bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectral STED microscopy improves spectral sensitivity with polarity-sensitive probes and enables correlative measurements of membrane order and anomalous lipid diffusion
Authors: Urbančič Iztok , Schneider Falk , Galiani Silvia , Sezgin Erdinc , Eggeling Christian
Published: February 2025

Molecular plasma membrane organization and dynamics play an important role in cellular signalling. Advances in our understanding of the nanoscale architecture of the plasma membrane heavily rely on the development of non-invasive experimental methods, particularly of advanced fluorescence microscopy and spectroscopy techniques with high spatio-temporal resolution and sensitivity to local molecular properties. However, it remains difficult to combine several of them for a multimodal characterisation that would reduce the possibility of misinterpretations. Here, we integrated a spectral detector into a super-resolution stimulated emission depletion (STED) microscope, achieving three goals. First, we show that compared to the standard ratiometric detection using fixed bandpass filters, the spectrally resolved acquisition together with spectral fitting or phasor analysis improves the accuracy of experiments determining membrane lipid order with polarity-sensitive probes multifold. Secondly, we demonstrate that this acquisition scheme allows the use of such probes in combination with other dyes with overlapping spectra, enabling co-localisation of the membrane order maps with other cellular structures of interest, e.g. fluorescently labelled proteins. Finally, we correlate the obtained membrane lipid order with the anomalous trapped diffusion properties of a fluorescent sphingomyelin lipid analogue in the plasma membrane of living cells, as determined by STED fluorescence correlation spectroscopy, and highlight that some of the most apparent trapping sites locate at the boundaries of local ordered environments discernible by the introduced spectral STED microscopy. With additional measurements in model membranes and Monte-Carlo simulations we conclude that for sub-100 nm ordered environments uneven probe partitioning cannot by itself explain the trapping diffusion of SM in cells.
Shoot at Site: Advancingin plantatransformation, regeneration and gene-editing through a cascade of wounding-mediated developmental regulators
Authors: Kshetry Arjun Ojha , Ghose Kaushik , Alok Anshu , Devkar Vikas , Raman Vidhyavathi , Stupar Robert M. , Herrera-Estrella Luis , Zhang Feng , Patil Gunvant B.
Published: February 2025

Developing transgenic and/or gene-edited plants largely depends on tedious, lengthy, and costlyin vitroregeneration protocols. While plants have remarkable regeneration ability, not all species, genotypes or even explants exhibit the same transformation and regeneration potential underin vitroconditions. To tackle this bottleneck, we have developed a seamless and user-friendly system to induce transgenic and gene-editedde novomeristems via a synthetic cascade comprising a wound-induced regeneration pathway, plant developmental regulators (DRs) and gene-editing reagents.WOUND INDUCED DEDIFFERENTIATION 1 (WIND1)is used as a transcriptional regulator to control the expression of various DR genes throughENHANCER OF SHOOT REGENERATION 1 (ESR1)promoter. This cascade was strategically appliedin plantato the non-meristematic internode ofN. benthamianato induce meristematic activity and regeneratede novoshoots with knock-out mutations of thephytoene desaturase(PDS) gene. This synthetic toolkit was further applied successfully to tomato and soybean. This methodology offers a transformative approach to overcome barriers in plant biotechnology, potentially accelerating the generation of transgenic and gene-edited plants without reliance on conventional tissue-culture intermediates.
Pan-cell type continuous chromatin state annotation of all IHEC epigenomes
Authors: Daneshpajouh Habib , Moghul Ismail , Wiese Kay C , Libbrecht Maxwell W
Published: February 2025

1

Understanding the mechanistic basis of genetic disease requires annotating the regulatory elements in the human genome. To this end, the International Human Epigenome Consortium (IHEC) has generated thousands of epigenomic datasets—including ChIP-seq, DNase-seq, and ATAC-seq—that measure various biochemical activities in the genome, including transcription factor binding, histone modification, and DNA accessibility. Currently, the predominant methods for integrating these data sets to annotate regulatory elements are segmentation and genome annotation (SAGA) algorithms such as ChromHMM and Segway. The majority of annotations generated by these methods are cell type-specific. However, as the number of profiled cell types has grown into the thousands, using thousands of cell type-specific chromatin state annotations proves undesirable for many applications. Therefore, recently, researchers have sought a single unified annotation of regulatory elements across all cell types, known as a “pan-cell type” annotation. Here, we present a pan-cell type annotation that summarizes all IHEC epigenomes using the recently-developed method, epigenome-ssm. This pan-cell type annotation comprises 33 genome-wide chromatin state feature signal tracks, each of which captures a regulatory program driving genomic activity in one or more cell types. We show that these feature maps constitute an intuitive and visualizable summary of epigenomic data.
Context-specific interaction of the lipid regulator DIP-2 with phospholipid synthesis in axon regeneration and maintenance
Authors: Park Seungmee , Jin Yishi , Chisholm Andrew D.
Published: February 2025

Neurons maintain their morphology over prolonged periods of adult life with limited regeneration after injury.C. elegansDIP-2 is a conserved regulator of lipid metabolism that affects axon maintenance and regeneration after injury. Here, we investigated genetic interactions ofdip-2with mutants in genes involved in lipid biosynthesis and identified roles of phospholipids in axon regrowth and maintenance. CEPT-2 and EPT-1 are enzymes catalyzing the final steps in thede novophospholipid synthesis (Kennedy) pathway. Loss of function mutants ofcept-2orept-1show reduced axon regrowth and failure to maintain axon morphology. We demonstrate that CEPT-2 is cell-autonomously required to prevent age-related axonal defects. Interestingly, loss of function indip-2led to suppression of the axon regrowth phenotype observed in eithercept-2orept-2mutants, suggesting that DIP-2 acts to counterbalance phospholipid synthesis. Our findings reveal the genetic regulation of lipid metabolism to be critical for axon maintenance under injury and during aging.

Article Summary

Little is known about how adult neurons live long with limited regenerative capacity. This study investigates the role of lipid metabolism in sustaining neuronal health inC. elegans.Mutating phospholipid synthetic genes impairs axon regrowth after injury. Lack of DIP-2, a lipid regulator, restores regrowth, suggesting DIP-2 counterbalances phospholipid synthesis. Moreover, neuronal phospholipid synthesis is essential for preventing age-dependent axonal defects. These findings reveal phospholipid biosynthesis is key to axon integrity during aging and injury. As lipid metabolism is implicated in neurological disorders, this study serves as an entry point into investigating neuronal lipid biology under various conditions.
Glutamatergic synaptic resilience to overexpressed human alpha-synuclein
Authors: Santos Patrícia I. , García-Plaza Inés Hojas , Shaib Ali , Rhee Jeong Seop , Chouaib Abed Alrahman , Rizzoli Silvio O. , Daniel James , Outeiro Tiago F.
Published: February 2025

Alpha synuclein (aSyn) is an abundant protein that, in the brain, is concentrated in neuronal presynaptic boutons and associates with synaptic vesicles. aSyn is strongly linked with Parkinson’s disease (PD) due to its accumulation in pathognomonic inclusions in neuronal cells, including in glutamatergic neurons. While increased expression of wild-type (WT) aSyn due to multiplications of the SNCA gene, or the expression of mutant forms of aSyn, can cause familial forms of PD, it is still unclear whether increased levels of aSyn alone are sufficient to impair synaptic function. Previous studies have used experimental systems that do not allow systematic characterisation of presynaptic physiology. To address this gap in research, we used lentiviral vectors to overexpress human aSyn (haSyn) in continental and autaptic glutamatergic neurons from rodent hippocampus and systematically analysed their presynaptic function. Virally-transduced neurons exhibited levels of expression of haSyn that mimic those associated with triplications of the SNCA gene in PD patients (2-fold increase in total aSyn), as determined using quantitative immunofluorescence imaging and immunoblots. Neuronal toxicity, neuronal morphology, and SNAP-25, a presynaptic protein, were not altered in continental cultures. Finally, a systematic characterization of autaptic neurons expressing haSyn exhibited no significant difference in any parameter of synaptic function, including basal properties of evoked and spontaneous neurotransmitter release, and synaptic plasticity compared to neurons infected with a control virus. These results indicate that rodent glutamatergic neurons are resilient to aSyn overexpression. In conclusion, our findings suggest neurotoxicity associated with aSyn overexpression is not universal, and that a deeper understanding of aSyn biology and pathobiology is necessary.
Cell signaling pathways discovery from multi-modal data
Authors: He Changhan , Simpson Claire , Cossentino Ian , Zhang Bin , Tkachev Sasha , Eddins Devon J. , Kosters Astrid , Yang Junkai , Sheth Shivani , Levy Tyler , Possemato Anthony , Huang Linglin , Tabatsky Evgeniy , Gregoretti Ivan , Ariss Majd , Dandekar Deepti , Ausekar Aniket , Ghosn Eliver E. B. , Colonna Marco , Rikova Klarisa , Nie Qing , Orlova Darya
Published: February 2025

Deciphering cell signaling pathways is essential for advancing our understanding of basic biology, disease mechanisms, and the development of innovative therapeutic interventions. Recent advancements in multi-omics technologies enable us to capture cell signaling information in a more meaningful context. However, omics data is inherently complex—high-dimensional, heterogeneous, and extensive—making it challenging for human interpretation. Currently, computational tools capable of inferring cell signaling pathways from multi-omics data are very limited, underscoring the urgent need to develop such methods.

To address this challenge, we developed Incytr, a method that facilitates the efficient discovery of cell signaling pathways by integrating diverse data modalities, including transcriptomics, proteomics, phosphoproteomics, and kinomics. We demonstrate Incytr’s application in elucidating cell signaling within the contexts of COVID-19, Alzheimer’s disease, and cancer. Incytr successfully rediscovered known subpathways in these diseases and generated novel hypotheses for cell-type-specific signaling pathways supported by multiple data modalities. We illustrate how overlaying Incytr-identified pathways with prior knowledge from biomarker and small molecule drug databases can be used to facilitate target and drug discovery. Overall, as we demonstrated here, with the use of simple natural language processing AI models, these pathways could serve as a discovery tool to deepen our understanding of cell-cell communication semantics and co-evolution.
Kinase-Inhibitor Binding Affinity Prediction with Pretrained Graph Encoder and Language Model
Authors: Guo Xudong , Ran Zixu , Li Fuyi
Published: February 2025

Motivation

The accurate prediction of inhibitor-kinase binding affinity is crucial in drug discovery and medical applications, especially in the treatment of diseases such as cancer. Existing methods for predicting inhibitor-kinase affinity still face challenges including insufficient data expression, limited feature extraction, and low performance. Despite the progress made through artificial intelligence (AI) methods, especially deep learning technology, many current methods fail to capture the intricate interactions between kinases and inhibitors. Therefore, it is necessary to develop more advanced methods to solve the existing problems in inhibitor-kinase binding prediction.

Results

This study proposed Kinhibit, a novel framework for inhibitor-kinase binding affinity predictor. Kinhibit integrates self-supervised pre-trained molecular encoders and protein language models (ESM-S) to extract features effectively. Kinhibit also employed a feature fusion approach to optimize the fusion of inhibitor and kinase features. Experimental results demonstrate the superiority of this method, achieving an accuracy of 92.6% in inhibitor prediction tasks of three MAPK signaling pathway kinases: Raf protein kinase (RAF), Mitogen-activated protein kinase kinase (MEK), and Extracellular Signal-Regulated Kinase (ERK). Furthermore, the framework achieves an impressive accuracy of 93.4% on a dataset containing over 200 kinases. This study provides promising and effective tools for drug screening and biological sciences.
Beyond Blacklists: A Critical Assessment of Exclusion Set Generation Strategies and Alternative Approaches
Authors: Wall Brydon P. G. , Ogata Jonathan D. , Nguyen My , McClay Joseph L. , Harrell J. Chuck , Dozmorov Mikhail G.
Published: February 2025

Short-read sequencing data can be affected by alignment artifacts in certain genomic regions. Removing reads overlapping these exclusion regions, previously known as Blacklists, help to potentially improve biological signal. Tools like the widely used Blacklist software facilitate this process, but their algorithmic details and parameter choices are not always clearly documented, affecting reproducibility and biological relevance. We examined the Blacklist software and found that pre-generated exclusion sets were difficult to reproduce due to variability in input data, aligner choice, and read length. We also identified and ad- dressed a coding issue that led to over-annotation of high-signal regions. We further explored the use of “sponge” sequences—unassembled genomic regions such as satellite DNA, ribosomal DNA, and mitochondrial DNA—as an alternative approach. Aligning reads to a genome that includes sponge sequences reduced signal correlation in ChIP-seq data comparably to Blacklist-derived exclusion sets while preserving biological signal. Sponge-based alignment also had minimal impact on RNA-seq gene counts, suggesting broader applicability beyond chromatin profiling. These results highlight the limitations of fixed exclusion sets and suggest that sponge sequences offer a flexible, alignment-guided strategy for reducing artifacts and improving functional genomics analyses.
The impact of ambient contamination on demultiplexing methods for single-nucleus multiome experiments
Authors: Li Terence , Alvarez Marcus , Liu Cuining , Abuhanna Kevin , Sun Yu , Ernst Jason , Plath Kathrin , Balliu Brunilda , Luo Chongyuan , Zaitlen Noah
Published: February 2025

Sample multiplexing has become an increasingly common design choice in droplet-based single-nucleus multi-omic sequencing experiments to reduce costs and remove technical variation. Genotype-based demultiplexing is one popular class of methods that was originally developed for single-cell RNA-seq, but has not been rigorously benchmarked in other assays, such as snATAC-seq and joint snRNA/snATAC assays, especially in the context of variable ambient RNA/DNA contamination. To address this, we develop ambisim, a genotype-aware read-level simulator that can flexibly control ambient molecule proportions and generate realistic joint snRNA/snATAC data. We use ambisim to evaluate demultiplexing methods across several important parameters: doublet rate, number of multiplexed donors, and coverage levels. Our simulations reveal that methods are variably impacted by ambient contamination in both modalities. We then applied the demultiplexing methods to two joint snRNA/snATAC datasets and found highly variable concordance between methods in both modalities. Finally, we develop a new metric,variant consistency, which we show is correlated with cell-level ambient molecule fractions in singlets. Applying our metric to two multiplexed joint snRNA/snATAC datasets reveals variable ambient contamination across experiments and modalities. We conclude that improved modelling of ambient material in demultiplexing algorithms will increase both sensitivity and specificity.
Loss of 5-HT2CReceptor Function Alters Motor Behavior in Male and Female Mice With and Without Spinal Cord Injury
Authors: Sim Margaret I. , Birch Derin , Mahrous Amr A. , Heckman C.J. , Tysseling Vicki M.
Published: February 2025

The 5-HT2Creceptor is involved in the regulation of spinal motor function, specifically in both volitional and involuntary motor behavior. It contributes to various aspects of voluntary movement, such as locomotion, gait, coordination, and muscle contraction, as well as to involuntary motor behavior like spasms, which affect many individuals with spinal cord injury. Despite its known involvement in motor function, little is known about the physiological role of the 5-HT2Creceptor and the changes it undergoes after spinal cord injury. In this study, we have investigated the volitional and involuntary motor behavior of male and female uninjured and spinal cord injured knock-out mice that lack the functional 5-HT2Creceptor by comparing these genetically manipulated mice to typical-functioning sex-matched wildtype mice. Behavioral assessments revealed differences in volitional muscle strength and coordination, as well as hyperreflexia, between the groups observed. Additionally,ex vivosacral cord preparation data suggest that 5-HT2Creceptor knock-out mice exhibit less spasm-like activity than wildtype mice, corroborating our results from behavioral testing in which the flexor withdrawal reflex of the hindlimbs was assessed. To investigate potential compensatory changes in 5-HT2Creceptor expression following spinal cord injury, western blot analysis was performed on lumbar and sacral spinal cord tissue from wildtype and 5-HT2Creceptor knock-out mice before and after injury. Both sex and injury status significantly influenced 5-HT2Creceptor relative expression and distribution of these receptors in both spinal cord regions. Through a comprehensive approach combining behavioral assessments, electrophysiological experiments, and whole-tissue protein analysis, our findings provide strong evidence that the 5-HT2Creceptor plays a critical role in both volitional motor function and involuntary motor behavior, and the relative expression of the 5-HT2Creceptor is influenced by both sex and injury status.
A biological growth curve is a sum of two distinct S-curves
Authors: Shruti I S
Published: February 2025

A growing population consists of successive generations of individuals interacting with each other and the environment. At every instant, these individual interactions can lead to either promotion or restriction of the population growth. Thus, within a growing population, differences arise over time due to both extrinsic and intrinsic factors such as resource utilization, competition, age distribution etc. These differences can lead to divergence within the population growth. The sum of these differences is manifested as the net population growth. In this work, we propose that these differences that arise over time within a growing population can be represented as two distinct S-curves. The sum of these two S-curves results in the growth curve of a population. These differences also arise in the growth curves of mean individual attributes such as height or weight. We demonstrate this by applying thea−mbiological growth model on (i) the population growth curves ofDrosophilaand yeast and (ii) the growth curves of mean individual height of sunflower plants and the mean individual body weight of male white rats. Finally, we also discuss the results using a coordinate system with the two distinct S-curves as the axes.
Nucleotide-specific RNA conformations and dynamics within ribonucleoprotein condensates
Authors: Wang Tong , Hu Qingyue , Fronhofer Scout , Pollack Lois
Published: February 2025

Ribonucleoprotein (RNP) condensates have distinct physiological and pathological significance, but the structure of RNA within them is not well understood. Using contrast-variation solution X-ray scattering, which discerns only the RNA structures within protein-RNA complexes, alongside ensemble-based structural modeling we characterize the conformational changes of flexible poly-A, poly-U and poly-C single stranded RNA as it interacts with polybasic peptides, eventually forming condensed coacervate mixtures. At high salt concentrations, where macromolecular association is weak, we probe association events that precede the formation of liquid-like droplets. Structural changes occur in RNA that reflect charge screening by the peptides as well asπ−πinteractions of the bases with basic residues. At lower salt concentrations, where association is enhanced, poly-A RNA within phase separated RNP mixtures exhibit a broad scattering peak, suggesting subtle ordering. Coarse-grained molecular dynamics simulations are used to elucidate the nucleotide-specific dynamics within RNP condensates. While adenine-rich condensates behave like stable semidilute solutions, uracil-rich RNA condensates appear to be compositionally fluctuating. This approach helps understand how RNA sequence contributes to the molecular grammar of RNA-protein phase separation.
Determinants of cholera fatalities in Malawi: A case-control study of patient-level and clinical management factors in the 2022-23 outbreak
Authors: Chitatanga Ronald , Thawani Alex , Chadwala Hope , Chirwa Amon , Mitambo Collins
Published: February 2025

Malawi experienced its deadliest cholera outbreak in 2022, reporting over 50,000 cases and more than 1,700 deaths, resulting in a case fatality rate of 3.1%. The outbreak was exacerbated by the devastation caused by Tropical Cyclones Freddy, Ana, and Gombe, which strained healthcare resources. Despite the severity of the outbreak, no real-time evaluations of patient-level risk factors influencing cholera mortality among hospitalized patients were conducted. This study characterizes patient-level factors and treatment practices associated with cholera mortality in Malawi. A multisite case-control study was conducted in August 2023 across four cholera treatment units. A retrospective review of 174 medical records (confirmed by rapid antigen test or stool culture) was performed by a team led by trained medical officers. Cases (deceased) and controls (survivors) were matched based on age and district of diagnosis. Conditional logistic regression was used to assess associations between patient characteristics and mortality. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated for all predictors, with statistical significance set at p < 0.05. The strongest predictor of mortality was inadequate intravenous (IV) fluid management, with 95% of deceased patients classified as inadequately managed. Inadequate fluid management was associated with significantly higher odds of mortality (OR = 117, 95% CI: 14.3–959, p < 0.001). This study highlights critical gaps in adherence to cholera treatment protocols in Malawi’s treatment units, emphasizing the need for timely and adequate IV fluid management to reduce cholera-related deaths.
OTULIN Interactome Reveals Immune Response and Autophagy Associated with Tauopathy in a Mouse Model
Authors: Li Ling , Li Mingqi , Zhou Yuyang , Kakhniashvili David , Wang Xusheng , Liao Francesca-Fang
Published: February 2025

Tauopathies are neurodegenerative diseases that are pathologically characterized by accumulation of misfolded microtubule-associated protein tau aggregates in the brain. Deubiquitination, particularly by OTULIN, a unique deubiquitinase targeting methionine-1 (M1) linkages from linear ubiquitin chain assembly complex (LUBAC)), is reportedly associated with the accumulation of neurotoxic proteins in several neurodegenerative diseases, likely including tauopathies. To investigate the potential roles of OTULIN in tauopathies, we analyzed the OTULIN interactome in hippocampal tissues from PS19 transgenic (Tg) mice and their non-transgenic (nTg) littermate controls using affinity purification-mass spectrometry (AP-MS). We identified 705 and 800 proteins enriched in Tg and nTg samples, respectively, with a protein false discovery rate (FDR) of <1%. Of these, 189 and 205 proteins were classified as probable OTULIN interactors in Tg and nTg groups, respectively, based on Significance Analysis of INTeractome (SAINT) score of ≥0.80 and FDR of ≤ 5%. A total of 84 proteins were identified as OTULIN interactors in the PS19 Tg group, while 100 proteins were associated with OTULIN in the nTg controls. Functional enrichment analyses revealed that OTULIN-interacting proteins in the nTg group were enriched in pathways related to spliceosome, complement and coagulation cascades, and ribosome, whereas those in the Tg group were associated with immune response and autophagy. These findings suggest that OTULIN-interacting proteins may play a critical role in the pathogenesis of tauopathy in this mouse model.

Highlights

OTULIN interactome in hippocampal tissues from PS19 transgenic (Tg) mice and their non-transgenic (nTg) littermate controls is analyzed.

OTULIN-interacting proteins in the nTg group are enriched in pathways related to spliceosome, complement and coagulation cascades, and ribosome.

OTULIN-interacting proteins in Tg are implicated in immune response and autophagy.

ATP2A2 is identified as an OTULIN-interacting protein specifically enhanced in Tg mice.
In vivobase editing reduces liver cysts in autosomal dominant polycystic kidney disease
Authors: Ibel Antonia , Bhardwaj Rishi , Yilmaz Duygu Elif , Kong Shuhan , Wendlinger Sarah , Papaioannou Dimitra , Diezemann Claudia , Eckardt Kai-Uwe , Hasan Fatima , Klämbt Verena , Halbritter Jan , Fedeles Sorin , Krappitz Matteus , Kaminski Michael M.
Published: February 2025

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder, affecting over 10 million individuals worldwide. Cystic expansion typically progresses to kidney failure and also involves the liver with limited treatment options. Pathogenic variants inPKD1orPKD2account for 85-90% of cases. Genetic re-expression ofPkd1orPkd2has been shown to partially reverse key characteristics of the disease phenotype in mice. Despite advancements in the understanding of the genetic basis, it remains unclear whether the correction of underlying pathogenic variants can effectively prevent, modify, or reverse the disease. Additionally, the feasibility of extrinsically delivered genome editing as a treatment option for ADPKD remains largely unexplored. In this study, we employed CRISPR base editing to correct a spectrum of representative pathogenicPKD1variants selected from a patient cohort achieving precise and efficient editingin vitro. Correction of a representative murine missense variant (c.6646C>T (R2216W)) in primary renal epithelial cells successfully increased polycystin-1 expression and reduced levels of the endoplasmic reticulum stress marker sXBP1.In vivo, base editor delivery to the c.6646C>T (R2216W) knock-in mouse enabled correction of the pathogenic variant, resulting in a significant reduction in liver cysts. These findings provide the first evidence of ADPKD reversibility through genome editing, opening promising novel therapeutic perspectives for affected patients and their families.
Motion processing in visual cortex of maculopathy patients
Authors: Michaud Célia , Guénot Jade , Faurite Cynthia , Gallice Mathilde , Chiquet Christophe , Vayssière Nathalie , Berry Isabelle , Trotter Yves , Soler Vincent , Peyrin Carole , Cottereau Benoit R.
Published: February 2025

Previous studies on animal models suggested that visual areas involved in motion processing could undergo important cortical reorganizations following retinal damages. This could have major implications for patients suffering from macular degeneration (MD), one leading cause of vision loss. Here, we performed fMRI recordings in a group of maculopathy patients (including individuals suffering from age-related macular degeneration or from Stargardt’s Disease) and a control group to characterize the motion processing cortical network in MD patients and determine whether this network undergoes significant large-scale reorganizations following the onset of the scotoma. We used an experimental protocol based on random-dot kinematograms (RDKs) classically employed to characterize motion-selective areas in the brain. To ensure that the visual information processed by the two groups was equivalent, the visual field in each control participant was masked using an artificial scotoma directly derived from clinical measurements in their paired patient. We found that in MD patients, translational motion elicited significant and robust activations in a restricted cortical network which included the human V5/MT+ complex (hMT+), areas V3A and V6, and a portion of primary visual areas (V1, V2 and V3) connected to peripheral vision. Importantly, the same patterns of responses were also observed in control participants. Moreover, the extent and strength of activation within these motion-selective areas did not differ significantly between the two groups. Altogether, these results suggest that in humans, the motion-selective network does not undergo significant large-scale cortical reorganizations following the onset of MD.

Significance statement

Motion processing in the visual cortex of patients with macular degeneration has never been characterized. Here, we performed fMRI recordings in 7 maculopathy patients and found robust motion-selective activations in a cortical network which included the human V5/MT+ complex (hMT+), areas V3A and V6, and a portion of primary visual areas connected to peripheral vision. These activations are in very good agreement with those reported in participants with normal vision in the literature and do not significantly differ from those measured in a group of age and gender-matched control participants. Altogether, our results suggest that the motion-selective network does not undergo significant large-scale reorganizations in maculopathy patients following the onset of the scotoma.
PI(3)P signaling regulates endosomal flux underlying developmental synaptic remodeling via Rab4
Authors: Singh Kamaldeep , Das Semanti , Rai Dipti , Sutradhar Sabyasachi , Sarkar Asmita , Howard Jonathon , Ray Krishanu
Published: February 2025

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) ofDrosophilalarvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes.

Author summary

Healthy brain function relies on the growth and remodeling of synaptic connections between nerve cells. This process involves carefully orchestrated movements of proteins and membranes inside the neurites. For instance, endosome movement in the nerve cell extensions helps guide neuronal growth, recycle parts of the synapse, and alter signal transmissions between neurons. Rab GTPases, a group of small G-proteins, are crucial for these processes. Here, we aimed to understand what triggers these processes and how the ’supply chain’ within nerve cell extensions is connected to changes at the synapse. In fruit fly larvae, we observed that the movement of Rab4 vesicles in the nerve cell extensions is inversely related to gross synapse content. A further examination revealed that onset of insulin signaling accelerates the Rab4 vesicles towards synapse through a lipid kinase called Vps34, which makes a signaling lipid called phospatidylinnositol-3-phosphate. Higher levels this lipid molecule on Rab4 vesicles attract a motor protein, Klp98A, speeding up their movement during the synaptic contraction phase. Our findings reveal a new mechanism that regulates Rab4 vesicle transport and its impact on synapse stability. The study further suggests potential targets for controlling age-related dementia likely to be caused by excessive insulin signaling in neurons.
CRISPR targeting of H3K4me3 activates gene expression and unlocks centromeric crossover recombination in Arabidopsis
Authors: Binenbaum Jenia , Adamkova Vanda , Fryer Hannah , Xu Linhao , Gorringe Nicola , Włodzimierz Piotr , Burns Robin , Papikian Ashot , Jacobsen Steven E. , Henderson Ian , Harris C. Jake
Published: February 2025

H3K4me3 is a fundamental and highly conserved chromatin mark across eukaryotes, playing a central role in many genome-related processes, including transcription, maintenance of cell identity, DNA damage repair, and meiotic recombination. However, identifying the causal function of H3K4me3 in these diverse pathways remains a challenge, and we lack the tools to manipulate it for agricultural benefit. Here we use the CRISPR-based SunTag system to direct H3K4me3 methyltransferases in the model plant,Arabidopsis thaliana. Targeting of SunTag-SDG2 activates the expression of the endogenous reporter gene,FWA. We show that SunTag-SDG2 can be employed to increase pathogen resistance by targeting the H3K4me3-dependent disease resistance gene,SNC1. Meiotic crossover recombination rates impose a limit on the speed with which new traits can be transferred to elite crop varieties. We demonstrate that targeting of SunTag-SDG2 to low recombining centromeric regions can significantly stimulate crossover formation. Finally, we reveal that the effect is not specific to SDG2 and is likely dependent on the H3K4me3 mark itself, as the orthogonal mammalian-derived H3K4me3 methyltransferase, PRDM9, produces a similar effect on gene expression with reduced off-target potential. Overall, our study supports an instructive role for H3K4me3 in transcription and meiotic recombination and opens the door to precise modulation of important agricultural traits.
Seven blood biomarkers are associated with TGF-β and VHL-HIF signaling in patients with clear cell renal cell carcinoma
Authors: Mallikarjuna Pramod , Erdem Cemal , Hedman Harald , Sitaram Raviprakash T. , Beorlegui Ruben Ilundain , Aripaka Karthik , Larsson Anders , Ljungberg Börje , Kamali-Moghaddam Masood , Landström Maréne
Published: February 2025

Clear cell renal cell carcinoma (ccRCC) is an aggressive kidney cancer subtype frequently associated with poor prognosis. Most ccRCC cases are asymptomatic in early stages and symptomatic mostly in advanced stages. Furthermore, the heterogeneity of ccRCC presents a challenge to design new treatments. In this study, using proximity extension assay (PEA), we analyzed blood samples from 134 patients with ccRCC and from 111 age- and gender-matched healthy donors. We identified a panel of seven proteins (ANXA1, ESM1, FGFBP1, MDK, METAP2, SDC1, and TFPI2) that are associated with clinicopathological parameters and patient survival. These biomarkers can differentiate patients with ccRCC from the control individuals with high diagnostic sensitivity and specificity. Moreover, by studying protein expression in solid tumors from the same ccRCC patients, we revealed associations between the panel biomarkers and proteins in the TGF-β and VHL-HIF signaling pathways. We found that most tumor promoting biomarkers were positively associated with TGF-β signaling and HIF-2α, and negatively associated with pVHL and HIF-1α. We also found that most tumor suppressing biomarkers were positively associated with pVHL and HIF-1α and negatively associated with TGF-β signaling and HIF-2α. For ccRCC patients, the blood protein biomarkers that were connected to poor prognosis and TGF-β/HIF-2α signaling, as identified in this study, are potentially important assets in personalized medicine.
De-novo DNA Methylation of Bivalent Promoters Induces Gene Activation through PRC2 Displacement
Authors: O’Geen Henriette , Mihalovits Alina , Brophy Benjamin D. , Yang He , Miller Maxwell W. , Lee Chelsey J. , Segal David J. , Tomkova Marketa
Published: February 2025

Promoter DNA methylation is a key epigenetic mark, commonly associated with gene silencing. However, we noticed that a positive association between promoter DNA methylation and expression is surprisingly common in cancer. Here, we use hit-and-run CRISPR/dCas9 epigenome editing to evaluate how deposition of DNA methylation can regulate gene expression dependent on pre-existing chromatin environment. While the predominant effect of DNA methylation in non-bivalent promoters is gene repression, we show that in bivalent promoters this often leads to gene activation. We demonstrate that gain of DNA methylation leads to reduced MTF2 binding and eviction of H3K27me3, a repressive mark that guards bivalent genes against activation. Our cancer patient data analyses reveal that in cancer, this mechanism likely leads to activation of a large group of transcription factors regulating pluripotency, apoptosis, and senescence signalling. In conclusion, our study uncovers an activating role of DNA methylation in bivalent promoters, with broad implications for cancer and development.
Endothelial cell Pannexin1 overexpression impairs ischemic stroke outcome in a sex-dependent manner
Authors: Tomàs-Gracia Maria , Mauro Amanda K , Duffy Colleen K , Dai Eric Y , Shahab Guleer , Medina Christopher B , Ravichandran Kodi S , Isakson Brant E , Good Miranda E
Published: February 2025

Ischemic stroke is a leading cause of morbidity and mortality. We have previously shown that deletion of endothelial cell (EC) Panx1 reduces ischemic stroke infarct volume and reduces cerebral arterial myogenic reactivity, which regulates cerebral blood flow. We hypothesized that EC Panx1 content dictates ischemic stroke outcome and thus increased EC Panx1 expression will worsen ischemic stroke outcomes due to exacerbated myogenic tone development and impaired cerebral blood flow recovery. To test this, we generated the Cdh5-CreERT2+ROSA26-hPanx1Tgmouse model that conditionally overexpresses the human isoform of Panx1 specifically in EC. We have found that cerebral myogenic reactivity is significantly increased with overexpression of EC Panx1 only in female mice, without alterations in peripheral vascular reactivity or blood pressure regulation. Similarly, we found that infarct size was increased and recovery of cerebral blood flow was reduced in female but not male EC Panx1 overexpressing mice. Our findings indicate a role for EC Panx1 as a mediator of ischemic stroke recovery. Furthermore, these data suggest a potential sex-dependent effect for EC Panx1, where females are more sensitive to increased EC Panx1 in cerebral vascular function and may provide a potential therapeutic target for the treatment of ischemic stroke in women.
Magnetic activation of electrically active cells
Authors: Duret Guillaume , Coffler Samantha , Avant Ben , Kim Wonjune , Peterchev Angel V. , Robinson Jacob
Published: February 2025

Magnetic control of cell activity has applications ranging from non-invasive neurostimulation to remote activation of cell-based therapies. Unlike other methods of regulating cell activity like heat and light, which are based on known receptors or proteins, no magnetically gated channel has been identified to date. As a result, effective approaches for magnetic control of cell activity are based on strong alternating magnetic fields able to induce electric fields or materials that convert magnetic energy into electrical, thermal, or mechanical energy to stimulate cells. In our investigations of magnetic cell responses, we found that a spiking HEK cell line with no other co-factors responds to a magnetic field that reaches a maximum of 500 mT within 200 ms using a permanent magnet. The response is rare, approximately 1 in 50 cells, but is fast and reproducible, generating an action potential within 200 ms of magnetic field stimulation. The magnetic field stimulation is over 10,000 times slower than the magnetic fields used in transcranial magnetic stimulation (TMS) and the induced electric field is more than an order of magnitude lower than necessary for neuromodulation, suggesting that induced electric currents do not drive the cell response. Instead, our calculation suggests that this response depends on mechanoreception pathways activated by the magnetic torque of TRP-associated lipid rafts. Despite the relatively rare response to magnetic stimulation, when cells form gap junctions, the magnetic stimulation can propagate to nearby cells, causing tissue-level responses. As an example, we co-cultured spiking HEK cells with beta-pancreatic MIN6 cells and found that this co-culture responds to magnetic fields by increasing insulin production. Together, these results point toward a method for the magnetic control of biological activity without the need for a material co-factor such as synthetic nanoparticles. By better understanding this mechanism and enriching for magneto-sensitivity it may be possible to adapt this approach to the rapidly expanding tool kit for wireless cell activity regulation.
Caecilians maintain a functional long-wavelength-sensitive cone opsin gene despite signatures of relaxed selection and more than 200 million years of fossoriality
Authors: Navarrete Méndez Maria José , Amini Sina S. , Santos Juan Carlos , Saal Jacob , Wake Marvalee H. , Ron Santiago R. , Tarvin Rebecca D.
Published: February 2025

Visual systems are tuned to animals’ ecologies, evolving in response to specific light environments and visual needs. Ecological transitions to fossorial lifestyles impose strong selective pressures favoring morphological adaptations for underground life, such as increased skull ossification and reduced eye protrusion. Fossoriality may simultaneously relax constraints on other aspects of vision leading to diminished visual capabilities. Caecilians (Gymnophiona)— specialized, fossorial amphibians—possess reduced eyes covered by skin or bone. For years, these traits, along with the presence of a single photoreceptor expressing one functional opsin gene, have been interpreted as evidence of limited visual capabilities, including an inability to focus or perceive color. Our results challenge these assumptions: we identified the long-wavelength-sensitive (LWS) opsin gene in 11 species of caecilians spanning 8 of 10 recognized families. Molecular evidence indicates thatLWSis intact and transcribed in the eye of at least one species (Caecilia orientalis). Anatomical observations from five caecilian families indicate highly organized retinae even in families with vestigial eyes. While the presence of cone cells in our study species remains uncertain, a putatively functionalLWSgene suggests that the visual capabilities of caecilians and the role of light perception in their ecology may be underestimated.
Joint single-cell profiling of CRISPR-Cas9 edits and transcriptomes reveals widespread off-target events and their effects on gene expression
Authors: Lorenzini Michael H. , Balderson Brad , Sajeev Karthyayani , Ho Aaron J. , McVicker Graham
Published: February 2025

A longstanding barrier in genome engineering with CRISPR-Cas9 has been the inability to measure Cas9 edit outcomes and their functional effects at single-cell resolution. Here we present Superb-seq, a new technology that leverages T7in situtranscription and single-cell RNA sequencing to jointly measure on- and off-target Cas9 edits and their effects on gene expression. We performed Superb-seq on 10,000 K562 cells, targeting four chromatin remodeler genes with seven guide RNAs. Superb-seq identified 11,891 edit events in 6,230 edited cells at all seven on-target sites and at an additional 36 off-target sites. Although the seven guides were selected for high specificity, six of them caused off-target edits, ranging in frequency from 0.03% to 18.6% of cells. A notable off-target edit within the first intron ofUSP9Xdisrupted the expression of this gene and over 150 downstream genes. In summary, Cas9 off-targeting is pervasive due to a combination of rare and common edit events, occurs primarily within introns of off-target genes, and can exert widespread effects on gene expression. Superb-seq uses off-the-shelf kits, standard equipment, and requires no virus, which will enable genome-wide CRISPR screens in diverse cell types as well as functional characterization of clinically-relevant guides.
Use of massive DNA barcoding to monitor biodiversity: a test on forest soil fauna
Authors: Jabot Franck , Auger Gwenaëlle , Bonnal Pauline , Pizaine Mathilde , Roncoroni Marilyn , Revaillot Sandrine , Pottier Julien
Published: February 2025

Biodiversity monitoring primarily focuses on particular taxonomic groups for which identification expertise is widely available, such as vascular plants or birds. As a result, the response of many taxonomic groups to forest management is much less documented. With the advent of low-cost next generation barcoding approaches, it is now possible to envisage monitoring strategies based on molecular approaches, beyond metabarcoding/eDNA strategies that do not give access to species abundances and integrate species presence over largely unknown spatio-temporal scales. In this contribution, we demonstrate the use of massive DNA barcoding – also referred as megabarcoding – as a promising solution to overcome identification difficulties in demanding taxonomic groups. We performed a proof of concept study in a mountainous beech forest in the Massif Central, France. We sampled soil macrofauna at 25 sampling sites and managed to visually identify at the species level 130 out of the 1413 sampled individuals. Using megabarcoding on the 1283 remaining non-identified individuals, we managed to assign 1124 additional individuals to an operational taxonomic species at a competitive cost. We present the summary statistics of barcoding success in the different taxonomic groups encountered and in larvae versus adult individuals. We demonstrate that larvae individuals, which can hardly be visually identified at the species level, make a substantial contribution to overall macrofauna diversity. We finally showcase how this megabarcoding approach provides a concrete avenue for forest biodiversity monitoring, by assessing the cost and labour intensity of this approach.