bluuebunny/crossref_metadata_2025 huggingface dataset:
Robust data-driven segmentation of pulsatile cerebral vessels using functional magnetic resonance imaging
Authors: Wright Adam M. , Xu Tianyin , Ingram Jacob , Koo John , Zhao Yi , Tong Yunjie , Wen Qiuting
Published: July 2024

Functional magnetic resonance imaging (fMRI) captures rich physiological and neuronal information that can offer insights into neurofluid dynamics, vascular health, and waste clearance function. The availability of cerebral vessel segmentation could facilitate fluid dynamics research in fMRI. However, without magnetic resonance angiography scans, cerebral vessel segmentation is challenging and time-consuming. This study leverages cardiac-induced pulsatile fMRI signal to develop a data-driven, automatic segmentation of large cerebral arteries and the superior sagittal sinus (SSS). The method was validated in a local dataset by comparing it to ground truth cerebral artery and SSS segmentations. Using the Human Connectome Project (HCP) aging dataset, the method’s reproducibility was tested on 422 participants aged 36 to 100 years, each with four repeated fMRI scans. The method demonstrated high reproducibility, with an intraclass correlation coefficient > 0.7 in both cerebral artery and SSS segmentation volumes. This study demonstrates that the large cerebral arteries and SSS can be reproducibly and automatically segmented in fMRI datasets, facilitating the investigation of fluid dynamics in these regions.
Gradations in protein dynamics captured by experimental NMR are not well represented by AlphaFold2 models and other computational metrics
Authors: Gavalda-Garcia Jose , Dixit Bhawna , Díaz Adrián , Ghysels An , Vranken Wim
Published: July 2024

The advent of accurate methods to predict the fold of proteins initiated by AlphaFold2 is rapidly changing our understanding of proteins and helping their design. However, these methods are mainly trained on protein structures determined with X-ray diffraction, where the protein is packed in crystals at often cryogenic temperatures. They can therefore only reliably cover well-folded parts of proteins that experience few, if any, conformational changes. Experimentally, solution nuclear magnetic resonance (NMR) is the experimental method of choice to gain insight into protein dynamics at near physiological conditions. Computationally, methods such as molecular dynamics and Normal Mode Analysis (NMA) allow the estimation of a protein’s intrinsic flexibility based on a single protein structure. This work addresses, on a large scale, the relationships for proteins between the AlphaFold2 pLDDT metric, the observed dynamics in solution from NMR metrics, interpreted MD simulations, and the computed dynamics with NMA from single AlphaFold2 models and NMR ensembles. We observe that these metrics agree well for rigid residues that adopt a single well-defined conformation, which are clearly distinct from residues that exhibit dynamic behavior and adopt multiple conformations. This direct order/disorder categorisation is reflected in the correlations observed between the parameters, but becomes very limited when considering only the likely dynamic residues. The gradations of dynamics observed by NMR in flexible protein regions are therefore not represented by these computational approaches. Our results are interactively available for each protein fromhttps://bio2byte.be/af_nmr_nma/.
Immune disease dialogue of chemokine-based cell communications as revealed by single-cell RNA sequencing meta-analysis
Authors: Rahman Mouly F. , Kurlovs Andre H. , Vodnala Munender , Meibalan Elamaran , Means Terry K. , Nouri Nima , Rinaldis Emanuele de , Savova Virginia
Published: July 2024

Immune-mediated diseases are characterized by aberrant immune responses, posing significant challenges to global health. In both inflammatory and autoimmune diseases, dysregulated immune reactions mediated by tissue-residing immune and non-immune cells precipitate chronic inflammation and tissue damage that is amplified by peripheral immune cell extravasation into the tissue. Chemokine receptors are pivotal in orchestrating immune cell migration, yet deciphering the signaling code across cell types, diseases and tissues remains an open challenge. To delineate disease-specific cell-cell communications involved in immune cell migration, we conducted a meta-analysis of publicly available single-cell RNA sequencing (scRNA-seq) data across diverse immune diseases and tissues. Our comprehensive analysis spanned multiple immune disorders affecting major organs: atopic dermatitis and psoriasis (skin), chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (lung), ulcerative colitis (colon), IgA nephropathy and lupus nephritis (kidney). By interrogating ligand-receptor (L-R) interactions, alterations in cell proportions, and differential gene expression, we unveiled intricate disease- specific and common immune cell chemoattraction and extravasation patterns. Our findings delineate disease-specific L-R networks and shed light on shared immune responses across tissues and diseases. Insights gleaned from this analysis hold promise for the development of targeted therapeutics aimed at modulating immune cell migration to mitigate inflammation and tissue damage. This nuanced understanding of immune cell dynamics at the single-cell resolution opens avenues for precision medicine in immune disease management.
Utilization of a Human Liver Tissue Chip for Drug-Metabolizing Enzyme Induction Studies of Perpetrator and Victim Drugs
Authors: Ohri Shivam , Parekh Paarth , Nichols Lauren , Rajan Shiny Amala Priya , Cirit Murat
Published: July 2024

Polypharmacy-related drug-drug interactions (DDIs) are a significant and growing healthcare concern. Increasing number of therapeutic drugs on the market underscores the necessity to accurately assess the new drug combinations during pre-clinical evaluation for DDIs. In vitro primary human hepatocyte (PHH)-only models are commonly used only for perpetrator DDI studies due to their rapid loss of metabolic function. But co-culturing non-human cells with human PHHs can stabilize metabolic activity and be utilized for both perpetrator and victim studies, though this raises concerns about human specificity for accurate clinical assessment. In this study, we evaluated Liver Tissue Chip (LTC) with PHH-only liver microphysiological system (MPS) for DDI induction studies. Liver MPS from three individual donors maintained their functional and metabolic activity for up to 4 weeks demonstrating suitability for long-term pharmacokinetics (PK) studies. The responses to rifampicin induction of three PHH donors were assessed using CYP activity and mRNA changes. Additionally, victim PK studies were conducted with midazolam (high clearance) and alprazolam (low clearance) following rifampicin-mediated induction which resulted in a 2-fold and a 2.6-fold increase in midazolam and alprazolam intrinsic clearance values respectively compared to the untreated liver MPS. We also investigated the induction effects of different dosing regimens of the perpetrator drug (rifampicin) on CYP activity levels, showing minimal variation in the intrinsic clearance of the victim drug (midazolam). This study demonstrates the utility of the LTC for in vitro liver-specific DDI induction studies, providing a translational experimental system to predict clinical clearance values of both perpetrator and victim drugs.
Genetic and selective constraints on the optimization of gene product diversity
Authors: Jiang Daohan , Kejiou Nevraj , Qiu Yi , Palazzo Alexander F. , Pennell Matt
Published: July 2024

RNA and protein expressed from the same gene can have diverse isoforms due to various post-transcriptional and post-translational modifications. For the vast majority of alternative isoforms, It is unknown whether they are adaptive or simply biological noise. As we cannot experimentally probe the function of each isoform, we can ask whether the distribution of isoforms across genes and across species is consistent with expectations from different evolutionary processes. However, there is currently no theoretical framework that can generate such predictions. To address this, we developed a mathematical model where isoform abundances are determined collectively bycis-acting loci,trans-acting factors, gene expression levels, and isoform decay rates to predict isoform abundance distributions across species and genes in the face of mutation, genetic drift, and selection. We found that factors beyond selection, such as effective population size and the number ofcis-acting loci, significantly influence evolutionary outcomes. Notably, suboptimal phenotypes are more likely to evolve when the population is small and/or when the number ofcis-loci is large. We also explored scenarios where modification processes have both beneficial and detrimental effects, revealing a non-monotonic relationship between effective population size and optimization, demonstrating how opposing selection pressures oncis- andtrans-acting loci can constrain the optimization of gene product diversity. As a demonstration of the power of our theory, we compared the expected distribution of A-to-I RNA editing levels in coleoids and found this to be largely consistent with non-adaptive explanations.
Spatial constraints and cell surface molecule depletion structure a randomly connected learning circuit
Authors: Thornton-Kolbe Emma M , Ahmed Maria , Gordon Finley R , Sieriebriennikov Bogdan , Williams Donnell L , Kurmangaliyev Yerbol Z , Clowney Eleanor Josephine
Published: July 2024

The brain can represent almost limitless objects to "categorize an unlabeled world" (Edelman, 1989). This feat is supported by expansion layer circuit architectures, in which neurons carrying information about discrete sensory channels make combinatorial connections onto much larger postsynaptic populations. Combinatorial connections in expansion layers are modeled as randomized sets. The extent to which randomized wiring exists in vivo is debated, and how combinatorial connectivity patterns are generated during development is not understood. Non-deterministic wiring algorithms could program such connectivity using minimal genomic information. Here, we investigate anatomic and transcriptional patterns and perturb partner availability to ask how Kenyon cells, the expansion layer neurons of the insect mushroom body, obtain combinatorial input from olfactory projection neurons. Olfactory projection neurons form their presynaptic outputs in an orderly, predictable, and biased fashion. We find that Kenyon cells accept spatially co-located but molecularly heterogeneous inputs from this orderly map, and ask how Kenyon cell surface molecule expression impacts partner choice. Cell surface immunoglobulins are broadly depleted in Kenyon cells, and we propose that this allows them to form connections with molecularly heterogeneous partners. This model can explain how developmentally identical neurons acquire diverse wiring identities.
Spike rate inference from mouse spinal cord calcium imaging data
Authors: Rupprecht Peter , Fan Wei , Sullivan Steve J. , Helmchen Fritjof , Sdrulla Andrei D.
Published: July 2024

Calcium imaging is a key method to record the spiking activity of identified and genetically targeted neurons. However, the observed calcium signals are only an indirect readout of the underlying electrophysiological events (single spikes or bursts of spikes) and require dedicated algorithms to recover the spike rate. These algorithms for spike inference can be optimized using ground truth data from combined electrical and optical recordings, but it is not clear how such optimized algorithms perform on cell types and brain regions for which ground truth does not exist. Here, we use a state-of-the-art algorithm based on supervised deep learning (CASCADE) and a non-supervised algorithm based on non-negative deconvolution (OASIS) to test spike rate inference in spinal cord neurons. To enable these tests, we recorded specific ground truth from glutamatergic and GABAergic somatosensory neurons in the superficial dorsal horn of spinal cord in mice of both sexes. We find that CASCADE and OASIS algorithms that were designed for cortical excitatory neurons generalize well to both spinal cord cell types. However, CASCADE models re-trained on our ground truth further improved the performance, resulting in a more accurate inference of spiking activity from spinal cord neurons. We openly provide re-trained models that can be applied to spinal cord data of variable noise levels and frame rates. Together, our ground-truth recordings and analyses provide a solid foundation for the interpretation of calcium imaging data from spinal cord dorsal horn and showcase how spike rate inference can generalize between different regions of the nervous system.

Significance Statement

Calcium imaging is a powerful method for measuring the activity of genetically identified neurons. However, accurate interpretation of calcium transients depends on having a detailed understanding of how neuronal activity correlates with fluorescence. Such calibration recordings have been performed for cerebral cortex but not yet for most other CNS regions and neuron types. Here, we obtained ground truth data in spinal cord by conducting simultaneous calcium and electrophysiology recordings in excitatory and inhibitory neurons. We systematically investigated the transferability of cortical algorithms to spinal neuron subpopulations and generated inference algorithms optimized to excitatory and inhibitory neurons. Our study provides a foundation for the rigorous interpretation of calcium imaging data from spinal cord.

Conflict of interest statement

The authors declare no competing financial conflicts of interest.
Comparing diploid and triploid apples from a diverse collection
Authors: Greaves Elaina , Davies Thomas , Myles Sean , Migicovsky Zoë
Published: July 2024

Apples (MalusX.domesticaBorkh.) are an economically important fruit species and the focus of continuing breeding efforts around the world. While most apple varieties are diploid, ploidy levels vary across the species, and triploids may be used in breeding despite poor fertility. The impact of ploidy on agricultural traits in apple is not well understood but is an important factor to consider when breeding new apple varieties. Here, we use mean heterozygosity values to categorize 970 apple accessions as diploid or triploid and then contrast apples of varying ploidy levels across 10 agriculturally important traits with sample sizes ranging from 427 to 928 accessions. After correction for multiple testing, we determine that triploids have significantly higher phenolic content. By examining historical release dates for apple varieties, our findings suggest that contemporary breeding programs are primarily releasing diploid varieties, and triploids tend to be older varieties. Ultimately, our results suggest that phenotypic differences between diploids and triploids are subtle and often insignificant indicating that triploids may not provide substantial benefit above diploids to apple breeding programs.
Viral delivery of an RNA-guided genome editor for transgene-free germline editing inArabidopsis
Authors: Weiss Trevor , Kamalu Maris , Shi Honglue , Li Zheng , Amerasekera Jasmine , Zhong Zhenhui , Adler Benjamin A. , Song Michelle , Vohra Kamakshi , Wirnowski Gabriel , Chitkara Sidharth , Ambrose Charlie , Steinmetz Noah , Sridharan Ananya , Sahagun Diego , Banfield Jillian F. , Doudna Jennifer A. , Jacobsen Steven. E.
Published: July 2024

Abstract

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. But most viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here, we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing ofArabidopsis thalianain a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.
RWRtoolkit: multi-omic network analysis using random walks on multiplex networks in any species
Authors: Kainer David , Lane Matthew , Sullivan Kyle A. , Miller J. Izaak , Cashman Mikaela , Morgan Mallory , Cliff Ashley , Romero Jonathon , Walker Angelica , Blair D. Dakota , Chhetri Hari , Wang Yongqin , Pavicic Mirko , Furches Anna , Noshay Jaclyn , Drake Meghan , Landry Natalie , Ireland AJ , Missaoui Ali , Kang Yun , Sedbrook John , Dehal Paramvir , Canon Shane , Jacobson Daniel
Published: July 2024

Leveraging the use of multiplex multi-omic networks, key insights into genetic and epigenetic mechanisms supporting biofuel production have been uncovered. Here, we introduce RWRtoolkit, a multiplex generation, exploration, and statistical package built for R and command line users. RWRtoolkit enables the efficient exploration of large and highly complex biological networks generated from custom experimental data and/or from publicly available datasets, and is species agnostic. A range of functions can be used to find topological distances between biological entities, determine relationships within sets of interest, search for topological context around sets of interest, and statistically evaluate the strength of relationships within and between sets. The command-line interface is designed for parallelisation on high performance cluster systems, which enables high throughput analysis such as permutation testing. Several tools in the package have also been made available for use in reproducible workflows via the KBase web application.
Integrating Phylogenies with Chronology to Assemble the Tree of Life
Authors: Barba-Montoya Jose , Craig Jack M , Kumar Sudhir
Published: July 2024

Reconstructing the global Tree of Life necessitates computational approaches to integrate numerous molecular phylogenies with limited species overlap into a comprehensive supertree. Our survey of published literature shows that individual phylogenies are frequently restricted to specific taxonomic groups due to the expertise of investigators and molecular evolutionary considerations, resulting in any given species present in a minuscule fraction of phylogenies. We present a novel approach, called the chronological supertree algorithm (Chrono-STA), that can build a supertree of species from such data by using node ages in published molecular phylogenies scaled to time. Chrono-STA builds a supertree of organisms by integrating chronological data from molecular timetrees. It fundamentally differs from existing approaches that generate consensus phylogenies from gene trees with missing taxa, as Chrono-STA does not impute nodal distances, use a guide tree as a backbone, or reduce phylogenies to quartets. Analyses of simulated and empirical datasets show that Chrono-STA can combine taxonomically restricted timetrees with extremely limited species overlap. For such data, approaches that impute missing distances or assemble phylogenetic quartets did not perform well. We conclude that integrating phylogenies via temporal dimension enhances the accuracy of reconstructed supertrees that are also scaled to time.
Multiomics-based assessment of the impact of airflow on diverse plant callus cultures
Authors: Kim June-Sik , Sato Muneo , Kojima Mikiko , Asrori Muchamad Imam , Uehara-Yamaguchi Yukiko , Takebayashi Yumiko , Do Thi Nhung , Do Thi Yen , Thi Kieu Oanh Nguyen , Sakakibara Hitoshi , Mochida Keiichi , Ogita Shijiro , Hirai Masami Yokota
Published: July 2024

Plant cell culture has multiple applications in biotechnology and horticulture, from plant propagation to the production of high-value biomolecules. However, the interplay between cellular diversity and ambient conditions influences the metabolism of cultured tissues; understanding these factors in detail will inform efforts to optimize culture conditions. This study presents multiomics datasets from callus cultures of tobacco (Nicotiana tabacum), rice (Oryza sativa), and two bamboo species (Phyllostachys nigraandP. bambusoides). Over four weeks, calli were cultured under continuous moisture without airflow or gradually reduced ambient moisture with airflow. For each sample, gene expression was profiled with high-throughput RNA sequencing, 442 metabolites were measured using liquid chromatography (LC) with triple-quadrupole mass spectrometry (LC–QqQMS), and 31 phytohormones were quantified using ultra-performance LC coupled with a tandem quadrupole mass spectrometer equipped with an electrospray interface (UPLC-ESI-qMS/MS) and ultra-high-performance LC– orbitrap MS (UHPLC-Orbitrap MS). These datasets highlight the impact of airflow on callus cultures, revealing differences between and within species, and provide a comprehensive resource to explore the physiology of callus growth.
OPUS-BFactor: Predicting protein B-factor with sequence and structure information
Authors: Xu Gang , Yang Yulu , Lv Ying , Luo Zhenwei , Wang Qinghua , Ma Jianpeng
Published: July 2024

Protein B-factor, also known as the Debye-Waller factor, measures the fluctuation of an atom around its average position. It serves as a crucial indicator of protein flexibility and dynamics. However, accurately predicting the B-factor of Cαatoms remains challenging. In this work, we introduce OPUS-BFactor, a tool for predicting the normalized protein B-factor. OPUS-BFactor operates in two modes: the first mode, OPUS-BFactor-seq, uses sequence information as input, allowing predictions based solely on protein sequence; the second mode, OPUS-BFactor-struct, uses structural information, requiring the 3D structure of the target protein. Evaluation on three test sets, including recently released targets from CAMEO and CASP15, demonstrates that OPUS-BFactor significantly outperforms other B-factor prediction methods. Therefore, OPUS-BFactor is a valuable tool for predicting protein properties related to the B-factor, such as flexibility, thermal stability, and region activity.
Is testing the bee-all and end-all ofvarroaeradication?
Authors: Abell Isobel R. , Flegg Jennifer A. , Le Thao P. , Baker Christopher M.
Published: July 2024

Varroa destructoris a significant European honeybee pest, impacting agricultural industries globally. Since arriving in 2022, Australia faces the possibility thatvarroawill become established in European honeybee colonies nationally. Australia initially pursued a strategy of testing and subsequently eliminating hives infested withvarroa. These management efforts raise interesting questions about the interplay between hive testing and elimination, and the spread ofvarroabetween hives. This study uses mathematical modelling to investigate how combined hive testing and elimination strategies impact the spread ofvarroathrough a network of European honeybee hives. We develop a model of both within-hive reproduction ofvarroaand hive testing, and between-hive movement ofvarroaon a network of hives. This model is used to assess the impact of various testing and hive elimination strategies on the total number of hives eliminated on the network of hives. Each model simulation starts with a single infested hive, and from this we observed one of two dynamics: either the infestation is caught before spreading, orvarroaspreads extensively through the network before being caught by testing. Within our model we implement two common hive testing methods – sugar shake and alcohol testing. A shared limitation of these testing methods is that they can only detect mites in a specific stage of their lifecycle. As such, testing is not only dependent on how manyvarroamites are in a hive, but what lifecycle stage the mites are in at the time of testing. By varying testing and movement parameters, we see that this testing limitation greatly impacts the number of hives eliminated in various scenarios. We find that there are largely two invasion possibilities: either there is only a small incursion, or thatvarroaachieves complete spread on the network. Furthermore, testing earlier, or testing more frequently, does not guarantee a smaller invasion. Our model results suggest irregular testing schedules, e.g. testing multiple times in close succession rather than just once in a given timeframe, may help overcome the limitations of common hive testing strategies.
Transcriptional, developmental, and functional parallels of lymphatic and venous smooth muscle
Authors: Arroyo-Ataz Guillermo , Yagüe Alejandra Carrasco , Breda Julia C. , Mazzilli Sarah A. , Jones Dennis
Published: July 2024

Lymphatic muscle cells (LMCs) are indispensable for lymphatic vessel contraction and their aberrant recruitment or absence is associated with both primary and secondary lymphedema. Despite their critical role in lymphatic vessel function, the transcriptomic and developmental basis that confer the unique contractile properties to LMCs are largely undefined. In this study, we employed single-cell RNA sequencing (scRNAseq), lineage tracing andin vivoimaging to investigate the basis for the hybrid cardiomyocyte and blood vascular smooth muscle cell (SMC) characteristics that have been described for LMCs. Using scRNAseq, the transcriptomes of LMC and venous SMCs from the murine hindlimb exhibited more similarities than differences, although both were markedly distinct from that of arteriole SMCs in the same tissue. Functionally, both lymphatic vessels and blood vessels in the murine hindlimb displayed pulsatile contractility. However, despite expressing genes that overlap with the venous SMC transcriptome, through lineage tracing we show that LMCs do not originate from Myh11+ SMC progenitors. Previous studies have shown that LMCs express cardiac-related genes, whereas in our study we found that arteriole SMCs, but not LMCs, expressed cardiac-related genes. Through lineage tracing, we demonstrate that a subpopulation of LMCs and SMCs originate from WT1+ mesodermal progenitors, which are known to give rise to SMCs. LMCs, however, do not derive from Nkx2.5+ cardiomyocyte progenitors. Overall, our findings suggest that venous SMCs and LMCs and may derive from a related mesodermal progenitor and adopt a similar gene expression program that enable their contractile properties.
Linking Molecular Mechanisms to their Evolutionary Consequences: a primer
Authors: Grah Rok , Guet Calin C. , Tkačik Gasper , Lagator Mato
Published: July 2024

A major obstacle to predictive understanding of evolution stems from the complexity of biological systems, which prevents detailed characterization of key evolutionary properties. Here, we highlight some of the major sources of complexity that arise when relating molecular mechanisms to their evolutionary consequences and ask whether accounting for every mechanistic detail is important to accurately predict evolutionary outcomes. To do this, we developed a mechanistic model of a bacterial promoter regulated by two proteins, allowing us to connect any promoter genotype to six phenotypes that capture the dynamics of gene expression following an environmental switch. Accounting for the mechanisms that govern how this system works enabled us to provide an in-depth picture of how regulated bacterial promoters might evolve. More importantly, we used the model to explore which factors that contribute to the complexity of this system are essential for understanding its evolution, and which can be simplified without information loss. We found that several key evolutionary properties – the distribution of phenotypic and fitness effects of mutations, the evolutionary trajectories during selection for regulation – can be accurately captured without accounting for all, or even most, parameters of the system. Our findings point to the need for a mechanistic approach to studying evolution, as it enables tackling biological complexity and in doing so improves the ability to predict evolutionary outcomes.
Navigating the Cold: Integrative Transcriptome Sequencing Approach Reveals Ionoregulatory and Whole-Body Responses to Cold Acclimation inDrosophila ananassae
Authors: Yılmaz Vera Miyase , Bao Zhihui , Grath Sonja
Published: July 2024

Understanding how species adapt to changing environments is a major goal in evolutionary biology and can elucidate the impact of climate change. Climate imposes inevitable effects on the geographical distribution of insects as their body temperature primarily depends on the environment. The vinegar flyDrosophila ananassaeexpanded from its tropical ancestral range to more temperate regions, which requires adaptation to colder temperatures. Transcriptome and genome-wide association studies focusing on the ancestral-range population identified the targets of selection related to ionoregulatory tissues. However, how cosmopolitanD. ananassaeadapted to colder environments, where low temperatures last longer, is still unknown. Here, we present a study on the effect of long-term cold exposure onD. ananassae, examining the gene expression variation in the whole body and the ionoregulatory tissues, namely the hindgut and the Malpighian tubule. To elucidate molecular mechanisms of cold adaptation during species expansion, we included cold-tolerant and cold-sensitive strains from the ancestral species range and cold-tolerant strains from the derived species range. We show that cold acclimation improves cold tolerance and results in differential expression of more than half of the transcriptome in the ionoregulatory tissues and the whole body. Notably, we provide complementary insight into molecular processes at four levels: strains, populations, phenotypes, and tissues. By determining the biochemical pathways of phenotypic plasticity underlying cold tolerance, our results enhance our understanding of how environmental changes affect thermal adaptation in natural populations.
A Hierarchical Method to Analyze Protein-DNA Interfaces
Authors: Tagad Amol , Patwari G. Naresh
Published: July 2024

The accessibility of genetic information and packaging of long chromosomal DNA into micron-sized nuclei is a great example of protein-DNA interaction. A large number of protein-DNA structures are available in the database and is continuously increasing. The analysis of such huge data to extract meaningful insights, several computational tools such as hydrogen bonding, SASA, all intermolecular heavy atom-to-atom contacts, as well as web-server and databases such as DNAproDB, PDIdb, PDB2PQR, Bio-python are available. Generally, the interaction between the protein and DNA is analyzed based on all heavy atom-to-atom contact matrices which are computationally expensive. Herein, a new and robust hierarchical approach is developed to analyze the protein-DNA interface. The present hierarchical method comprises two steps; the first step is to recognize the protein residue-nucleotide pairs at the interface by employing pairwise distance cut-off between the Cα of the protein residues and 05′ of the nucleotide and the second step is to calculate heavy atom-to-atom contact matrix using the first step as a qualifier. This method reduces the computational cost by three orders of magnitude making it tractable even on personal computers. On the whole, the protein-DNA interface is dominated by the arginine residues with a notable presence of lysine, serine, and tyrosine, highlighting the pivotal role of electrostatic interactions and hydrogen bonding in aggregation.

Abstract Figure
Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles
Authors: Candelaria Juliana I. , Botigelli Ramon C. , Guiltinan Carly , Shikanov Ariella , Denicol Anna C.
Published: July 2024

STRUCTURED ABSTRACT

Purpose

Here we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment.

Methods

Bovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate.

Results

Primary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm (LHX1) and early coelomic epithelium/bipotential gonad markers (OSR1,GATA4,WT1). Higher BMP4 concentrations upregulated the lateral plate mesoderm markerFOXF1.PAX3expression was not induced, indicating absence of the paraxial mesoderm lineage.

Conclusions

Culture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here.

CAPSULE SUMMARY

We demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.
A selective S-acyltransferase inhibitor suppresses tumor growth
Authors: Lee Jia-Ying , Dilones Sebastian , Maujean Timothe , Asad Mohammad , Mohd Altaf , Auslander Noam , Brady Donita C. , Burslem George M. , Witze Eric S.
Published: July 2024

S-acyltransferases play integral roles in essential physiological processes including regulation of oncogenic signaling pathways. While discovered over 40 years ago the field still lacks specific S-acylation inhibitors thus the potential benefit of pharmacologically targeting S-acyltransferases for human disease is still unknown. Here we report the identification of an orally bioavailable acyltransferase inhibitor SD-066-4 that inhibits the acyltransferase ZDHHC20. We identified a specific alanine residue that accommodates the methyl group of SD-066-4, thus providing isoform selectivity. SD-066-4 stably reduces EGFR S-acylation in Kras mutant cells and blocks the growth of Kras mutant lung tumors extending overall survival. We find that lung cancer patients harboring deletions in ZDHHC20 or ZDHHC14 concurrent with Kras alterations have a significant survival benefit, underscoring the translational importance of these enzymes.
SARS-CoV-2 dynamics in New York City during March 2020–August 2023
Authors: Yang Wan , Parton Hilary , Li Wenhui , Watts Elizabeth A. , Lee Ellen , Yuan Haokun
Published: July 2024

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widespread since 2020 and will likely continue to cause substantial recurring epidemics. However, understanding the underlying infection burden (i.e., including undetected asymptomatic/mild infections) and dynamics, particularly since late 2021 when the Omicron variant emerged, is challenging due to the potential for asymptomatic and repeat SARS-CoV-2 infection, changes in testing practices, and changes in disease reporting. Here, we leverage extensive surveillance data available in New York City (NYC) and a comprehensive model-inference system to reconstruct SARS-CoV-2 dynamics therein from the pandemic onset in March 2020 to August 2023, and further validate the estimates using independent wastewater surveillance data. The validated model-inference estimates indicate a very high infection burden totaling twice the population size (>5 times documented case count) but decreasing infection-fatality risk (a >10-fold reduction) during the first 3.5 years. The detailed estimates also reveal highly complex variant dynamics and immune landscape, changing virus transmissibility, and higher infection risk during winter in NYC over this time period. These transmission dynamics and drivers, albeit based on data in NYC, may be relevant to other populations and inform future planning to help mitigate the public health burden of SARS-CoV-2.
Ecological risk-taking across age in Barbary macaques
Authors: Bosshard Tiffany Claire , Mundry Roger , Fischer Julia
Published: July 2024

When foraging, animals face decisions regarding unpredictable resources and varying predation risk, in which the cost-benefit trade-off must be assessed. Life-history theory focuses on resource allocation with regard to development and suggests there should be a variation in risk-taking propensity across age, manifested by a shift in goals from gain acquisition to loss aversion over time. Consequently, we aimed to assess age-related variation in decision-making strategies under risk in Barbary macaques (Macaca sylvanus) living at “La Forêt des Singes” in Rocamadour, France. We experimentally varied the perceived risk and potential gains in a 2×2 design. The high-risk stimulus was a rubber snake; the low-risk stimulus a cube with a snakeskin pattern. The high-value food reward was a peanut; the low-value food reward a popcorn. In 2022, we presented 83 subjects with one of the four possible factor combinations, and in 2023, we presented 70 individuals with the inverted combination (e.g. snake-popcorn in 2022, cube-peanut in 2023). We took each monkey’s propensity to retrieve the reward as a measure of risk-taking, and the latency to retrieve the reward as motivation to engage in the risky choice. Regardless of age, subjects generally retrieved the reward when placed next to the cube, whether peanut or popcorn, but were less likely to retrieve the reward placed next to the snake when this was popcorn rather than peanut. However, older individuals were faster than younger conspecifics to retrieve the reward when it was popcorn, whether paired with the cube or snake. Our results do not comply with the notion that risk-taking propensity declines with age, but rather suggest that in this paradigm, the animals assess the costs and benefits of a risky situation across age, and that the subjective value of a resource may change with age.
Altered calcium responses and antioxidant properties in Friedreich’s ataxia-like cerebellar astrocytes
Authors: Marullo Chiara , Croci Laura , Giupponi Iris , Rivoletti Claudia , Zuffetti Sofia , Bettegazzi Barbara , Casoni Filippo , Cremona Ottavio , Consalez Gian Giacomo , Codazzi Franca
Published: July 2024

Friedreich’s ataxia (FRDA) is a neurodegenerative disorder characterized by severe neurological signs affecting both the peripheral and central nervous system, caused by reduced levels of the frataxin protein (FXN). While several studies highlight cellular dysfunctions in neurons and various other cell types, there is limited information on the effects of FXN depletion in astrocytes and on the potential non-cell autonomous mechanisms affecting neurons in FRDA. In this study, we generated a model of FRDA cerebellar astrocytes to unveil phenotypic alterations that might contribute to cerebellar atrophy and the degeneration of glutamatergic neurons observed in cerebellar dentate nuclei. We treated primary cerebellar astrocytes with an RNA interference-based approach, to achieve a reduction of FXN comparable to that observed in patients. These FRDA-like astrocytes display some typical features of the disease, such as an increase of oxidative stress, as well as specific functional alterations. Notably, cerebellar astrocytes deplete their reduced glutathione content, becoming more susceptible to oxidative insults. Moreover, FRDA-like astrocytes exhibit alterations of calcium homeostasis, with a reduction in calcium content in the intracellular stores and a corresponding change of calcium responses to purinergic stimuli. Our findings shed light on cellular changes caused by FXN downregulation in cerebellar astrocytes, which can interfere with their physiological and complex interaction with neurons. The potentially impaired ability to provide neuronal cells with glutathione or to release neuromodulators and bioactive molecules in a calcium-dependent manner could impact neuronal function and contribute to neurodegeneration.
Revisiting Sodium Phosphotungstate and Ammonium Molybdate as non-radioactive negative staining agents for single particle analysis
Authors: Gunkel Monika , Macha Arthur , Behrmann Elmar
Published: July 2024

This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate for negative staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step both stains yield comparable images to uranyl formate. Subsequently, using β-galactosidase, it is shown that both stains can also successfully be employed for single particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are non-radioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample grid preparation – thus directly after purification – accessible to a broader range of scientists.
Enhancing gRNA Transcript levels by Reducing the Scaffold Poly-T Tract for Optimal SpCas9- and SaCas9-mediated Gene Editing
Authors: Chey Yu C. J. , Gierus Luke , Lushington Caleb , Arudkumar Jayshen C. , Geiger Ashleigh , Staker Lachlan G. , Robertson Louise J. , Pfitzner Chandran , Kennedy Jesse G. , Lee Ryan H. B. , Godahewa Gelshan I. , Thomas Paul Q. , Adikusuma Fatwa
Published: July 2024

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes.
The Acheulean niche: climate and ecology predict handaxe production in Europe
Authors: Leonardi Michela , Lycett Stephen J. , Manica Andrea , Key Alastair
Published: July 2024

The Acheulean was the most spatially and temporally vast stone tool industry produced by early humans, persisting for >1.6 million years across three continents. Understanding how behavioral and artifactual variation in these populations relates to climate change and ecology is vital to correctly interpreting the archaeological record, especially given the major climatic and cultural fluctuations during the Pleistocene. Bifacially flaked core tools - which technologically define the tradition - are sometimes regionally and periodically absent within the Acheulean's boundaries. Biface presence is broadly associated with, but rarely formally tested against, environmental conditions. Here, we investigate biface presence/absence and climate/ecological variation at a continental scale between 726 and 130ka. Using a comprehensive sample of European sites matched to paleoenvironmental reconstructions, we demonstrate clear associations between Acheulean presence and specific environments. Acheulean habitat suitability was most strongly associated with mean winter temperatures above -5/-10 degrees C, and precipitation of the driest month above 15 mm. Two major habitats, warm forest/woodland and shrubland, comprised the European Acheulean niche. Acheulean spatial presence was reconstructed through time: during interglacial and glacial periods it covered most of southwestern Europe, but extended along northern coastal routes during interglacials. Central and Eastern Europe always remained unsuitable. The Acheulean was excluded from areas experiencing both low winter temperatures and low summer precipitation, but reached areas including only one of those two climatic extremes, suggesting seasonal migrations to avoid the harshest seasons. These analyses explain current Acheulean site distributions in Europe and suggest strong links between handaxes, specific ecological conditions, and prehistoric demographic patterns.
Chemically di-ubiquitylated H1 variants reveal position-dependent stimulation of the DNA repair protein RNF168
Authors: Franz Pauline , Delvaux de Fenffe Charlotte , Fierz Beat
Published: July 2024

Ubiquitylation of histone H2A at lysines 13 and 15 by the E3 ligase RNF168 plays a key role in orchestrating DNA double-strand break (DSB) repair, which is often deregulated in cancer. RNF168 activity is triggered by DSB signaling cascades, reportedly through K63-linked poly-ubiquitylation of linker histone H1. However, direct experimental evidence of this mechanism has been elusive, primarily due to the lack of methods to specifically poly-ubiquitylate H1. Here, we developed a versatile click-chemistry approach to covalently link multiple proteins in a site-specific, controlled, and stepwise manner. Applying this method, we synthesized H1 constructs bearing triazole-linked di-ubiquitin on four DNA repair-associated ubiquitylation hotspots (H1KxUb2, at K17, 46, 64 and 96). Integrated into nucleosome arrays, the H1KxUb2variants stimulated H2A ubiquitylation by RNF168 in a position-dependent manner, with H1K17Ub2showing the strongest RNF168 activation effect. Moreover, we show that di-ubiquitin binding is the driving force underlying RNF168 recruitment, introducing H1K17Ub2into living U-2 OS cells. Together, our results support the hypothesis of poly-ubiquitylated H1 guiding RNF168 recruitment to DSB sites. Moreover, we demonstrate how the streamlined synthesis of H1KxUb2variants enables mechanistic studies into RNF168 regulation, with potential implications for its inhibition in susceptible cancers.