bluuebunny/crossref_metadata_2025 huggingface dataset:
Contributions of temporal and spatial masking signals in perception of sequential visual events
Authors: Buonocore Antimo , Cuomo Maria , Maresca Martina , Fracasso Alessio
Published: December 2024

Accurate perception of time and space is essential for moment-to-moment interactions with our surroundings. This process requires flexibility, as it integrates information from our actions and the external context. Probing the visual system during the updating process reveals spatiotemporal distortions, where sequential stimuli appear closer in time and space than they are. These effects occur perisaccadically or when a visual mask follows the stimuli. The study investigated whether non-overlapping visual masks could influence temporal inversion judgments (TOJs), suggesting that a temporal signal might act as an anchor during updating. In Experiment 1, participants judged the temporal order of two stimuli under three conditions: no mask, a full-field mask, or a partial mask avoiding stimuli’s locations. Compared to no mask, both masks triggered TOJs when presented within 30 milliseconds of the second stimulus. In a control experiment, delaying mask onset by 30 milliseconds eliminated the inversion effect. In Experiment 2, TOJs were observed for both ipsilateral and contralateral masks, suggesting that long range inhibitory signals might also contribute to the effect. Together, these findings indicate that temporal inversions can occur with non-overlapping stimuli masks configuration, pointing to a non-spatial signal related to mask timing as the underlying mechanism.
Critical periods support representation learning in a model of cortical processing
Authors: Delrocq Ariane , Wu Zihan , Bellec Guillaume , Gerstner Wulfram
Published: December 2024

Exposure of the brain to multiple stimuli drives the development of cortical representations, likely controlled by rules of synaptic plasticity. However, the type of developmental plasticity rules that lead to high-level representations of objects are unknown. Here we study a generalized Hebbian plasticity model that includes a predictive component. The learning rule uses only quantities that are locally available at the site of the synapse, is consistent with recent plasticity experiments in pyramidal neurons, and, as opposed to backpropagation learning, does not need a detailed feedback architecture. Our model shows that limiting plasticity in time to critical periods of development improves the quality and stability of sensory representation across different cortical areas described as layers of an artificial neural network. Our model achieves state-of-the-art performance for bio-plausible plasticity models on both an abstract hierarchical object database and a large image dataset designed for unsupervised learning.
Multiomics plasticity in seed traits of pan-genome wheat cultivars
Authors: Bose Utpal , Winkler Jana Barbro , Sorg Elisa , Mitu Shahida A. , Huber Gregor , Koller Robert , Beale David J. , Dawson Amanda L. , Escobar-Correas Sophia , Biswas Bhabananda , Rahman Mohammad M. , Stockwell Sally , Byrne Keren , Broadbent James , Neerukonda Manjusha , Buegger Franz , Sigalas Alexandros , Mayer Klaus F. X. , Schuppan Detlef , Pozniak Curtis , Colgrave Michelle L. , Spannagl Manuel , Juhász Angéla , Schnitzler Jörg-Peter
Published: December 2024

The molecular basis of cultivar-level variations in polyploid wheat that enables environmental adaptation while maintaining yield and quality in polyploid wheat remains poorly understood. We conducted a detailed phenotypic assessment and multiomics analysis of nine pan-genome polyploid wheat cultivars grown under control and drought conditions. We aimed to investigate the subgenome-level variations, cultivar differences and biochemical mechanisms affecting plant fitness under moderate drought stress. Intrinsic water use efficiency, grain yield, and grain protein content and quality differed among cultivars, supporting the plasticity of drought stress responses. Biased proteome and metabolome abundance changes in response to moderate drought stress during the vegetative stage indicate different strategies for the utilization of homeologous protein isoforms assigned to the A, B, and D subgenomes. Drought effects were detected at the protein level, but significant changes were observed in central carbon pathway metabolites and micronutrient profiles. The subgenomic localization of seed storage proteins highlight differences in nutrient reservoir accumulation and emphasizes the enhanced role of S-rich prolamins in the stress response. Subgenomic variations define cultivar phenotypes by producing molecules that accumulate and enable the underlying trade-offs between environmental adaptation and yield- or quality-related traits. These variations can be used to select crops with increased stress resistance without compromising yield.
Sound-Evoked Plasticity Differentiates Tinnitus from Non-Tinnitus Mice
Authors: Fabrizio-Stover Emily M. , Lee Christopher M. , Oliver Douglas L. , Burghard Alice L.
Published: December 2024

Tinnitus is the perception of non-meaningful sound in the absence of external stimuli. Although tinnitus behavior in animal models is associated with altered central nervous system activity, it is not currently possible to identify tinnitus using neuronal activity alone. In the mouse inferior colliculus (IC), a subpopulation of neurons demonstrates a sustained increase in spontaneous activity after a long-duration sound (LDS). Here, we use the “LDS test” to reveal tinnitus-specific differences in sound-evoked plasticity through IC extracellular recordings and the auditory brainstem response (ABRLDS) in CBA/CaJ mice after sound exposure and behavioral tinnitus assessment. Sound-exposed mice showed stronger and shorter tone-evoked responses in the IC compared to unexposed controls, but these differences were not strong predictors of tinnitus. In contrast, in the LDS test, non-tinnitus mice had a significantly stronger suppression in tone-evoked spike rate compared to tinnitus and unexposed control mice. ABR peak amplitudes also revealed robust differences between tinnitus and non-tinnitus mice, with ABR peaks from non-tinnitus mice exhibiting significantly stronger suppression in the LDS test compared to tinnitus and control mice. No significant differences were seen between cohorts in ABR amplitude, latency, wave V:I ratio, wave V:III ratio, I-V intra-peak latency, and I-VI intra-peak latency. We found high-frequency tone stimuli better suited to reveal tinnitus-specific differences for both extracellular IC and ABR recordings. We successfully used the LDS test to demonstrate that tinnitus-specific differences in sound-evoked plasticity can be shown using both multi-unit near-field recordings in the IC and non-invasive far-field recordings, providing a foundation for future electrophysiological research into the causes and treatment of tinnitus.
Glycosomal phosphoenolpyruvate carboxykinase CRISPR/Cas9-deletion and its role inTrypanosoma cruzimetacyclogenesis and infectivity in mammalian host
Authors: Vieira Carolina Silva Dias , Wang Wei , Sanchez-Valdez Fernando , Lim Jihyun , White Brooke E , da Silva Souza Camilla Garcia , Tarleton Rick L , Paes Marcia Cristina , Nogueira Natalia Pereira
Published: December 2024

Trypanosoma cruzi, the causative agent of Chagas disease, possesses glycosomes - unique organelles that house key metabolic enzymes, several of which are promising therapeutic targets. Among them, phosphoenolpyruvate carboxykinase (PEPCK) plays a central role in succinic fermentation, the main pathway for NAD+regeneration within the organelle. Using CRISPR/Cas9 editing, PEPCK gene was disrupted inT. cruzi, producing single- allele knockout epimastigotes (TcPEPCK-sKO) with reduced enzyme activity. This disruption impaired glucose consumption and mitochondrial respiration, particularly oxidative phosphorylation, reducing dependence on mitochondrial ATP production. To compensate, pyruvate phosphate dikinase was upregulated, increasing alanine production, possibly to maintain redox balance. Although TcPEPCK-sKO epimastigotes exhibited a minor reduction in growth, their differentiation (metacyclogenesis) and invasion were severely compromised. However, once inside the host cell, TcPEPCK-sKO amastigotes increased their replication, leading to enhanced trypomastigote production. The same was observed inin vivoinfection, where TcPEPCK-sKO infection in IFNγ-deficient mice caused uncontrolled parasitemia and severe pathology, highlighting the PEPCK critical role in host-pathogen interactions. However, an intact immune system effectively contained TcPEPCK-sKO infection. Taken together, our findings demonstrate that PEPCK is crucial forT. cruzienergy metabolism, enabling the parasite differentiation within the insect vector and controlling the infection of mammalian host cells.
Frataxin deficiency in the astrocytes drives neurocognitive impairment in sickle cell disease mice
Authors: Novelli Enrico M. , Lenhart Shane C , Foley Lesley M. , Sekar Nandinii , Mondal Paritosh , Wang Hong , Hitchens T. Kevin , Ghosh Samit , Chan Stephen Y. , Hu Xiaoming , Hazra Rimi
Published: December 2024

Individuals with sickle cell disease (SCD) suffer from a high burden of cerebrovascular lesions and cognitive impairment that vastly impact quality of life. Cerebrovascular lesions are characterized by microstructural neuroaxonal damage, but their pathogenesis has not been fully elucidated. Herein, we report that SCD mice (SS) have reduced expression of frataxin (FXN), a mitochondrial protein, in their astrocytes compared to control (AA) mice. Next, we generated chimeric mice with SS bone marrow and astrocyte-specific deletion of FXN (SSFXN-KO). Ex-vivo diffusion tensor magnetic resonance imaging and immunohistopathology of the brain showed that the SSFXN-KOmice have increased white matter neuroaxonal damage compared to the SS bone marrow chimera mice with wild-type FXN expression (SSFXN-WT). The SSFXN-KOmice also displayed poorer cognitive function as measured by the functional Y-maze and novel object recognition tests. Pharmacological induction of FXN by administration of insulin growth factor-1 improved cognitive function in the SSFXN-KOmice. Overall, our data demonstrate that FXN is a critical factor regulating neuroaxonal health and cognitive function in SCD mice. FXN may therefore be a novel pharmacologic target to prevent cerebrovascular complications in SCD.
Comparison and evaluation of methods to infer gene regulatory networks from multimodal single-cell data
Authors: Badia-i-Mompel Pau , Casals-Franch Roger , Wessels Lorna , Müller-Dott Sophia , Trimbour Rémi , Yang Yunxiao , Ramirez Flores Ricardo O. , Saez-Rodriguez Julio
Published: December 2024

Cells regulate their functions through gene expression, driven by a complex interplay of transcription factors and other regulatory mechanisms that together can be modeled as gene regulatory networks (GRNs). The emergence of single-cell multi-omics technologies has driven the development of several methods that integrate transcriptomics and chromatin accessibility data to infer GRNs. While these methods provide examples of their utility in discovering new regulatory interactions, a comprehensive benchmark evaluating their mechanistic and predictive properties as well as their ability to recover known interactions is lacking. To address this, we built a comprehensive framework, Gene Regulatory nETwork Analysis (GRETA), available as a Snakemake pipeline, that includes state of the art methods decomposing their different steps in a modular manner. With it, we found that the GRNs were highly sensitive to methods’ choices, such as changes in random seeds, or replacing steps in the inference pipelines, as well as whether they use paired or unpaired multimodal data. Although the obtained networks performed well in predictive evaluation tasks and partially recovered known interactions, they struggled to capture causal relationships from perturbation assays. Our work brings attention to the challenges of inferring GRNs from single-cell omics, offers guidelines, and presents a flexible framework for developing and testing new approaches.

Graphical Abstract
Design of multimodal antibiotics against intracellular infections using deep learning
Authors: Cesaro Angela , Wan Fangping , Torres Marcelo D. T. , de la Fuente-Nunez Cesar
Published: December 2024

The rise of antimicrobial resistance has rendered many treatments ineffective, posing serious public health challenges. Intracellular infections are particularly difficult to treat since conventional antibiotics fail to neutralize pathogens hidden within human cells. However, designing molecules that penetrate human cells while retaining antimicrobial activity has historically been a major challenge. Here, we introduce APEXDUO, a multimodal artificial intelligence (AI) model for generating peptides with both cell-penetrating and antimicrobial properties. From a library of 50 million AI-generated compounds, we selected and characterized several candidates. Our lead, Turingcin, penetrated mammalian cells and eradicated intracellularStaphylococcus aureus. In mouse models of skin abscess and peritonitis, Turingcin reduced bacterial loads by up to two orders of magnitude. In sum, APEXDUOgenerated multimodal antibiotics, opening new avenues for molecular design.
ELONGATED HYPOCOTYL 5 regulates light-mediated squalene biosynthesis and development inArabidopsis thaliana
Authors: Pathak Pranshu Kumar , Khan Aruba , Sharma Ashish , Singh Nivedita , Sandhu Gurpreet , Trivedi Prabodh Kumar
Published: December 2024

Terpenoids are diverse groups of metabolite families that are crucial for plant development and required in cosmetics and pharmacological industries. Various development processes and environmental factors, including light, have been shown to affect terpenoid biosynthesis. However, regulatory factors involved in such regulation have not been explored much. Squalene synthases (SQSs), key enzymes in the terpenoid pathway, are pivotal for sterol and triterpene biosynthesis across various organisms. In this study, we report thatAtSQS1expression and squalene content are higher in the dark, and light through ELONGATED HYPOCOTYL 5 (HY5) negatively regulates theAtSQS1gene and squalene biosynthesis inArabidopsis thaliana. Our study shows that theAtSQS1gene is unaffected in thehy5-215mutant during light and dark conditions, but it is down-regulated in WT and HY5OX lines. The histochemical GUS assays and GFP expression patterns indicate a negative regulation of squalene biosynthesis by AtHY5. Yeast one-hybrid assays, EMSA, and ChIP experiments have confirmed the physical binding of AtHY5 on light-responsive elements of theAtSQS1promoter. We have validated our results by developingAtSQS1promoter: reporter transgenic lines in WT,hy5-215mutants, and HY5OX backgrounds and conducted expression, metabolite quantification, and histochemical GUS assays. The metabolites quantification of squalene and phytosterols through GC-MS further confirms that AtHY5 negatively regulates the squalene biosynthesis in a light-dependent manner in Arabidopsis.
Transport kinetics across interfaces between coexisting liquid phases
Authors: Hubatsch Lars , Bo Stefano , Harmon Tyler S. , Hyman Anthony A. , Weber Christoph A. , Jülicher Frank
Published: December 2024

We investigate the kinetics of material transport across interfaces in phase-separated mixtures. Using non-equilibrium thermodynamics we derive the interfacial kinetics, describing the movement of the interface, and of molecular transport across the interface. In the limit of a thin interface, we show that breaking local equilibrium at the interface introduces a local transport resistance. Subsequently, using a continuum approach, we explore two physical scenarios where such a resistance emerges: a mobility minimum and a potential barrier at the interface. These scenarios lead to the same effective transport model in the sharp interface limit, characterized by a single interface resistance parameter. The experimental conditions are discussed for which interface resistance could be observed and how these scenarios could be distinguished.
Early-life Oxytocin Rescues Hippocampal Synaptic Plasticity and Episodic Memory in a Mouse Model of Fragile X Syndrome
Authors: Chavez Jasmine , Le Aliza A. , Lauterborn Julie C. , Cox Brittney M. , Jia Yousheng , Lynch Gary , Gall Christine M.
Published: December 2024

Cognitive disabilities including impairments to episodic memory are debilitating features of autism spectrum disorder (ASD). Here we report that early-life treatment with oxytocin (OXT) fully restores episodic memory and associated synaptic plasticity in a rodent model of an ASD.Fmr1-knockout (KO) mice -- which mimic the single gene mutation in Fragile X Syndrome -- failed to encode three basic elements of an episode (identity, location, and temporal order) during a first time, unrewarded encounter with a set of cues. Intranasal administration of OXT during the second postnatal week eliminated each of these impairments in mice tested in adulthood. OXT treatment during the second, but not fifth, postnatal week also corrected pronounced defects in two distinct forms of hippocampal Long-Term Potentiation (LTP). Rescue of LTP in lateral perforant path (LPP) to dentate gyrus synapses was linked to recovery of NMDAR-gated synaptic responses, which are otherwise profoundly reduced in the mutant LPP. LTP induced by a threshold theta burst stimulation protocol in CA3-CA1 synapses was severely impaired in adultFmr1-KOs as was a previously unreported post-induction growth phase for potentiation. Both effects were restored in adult Fmr1-KOs given early OXT treatment. Infusion of OXT into adultFmr1-KO hippocampal slices normalized LTP in CA1 but had no effect on the defective potentiation (or NMDAR-mediated EPSCs) at LPP-dentate gyrus synapses. These results show that severe, autism-related defects in cognition critical to memory, as evident in a rodent model, are reversible and that an early-life therapeutic intervention can effect an enduring restoration of function.
Coordinated spinal locomotor network dynamics emerge from cell-type-specific connectivity patterns
Authors: Wandler F David , Lemberger Benjamin K , McLean David L , Murray James M
Published: December 2024

Even without detailed instruction from the brain, spinal locomotor circuitry generates coordinated behavior characterized by left-right alternation, segment-to-segment propagation, and variable-speed control. While existing models have emphasized the contributions of cellular- and network-level properties, the core mechanisms underlying rhythmogenesis remain incompletely understood. Further, neither family of models has fully accounted for recent experimental results in zebrafish and other organisms pointing to the importance of long-range, cell-type-specific connectivity patterns and recruitment of speed-selective subpopulations of interneurons. Informed by these experimental findings and others, we developed a hierarchy of increasingly detailed models of the locomotor circuit. Coordinated locomotion emerged in an inhibition-dominated network in which connectivity is determined by desired phase relationships among interneurons and variable-speed control is implemented by recruitment of speed-selective subpopulations. Further, while structured excitatory connections were not necessary for rhythmogenesis, they were useful for increasing the peak locomotion frequency, albeit at the cost of losing some control at intermediate frequencies, suggesting a basic computational trade-off between speed and control. Together, this family of models shows that network-level interactions are sufficient to generate coordinated, variable-speed locomotion, providing new interpretations of intersegmental excitatory and inhibitory connectivity, as well as the basic, recruitment-based mechanism of speed control.
RNA-SeqEZPZ: A Point-and-Click Pipeline for Comprehensive Transcriptomics Analysis with Interactive Visualizations
Authors: Taslim Cenny , Zhang Yuan , Kendall Genevieve C. , Theisen Emily R.
Published: December 2024

RNA-Seq analysis has become a routine task in numerous genomic research labs, driven by the reduced cost of bulk RNA sequencing experiments. These generate billions of reads that require accurate, efficient, effective, and reproducible analysis. But the time required for comprehensive analysis remains a bottleneck. Many labs rely on in-house scripts, making standardization and reproducibility challenging. To address this, we developed RNA-SeqEZPZ, an automated pipeline with a user-friendly point-and-click interface, enabling rigorous and reproducible RNA-Seq analysis without requiring programming or bioinformatics expertise. For advanced users, the pipeline can also be executed from the command line, allowing customization of steps to suit specific requirements.

This pipeline includes multiple steps from quality control, alignment, filtering, read counting to differential expression and pathway analysis. We offer two different implementations of the pipeline using either (1) bash and SLURM or (2) Nextflow. The two implementation options allow for straightforward installation, making it easy for individuals familiar with either language to modify and/or run the pipeline across various computing environments.

RNA-SeqEZPZ provides an interactive visualization tool using R shiny to easily select the FASTQ files for analysis and compare differentially expressed genes and their functions across experimental conditions. The tools required by the pipeline are packaged into a Singularity image for ease of installation and to ensure replicability. Finally, the pipeline performs a thorough statistical analysis and provides an option to perform batch adjustment to minimize effects of noise due to technical variations across replicates.

RNA-SeqEZPZ is freely available and can be downloaded fromhttps://github.com/cxtaslim/RNA-SeqEZPZ.
Combined inhibition of SHP2 overcomes adaptive resistance to type 1 BRAF inhibitors in BRAF V600E-driven high-grade glioma
Authors: Ayanlaja Abiola A. , Chang Michael , Lalwani Kriti , Ioannou Maria , Wang Jiawan , Jagtap Shreya , Yang Yanbo , Gartrell Robyn D. , Pratilas Christine A. , Schreck Karisa C.
Published: December 2024

BRAF-mutant gliomas can be therapeutically targeted with BRAF mutant-selective inhibitors, yet responses are often transient due to short-term adaptive or long-term treatment-emergent resistance. We hypothesized that vertical inhibition of multiple signaling nodes could improve the durability of BRAF inhibition and prevent or overcome adaptive resistance. Using human tissue samples, we identified frequent RAS pathway reactivation in gliomas resistant to BRAF inhibitors, suggesting a common escape mechanism. Using patient-derived cell lines, we observed that upregulation of RAS activity was an adaptive response to BRAFi and that knockdown of SHP2, a central regulator of RAS activity, resulted in enhanced sensitivity to BRAF or MEK inhibition. Moreover, combined small molecule inhibition with SHP2 and BRAF or MEK inhibitors increased the depth and durability of ERK pathway inhibition, as well as prevented paradoxical upregulation of RAS activity. RNA sequencing analysis revealed deeper suppression of ERK transcriptional output with combined therapy, along with decreased reactivation of EGFR. Combined SHP2/BRAF small molecule inhibitors prevented growth and induced cell death in some cell line models. In cell lines with treatment-emergent resistance, moreover, combined SHP2 and BRAF inhibition overcame resistance to BRAF inhibitor monotherapy.In vivoorthotopic and patient-derived xenograft models confirmed enhanced tumor growth inhibition with combined therapy. Together, our findings demonstrate the critical role of RAS/ERK signaling reactivation in driving resistance to BRAF inhibition in glioma, and demonstrate the potential utility for adding SHP2 inhibitors to overcome resistance inBRAFV600E mutant glioma.

Significance

The addition of a SHP2i to BRAFi in BRAF-V600E glioma cells prevents tumor growth and can overcome resistance to BRAFi in preclinical models in vitro and in vivo.
The biophysical mechanism of mitochondrial pearling
Authors: Sturm Gabriel , Hake Kayley , Lefebvre Austin E.Y.T. , Rux Caleb J. , Ivanova Daria , Millett-Sikking Alfred , Tharp Kevin M. , Rao Beiduo , Closser Michael , Waite Adam , Precido-Lopez Magdalena , Dumont Sophie , Lu Wen , Manley Suliana , Landoni Juan C. , Marshall Wallace F.
Published: December 2024

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, “beads-on-a-string” morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of “pearls” connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation and neuronal firing. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling regulated by ionic flux, and ii) external tension-driven pearling regulated by molecular motors. By applying live-cell STED microscopy we document the structural reorganization of inner cristae membranes during mitochondrial pearling. We then compare numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux amplifying internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.
Expanding the capillarics toolbox: 3D-printed microfluidic phaseguides and self-coalescence modules
Authors: Craig Cosette , Leong Kelsey , Lin Carrie , Chang Megan , Olanrewaju Ayokunle
Published: December 2024

Capillarics are microfluidic circuits that are assembled from individual fluidic elements, powered by surface tension forces encoded by microchannel geometry and surface chemistry, and enable instrument-free pre-programmed automation of multi-step liquid handling processes. 3D printing has recently transformed capillarics by enabling rapid and cost-effective prototyping, provided addition geometric degrees of freedom in multi-level fabrication, and facilitated new design paradigms with greater capabilities than traditional cleanroom fabrication. Despite widespread interest in 3D printing and development of custom high-resolution stereolithography printers for microfluidic applications, fluidic elements that require precise and tunable control over capillary pinning lines – such as fluidic phaseguides and self-coalescence modules (SCMs) – have so far only been manufactured with centralized and expensive cleanroom methods. Not only does this limit access to versatile capillaric features to only well-resourced settings, but it also slows innovation and widespread application of these fluid handling technologies. Here we expand the toolbox of 3D-printed capillarics to include phaseguides and SCMs, demonstrating their potential for precise instrument-free control over multi-step liquid handling and reagent rehydration. We employed benchtop stereolithography printers to prototype (up to 50X) scaled-up phaseguides and SCMs and integrated them into a capillaric circuit for inline reagent reconstitution, dynamic fluid control, and sequential drainage. We showcased scalable designs, customizable geometries, and robust self-coalescing flow for larger liquid volumes – up to 50 µL compared with 1.25 µL in cleanroom-fabricated SCMs. This work represents a significant advance in democratizing access to microfluidics, with potential for broad applications in diagnostics, assay automation, and organ-on-chip systems.
Developmental delay ensures global tissue size robustness upon local induction of apoptosis
Authors: Staneva Ralitza , Sobczyk-Moran Gabriel , Levillayer Florence , Villars Alexis , Davidović Anđela , Levayer Romain
Published: December 2024

The capacity of our tissues to cope with external and internal stress relies on the tight coupling between cell proliferation, cell growth and cell death. This coupling is assumed to be based on compensatory proliferation, where local mitogenic signals and mechanical inputs generated by dying cells promote neighbouring cell proliferation. However, compensatory proliferation was mostly studied in the context of massive death induction, irradiation, surgical tissue ablation or upon genetic perturbation of apoptosis execution. It remains thus unclear whether the same mechanism operates during physiological programmed cell death or upon mild induction of apoptosis, especiallyin vivo. Here, we use theDrosophilaprospective wing (the larval wing disc), to study the impact of local induction of apoptosis on tissue size and proliferation pattern. We first confirmed that the wing could recover its final size and compensate for mild induction of apoptosis. However, using spatial statistics we found surprisingly that local induction of death is not associated with any local increase of proliferation, could it be upon clonal or compartment induction of apoptosis. Compensation is driven instead by a JNK dependant delay of growth and lengthening of the larval stage which is required to reach the final tissue target size. These results suggest that compensation is here driven by a global response rather that a local proliferation induction. Accordingly, while total tissue size is maintained despite local induction of apoptosis, this mechanism fails to correct the local reduction of cell number, hence modulating wing shape and proportion. Overall, this study opens novel perspectives on tissue size regulation and outlines the context-dependency of compensatory mechanisms.
Field monitoring and hydraulic modelling quantify untreated wastewater as dominant source of AMR in a small river running through a big city
Authors: Sonkar Vikas , Kashyap Arun , Pallarés-Vega Rebeca , Sasidharan Sai Sugitha , Modi Ankit , Uluseker Cansu , Jambu Sangeetha Chandrakalabai , Mohapatra Pranab Kumar , Larsen Joshua , Graham David , Thatikonda Shashidhar , Kreft Jan-Ulrich
Published: December 2024

Antimicrobial resistance (AMR) is a silent pandemic, which is transmitted and spread through the environment. Few of many studies on waterborne AMR spread have characterised the prevalence, sources, and transport of AMR in adequate detail to inform mathematical modelling and exposure risk assessments. Here, we quantified taxonomic and resistance genes (ARGs), sensitive and resistant bacteria (ARBs), and environmental conditions in water and sediment samples at ten locations along the Musi River through Hyderabad, a city renowned for antimicrobial manufacturing, to develop a hydraulic model for AMR spread in the catchment. In the dry season, absolute water column ARG and ARB abundances increased rapidly upon entering the city, then declined downstream. In the wet season, the increase and decline of AMR levels was more gradual. Clustering and ordination of ARGs separated the river into three stretches: upstream, city, and downstream. Most ARGs and water quality indicators differed significantly between stretches and seasons (p<0.05; PERMANOVA). Linear Discriminant Analysis showed that various water quality parameters, such as lower dissolved oxygen and elevated total nitrogen, can distinguish between AMR-polluted and less-polluted sites and might be good proxies to detect AMR hotspots. Modelling showed that 60%-80% of river water in the city was untreated wastewater, suggesting municipal wastewater dominated AMR in the river, not manufacturing sources. This will be a typical situation where wastewater treatment is insufficient and dilution in receiving water bodies limited, highlighting the urgent need for improved wastewater management to make rivers like the Musi safer for use, such as irrigation, reducing AMR exposures across wider environments.

Highlights

Within the city, the concentration of ARGs and ARBs increases in the dry season

Clustering and ordination of ARGs separates upstream, city and downstream stretches

PERMANOVA shows stretches and seasons are significantly different

LDA can discriminate stretches and seasons based on water quality parameters

Point source load calculations indicate 60-80% of river water is untreated wastewater
HYDRA: HYdrogel Dispensing with Robotic Automation for high throughput drug testing
Authors: Torchia Eloisa , Di Sante Moises , Horda Bohdana , Mihajlovic Marko , Zimmermann Julius , Pezzotti Melissa , Cimetta Elisa , Gabriele Sylvain , Auricchio Ferdinando , Lind Johan Ulrik , Enrico Alessandro , Pasqualini Francesco Silvio
Published: December 2024

The poor predictivity of traditional cell culture platforms hampers drug research and development efficiency. While organoids and organs-on-chip mitigate this problem by introducing biomimetic in-vitro microenvironments in late pre-clinical stages, there is a lack of biomimetic platforms in earlier stages, particularly for high-throughput imaging screening (HTS). Something as simple as a thin hydrogel layer would provide physiological stiffness and native-like biochemical cues. However, casting hydrogels inside the small-diameter wells of HTS plates creates high-curvature menisci that negatively affect cell seeding and microscopy readouts. Here, we present a solution to this problem: HYdrogel Dispensing with Robotic Automation or HYDRA. This method allows automatic dispensing and aspiration of hydrogel precursors while preventing meniscus formation. Using this approach, we can produce gelatin hydrogels with tunable stiffnesses and thickness at room temperature and in commercial HTS plates. Then, we validate HYDRA in dose-response studies for anti-cancer drugs nocodazole and paclitaxel using genetically engineered epithelial cells (HaCaT) and imaging-based HTS readout methods such as phase holography and fluorescence microscopy. Taken together, this work shows that pharmaceutical companies can readily apply HYDRA with their current infrastructure to produce more biomimetic and predictive HTS platforms.
Synergistic activation of the human phosphate exporter XPR1 by KIDINS220 and inositol pyrophosphate
Authors: Zuo Peng , Wang Weize , Dai Zonglin , Zheng Jiye , Yu Shang , Wang Guangxi , Yin Yue , Liang Ling , Yin Yuxin
Published: December 2024

Inorganic phosphate (Pi) is fundamental to life, and its intracellular concentration must be tightly regulated to prevent toxicity. XPR1, the only known phosphate exporter, plays a crucial role in maintaining this delicate balance. However, the mechanisms underlying the function and regulation of XPR1 remain elusive until now. Here we present cryo-electron microscopy structures of the human XPR1-KIDINS220 complex in both substrate-free closed states and substrate-bound outward-open states, as well as the structure of an XPR1 mutant alone in a substrate-bound inward-facing state. In the presence of inositol hexaphosphate (InsP6) and phosphate, the XPR1-KIDINS220 complex adopts an outward-open conformation. InsP6 binds both the SPX domain and the peripheral juxtamembrane regions of XPR1, indicative of an active phosphate-export state. Conversely, in the absence of either phosphate or InsP6, the complex assumes a closed state, where the extracellular half of transmembrane 9 occupies the outward cavity, and a C-terminal plug-in loop blocks the intracellular cavity. Notably, XPR1 without KIDINS220 adopts a closed state despite the presence of phosphate and InsP6. The functional mutagenesis experiments further demonstrate that InsP6, whose concentrations fluctuate in response to Pi availability, functions synergistically with KIDINS220 to regulate the phosphate export activity of XPR1. These findings not only elucidate the intricate mechanisms of cellular phosphate regulation but also hold promise for the development of targeted therapies for ovarian cancer, where XPR1 plays a significant role.
Whole-transcriptome analysis of BLV-infected cows reveals downregulation of immune response genes in high proviral loads cows
Authors: Petersen MI , Suarez Archilla G , Miretti MM , Trono KG , Carignano HA
Published: December 2024

Bovine leukemia virus (BLV) is a retrovirus that infects cattle, causing bovine enzootic leukosis, a chronic disease characterized by the proliferation of infected B cells. BLV proviral load (PVL) is a key determinant of disease progression and transmission risk. Cattle can exhibit distinct phenotypes of low PVL (LPVL) or high PVL (HPVL), which remain stable throughout their lifetime. Differential expression analysis revealed 1,908 differentially expressed genes (DEGs) between HPVL and LPVL animals, including 774 downregulated (DReg) and 1134 upregulated (UReg) genes. Functional enrichment analysis revealed that DReg genes were associated primarily with immune response pathways. Conversely, the UReg genes were enriched in processes related to cell cycle regulation, mitotic division, and DNA biosynthesis. Protein□protein interaction analysis revealed six highly interconnected clusters. Interestingly, a cluster was enriched for sphingolipid metabolism, a process critical to enveloped virus infection and immune receptor signaling. These findings provide valuable insights into the molecular mechanisms of BLV infection, suggesting potential markers for disease monitoring and targets for therapeutic intervention.
Functional specialization of MITF, TFEB and TFE3 drives radically distinct adaptive gene expression programs in melanoma
Authors: Dias Diogo , Oliveira Erica , Martí-Díaz Román , Andrews Sarah , Chocarro-Calvo Ana , Bellini Alice , Mosteo Laura , Vivas García Yurena , Chauhan Jagat , Li Linxin , García-Martinez José Manuel , Rodriguez-López José Neptuno , Maria-Engler Silvya Stuchi , García-Jiménez Custodia , Sanchez-del-Campo Luis , Louphrasitthiphol Pakavarin , Goding Colin R
Published: December 2024

Within cells multiple related transcription factors targeting the same sequences may co-exist, leading to potential regulatory cooperativity, redundancy or competition. Yet the differential roles and biological functions of co-targeting transcription factors is poorly understood. In melanoma, three highly-related transcription factors are co-expressed: The mTORC1-regulated TFEB and TFE3, that are key effectors of a wide range of metabolic and microenvironmental cues; and MITF, that controls melanoma phenotypic identity. Here we reveal the functional specialization of MITF, TFE3 and TFEB and their impact on cancer progression. Notably, although all bind the same sequences, each regulates radically different and frequently opposing gene expression programs to coordinate differentiation, metabolic reprogramming, protein synthesis, and expression of immune modulators. The results uncover a hierarchical cascade in which microenvironmental stresses, including glucose limitation, lead MITF, TFEB and TFE3 to drive distinct biologically important transcription programs that underpin phenotypic transitions in cancer.
Ephrin-A5 or EphA7 stimulation is anti-proliferative for human rhabdomyosarcomain vitro
Authors: Cecchini A. , Ceccon L. , Chen A. , Schwesig J. C. , Cornelison DDW
Published: December 2024

Rhabdomyosarcoma (RMS) is a tumor which resembles skeletal muscle. Current treatments are limited to surgery and non-targeted chemotherapy, highlighting the need for alternative therapies. Differentiation therapy uses molecules that act to shift the tumor cells’ phenotype from proliferating to differentiated, which in the case of skeletal muscle includes exit from the cell cycle and potentially fusion into myofibers. We previously identified EphA7 expressed on terminally differentiated myocytes as a potent driver of skeletal muscle differentiation: stimulation of ephrin-A5-expressing myoblasts with EphA7 causes them to undergo rapid, collective differentiation. We therefore tested EphA7 as a candidate molecule for differentiation therapy on human RMS (hRMS) cell lines. Surprisingly, EphA7 had a lesser effect than ephrin-A5, a difference explained by the divergent suite of Ephs and ephrins expressed by hRMS. We show that in hRMS ephrin-A5 binds and signals to EphA8 and EphA7 binds and signals to ephrin-A2, and that Fc chimeras of both molecules are potent inhibitors of hRMS proliferation. These results identify key differences between hRMS and normal muscle cells and support further research into Eph:ephrin signaling as potential differentiation therapies.

Summary statement

This study identifies EphA7 and ephrin-A5 as external regulators of rhabdomyosarcoma proliferation, highlighting ephrin-A5 as a potential candidate for differentiation therapy in future cancer treatments.
Auditory brainstem responses to speech-in-noise reflect selective attention, comprehension, and subjective listening effort
Authors: Mankel K. , Comstock D. C. , Bormann B. M. , Das S. , Sagiv D. , Brodie H. , Miller L. M.
Published: December 2024

The auditory brainstem plays a crucial role in speech-in-noise listening, refining numerous acoustic features such as pitch and spatial location under continual descending influence from cortex. However, the difficulty of characterizing brainstem activity during continuous speech listening has obscured its functional role in ecologically valid contexts—not only the effects of selective attention on neural responses, but also their impact on comprehension and listening effort. Here, we evaluate the role of the brainstem on speech-in-noise perception and selective attention using a continuous, speech-based stimulus with embedded chirps (Cheech) that rapidly and effectively evokes auditory brainstem responses (ABRs) while participants listen to short story narratives. The Cheech-modified stories were presented alone or in the presence of another spatially separated talker, and neural responses were measured throughout by EEG. Both word-level detection performance and narrative-level comprehension were evaluated, as well as subjective reports of listening effort. ABR wave V was modulated by the presence of a competing talker, selective attention for the target versus masker, and the talker gender. Additionally, faster wave V peak latencies and larger amplitudes were associated with identification of the target words embedded within the stories, while faster latencies additionally related to better comprehension question accuracy and lower subjective listening effort. Collectively, our results provide clear evidence for the influence of brainstem encoding processes on individual speech-listening behaviors, including the ability to selectively attend to and comprehend target speech in the presence of a competing talker.

Significance statement

This study highlights the crucial role of the brainstem in speech-in-noise perception and higher-level language abilities like comprehension and subjective listening effort. Through the use of a novel speech stimulus blended with embedded chirps (Cheech) to evoke brainstem responses while listening to short story narratives, we show how selective attention influences subcortical processes. Faster and more robust subcortical processes, in turn, were associated with more successful speech-listening performance across multiple behavioral metrics. These findings advance our understanding of how the brainstem supports selective attention and speech perception in challenging listening environments.
More fungi than legs: the first fungal microbiome for a fungus-eating millipede (Colobognatha)
Authors: Macias Angie , Lovett Brian , Jusino Michelle , Cole Lauren , Kasson Matt T.
Published: December 2024

Fungi are widely consumed across the animal kingdom for nutritional and defensive benefits. Millipedes, among the first air-breathing land animals, were also among the first terrestrial fungivores. As detritus-eating omnivores, most millipedes regularly consume fungi. Millipede diets diverged ∼200 million years ago, when obligate fungivorous millipedes (subterclass Colobognatha) diverged from their detritivorous counterparts. Despite their global distribution and uncommon diet, little is known about the association between Colobognaths and the fungi they consume. In 2019, surveys of fungal communities associated with the ColobognathBrachycybe lecontiirevealed associations with at least 176 genera of culturable fungi. Given the known biases of culture-based approaches, a more comprehensive survey ofB. lecontii’s fungal microbiome using amplicon sequencing was undertaken. In this study, we generated amplicon sequence data to look for associations between fungi andB. lecontii, and to determine if patterns of fungal diversity are millipede- or habitat-driven. Altogether, the fungal microbiome ofB. lecontiiencompassed 620 fungal genera and 100 orders. Despite much greater observed fungal diversity in the amplicon-based study, sampling was likely not sufficient to capture the full diversity of fungi associated withB. lecontii. Taxonomic and functional diversity were significantly influenced by site and colony, indicating that community structure is shaped by geography and habitat. It remains unknown whether these findings are representative of the larger patterns of fungal diversity for the entire millipede subterclass. Nevertheless, the obligate fungivorous lifestyle employed by these long-extant animals may provide important clues regarding fungal diversity and function.