust metagenomic classification
Authors: Martí Jose Manuel , Kok Car Reen , Thissen James B. , Mulakken Nisha J. , Avila-Herrera Aram , Jaing Crystal J. , Allen Jonathan E. , Be Nicholas A.
Published: June 2024

Background

Accurate metagenomic classification relies on comprehensive, up-to-date, and validated reference databases. While the NCBI BLAST Nucleotide (nt) database, encompassing a vast collection of sequences from all domains of life, represents an invaluable resource, its massive size —currently exceeding 1012 nucleotides— and exponential growth pose significant challenges for researchers seeking to maintain current nt-based indices for metagenomic classification. Recognizing that no current nt-based indices exist for the widely used Centrifuge classifier, and the last public version was released in 2018, we addressed this critical gap by leveraging advanced high-performance computing resources.

Results

We present new Centrifuge-compatible nt databases, meticulously constructed using a novel pipeline incorporating different quality control measures, including reference decontamination and filtering. These measures demonstrably reduce spurious classifications, and through temporal comparisons, we reveal how this approach minimizes inconsistencies in taxonomic assignments stemming from asynchronous updates between public sequence and taxonomy databases. These discrepancies are particularly evident in taxa such asListeria monocytogenesandNaegleria fowleri, where classification accuracy varied significantly across database versions.

Conclusions

These new databases, made available as pre-built Centrifuge indexes, respond to the need for an open, robust, nt-based pipeline for taxonomic classification in metagenomics. Applications such as environmental metagenomics, forensics, and clinical metagenomics, which require comprehensive taxonomic coverage, will benefit from this resource. Our new nt-based index highlights the importance of treating reference databases as dynamic entities, subject to ongoing quality control and validation akin to software development best practices. This dynamic update approach is crucial for ensuring the accuracy and reliability of metagenomic analysis, especially as databases continue to expand in size and complexity.
On the potential for GWAS with phenotypic population means and allele-frequency data (popGWAS)
Authors: Pfenninger Markus
Published: June 2024

It is vital to understand the genomic basis of differences in ecologically important traits if we are to understand the impact of global change on biodiversity and enhance our ability for targeted intervention. This study explores the potential of a novel genome-wide association study (GWAS) approach for identifying loci underlying quantitative polygenic traits in natural populations, based on phenotypic population means and genome-wide allele frequency data as obtained e.g. by PoolSeq approaches. Extensive population genetic forward simulations demonstrate that the approach is generally effective for oligogenic and moderately polygenic traits and relatively insensitive to low heritability. However, applicability is limited for highly polygenic architectures and pronounced population structure. The required sample size is moderate with very good results being obtained already for a few dozen populations scored. When combined with machine learning for feature selection, the method performs very well in predicting population means. The data efficiency of the method, particularly when using pooled sequencing and bulk phenotyping, makes GWAS studies more accessible for research in biodiversity genomics. Moreover, in a direct comparison to individual based GWAS, the proposed method performed constistently better with regard to the number of true positive loci identified and prediction accuracy. Overall, this study highlights the promise of popGWAS for dissecting the genetic basis of complex traits in natural populations.
Enhancing the biological insight from limited proteolysis-coupled mass spectrometry
Authors: Nagel L. , Grossbach J. , Cappelletti V. , Doerig C. , Picotti P. , Beyer A.
Published: June 2024

Limited proteolysis combined with mass spectrometry (LiP-MS) facilitates probing structural changes on a proteome-wide scalein situ. Distinguishing the different signal contributions, such as changes in protein abundance, from protein abundance changes remains challenging. We propose a two-step approach, first removing unwanted variations from the LiP signal that are not caused by protein structural effects and subsequently inferring the effects of variables of interest on the remaining signal. Using LiP-MS data from three species we demonstrate that our framework provides a uniquely powerful approach for deconvolving LiP-MS signals and inferring protein structural changes.
Intra-genomic genes-to-genes correlation enables genome representation
Authors: Chen Jingjie , Ma Xuchuan , He Junwei , Wang Yingxia , Ren Yuji , Qi Li , Song Liuyang , Ruan Lin , Fan Cun , Jiang Jun , Bao Yongjia , Huang Tao , Cheng Jingbo , Liu Xing , Chen Fang , Huang Yichen , Zhao Na , Guo Chunming
Published: June 2024

The pan-genome consists of core genes shared by all members of a taxonomy and accessory genes found in only a subset, holding the keys to advancing our understanding of evolution and tackling medical challenges. Here, we discovered a strong intra-genomic correlation among bacterial genes within each ofEscherichia coli,Listeria monocytogenes,Staphylococcus aureus, andCampylobacter jejuni. With a convolutional neural network assisted core genome knock-out simulation, we found that different combinations of fewer than 20 highly variable core genes could recover the sub-species type classified by complete core genome with accuracy >95%. This observation led us to test the genes-to-genes predictability: with more than 52,000 assemblies from each species, combinations of highly variable core genes could predict the sequence variants of other core genes (average accuracy >94%) within the same genome and could also predict sequence variants (average accuracy >91%) as well as the presence (average AUROC >0.91) of some accessory genes. Furthermore, combinations of highly variable core genes could also predict multiple antibiotic resistances (AUROC >0.80) in large published datasets ofE. coli,S. aureus, andMycobacterium tuberculosis. Collectively, we propose that genes within the same genome can strongly correlate with each other. Therefore, the strain phylogeny and the stauts of other genes could be uniformly represented by combinations of highly variable core genes, which could further represent certain phenotypes includingin vitroresistance.
Interferon signaling is enhanced by ATR inhibition in glioblastoma cells irradiated with X-rays, protons or carbon ions
Authors: Rødland Gro Elise , Temelie Mihaela , Serban Ana Maria , Eek Mariampillai Adrian , Edin Nina Frederike Jeppesen , Malinen Eirik , Gilbert Antoine , Chevalier François , Savu Diana I. , Syljuåsen Randi G.
Published: June 2024

Background and purpose

Interferon signaling plays an important role in antitumor immune responses. Inhibitors of the DNA damage response, such as ATR inhibitors, can increase interferon signaling upon conventional radiotherapy with X-rays. However, whether such inhibitors also increase interferon (IFN) signaling after high linear energy transfer (LET) particle irradiation is not known.

Materials and methods

Human glioblastoma U-251 and T98G cells were treated with X-rays, protons (linear energy transfer (LET): 7 and 38 keV/μm) and carbon ions (LET: 28 and 73 keV/μm), with and without ATR inhibitor (VE822) or ATM inhibitor (AZD1390). DNA damage signaling and cell cycle distribution were assayed by immunoblotting and flow cytometry, and radiosensitivity by clonogenic survival. IFN-β secretion was measured by ELISA and STAT1 activation by immunoblotting.

Results

High-LET protons and carbon ions caused stronger activation of the DNA damage response compared to low-LET protons andX-rays at similar radiation dose. G2 checkpoint arrest was abrogated by the ATR inhibitor and prolonged by the ATM inhibitor after all radiation types. The inhibitors increased radiosensitivity, as measured after X- and carbon-ion-irradiation. ATR inhibition increased IFN signaling after both low-LET and high-LET irradiation in both cell lines. In T98G, IFN signaling was also enhanced by ATM inhibition. Notably, T98G cells secreted markedly more IFN-β when the inhibitors were combined with high-LET compared to low-LET irradiation.

Conclusion

Our results show that ATR inhibition can increase IFN signaling after both X-, proton- and carbon-ion-irradiation. Additionally, IFN induction is strongly dependent on LET in one of the tested cell lines.
The human pathogenMycobacterium tuberculosisand the fish pathogenMycobacterium marinumtrigger the same core set of late innate immune response genes in zebrafish larvae
Authors: Dirks Ron P. , Ordas Anita , Jong-Raadsen Susanne , Brittijn Sebastiaan A. , Haks Mariëlle C. , Henkel Christiaan V. , Oravcova Katarina , Racz Peter I. , Tuinhof-Koelma Nynke , Korzeniowska nee Wiweger Malgorzata I. , Gillespie Stephen H. , Meijer Annemarie H. , Ottenhoff Tom H.M. , Jansen Hans J. , Spaink Herman P.
Published: June 2024

Zebrafish is a natural host of variousMycobacteriumspecies and a surrogate model organism for tuberculosis research.Mycobacterium marinumis evolutionarily one of the closest non-tuberculous species related toM. tuberculosisand shares the majority of virulence genes. Although zebrafish is not a natural host of the human pathogen, we have previously demonstrated successful robotic infection of zebrafish embryos withM. tuberculosisand performed drug treatment of the infected larvae. In the present study we examined for how longM. tuberculosiscan be propagated in zebrafish larvae and tested a time series of infected larvae to study the transcriptional response via Illumina RNA deep sequencing (RNAseq). Granuloma-like structures carrying fluorescently labeledM. tuberculosiscould be detected up to 9 days post infection. The continued presence of viableM. tuberculosisin the zebrafish larvae was further confirmed using the molecular bacterial load (MBL) assay. The infected larvae showed a clear and specific transcriptional immune response with a high similarity to the response of zebrafish larvae infected with the surrogate speciesM. marinum. We conclude thatM. tuberculosiscan be propagated in zebrafish larvae for at least one week after infection and provide further evidence thatM. marinumis a good surrogate model forM. tuberculosis.
T cell responsiveness to IL-10 defines the immunomodulatory effect of costimulation blockade via anti-CD154 and impacts transplant survival
Authors: Iglesias Marcos , Bibicheff Darrel , Komin Alexander , Chicco Maria , Guinn Samantha , Foley Brendan , Raimondi Giorgio
Published: June 2024

Costimulation blockade (CoB)-based immunotherapy is a promising alternative to immunosuppression for transplant recipients; however, the current limited understanding of the factors that impact its efficacy restrains its clinical applicability. In this context, pro- and anti-inflammatory cytokines are being recognized as having an impact on T cell activation beyond effector differentiation. This study aims at elucidating the impact of direct IL-10 signaling in T cells on CoB outcomes. We used a full-mismatch skin transplantation model where recipients had a T cell-restricted expression of a dominant negative IL-10 receptor (10R-DN), alongside anti-CD154 as CoB therapy. Unlike wild-type recipients, 10R-DN mice failed to benefit from CoB. This accelerated graft rejection correlated with increased accumulation of T cells producing TNF-α, IFN-γ, and IL-17. In vitro experiments indicated that while lack of IL-10 signaling did not change the ability of anti-CD154 to modulate alloreactive T cell proliferation, the absence of this pathway heightened TH1 effector cell differentiation. Furthermore, deficiency of IL-10 signaling in T cells impaired Treg induction, a hallmark of anti-CD154 therapy. Overall, these findings unveil an important and novel role of IL-10 signaling in T cells that defines the success of CoB therapies and identifies a target pathway for obtaining robust immunoregulation.
Signals in the Cells: Multimodal and Contextualized Machine Learning Foundations for Therapeutics
Authors: Velez-Arce Alejandro , Lin Xiang , Li Michelle M. , Huang Kexin , Gao Wenhao , Fu Tianfan , Pentelute Bradley L. , Kellis Manolis , Zitnik Marinka
Published: June 2024

Drug discovery AI datasets and benchmarks have not traditionally included single-cell analysis biomarkers. While benchmarking efforts in single-cell analysis have recently released collections of single-cell tasks, they have yet to comprehensively release datasets, models, and benchmarks that integrate a broad range of therapeutic discovery tasks with cell-type-specific biomarkers. Therapeutics Commons (TDC-2) presents datasets, tools, models, and benchmarks integrating cell-type-specific contextual features with ML tasks across therapeutics. We present four tasks for contextual learning at single-cell resolution: drug-target nomination, genetic perturbation response prediction, chemical perturbation response prediction, and protein-peptide interaction prediction. We introduce datasets, models, and benchmarks for these four tasks. Finally, we detail the advancements and challenges in machine learning and biology that drove the implementation of TDC-2 and how they are reflected in its architecture, datasets and benchmarks, and foundation model tooling.
Entropy Changes in Water Networks Promote Protein Denaturation
Authors: Wang Yichong , Liu Junlang , Peters Michael M , Ishii Ryoma , Wang Dianzhuo , Chowdhury Sourav , Parker Kevin Kit , Shakhnovich Eugene I
Published: June 2024

For over a century, an explanation for how concentrated ions denature proteins has proven elusive. Here, we report a novel mechanism of protein denaturation driven by entropy changes in water networks. Experiments and simulations show that ion pairs like LiBr and LiCl localize water molecules and disrupt the water network's structure, while others exert a more global effect without compromising network integrity. This disruption reduces the entropy penalty when proteins sequester water molecules during unfolding, resulting in a peculiar yet universal "inverse hydrophobic effect" that potentiates protein denaturation. Through successful isolation and systematic study of indirect solute effects, our findings offer a universal approach to salt induced protein denaturation and provide a unified framework for the decoding of the protein-water-solute nexus.
In VivoProximity Labeling Identifies a New Function for the Lifespan and Autophagy-regulating Kinase Pef1, an Ortholog of Human Cdk5
Authors: Zhang Haitao , Zhang Dongmei , Li Ling , Willard Belinda , Runge Kurt W.
Published: June 2024

Cdk5 is a highly-conserved, noncanonical cell division kinase important to the terminal differentiation of mammalian cells in multiple organ systems. We previously identified Pef1, theSchizosaccharomyces pombeortholog of cdk5, as regulator of chronological lifespan. To reveal the processes impacted by Pef1, we developed APEX2-biotin phenol-mediated proximity labeling inS. pombe.Efficient labeling required a short period of cell wall digestion and eliminating glucose and nitrogen sources from the medium. We identified 255 high-confidence Pef1 neighbors in growing cells and a novel Pef1-interacting partner, the DNA damage response protein Rad24. The Pef1-Rad24 interaction was validated by reciprocal proximity labeling and co-immunoprecipitation. Eliminating Pef1 partially rescued the DNA damage sensitivity of cells lacking Rad24. To monitor how Pef1 neighbors change under different conditions, cells induced for autophagy were labeled and 177 high-confidence Pef1 neighbors were identified. Gene ontology (GO) analysis of the Pef1 neighbors identified proteins participating in processes required for autophagosome expansion including regulation of actin dynamics and vesicle-mediated transport. Some of these proteins were identified in both exponentially growing and autophagic cells. Pef1-APEX2 proximity labeling therefore identified a new Pef1 function in modulating the DNA damage response and candidate processes that Pef1 and other cdk5 orthologs may regulate.
Uncovering topologically associating domains from three-dimensional genome maps with TADGATE
Authors: Dang Dachang , Zhang Shao-Wu , Dong Kangning , Duan Ran , Zhang Shihua
Published: June 2024

Topologically associating domains (TADs) emerge as indispensable units in three-dimensional (3D) genome organization, playing a critical role in gene regulation. However, accurately identifying TADs from sparse chromatin contact maps and exploring the structural and functional elements within TADs remain challenging. To this end, we develop a graph attention auto-encoder, TADGATE, to accurately identify TADs even from ultra-sparse contact maps and generate the imputed maps while preserving or enhancing the underlying topological structures. TADGATE can capture specific attention patterns, pointing to two types of units with different characteristics in TADs. Moreover, we find that the organization of TADs is closely associated with chromatin compartmentalization, and TAD boundaries in different compartmental environments exhibit distinct biological properties. We also utilize a two-layer Hidden Markov Model to functionally annotate the TADs and their internal regions, revealing the overall properties of TADs and the distribution of the structural and functional elements within TADs. At last, we apply TADGATE to highly sparse and noisy Hi-C contact maps from 21 human tissues or cell lines, enhancing the clarity of TAD structures, investigating the nature of conserved and cell type-specific boundaries, and unveiling the cell type-specific transcriptional regulatory mechanisms associated with topological domains.
Promoting Human Intestinal Organoid Formation and Stimulation Using Piezoelectric Nanofiber Matrices
Authors: Poling Holly M. , Singh Akaljot , Krutko Maksym , Reza Abid A. , Srivastava Kalpana , Wells James M. , Helmrath Michael A. , Esfandiari Leyla
Published: June 2024

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology three days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.
Synergy Between TNFα and Proteostatic Stress Drives Cell Death and Guard Immunity
Authors: Sonnett Matthew , Centofanti Edoardo , Boersma Sanne , Brambach Max , Peshkin Leon , Tatavosian Roubina , Oyler-Yaniv Alon , Oyler-Yaniv Jennifer
Published: June 2024

The production and sensing of type I interferons (IFN-I) are critical for antiviral defense, yet most virus-infected cells do not produce IFN-I or upregulate IFN-stimulated genes. Using quantitative proteomics and global protein synthesis measurements, we show that productive viral infection globally down-regulates protein synthesis, restricting the IFN response. Guard immunity, which responds to disruptions in essential cellular processes, might compensate for the lack of IFN-I response by rapidly killing infected cells. However, non-pathological stressors can also disrupt proteostasis, making it unclear how cells decide to trigger guard immunity. We hypothesized that TNFα, produced by macrophages, provides a contextual signal allowing specificity. Using live-cell fluorescence microscopy and mathematical modeling, we showed that TNFα synergizes with the rapid decay of the anti-apoptotic protein c-FLIP to induce cell death and prevent viral spread. Our findings demonstrate that TNFα contextualizes proteostasis loss as non-sterile, enabling the activation of guard immunity to counteract viral infection.
An unbiased comparison of immunoglobulin sequence aligners
Authors: Konstantinovsky Thomas , Peres Ayelet , Polak Pazit , Yaari Gur
Published: June 2024

Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) is critical for our under-standing of the adaptive immune system’s dynamics in health and disease. Reliable analysis of AIRR-seq data depends on accurate Immunoglobulin (Ig) sequence alignment. Various Ig sequence aligners exist, but there is no unified benchmarking standard representing the complexities of AIRR-seq data, obscuring objective comparisons of aligners across tasks. Here, we introduce GenAIRR, an efficient simulation framework for generating Ig sequences alongside their ground truths. GenAIRR realistically simulates the intricacies of V(D)J recombination, somatic hypermutation, and an array of sequence corruptions. We comprehensively assessed prominent Ig sequence aligners across various metrics, unveiling unique performance characteristics for each aligner. The GenAIRR-produced datasets, combined with the proposed rigorous evaluation criteria, establish a solid basis for unbiased benchmarking of immunogenetics computational tools. It sets up the ground for further improving the crucial task of Ig sequence alignment, ultimately enhancing our understanding of adaptive immunity.
Transcriptome-wide analysis of circRNA and RBP profiles and their molecular and clinical relevance for GBM
Authors: Latowska-Łysiak J , Zarębska Ż , Sajek MP , Grabowska A , Buratin A , Misiorek JO , Kuczyński K , Bortoluzzi S , Żywicki M , Kosiński JG , Rybak-Wolf A , Piestrzeniewicz R , Barciszewska AM , Rolle K
Published: June 2024

Glioblastoma (GBM) is the most aggressive and lethal type of glioma, characterized by aberrant expression of non-coding RNAs including circular RNAs (circRNAs). They might impact cellular processes by interacting with other molecules – like microRNAs or RNA-binding proteins (RBPs). The diagnostic value of circRNAs and circRNAs/RBPs complexes is still largely unknown. To explore circRNAs and RBPs transcripts expression in GBM, we performed and further analyzed RNA-seq data from GBM patients’ primary and recurrent tumor samples. We identified circRNAs differentially expressed in primary tumors, the circRNA progression markers in recurrent GBM samples as well as the expression profile of RBP transcripts. Subsequent analysis allowed us to generate a comprehensive catalog of circRNA-RBP interactions regarding both the RBPs sequestration by circRNA as well as the RBPs involvement in circRNA biogenesis. Furthermore, we demonstrated the clinical potential of circRNAs and RBPs in GBM and proposed them as the stratification markers in the de novo assembled tumor subtypes. Therefore, our transcriptome-wide study specified circRNA-RBP interactions that could play a significant regulatory role in gliomagenesis and GBM progression.
Accelerated phenology fails to buffer fitness loss from delayed rain onset in a clade of wildflowers
Authors: Worthy Samantha J. , Ashlock Sarah R. , Miller Arquel , Maloof Julin N. , Strauss Sharon Y. , Gremer Jennifer R. , Schmitt Johanna
Published: June 2024

The timing of early life cycle events has cascading effects on phenology and fitness. These effects may be critical for climate resilience of plant populations, especially in Mediterranean environments, where delayed rainfall onset causes delayed germination. To examine impacts of germination timing on ten species of theStreptanthus/Caulanthusclade, we induced germination across a range of dates in ambient seasonal conditions and recorded phenological and fitness traits. Later germination cohorts accelerated flowering, partially stabilizing flowering date, but the degree of this compensatory plasticity differed across species. Fitness declined with later germination; the magnitude of this decline depended on the balance between direct negative effects of later germination and compensatory positive effects of accelerated flowering. The resulting species’ differences in fitness responses suggest differential vulnerability to climate change. Species from wetter, cooler, less variable habitats accelerated flowering more and declined less in seed set with later germination, suggesting climate adaptation. However, other fitness responses to germination timing, like first year fitness, were evolutionarily labile across the clade and unrelated to climate. Although compensatory phenological plasticity may buffer the impacts of delayed germination, it cannot prevent long term declines in population fitness as fall rains come later with climate change.
Near Zero Photon Bioimaging
Authors: Sheneman Lucas , Balogun Sulaimon , Johnson Jill L. , Harrison Maria J. , Vasdekis Andreas E.
Published: June 2024

Enhancing the reliability and reproducibility of optical microscopy by reducing specimen irradiance continues to be an important biotechnology target. As irradiance levels are reduced, however, the particle nature of light is heightened, giving rise to Poisson noise, or photon sparsity that restricts only a few (0.5%) image pixels to comprise a photon. Photon-sparsity can be addressed by collecting more than 200 photons per pixel; this, however, requires extended acquisition durations and, thus, suboptimal imaging rates. Here, we introduce near-zero photon imaging, a method that operates at kHz rates and 10,000-fold lower irradiance than modern microscopy. To achieve this performance, we deployed a judiciously designed epi-fluorescence microscope enabling ultralow background and artificial intelligence that learns to reconstruct biological images from as low as 0.01 photons per pixel. We demonstrate that near-zero photon imaging captures the structure of both multicellular and subcellular targets with high fidelity, including features represented by nearly zero photons. Beyond optical microscopy, the near-zero photon imaging paradigm can be applied in remote sensing, covert applications, and biological or biomedical imaging that utilize damaging or quantum light.
A humoral immune response to parasitoid wasps inDrosophilais regulated by JAK/STAT, NF-κB and GATA
Authors: Zhou Shuyu Olivia , Day Jonathan P. , Deplancke Bart , Leitão Alexandre B. , Jiggins Francis M.
Published: June 2024

The two arms of innate immunity consist of the cell-mediated cellular defenses and the systemic humoral immune responses.Drosophilahumoral immune defenses in the context of antimicrobial immunity, particularly the regulation and activation of antimicrobial peptide secretion from the fat body, have been studied extensively. HowDrosophilaregulates humoral immunity against another major natural enemy, the parasitoid wasp, is less well-characterized. In this study, we focused on a gene crucial in anti-parasitoid immunity,lectin-24A, which is specifically induced following parasitization. We found that a fluorescent reporter driven by the region upstream oflectin-24Ashowed localized posterior expression in the larval fat body, theDrosophilatissue mediating humoral immunity. Furthermore, with RNA sequencing of the anterior and posterior fat body sections, we found that components of JAK/STAT, GATA, and Toll pathways were regulated differentially in the anterior-posterior axis of the fat body and/or by infection. Predicted binding motifs for transcription factors in all three of these pathways were identified in the 444bp upstream region of thelectin-24Agene, where scrambling these motifs leads to reduced basal or induced expression of the fluorescent reporter. Investigating each of these pathways, we found that JAK/STAT, the GATA factor Pannier, and the NF-κB factor dorsal all modulate the expression oflectin-24A. The binding motifs associated with these transcription factors were also enriched in the upstream sequences of parasitism-induced genes in the fat body. Taken together, these results indicate that JAK/STAT, Pannier, and NF-κB signaling are involved in the regulation oflectin-24Aand, more generally,Drosophilahumoral anti-parasitoid immunity after infection.
Interplay between phosphorylation and oligomerization tunes the conformational ensemble of SWEET transporters
Authors: Weigle Austin T. , Shukla Diwakar
Published: June 2024

SWEET sugar transporters are desirable biotechnological targets for improving plant growth. One engineering strategy includes modulating how SWEET transporters are regulated. Phosphorylation and oligomerization have been shown to positively regulate SWEET function, leading to increased sugar transport activity. However, constitutive phosphorylation may not be beneficial to plant health under basal conditions. Structural and mechanistic understanding of the interplay between phosphorylation and oligomerization in functional regulation of SWEETs remains limited. Using extensive molecular dynamics simulations coupled with Markov state models, we demonstrate the thermodynamic and kinetic effects of SWEET phosphorylation and oligomerization using OsSWEET2b as a model. We report that the beneficial effects of these SWEET regulatory mechanisms bias outward-facing states and improved extracellular gating, which complement published experimental findings. Our results offer molecular insights to SWEET regulation and may guide engineering strategies throughout the SWEET transport family.
Regeneration-specific promoter switching facilitates Mest expression in the mouse digit tip to modulate neutrophil response
Authors: Jou Vivian , Peña Sophia M. , Lehoczky Jessica A.
Published: June 2024

The mouse digit tip regenerates following amputation, a process mediated by a cellularly heterogeneous blastema. We previously found the gene Mest to be highly expressed in mesenchymal cells of the blastema and a strong candidate pro-regenerative gene. We now show Mest digit expression is regeneration-specific and not upregulated in post-amputation fibrosing proximal digits. Mest homozygous knockout mice exhibit delayed bone regeneration though no phenotype is found in paternal knockout mice, inconsistent with the defined maternal genomic imprinting of Mest. We demonstrate that promoter switching, not loss of imprinting, regulates biallelic Mest expression in the blastema and does not occur during embryogenesis, indicating a regeneration-specific mechanism. Requirement for Mest expression is tied to modulating neutrophil response, as revealed by scRNAseq and FACS comparing wildtype and knockout blastemas. Collectively, the imprinted gene Mest is required for proper digit tip regeneration and its blastema expression is facilitated by promoter switching for biallelic expression.
Escalation of genome defense capacity enables control of an expanding meiotic driver
Authors: Chen Peiwei , Pan Katherine C. , Park Eunice H. , Luo Yicheng , Lee Yuh Chwen G. , Aravin Alexei A.
Published: June 2024

From RNA interference to chromatin silencing, diverse genome defense pathways silence selfish genetic elements to safeguard genome integrity1,2. Despite their diversity, different defense pathways share a modular organization, where numerous specificity factors identify diverse targets and common effectors silence them. In the PIWI-interacting RNA (piRNA) pathway, which controls selfish elements in the metazoan germline, diverse target RNAs are first identified by complementary base pairing with piRNAs and then silenced by PIWI-clade nucleases via enzymatic cleavage1,3. Such a binary architecture allows the defense systems to be readily adaptable, where new targets can be captured via the innovation of new specificity factors4,5. Thus, our current understanding of genome defense against lineage-specific selfish genes has been largely limited to the evolution of specificity factors, while it remains poorly understood whether other types of innovations are required. Here, we describe a new type of innovation, which escalates the defense capacity of the piRNA pathway to control a recently expanded selfish gene inDrosophila melanogaster. Through anin vivoRNAi screen for repressors ofStellate—a recently evolved and expanded selfish meiotic driver6–8—we discovered a novel defense factor, Trailblazer. Trailblazer is a transcription factor that promotes the expression of two PIWI-clade nucleases, Aub and AGO3, to matchStellatein abundance. Recent innovation in the DNA-binding domain of Trailblazer enabled it to drastically elevate Aub and AGO3 expression in theD. melanogasterlineage, thereby escalating the silencing capacity of the piRNA pathway to control expandedStellateand safeguard fertility. As copy-number expansion is a recurrent feature of diverse selfish genes across the tree of life9–12, we envision that augmenting the defense capacity to quantitatively match selfish genes is likely a repeatedly employed defense strategy in evolution.
Understanding the heterogeneous performance of variant effect predictors across human protein-coding genes
Authors: Fawzy Mohamed , Marsh Joseph A.
Published: June 2024

Variant effect predictors (VEPs) are computational tools developed to assess the impacts of genetic mutations, often in terms of likely pathogenicity, employing diverse algorithms and training data. Here, we investigate the performance of 35 VEPs in the discrimination between pathogenic and putatively benign missense variants across 963 human protein-coding genes, revealing considerable gene-level heterogeneity as measured by the widely used area under the receiver operating characteristic curve (AUROC) metric. To investigate the origins of this heterogeneity and the extent to which gene-level VEP performance is predictable, we train random forest models to predict the gene-level AUROC for each VEP. We find that performance as measured by AUROC is related to factors such as gene function, protein structure, and evolutionary conservation. Notably, intrinsic disorder in proteins emerged as a significant factor influencing apparent VEP performance, often leading to inflated AUROC values due to their enrichment in weakly conserved putatively benign variants. While our results suggest that gene-level features may be useful for identifying genes where VEP predictions are likely to be more or less reliable, they also highlight the limitations of AUROC for comparing VEP performance across different genes.
Mechanisms of sustaining oxygen extraction efficiency in dragonfly nymphs during aquatic hypoxia
Authors: Lee Daniel J. , Matthews Philip G.D.
Published: June 2024

Despite breathing water using their tidally ventilated rectal gills, dragonfly nymphs show a surprising ability to maintain oxygen (O2) extraction from the water during hypoxia. However, an increase in convective O2transfer is insufficient to sustain aerobic demands by itself, which suggests that diffusive mechanisms must also be involved. This study examines the contributions of changing the O2partial pressure gradient (PO2) and/or O2conductance across the rectal gill in maintaining O2extraction efficiency (OEE) of dragonfly nymphs during hypoxia. Data were collected using the same custom-designed respiro-spirometer described in a previous study with the addition of an implanted O2sensor to measure hemolymph PO2. Results show that the implantation of the O2sensor does not affect the respiratory and ventilatory response of nymphs to hypoxia. Hemolymph PO2fell from 6.3 ± 1.6 kPa at normoxia to 2.5 ± 0.6 kPa at 16.0 kPa, which resulted in the PO2diffusion gradient remaining statistically constant at these two water PO2s (17.5 ± 1.7 and 15.4 ± 0.7 kPa during normoxia and 16.0 kPa respectively). Beyond 16.0 kPa, a progressive reduction in hemolymph PO2was unable to sustain the diffusion gradient. Mathematical modeling revealed that while the addition of hemolymph PO2in tandem with ventilation frequency was able to elevate OEE during 16.0 kPa to that of normoxia, both were still insufficient during severe hypoxia and required an increase in O2conductance. Estimating the change in whole-gill conductance showed that nymphs are indeed increasing their conductance as the water becomes hypoxic, demonstrating a reliance on both diffusion gradient and O2conductance to enhance diffusive O2transfer in conjunction with convective mechanisms to maintain O2extraction during hypoxia.
Seasonal analysis of leaf antioxidant activity in Colombian eliteHevea brasiliensisgenotypes as a breeding strategy to enhance water deficit tolerance under Amazonian environmental conditions
Authors: Guaca-Cruz Lised , Sterling Armando , Clavijo Andrés , Suárez-Salazar Juan Carlos
Published: June 2024

This study evaluated the foliar antioxidant activity in nineHevea brasiliensisgenotypes from the ECC-1 (Élite Caquetá Colombia) selection and the IAN 873 cultivar (control) in trees in the growth stage in two large-scale clonal trials in response to different climatic (semi-humid warm and humid warm sites) and seasonal (dry and rainy periods) conditions in the Colombian Amazon. The results indicated that ROS production increased under conditions of lower water availability (dry period), leading to lipid peroxidation, high defense of photosynthetic pigments, and development of better osmotic adjustment capacity in the ECC 64, IAN 873, ECC 90, and ECC 35 genotypes due to high concentrations of carotenoids (0.40 mg g-1), reducing sugars (65.83 µg mg-1), and MDA (2.44 nmol ml-1). In contrast, during the rainy period, a post-stress action was observed due to high contents of proline and total sugars (39.43 µg g-1and 173.03 µg g-1, respectively). At the site level, with high PAR values (1143 moles photons m-2s-1), temperature (32.11 °C), and lower precipitation (135 mm), higher antioxidant activity (chlorophylls a, b and total, carotenoids, and proline) was recorded at the humid warm site, demonstrating that the ECC 90, ECC 64, and ECC 66 genotypes are tolerant to water deficit compared to IAN 873. The ECC 64 genotype, independent of seasonal changes and site conditions, presented the highest contents in Chl a, total Chl, reducing sugars, total sugars, and MDA, showing a tendency to adapt to fluctuating conditions. This study showed that water fluctuations do not cause the same metabolic responses, and these vary intraspecifically, depending on their developmental stage and the climatic and seasonal variations characteristic of the Colombian Amazon.
Continuous exposure to 60 Hz extremely low frequency electromagnetic field at 10 to 16 mT promotes various human cell proliferation by activating extracellular-signal-regulated kinase
Authors: Goh Jaeseong , Suh Donghwa , Um Dae Yong , Chae Seung Ahn , Park Gwan Soo , Song Kiwon
Published: June 2024

We previously showed that continuous exposure to 60 Hz extremely low-frequency electromagnetic fields (ELF-EMF) at 6 mT promotes cell proliferation. Here, we investigated the cellular effect of 60 Hz ELF-EMF at over 10 mT. We revised the ELF-EMF-generating device to increase the magnetic flux density of the ELF-EMF stably without thermal effect. We investigated the cellular effect of 10-16 mT ELF-EMF on various mammalian cells including human cervical carcinoma HeLa, rat neuroblastoma B103, liver cancer stem cells Huh7 and Hep3B, immortalized normal hepatic cell MIHA, and normal fibroblast IMR-90. Cell proliferation was promoted around 20% or more in all cells through continuous ELF-EMF exposure at 10 and 14 mT for 72 h, compared with the sham exposure group. In the cells whose proliferation was activated by 14 mT ELF-EMF, the MEK-ERK pathway and NF-κB were activated but not Akt. These cells showed a slight increase in the S phase population in BrdU incorporation and Ki-67 expression. In these cells, intracellular and mitochondrial ROS levels were not changed, and the proliferation-activating cellular effects of ELF-EMF were maintained even when oxidative phosphorylation was interrupted by CCCP. Additionally, no changes in intracellular calcium levels were observed in ELF-EMF-exposed cells and the proliferation-activating cellular effects of ELF-EMF were maintained in the presence of a calcium chelator, BAPTA-AM. These observations suggested that ROS and intracellular calcium do not mediate ELF-EMF’s proliferation-activating physiological effect. Altogether, we demonstrated that 60 Hz ELF-EMF at 10 to 14 mT promotes cell proliferation by activating ERK1/2 and does not affect intracellular ROS and calcium levels.
Personalizing a computational upper body model improves kinematic tracking in high range-of-motion arm movements
Authors: Maier Jennifer N , Bianco Nicholas August , Ong Carmichael , Muccini Julie , Kuhl Ellen , Delp Scott
Published: June 2024

Musculoskeletal models of the shoulder are needed to understand the mechanics of overhead motions. Existing models can be scaled to represent the size of an individual person, but the kinematics are generic. We introduce a method to personalize the shoulder kinematics of a computational model of the upper body that defines the orientations of the clavicle and scapula based on glenohumeral joint angles. During five static calibration poses, we palpate and measure the orientation of the scapula. We explore the importance of representing shoulder elevation by introducing clavicle elevation as a degree of freedom that is independent of the glenohumeral angles. For ten subjects, we record the five calibration poses, ten additional static poses, and dynamic arm raises using optical motion capture. We examine the data using a dynamically-constrained inverse kinematics analysis. Personalization, independent clavicle elevation, and both in combination reduce the average upper body marker tracking error compared to the generic model in the static poses (26 mm to 17-20 mm) and in the dynamic trials (22 mm to 14-17 mm). Only personalization reduces the average scapula marker error (51 mm to 36-38 mm) and scapula axis-angle error (15° to 10°) in the static poses, and in the dynamic trials at instances that best match the static poses (53 mm to 37-40 mm, 15° to 9°). Our results show that personalizing upper body models improves kinematic tracking. We provide our experimental data, model, and methods to allow researchers to reproduce and build upon our results.
Experimental evolution under combined thermal-nutritional stress inDrosophila melanogasterresults in evolved shifts in larval thermotolerance and thermal plasticity
Authors: Choy Yeuk Man Movis , Kutz Teresa , Cockerell Fiona E. , Lombardi Emily , Hangartner Sandra , Mirth Christen K. , Sgrò Carla M.
Published: June 2024

Animals are facing increasing thermal and nutritional stress under climate change. Genetic adaptation is necessary to buffer long-term stress. However, it’s unclear whether adaptation to combined stress can occur without compromising viability and thermal plasticity. We tested thermotolerance and thermal plasticity in larvalDrosophila melanogasterselected under different temperatures (18°C, 25°C, 28°C) and diets (standard, diluted, low protein:carbohydrate). Basal cold tolerance was affected by both low protein concentration and temperature; larvae evolved higher basal cold tolerance on diluted and low protein:carbohydrate diets at 18°C and 28°C. Hardening increased cold tolerance for most lines, except those selected at 18°C and 28°C on low protein:carbohydrate diets and 25°C on standard diets. Basal heat tolerance was affected by selection temperature, with higher tolerance in 25°C selection lines. An interaction between selection temperature, selection diet and hardening treatment affected plasticity in heat tolerance, with most selection lines showing reduced heat tolerance upon hardening, except for lines selected at 25°C on low protein diets and at 28°C on standard diets. Our results indicate that adaptation to combined stress allows basal cold tolerance and plasticity in cold tolerance to evolve independently, but not heat tolerance, highlighting the increased vulnerability of ectotherms to long-term climate change.