bluuebunny/crossref_metadata_2025 huggingface dataset:
Demonstrating the need for long inter-stimulus intervals when studying the post-movement beta rebound following a simple button press
Authors: Bailey Lyam M. , Bardouille Timothy
Published: January 2025

Voluntary movements reliably elicit event-related synchronization of oscillatory neuronal rhythms in the beta (15-30 Hz) range immediately following movement offset, as measured by magneto/electroencephalography (M/EEG). This response has been termed the post-movement beta rebound (PMBR). While early work on the PMBR advocated for long inter-stimulus intervals (ISIs)—arguing that the PMBR might persist for several seconds—these concerns have since fallen by the wayside, with many recent studies employing very short (< 5 s) ISIs. In this work we interrogated sensor-level MEG time courses in 635 individuals who participated in a cued button- pressing paradigm as part of the Cambridge Centre for Ageing and Neuroscience (Cam-CAN) project. We focussed on a subset of trials in which button presses were separated by at least 15 seconds and, using curve modelling and Bayesian inference, estimated the point at which beta power returned to baseline levels. We show that beta power takes around 4-5 seconds to return to baseline levels following movement. These results have important implications for experimental design. The PMBR is ubiquitously defined relative to a preceding baseline period; we argue that short ISIs preclude true baseline estimation and, in turn, accurate estimation of PMBR magnitude. We recommend that future studies targeting the PMBR use ISIs of at least 7 seconds—5 seconds for beta power to return to baseline, plus a 1-2 second period for proper baseline estimation. Further work is needed to clarify PMBR duration in the context of different sensorimotor paradigms and clinical populations.
AC electro-osmosis in bacterial biofilms: a cautionary tale for electrophysiology experiments
Authors: Carneiro da Cunha Martorelli Victor , Akabuogu Emmanuel , Tank Raveen , Krašovec Rok , Roberts Ian S. , Waigh Thomas A.
Published: January 2025

Synthetic cationic fluorophores are used widely as probes to measure the membrane potentials of bacterial cells, eukaryotic cells and organelles, such as mitochondria. An external oscillating electric field was applied toEscherichia colicells using microelectrodes and AC electro-osmosis was observed for the fluorophores, independent of the electrophysiology of the bacteria, giving rise to phantom action potentials. The fluorophores migrate around the microfluidic device in vortices modulating their concentration having decreases or dips in fluorescence. We show that the fluorescent dips are universally present when using cationic fluorophores, such as thioflavin-T, propidium iodide, Syto9 and Sytox Green, with or withoutE. colicells in the inoculum, when stimulated with AC voltages. This is in contrast to the study of Stratford et al (PNAS, 2019) who claim the existence of action potentials. Furthermore,E. colibiofilms also demonstrated similar phenomena with dips in the fluorescence. We measured the relaxation times of the fluorophores experiencing AC electro-osmosis, which depended on the biofilm, the cells and the fluorophores used. PI had the smallest relaxation time and Syto9 the highest. Removing the cells resulted in longer relaxation times and introducing biofilm did not significantly change the relaxation times compared with the single cell experiments. Furthermore, fluorescently labelled DNA and fluorescent colloidal beads also demonstrate fluorescent dips through AC electro-osmosis, showing that these particles can be driven through biofilms. This is the first study of AC electro-osmosis in bacterial biofilms, indicating a surprisingly high mobility of charged molecules within the extracellular polymeric substance, which could be used to treat biofilms i.e. to increase the kinetics of delivery of antibiotics.
Intraluminal pressure triggers a rapid and persistent reinforcement of endothelial barriers
Authors: Bancaud Aurélien , Nakajima Tadaaki , Suehiro Jun-Ichi , Alric Baptiste , Morfoisse Florent , Cacheux Jean , Matsunaga Yukiko T.
Published: January 2025

In response to mechanical cues, endothelial cells elicit highly sensitive cellular response pathways that contribute to the regulation of the physiology and disorders of the vascular system. However, it remains relatively unexplored how endothelial tissues process and integrate the intraluminal pressure, and in turn regulate the permeation flow across the vessel wall. Leveraging a tissue engineering approach to create microvessels (MVs), we measured real-time permeation flow induced by intraluminal pressures ranging from 0.1 to 2.0 kPa. Our findings reveal that mechanically stimulated MVs strengthen their barrier function within seconds of exposure to pressures below 1 kPa, with this enhanced barrier function persisting for 30 minutes. We demonstrate that this barrier reinforcement is linked to the closure of paracellular gaps. Additionally, we observe that it is associated with, and depends on, actin cytoskeleton reorganization, including the accumulation of stress fibers near intercellular junctions and the broadening of adherence junction protein localization. These findings provide insights into the ability of endothelial tissues to regulate interstitial fluid flow in response to sudden increases in blood pressure.
Integrative spatial transcriptomic analysis pinpoints the role of TaMCO3 encoding ferroxidase in wheat root tip iron mobilization
Authors: Joon Riya , Singh Gourav , Tyagi Deepshikha , Meena Varsha , Shukla Vishnu , Agrwal Kanupriya , Banoo Hamida , Satbhai Santosh , Singh Jagtar , Long Terri , Ramireddy Eswarayya , Pandey Ajay K
Published: January 2025

Iron (Fe) deficiency triggers molecular responses in plant roots, but the specific involvement of root tips in nutrient responses remains unclear. Therefore, in this study, we conducted a transcriptome analysis of wheat root tips under Fe deficiency, and a comparative transcriptome analysis was performed with the total root dataset. Gene ontology analysis highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip tissue, which are critical for iron mobilisation. Interestingly, wheat displayed varying gene expression levels arising from the three genomes (A, B, and D) yet contributed to similar molecular functions. Detailed analysis of the oxidoreductase function in the root tip identified multiplemulti-copperoxidase (MCO) proteins possibly contributing to the total ferroxidase activity. Detailed characterisation of Fe-responsiveTaMCO3shows that it complements the yeast FET3 mutant and rescues the Fe-deficiency sensitivity phenotype ofArabidopsis atmco3mutants by enhancing vascular iron loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root iron accumulation and improved tolerance to Fe deficiency by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in Fe deficiency responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.
Towards a monograph of marine Verrucariaceae in Southern South America and maritime Antarctica: Integrative taxonomy reveals a previously unknown diversity
Authors: Fernández-Costas Daniel , Schiefelbein Ulf , Sancho Leopoldo G. , de los Ríos Asunción , Pérez-Ortega Sergio
Published: January 2025

The family of predominantly lichen-forming Verrucariaceae, contains several lineages that have adapted to live in the stressful conditions of the intertidal and supralittoral zones of rocky seashores in both hemispheres. The marine Verrucariaceae has been the subject of several systematic and taxonomic studies over the past few decades, although these studies have primarily concentrated on groups from the northern hemisphere. This study aims to address the paucity of taxonomic studies in the southern hemisphere by examining a substantial number of specimens collected in Southern South America and maritime Antarctica over the past two decades by the authors. We opted for an integrative taxonomic approach that joins the characterization and measurement of morpho-anatomical characters, coupled together with molecular barcoding to produce species delimitations to overcome the difficulties derived by the scarcity of taxonomic characters and the high plasticity of those characters. We barcoded a total of 301 specimens using the universal barcode un fungi (nrITS) and used single-locus species delimitation algorithms (ASAP, PTP, GMYC) to produce candidate species hypothesis that were corroborated with the morphological data. Our findings indicate that the taxonomic diversity of the Southern HemisphereVerrucariamarina group has been significantly underestimated in previous studies. A total of 27 species were found in the region, 21 of which represent new taxa for science. Thorough descriptions together with illustrations showing the main characters of the species are provided for each taxon. In addition, we explored the systematics of the new taxa. We reconstructed the phylogenetic relationships of the family based on on six molecular markers (ITS, mcm7, nrSSU, nrLSU, mtSSU, RPB1). Our results showed that marine Verrucariaceae from the studied area belong to two distinct and non-related clades. A small group of species were related to the northern hemisphere speciesTurgidosculum ulvae. The second and more numerous group, which included the characteristic speciesMastodia tessellatafor a clade sister to the European genusVerrucariopsis. The systematic consequences of our findings are discussed. None of the previously reported species from the northern hemisphere and considered bipolar were found during our study.
Simulation and quantitative analysis of spatial centromere distribution patterns
Authors: Keikhosravi Adib , Guin Krishnendu , Pegoraro Gianluca , Misteli Tom
Published: January 2025

A prominent feature of eukaryotic chromosomes are centromeres, which are specialized regions of repetitive DNA required for faithful chromosome segregation during cell division. In interphase cells centromeres are non-randomly positioned in the three-dimensional space of the nucleus in a cell-type specific manner. The functional relevance and the cellular mechanisms underlying this observation are unknown, and quantitative methods to measure distribution patterns of centromeres in 3D space are needed. Here we have developed an analytical framework that combines robust clustering metrics and advanced modeling techniques for the quantitative analysis of centromere distributions at the single cell level. To identify a robust quantitative measure for centromere clustering, we benchmarked six metrics for their ability to sensitively detect changes in centromere distribution patterns from high-throughput imaging data of human cells, both under normal conditions and upon experimental perturbation of centromere distribution. We find that Ripley’s K Score has the highest accuracy with minimal sensitivity to variations in centromeres number, making it the most suitable metric for measuring centromere distributions. As a complementary approach, we also developed and validated spatial models to replicate centromere distribution patterns, and we show that a radially shifted Gaussian distribution best represents the centromere patterns seen in human cells. Our approach creates tools for the quantitative characterization of spatial centromere distributions with applications in both targeted studies of centromere organization as well as in unbiased screening approaches.
Metagenomic, metabolomic and sensorial characteristics of fermentedCoffea arabicaL. var. Castillo beans inoculated with microbial starter cultures
Authors: Madrid-Restrepo Maria A. , León-Inga Ana M. , Peñuela-Martínez Aida Esther , Cala Mónica P. , Reyes Alejandro
Published: January 2025

Coffee is one of the most important and widely consumed drinks around the world, and fermentation plays a pivotal role in shaping its quality. This research explores the impact of co-fermentation with “starter cultures” on the sensory and metabolic profiles, as well as in the microbial communities involved in coffee processing. Freshly harvested Arabica coffee beans were subjected to two wet-fermentation processes, where one was inoculated with a microbial starter culture and the other, spontaneous fermentation was allowed to occur. Quantitative descriptive analysis revealed that the inoculated coffee outperformed the spontaneous fermentation in all sensory attributes, boasting a higher sweetness, reduced acidity and bitterness, and presence of consumer-preferred notes. Untargeted metabolomic analysis identified over a hundred differential metabolites distinguishing both fermentation processes in green and roasted coffee beans. Inoculated coffee displayed elevated levels of compounds such as sucrose, mannitol, methyl phenylacetate, and organic acids like malic, citric, and quinic acid, compounds likely associated with a better sensory perception. The inoculated process featured abundant lactic acid bacteria andKazachstaniayeasts, which are linked to desirable metabolites like lactic, acetic, isobutyric, and hexanoic acids. Our results strongly suggest that the use of starter cultures can enhance coffee beverage quality as determined by standardized cupping and reflected by changes in the metabolic profile and microbial community structures. Future studies should focus on microbial identification and the association between species and resulting metabolic compounds, where specific bacterial and fungal groups could be isolated to be used as commercially available starter cultures for coffee fermentation.

IMPORTANCE

Our study demonstrates that inoculating coffee fermentation alters the sensory qualities of coffee and affects the dynamics of bacterial and fungal communities during this process. We identified specific changes in microbial diversity and associated metabolite profiles resulting from inoculation. Notably, we found that inoculation influences the metabolite composition of coffee beans and identified key compounds, such as aminophenol and phenol, which are likely linked to phenolic defects. This suggests that inoculation could serve as a tool to control cup defects by modifying the fermentation microbiota. Our findings advance the understanding of coffee fermentation and open opportunities for developing techniques to produce coffee with unique and desirable qualities.
CD4 T cell dysfunction is associated with bacterial recrudescence during chronic tuberculosis
Authors: Chang Evelyn , Cavallo Kelly , Behar Samuel M.
Published: January 2025

While most people containMycobacterium tuberculosisinfection, some individuals develop active disease, usually within two years of infection. Why immunity fails after initially controlling infection is unknown. C57BL/6 mice controlMycobacterium tuberculosisfor up to a year but ultimately succumb to disease. We hypothesize that the development of CD4 T cell dysfunction permits bacterial recrudescence. We developed a reductionist model to assess antigen-specific T cells during chronic infection and found evidence of CD4 T cell senescence and exhaustion. In C57BL/6 mice, CD4 T cells upregulate coinhibitory receptors and lose effector cytokine production. Single cell RNAseq shows that only a small number of CD4 T cells in the lungs of chronically infected mice are polyfunctional. While the origin and causal relationship between T- cell dysfunction and recrudescence remains uncertain, we propose T cell dysfunction leads to a feed-forward loop that causes increased bacillary numbers, greater T cell dysfunction, and progressive disease.
Dual Probe Ligation In Situ Hybridization with Rolling-Circle Amplification for High-Plex Spatial Transcriptomics
Authors: Maguire Sarah E. , Credle Joel , Bertelson Elizabeth M.W. , Lee Sunny , Cha Boyoung , Xie Dan , Kirk Greg , Ray Debjit , George Logan , Suru Aditya , Maalouf Alexandre , Ikenaga Chiseko , Lloyd Thomas , Llosa Nicolas J. , Larman H. Benjamin
Published: January 2025

New biological insights are increasingly dependent upon a deeper understanding of tissue architectures. Critical to such studies are spatial transcriptomics technologies, especially those amenable to analysis of the most widely available human tissue type, formalin-fixed and paraffin-embedded (FFPE) clinical specimens. Here we build on our previous oligonucleotide probe ligation-based approach to accurately analyze FFPE mRNA, which suffers from variable levels of degradation. LigationIn SituHybridization followed by rolling circle amplification (LISH-Lock’n’Roll or LISH-LnR), provides a streamlined method to detect the spatial location of specific mRNA isoforms within FFPE tissue architectures. Iterative fluorescent probe hybridization and imaging enables highly multiplexed spatial transcriptomic studies, as demonstrated herein for fixed specimens from inclusion body myositis patients and pediatric rhabdomyosarcoma patients. We additionally demonstrate a system of molecular rheostats that can be used to fine tune the performance of the LISH-LnR assay. Combined with LISH-seq and LISH-QC, the LISH-LnR methodology provides a powerful approach to spatial transcriptomics.
Smooth Muscle Cells and Fibroblasts in the Proximal Thoracic Aorta Exhibit Minor Differences Between Embryonic Origins in Angiotensin II-driven Transcriptional Alterations
Authors: Ito Sohei , Graf David B. , Katsumata Yuriko , Moorleghen Jessica J. , Zhang Chen , Li Yanming , LeMaire Scott A. , Shen Ying H. , Lu Hong S. , Daugherty Alan , Sawada Hisashi
Published: January 2025

Background

Thoracic aortopathy is influenced by angiotensin II (AngII) and exhibits regional heterogeneity with the proximal region of the thoracic aorta being susceptible. Smooth muscle cells (SMCs) and selected fibroblasts in this region are derived from two embryonic origins: second heart field (SHF) and cardiac neural crest (CNC). While our previous study revealed a critical role of SHF-derived cells in AngII-mediated aortopathy formation, the contribution of CNC-derived cells remains unclear.

Methods

Mef2c-Cre R26RmT/mGmice were infused with AngII (1,000 ng/kg/min). Proximal thoracic aortas were harvested at baseline or after 3 days of infusion, representing the prepathological phase. Cells were sorted by origins using mGFP (SHF-derived) and mTomato (other origins, nSHF-derived) signals, respectively. After sorting cells by origin, single-cell RNA sequencing was performed and analyzed.

Results

Short-term AngII infusion induced significant transcriptomic changes in both SHF- and nSHF-derived SMCs, but differences between origins were modest. Fibroblast transcriptomes also underwent notable changes by AngII infusion, but differences between SHF and nSHF origins remained modest. Interestingly, AngII infusion resulted in the emergence of a new fibroblast sub-population. Several molecules related to the extracellular matrix, such asElnandCol3a1, were downregulated in SHF-derived fibroblasts compared to nSHF-derived fibroblasts in the new subcluster.

Conclusion

Fibroblasts in the new subcluster exhibited lineage-specific differences in extracellular matrix-related genes; however, overall transcriptomic differences between origins in SMCs and fibroblasts in response to AngII were modest in the pre-pathological phase of AngII-induced thoracic aortopathy.

GRAPHIC ABSTRACT
The fractionation dependence of tumor control in proton therapy for early-stage non-small cell lung cancer
Authors: Jeong Jeho , Taasti Vicki T. , Jackson Andrew , Gouw Zeno A. R. , Simone Charles B. , Lambin Philippe , Deasy Joseph O.
Published: January 2025

Purpose

The relative biological effectiveness (RBE) of tumor control for proton beam therapy (PBT) compared to photon radiotherapy (RT) is typically assumed to be independent of fractionation. To test this, we modeled published PBT outcome results for early-stage non-small cell lung cancer (NSCLC) treatments across a range of fractionation schedules.

Materials and Methods

All published and analyzable cohorts were included (399 patients, 413 treated lesions). Two models were used to fit the data: a previously published tumor simulation model that fits photon RT results of NSCLC across all fractionation regimes and the Fowler LQ model with a kick-off time term. The treatment effect of each cohort was referenced to the photon equivalent dose through mechanistic model simulations in a 2 Gy/weekday scenario, with radiobiological parameters determined to simultaneously best-fit all fractionation results. The tumor control RBE of each published treatment schedule, compared to the modeled photon RT effect of the same schedule, was then estimated.

Results

For cohorts whose treatments lasted less than three weeks (i.e., 12 fractions or less), the RBE of PBT was in the range of 1.08 to 1.11. However, for fractionated treatments stretching over four weeks or more (20-25 fractions), the relative effectiveness was much lower, with RBEs in the range of 0.82-0.89. This conclusion was unchanged using the simpler Fowler LQ + time model.

Conclusions

The proton RBE for hypo-fractionated schedules was 20-30% higher than for conventional schedules. The derived radiobiological parameters of PBT differ significantly from those of photon RT, indicating that PBT is influenced differentially by radiobiological mechanisms which require further investigation.
Experimental wildflower additions increase pollination in urban agroecosystems
Authors: Ellis Emilie E. , Edmondson Jill L. , Campbell Stuart A.
Published: January 2025

The addition of nectar-rich flower patches in human-modified ecosystems is a common practice to mitigate pollinator declines and boost pollination. However, the benefits of these additions for pollinator communities and pollination services are rarely tested, especially in urban environments.

In a city-scale experiment we added floral resources to urban allotments and monitored the effects on bees, hoverflies and moths, and tested for improved seed set in a model crop (tomato,Solanum lycopersicum).

The addition of wildflowers did not benefit all insect communities. Only social bee abundance (BombusandApis) benefitted from increasing floral resource area whereas other insect taxa showed no changes in abundance potentially due to the divergence in foraging patterns of moths, hoverflies, social bees and solitary bees. The addition of wildflower patches enhanced pollination by supporting a 25.3% increase in tomato seed set, providing evidence that wildflower interventions can improve urban pollination. Seed set was higher in more urban sites, suggesting an “oasis effect” where pollinating insects are concentrated into limited greenspaces. This highlights the precarity of pollination services in highly urban areas.

Our results suggest that the practice of planting wildflower patches can positively affect pollination services in urban areas. The continued promotion of flower patch addition is likely to benefit some key insect taxa, however, the common wildflower species in seed mixes may not benefit hoverflies and moths compared to bees. The taxon-specific foraging patterns we observed should inform the design and development of pollinator-friendly wildflower seed mixes.

Societal impact statement

Enhancing urban greenspaces for pollinator communities by planting wildflower patches is increasingly common, but their efficacy for different groups of insects (bees, hoverflies and moths) is unclear. Our city-scale experiment demonstrated that wildflower patches benefitted the abundance of social bees, but did not increase other groups. Wildflower addition increased pollination services, with an increase in seed-set in our model crop, particularly in more urban areas. Wildflower patches do benefit pollinator communities and in turn humans, through the pollination services, but the species mix may not benefit some insect groups. This suggests there is potential to improve the benefit provided by wildflower patches by redesigning pollinator-friendly seed mixes.
Interplay between high-energy quenching and state transitions inChlamydomonas reinhardtii: a single-cell approach
Authors: Lahlou Aliénor , Orlando Marcelo , Bujaldon Sandrine , Gaultier William , Israelievitch Eliora , Hanappe Peter , Le Saux Thomas , Jullien Ludovic , Colliaux David , Bailleul Benjamin
Published: January 2025

Studying cell-to-cell heterogeneity is essential to understand how unicellular organisms respond to stresses. Here, we combine single-cell measurements of chlorophyllafluorescence with machine learning to study light stress responses in the model green algaChlamydomonas reinhardtii. This framework allows us to score the extent of three high-light responses at the single cell level – state transitions, high-energy quenching, and photoinhibition – and reveals significant cell-to-cell response heterogeneity. The unexpectedly strong correlation between state transitions and high-energy quenching also indicates a cellular control over the relative extent of these processes that cannot be detected by bulk measurements. Accordingly, new models were derived for the coordinated functional response to light stress inChlamydomonas. The improved phenotypic characterization allowed by this approach will facilitate the identification of genetic determinants of intercellular heterogeneity through single cell–omics and isolation of cells displaying traits of interest for biotechnology.

Teaser

Scoring of NPQ components in single cells algae unveils the general orchestration of high light photosynthetic responses.
Unsupervised learning reveals rapid gait adaption after leg loss and regrowth in spiders
Authors: Kane Suzanne Amador , Quinn Brooke L. , Wu Xuanyi Kris , Xi Sarah Y. , Ochs Michael F. , Hsieh S. Tonia
Published: January 2025

Many arthropods and some vertebrates can voluntarily lose (autotomize) limbs during antagonistic encounters, and some can regenerate functional replacements. Spiders in particular frequently autotomize one or more legs. In this study, we investigated the time course of locomotor recovery after leg loss and regeneration in juvenile tarantulas (Arachnida: Araneae) with no prior experience of autotomy. We recorded high-speed video of spiders running with all legs intact, then immediately after, and again one day after, they autotomized two legs. The legs were allowed to regenerate, and the same sequence of experiments repeated. Running performance, posture, and path tortuosity were measured from video tracking. Spiders were found to resume their pre-autotomy speed and stride frequency after leg regeneration and in ≤1 day after both autotomies; furthermore, path tortuosity was unaffected by these treatments. They adjusted their posture to compensate for missing legs, spreading their remaining legs and running with their bodies rotated 11-15 deg from their velocity. To analyze gaits, we applied unsupervised machine learning for the first time to measured kinematic data in combination with gait space metrics. Spiders were found to robustly adopt new gait patterns immediately after losing legs, with no evidence of learning. This novel clustering approach both demonstrated concordance with previously-hypothesized gaits and revealed transitions between and variations within these patterns. More generally, clustering in gait space enables the identification of patterns of leg motions in large datasets that correspond to either known gaits or undiscovered behaviors.
Impact of missing data correlated with labels to be predicted in neurodegeneration classification tasks
Authors: Prakash Mithilesh , Tohka Jussi ,   Alzheimer’s Disease Neuroimaging Initiative
Published: January 2025

We introduce a new subtype of ‘Missing Not at Random’ (MNAR) data, where the missingness is correlated with the labels (y) to be predicted, termed(y)-dependent MNAR. We demonstrate that this subtype can significantly bias the estimation of performance metrics in typical machine learning tasks. Unbiased error estimation is crucial in predictive modeling to accurately assess model performance, identify potential biases, and ensure generalizability to new, unseen data.

We explore the effects of imputing this new subtype of MNAR and compare it with general missing types, namely Missing at Random (MAR) and Missing Completely at Random (MCAR). Our comparison analysis employs both synthetic and clinical datasets, including the Alzheimer’s Disease Neuroimaging Initiative (ADNI) dataset, the Parkinson’s Progression Markers Initiative (PPMI) dataset, and the Anti-Amyloid Treatment in Asymptomatic Alzheimer’s Disease (A4) dataset. After introducing missingness into the datasets, we trained different classifiers paired with various imputation methods and measured repeated cross-validation test metrics.

Our findings reveal that datasets with non-ignorable missing types (MNAR) exhibit a strong bias compared to ignorable types (MAR and MCAR) in downstream analysis. Non-linear classifiers tend to exploit patterns from imputed data, particularly when the imputed values correlate with the target label (y), which can lead to unreliable estimation of the generalization error. Mean and median imputations proved to be more robust than tree-based or gradient boosting methods.
Exploring Antibiotic Resistance in Diverse Homologs of the Dihydrofolate Reductase Protein Family through Broad Mutational Scanning
Authors: Romanowicz Karl J. , Resnick Carmen , Hinton Samuel R. , Plesa Calin
Published: January 2025

Current antibiotic resistance studies often focus on individual protein variants, neglecting broader protein family dynamics. Dihydrofolate reductase (DHFR), a key antibiotic target, has been extensively studied using deep mutational scanning, yet resistance mechanisms across this diverse protein family remain poorly understood. Using DropSynth, a scalable gene synthesis platform, we designed a library of 1,536 synthetic DHFR homologs representing 778 species of bacteria, archaea, and viruses, including clinically relevant pathogens. A multiplexedin vivoassay tested their ability to restore metabolic function and confer trimethoprim resistance in anE. coliΔfolAstrain. Over half of the synthetic homologs rescued the phenotype without supplementation, and mutants with up to five amino acid substitutions increased the rescue rate to 90%, highlighting DHFR’s evolutionary resilience. Broad Mutational Scanning (BMS) of homologs and 100,000 mutants provided critical insights into DHFR’s fitness landscape and resistance pathways, representing the most extensive analysis of homolog complementation and inhibitor tolerance to date and advancing our understanding of antibiotic resistance mechanisms.

Graphical Abstract

Teaser

DropSynth technology enables scalable and cost-effective exploration of antibiotic resistance across the DHFR protein family.
Nucleoporins shape germ granule architecture and balance small RNA silencing pathways
Authors: Shi Kun , Zhang Ying , Du Zhenzhen , Liu Symonne C , Fan Xinyu , Lee Heng-Chi , Zhang Donglei
Published: January 2025

Animals have evolved distinct small RNA pathways, including piRNA and siRNA, to silence invasive and selfish nucleic acids. piRNA pathway factors are concentrated in perinuclear germ granules that frequently associate with nuclear pore complexes (NPCs). However, the factors mediating germ granule-NPC association and the functional relevance of such association remain unknown. Here we show that the conserved nucleoporins NPP-14 (NUP-214) and NPP-24 (NUP-88), components of the cytoplasmic filaments of NPC, play critical roles in anchoring germ granule to NPC and in attenuating piRNA silencing InC. elegans. Proximity labeling experiments further identified EPS-1 (enhanced piRNA silencing) as a key germ granule factor contributing to germ granule-NPC interaction. Innpp-14,npp-24,oreps-1mutant animals, we observed fewer but enlarged, unorganized germ granules, accompanied by the over-amplification of secondary small RNAs at piRNA targeting sites. Nonetheless, we found this enhancement of piRNA silencing comes at the cost of dampened RNAi efficiency and RNAi inheritance. Together, our studies uncovered factors contributing to germ granule-NPC association and underscored the importance of spatial organization of germ granules in balancing small RNA silencing pathways.
An evolutionarily conserved scheme for reformatting odor concentration in early olfactory circuits
Authors: Shen Yang , Banerjee Arkarup , Albeanu Dinu F. , Navlakha Saket
Published: January 2025

Understanding how stimuli from the sensory periphery are progressively reformatted to yield useful representations is a fundamental challenge in neuroscience. In olfaction, assessing odor concentration is key for many behaviors, such as tracking and navigation. Initially, as odor concentration increases, the average response of first-order sensory neurons also increases. However, the average response of second-order neurons remains flat with increasing concentration – a transformation that is believed to help with concentration-invariant odor identification, but that seemingly discards concentration information before it could be sent to higher brain regions. By combining neural data analyses from diverse species with computational modeling, we propose strategies by which second-order neurons preserve concentration information, despite flat mean responses at the population level. We find that individual second-order neurons have diverse concentration response curves that are unique to each odorant — some neurons respond more with higher concentration and others respond less — and together this diversity generates distinct combinatorial representations for different concentrations. We show that this encoding scheme can be recapitulated using a circuit computation, called divisive normalization, and we derive sufficient conditions for this diversity to emerge. We then discuss two mechanisms (spike rate vs. timing based) by which higher order brain regions may decode odor concentration from the reformatted representations. Since vertebrate and invertebrate olfactory systems likely evolved independently, our findings suggest that evolution converged on similar algorithmic solutions despite stark differences in neural circuit architectures. Finally, in land vertebrates a parallel olfactory pathway has evolved whose second-order neurons do not exhibit such diverse response curves; rather neurons in this pathway represent concentration information in a more monotonic fashion on average, potentially allowing for easier odor localization and identification at the expense of increased energy use.
Gating of hair cell Ca2+channels governs the activity of cochlear neurons
Authors: Karagulyan Nare , Thirumalai Anupriya , Michanski Susann , Qi Yumeng , Fang Qinghua , Wang Haoyu , Ortner Nadine J. , Striessnig Jörg , Strenzke Nicola , Wichmann Carolin , Hua Yunfeng , Moser Tobias
Published: January 2025

Our sense of hearing processes sound intensities spanning six orders of magnitude. In the ear, postsynaptic spiral ganglion neurons (SGNs) tile this intensity range with their firing rate codes. Presynaptic inner hair cells (IHCs) vary Ca2+-influx among their active zones (AZs) diversifying glutamate release and likely contributing to SGN firing diversity. Here we show that low-voltage activation of IHC-Ca2+-influx of mice, modeling the human CaV1.3A749Gmutation, increases spontaneous SGN-firing and lowers sound threshold. Altered synaptic morphology in CaV1.3A749G/A749Gmice already at ambient sound levels of standard mouse husbandry indicates a risk for noise-induced alterations in CaV1.3A749Gpatients. We conclude that heterogeneous voltage-dependence of CaV1.3 activation among IHC-AZs contributes to the diversity of SGN firing for sound intensity coding and synaptic vulnerability.
Seasonal Enhancement of the Viral Shunt Catalyzes a Subsurface Oxygen Maximum in the Sargasso Sea
Authors: Gilbert Naomi Elizabeth , Muratore Daniel , Shopen Gochev Camelia , LeCleir Gary R. , Cagle Shelby M. , Pound Helena L. , Sun Christine L. , Carillo Alfonso , Ndlovu Kimberley S. , Maidanik Ilia , Coenen Ashley R. , Chittick Lauren , DeBruyn Jennifer M. , Buchan Alison , Lindell Debbie , Sullivan Matthew B. , Weitz Joshua S. , Wilhelm Steven W.
Published: January 2025

Subsurface oxygen maxima (SOMs) occur directly beneath the mixed layer of stratified water columns across oligotrophic open ocean basins. SOMs occur seasonally and are hypothesized to result from elevated microbial net primary productivity (NPP). Here, we set out to identify mechanistic drivers of the SOM near the Bermuda Atlantic Time Series (BATS) site in the Sargasso Sea in October 2019. Coupled time-series analysis of metatranscriptomics, flow cytometry, and family-specific cyanophage quantification revealed elevated Prochlorococcus abundances, cyanophage abundances, and cyanophage-specific transcriptional activity in the SOM. These findings spurred us to analyze historical oxygen saturation profiles at BATS - identifying a repeated, seasonal cycle in SOM emergence associated with elevated virus-like particles and Prochlorococcus numbers. Returning to the 2019 study site, we found that transcriptional markers for increased dissolved organic matter uptake by copiotrophic bacteria were enriched in the SOM, consistent with enhanced catabolic activity due to the viral shunt. In addition, Prochlorococcus exhibited enrichment in ammonium transport transcripts at the SOM, consistent with increased responsiveness to remineralization activity by heterotrophs. Altogether, these findings suggest that enhanced viral lysis leads to locally elevated nutrient recycling and oxygen production, further reinforcing hypotheses that viruses may play a critical role in the emergence of SOMs in the oligotrophic ocean.
Deep learning models for unbiased sequence-based PPI prediction plateau at an accuracy of 0.65
Authors: Reim Timo , Hartebrodt Anne , Blumenthal David B. , Bernett Judith , List Markus
Published: January 2025

As most proteins interact with other proteins to perform their respective functions, methods to computationally predict these interactions have been developed. However, flawed evaluation schemes and data leakage in test sets have obscured the fact that sequence-based protein-protein interaction (PPI) prediction is still an open problem. Recently, methods achieving better-than-random performance on leakage-free PPI data have been proposed. Here, we show that the use of ESM-2 protein embeddings explains this performance gain irrespective of model architecture. We compared the performance of models with varying complexity, per-protein, and per-token embeddings, as well as the influence of self- or cross-attention, where all models plateaued at an accuracy of 0.65. Moreover, we show that the tested sequence-based models cannot implicitly learn a contact map as an intermediate layer. These results imply that other input types, such as structure, might be necessary for producing reliable PPI predictions.
CD8+T cells mediate vaccination-induced lymphatic containment of latentMycobacterium tuberculosisinfection following immunosuppression, while B cells are dispensable
Authors: Miranda-Hernandez Socorro , Kumar Manoharan , Henderson Alec , Graham Erin , Tan Xiao , Taylor Jim , Meehan Michael , Ceja Zuriel , del Pozo-Ramos Lidia , Pan Yi , Tsui Ellen , Donovan Meg L , Rentería Miguel E , Flores-Valdez Mario Alberto , Blumenthal Antje , Nguyen Quan , Subbian Selvakumar , Field Matt A , Kupz Andreas
Published: January 2025

It is estimated that two billion people are latently infected withMycobacterium tuberculosis(Mtb), the causative agent of tuberculosis (TB). LatentMtbinfection (LTBI) can occur in multiple organs, including the lymphatics. The risk of LTBI reactivation increases in immunocompromised conditions, such as coinfection with human immunodeficiency virus (HIV), and during treatment of autoimmune diseases and organ transplantation. The immunological correlates of protection against TB, including against reactivation of LTBI, remain largely elusive. Here, we used a mouse model of latent lymphaticMtbinfection to dissect the immunological mechanisms underlying LTBI containment versus reactivation. We show that immunosuppression-mediated reactivation of lymphatic LTBI and the subsequent spread to non-lymphatic organs can be prevented by vaccination with multiple recombinant BCG (rBCG) strains despite the deficiency of CD4+T cells. Using spatial transcriptomics, multi-parameter imaging, network analysis and bioinformatic integration of histopathological images, we reveal that immunosuppression is associated with a distinct repositioning of non-CD4 immune cells at the edge of TB lesions within the infection-draining cervical lymph nodes. While B cells increased in numbers, they are dispensable for the containment of LTBI. LymphaticMtbinfection in different immune cell-deficient mouse strains, antibody-mediated cell depletion and adoptive transfer experiments into highly susceptible mice unequivocally show that vaccination-mediated prevention of LTBI reactivation is critically dependent on CD8+T cells. These findings have profound implications for our understanding of immunity to TB and the management of LTBI.

Graphical Abstract
A Robust Proteomics-Based Method for Identifying Preferred Protein Targets of Synthetic Glycosaminoglycan Mimetics
Authors: Afosah Daniel K. , Ongolu Ravikumar , Fayyad Rawan M. , Hawkridge Adam , Desai Umesh R.
Published: January 2025

A robust technology is critically needed for identifying preferred protein targets of glycosaminoglycans (GAGs), and synthetic mimetics thereof, in biological milieu. We present a robust 10-step strategy for identification and validation of preferred protein targets of highly sulfated, synthetic, small, GAG-like molecules using diazirine-based photoaffinity labeling– proteomics approach. Our work reveals that optimally designed, homogeneous probes based on minimalistic photoactivation and affinity pulldown groups coupled with rigorous proteomics, biochemical and orthogonal validation steps offer excellent potential to identify preferred targets of GAG mimetics from the potentially numerous possible targets that cloud GAG interaction studies. Application of this 10-step strategy for a promising highly sulfated, small GAG mimetic led to identification of only a handful of preferred targets in human plasma. This new robust strategy will greatly aid drug discovery and development efforts involving GAG sequences, or sulfated small mimetics thereof, as leads.
Evaluating genotyping strategies for a small managed population with simulation
Authors: Martin Audrey AA , Schoenebeck Jeffrey , Clements Dylan N. , Lewis Tom , Wiener Pamela , Gorjanc Gregor
Published: January 2025

Background

Collecting genomic information is crucial to advance breeding for complex traits such as health, welfare, and behaviour in domesticated populations. For that purpose, different data collection scenarios can be envisioned based on the number of individuals, the number of markers, and the genotyping technology. This study developed a simulation framework, based on a service dog population, aiming to identify an optimal and cost-effective genotyping strategy that would support the implementation of genomic selection, investigation of the genetic architecture of traits of interest, and track loci of interest.

Methods

We simulated a population based on the existing pedigree, using the gene drop method in AlphaSimR. The existing pedigree was extended with additional progeny generations to evaluate the outcomes of different genotyping strategies in the future. We generated genotype data based on existing high-coverage whole-genome sequences (WGS) for the current breeding dogs and evaluated different scenarios for genotyping the progeny. The genotyping options considered SNP arrays of various densities and WGS callsets produced from different sequencing depths. We then phased and imputed the genotype data to high-coverage WGS using AlphaPeel.

Results

All scenarios were compared based on individual imputation accuracy against the simulated true whole-genome genotype. Averaged over five generations of simulated progeny, low-pass sequencing (0.5 to 2X depth) achieved accuracies of 0.998 to 0.999. The accuracy of SNP array genotyping (25K to 710K markers) was lower, with means of 0.911 to 0.938.

Conclusions

Our simulation was tailored to identify the most cost-effective and efficient strategy for downstream use in genomic selection and genetic research into traits and loci of interest. Low-pass sequencing outperformed SNP array genotyping in imputation accuracy of whole-genome genotypes as expected. Additionally, low-pass sequencing technology was the most affordable genotyping approach currently available for dogs. Thus, it appears to be the optimal choice for balancing the goals of regimented breeding programmes such as those that produce service dogs. This simulation framework could also be adapted to address other objectives for breeding organisations working with small populations.
Multiple lesion inductions intensify central sensitization driven by neuroinflammation in a mouse model of endometriosis
Authors: Harvey Madeleine E. , Shi Mingxin , Oh Yeongseok , Mitchell Debra A. , Slayden Ov D. , MacLean James A. , Hayashi Kanako
Published: January 2025

Introduction

Endometriosis is an inflammatory disease associated with chronic pelvic pain (CPP). Growing evidence indicates that endometriotic lesions are not the sole source of pain. Instead, central nervous system (CNS) dysfunction created by prolonged peripheral and central sensitization plays a role in developing endometriosis-associated CPP. This study investigated how CPP is established using a multiple lesion induction mouse model of endometriosis, as repeated retrograde menstruation is considered underlying endometriosis pathogenesis.

Methods

We generated endometriosis-like lesions by injecting endometrial tissue fragments into the peritoneal cavity in mice. The mice received a single (1x) or multiple inductions (6x) to simulate recurrent retrograde menstruation. Lesion development, hyperalgesia by behavioral testing, signs of peripheral sensitization, chronic inflammation, and neuroinflammation were examined with lesions, peritoneal fluids, dorsal root ganglia (DRG), spinal codes, and brain.

Results

Multiple lesion inductions increased lesion numbers and elevated abdominal and hind paw hypersensitivity compared to single induction mice. Elevated persistent glial cell activation across several brain regions and/or spinal cords was found in the multiple induction mice.

Specifically, IBA1+ microglial soma size was increased in the hippocampus and thalamus. IBA1+ cells were abundant in the cortex, hippocampus, thalamus, and hypothalamus of the multiple induction mice. GFAP+ astrocytes were mainly elevated in the hippocampus. Elevated TRPV1, SP, and CGRP expressions in the DRG were persistent in the multiple induction mice. Furthermore, multiple inductions induced the severe disappearance of TIM4hiMHCIIloresidential macrophages and the influx of increased proinflammatory TIM4loMHCIIhimacrophages in the peritoneal cavity. The single and multiple inductions elevated secreted TNFα, IL-1β, and IL-6 levels in the peritoneal cavity at 2 weeks. Elevated cytokine levels returned to the pre-induction levels in the single induction mice at 6 weeks; however, they remained elevated in the multiple induction mice.

Conclusions

Our results indicate that the repeatedly occurring lesion inductions (=mimic retrograde menstruation) can be a peripheral stimulus that induces nociceptive pain and creates composite chronic inflammatory stimuli to cause neuroinflammation and sensitize the CNS. The circuits of neuroplasticity and stimulation of peripheral organs via a feedback loop of neuroinflammation may mediate widespread endometriosis-associated CPP.