bluuebunny/crossref_metadata_2025 huggingface dataset:
Policy complexity suppresses dopamine responses
Authors: Gershman Samuel J. , Lak Armin
Published: September 2024

Limits on information processing capacity impose limits on task performance. We show that animals achieve performance on a perceptual decision task that is near-optimal given their capacity limits, as measured by policy complexity (the mutual information between states and actions). This behavioral profile could be achieved by reinforcement learning with a penalty on high complexity policies, realized through modulation of dopaminergic learning signals. In support of this hypothesis, we find that policy complexity suppresses midbrain dopamine responses to reward outcomes, thereby reducing behavioral sensitivity to these outcomes. Our results suggest that policy compression shapes basic mechanisms of reinforcement learning in the brain.
Pipeline Olympics: continuable benchmarking of computational workflows for DNA methylation sequencing data against an experimental gold standard
Authors: Lin Yu-Yu , Breuer Kersten , Weichenhan Dieter , Lafrenz Pascal , Wilk Agata , Chepeleva Marina , Mücke Oliver , Schönung Maximilian , Petermann Franziska , Kensche Philipp , Weiser Lena , Thommen Frank , Giacomelli Gideon , Nordstroem Karl , Gonzalez-Avalos Edahi , Merkel Angelika , Kretzmer Helene , Fischer Jonas , Krämer Stephen , Iskar Murat , Wolf Stephan , Buchhalter Ivo , Esteller Manel , Lawerenz Chris , Twardziok Sven , Zapatka Marc , Hovestadt Volker , Schlesner Matthias , Schulz Marcel , Hoffmann Steve , Gerhauser Clarissa , Walter Jörn , Hartmann Mark , Lipka Daniel B. , Assenov Yassen , Bock Christoph , Plass Christoph , Toth Reka , Lutsik Pavlo
Published: September 2024

DNA methylation is a widely studied epigenetic mark and a powerful biomarker of cell type, age, environmental exposures, and disease. Whole-genome sequencing following selective conversion of unmethylated cytosines into thymines via bisulfite treatment or enzymatic methods remains the reference method for DNA methylation profiling genome-wide. While numerous software tools facilitate processing of DNA methylation sequencing reads, a comprehensive benchmarking study has been lacking thus far. In this study, we systematically compared complete computational workflows for processing DNA methylation sequencing data using a dedicated benchmarking dataset generated with five genome-wide profiling protocols. As an evaluation reference, we employed highly quantitative locus-specific measurements from our preceding benchmark of targeted DNA methylation assays. Based on this experimental gold-standard assessment and several comprehensive metrics, we identified workflows that consistently demonstrated superior performance and revealed major workflow development trends. To facilitate the sustainability of our benchmark, we implemented an interactive workflow execution and data presentation platform, adaptable to user-defined criteria and seamlessly expandable to future software.
Heritability of movie-evoked brain activity and connectivity
Authors: Gruskin David C. , Vieira Daniel J. , Lee Jessica K. , Patel Gaurav H.
Published: September 2024

The neural bases of sensory processing are conserved across people, but no two individuals experience the same stimulus in exactly the same way. Recent work has established that the idiosyncratic nature of subjective experience is underpinned by individual variability in brain responses to sensory information. However, the fundamental origins of this individual variability have yet to be systematically investigated. Here, we establish a genetic basis for individual differences in sensory processing by quantifying (1) the heritability of high-dimensional brain responses to movies and (2) the extent to which this heritability is grounded in lower-level aspects of brain function. Specifically, we leverage 7T fMRI data collected from a twin sample to first show that movie-evoked brain activity and connectivity patterns are heritable across the cortex. Next, we use hyperalignment to decompose this heritability into genetic similarity inwherevs.howsensory information is processed. Finally, we show that the heritability of brain activity patterns can be partially explained by the heritability of the neural timescale, a one-dimensional measure of local circuit functioning. These results demonstrate that brain responses to complex stimuli are heritable, and that this heritability is due, in part, to genetic control over stable aspects of brain function.
Analysis of coordinated group behavior based on role-sharing: Practical application from an experimental task to a 3-on-3 basketball game as a pilot study
Authors: Ichikawa Jun , Yamada Masatoshi , Fujii Keisuke
Published: September 2024

A human group shares a common goal and distributes roles to implement activities that are difficult to accomplish alone and higher performance than an individual. In such coordination, nonverbal behavior among three or more members makes it difficult to explain the mechanism because of complex and dynamic interactions. In cognitive science, a crucial role is indicated, which intervenes moderately with others and adjusts the whole balance without interrupting their main smooth interactions, using an experimental task. It suggests that the third role and related playing supports to coordination. This is similar to off-ball movements in team sports, which are not involved directly with the ball and are focused on mainly in sports science because common statistical data do not reflect. A new perspective for discussing coordination has arisen because existing theories, such as synchronization, cannot explain the mentioned role; however, there is room for its usefulness. Therefore, this study applied the experimental findings to the sports field. We designed a 3-on-3 basketball game in which the relevant offensive role is key and introduced it to the practice of the university team as a pilot study. Participants repeatedly engaged in the mini-game, and the playing on the offensive team was compared before and after receiving tips on this role. Consequently, in the bins of the relatively large distance between the participant required in this role and each defensive player, the frequencies after the tips were significantly higher, and the winning rate on the offensive team improved temporarily. It suggests that the spacing skill, which indicates a reasonable distance for intervening with the other offensive players, emerged. This study may provide findings that satisfy usefulness and ecological validity, and make an educational contribution about giving the tips of off-ball movements.
Predicting Degradation Potential of Protein Targeting Chimeras
Authors: Petrou Andreas , Minhas Fayyaz
Published: September 2024

PRoteolysis TArgeting Chimeras (PROTACs) can inhibit protein activity by utilizing natural proteasomal degradation pathways for the degradation of target proteins. Being able to determine the degradation potential of PROTACs is crucial in drug development as it can lead to time, labor and cost savings. In this paper, we present a novel machine-learning pipeline that utilizes common compound fingerprints and a pre-trained graph neural network for the prediction of half-maximal degradation concentration of PROTACs by benchmarking a variety of protein tertiary structures and chemical features. Based on critical analysis of our cross-validation and independent test results, we have highlighted several key challenges underlying this prediction problem that need to be addressed to improve the generalization of predictive models in this domain. Moreover, we demonstrate the effectiveness of our approach by testing it on two different datasets and show that it performs better than the current state of the art with an AUC-ROC of 0.85 and accuracy of 0.875 on the DeepPROTACs test dataset.
Notch and LIM-homeodomain protein Arrowhead regulate each other in a feedback mechanism to play a role in wing and neuronal development inDrosophila
Authors: Singh Jyoti , Verma Dipti , Sarkar Bappi , Paul Maimuna Sali , Mutsuddi Mousumi , Mukherjee Ashim
Published: September 2024

Notch pathway is an evolutionarily conserved signaling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. To identify novel effectors of Notch signaling, we analyzed the whole transcriptome ofDrosophilawing and eye imaginal discs in which an activated form of Notch was overexpressed. A LIM homeodomain protein Arrowhead (Awh) was identified as a novel candidate which plays a crucial role in Notch mediated developmental events.Awhalleles show strong genetic interaction with Notch pathway components. Awh loss-of-function upregulates Notch targets Cut and Wingless. Awh gain-of-function downregulates Notch targets by reducing the expression of ligand, Delta. Consequently, the expression of Wingless effector molecule Armadillo and its downstream targets, Senseless and Vestigial, also gets downregulated. Awh overexpression leads to ectopicexpression ofengrailed, a segment polarity gene in the anterior region of wing disc, leading to patterning defects. Additionally, Notch gain-of-function mediated neuronal defects get significantly rescued with Awh overexpression. Activated Notch inhibits Awh activity, suggesting a regulatory loop between Awh and Notch. Additionally, the defects caused by Awh gain-of-function were remarkably rescued by Chip, a LIM interaction domain containing transcriptional co-factor. The present study highlights the novel feedback regulation between Awh and Notch.
Auditory Cortex Learns to Discriminate Audiovisual Cues through Selective Multisensory Enhancement
Authors: Chang Song , Zheng Beilin , Keniston Les , Xu Jinghong , Yu Liping
Published: September 2024

Multisensory object discrimination is essential in everyday life, yet the neural mechanisms underlying this process remain unclear. In this study, we trained rats to perform a two-alternative forced-choice task using both auditory and visual cues. Our findings reveal that multisensory perceptual learning actively engages auditory cortex (AC) neurons in both visual and audiovisual processing. Importantly, many audiovisual neurons in the AC exhibited experience-dependent associations between their visual and auditory preferences, displaying a unique integration model. This model employed selective multisensory enhancement for the auditory-visual pairing guiding the contralateral choice, which correlated with improved multisensory discrimination. Furthermore, AC neurons effectively distinguished whether a preferred auditory stimulus was paired with its associated visual stimulus using this distinct integrative mechanism. Our results highlight the capability of sensory cortices to develop sophisticated integrative strategies, adapting to task demands to enhance multisensory discrimination abilities.
Direct measurement of the mutation rate and its evolutionary consequences in a critically endangered mollusk
Authors: Wooldridge Brock , Ford Sarah , Conwell Holland , Hyde John , Harris Kelley , Shapiro Beth
Published: September 2024

The rate at which mutations arise is a fundamental parameter of biology. Despite recent progress in measuring germline mutation rates across diverse taxa, such estimates are missing for much of Earth's biodiversity. We present the first estimate of a germline mutation rate from the phylum Mollusca, which is diverged by more than 1200 Ma years from the closest relative for which a mutation rate estimate exists. We sequenced three pedigreed families of the white abalone Haliotis sorenseni, a long-lived, large-bodied, and critically endangered mollusk, and estimated a de novo mutation rate of 8.60e-09 single nucleotide mutations per site per generation. This mutation rate is similar to rates measured in vertebrates with similar generation times and longevity to abalone, and higher than mutation rates measured in faster-reproducing invertebrates. We use our estimated rate to infer baseline effective population sizes (Ne) across multiple Pacific abalone and find that abalone persisted over most of their evolutionary history as large and stable populations, in contrast to extreme fluctuations over recent history and small census sizes in the present day. We then use our mutation rate to infer the timing and pattern of evolution of the abalone genus Haliotis, which was previously unknown due to few fossil calibrations. Our results are an important step toward understanding mutation rate evolution and establish a key parameter for conservation and evolutionary genomics research in mollusks.
Interphase chromosome conformation is specified by distinct folding programs inherited via mitotic chromosomes or through the cytoplasm
Authors: Schooley Allana , Venev Sergey V. , Aksenova Vasilisa , Navarrete Emily , Dasso Mary , Dekker Job
Published: September 2024

Identity-specific interphase chromosome conformation must be re-established each time a cell divides. To understand how interphase folding is inherited, we developed an experimental approach that physically segregates mediators of G1 folding that are intrinsic to mitotic chromosomes from cytoplasmic factors. Proteins essential for nuclear transport, RanGAP1 and Nup93, were degraded in pro-metaphase arrested DLD-1 cells to prevent the establishment of nucleo-cytoplasmic transport during mitotic exit and isolate the decondensing mitotic chromatin of G1 daughter cells from the cytoplasm. Using this approach, we discover a transient folding intermediate entirely driven by chromosome-intrinsic factors. In addition to conventional compartmental segregation, this chromosome-intrinsic folding program leads to prominent genome-scale microcompartmentalization of mitotically bookmarked and cell type-specific cis-regulatory elements. This microcompartment conformation is formed during telophase and subsequently modulated by a second folding program driven by factors inherited through the cytoplasm in G1. This nuclear import-dependent folding program includes cohesin and factors involved in transcription and RNA processing. The combined and inter-dependent action of chromosome-intrinsic and cytoplasmic inherited folding programs determines the interphase chromatin conformation as cells exit mitosis.
Shared subcortical arousal systems across sensory modalities during transient modulation of attention
Authors: Khalaf Aya , Lopez Erick , Li Jian , Horn Andreas , Edlow Brian L. , Blumenfeld Hal
Published: September 2024

Subcortical arousal systems are known to play a key role in controlling sustained changes in attention and conscious awareness. Recent studies indicate that these systems have a major influence on short-term dynamic modulation of visual attention, but their role across sensory modalities is not fully understood. In this study, we investigated shared subcortical arousal systems across sensory modalities during transient changes in attention using block and event-related fMRI paradigms. We analyzed massive publicly available fMRI datasets collected while 1,561 participants performed visual, auditory, tactile, and taste perception tasks. Our analyses revealed a shared circuit of subcortical arousal systems exhibiting early transient increases in activity in midbrain reticular formation and central thalamus across perceptual modalities, as well as less consistent increases in pons, hypothalamus, basal forebrain, and basal ganglia. Identifying these networks is critical for understanding mechanisms of normal attention and consciousness and may help facilitate subcortical targeting for therapeutic neuromodulation.
RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis
Authors: Yang Moua , Boye-Doe Alexandra , Abosabie Salma A.S. , Barr Alexandra M. , Mendez Lourdes M. , Sharda Anish V.
Published: September 2024

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.
More than fruity scents: floral biology, scent and spectral reflectance of Annonaceae species
Authors: Liu Ming-Fai , Chen Junhao , Pang Chun-Chiu , Scharaschkin Tanya , Saunders Richard M. K.
Published: September 2024

Premise

The family Annonaceae possesses a broad array of floral phenotypes and pollination specialisations, and are important in the plant-pollinator interactions of tropical rainforests. Although there has been considerable effort to assess their interactions with pollinators, attempts to characterise their visual and olfactory communication channels are scarce.

Methods

Here, we investigated the pollination biology of 12 Annonaceae species from five genera, viz.Meiogyne,Monoon,Polyalthia,Pseuduvaria, andUvaria. Furthermore, their floral colour was characterised by reflectance spectroscopy and floral odour chemistry was assessed using gas chromatography-mass spectrometry. Floral scent was further compared across the whole family using non-metric dimensional scaling plots to identify specialisation in floral odour.

Results

TheMeiogynespecies are likely pollinated by small beetles; thePolyalthiaandPseuduvariaspecies are likely pollinated by beetles and flies; and theUvariaspecies is likely pollinated by beetles and bees. Flowers of most species are UV non-reflective, and have various spectral reflectance profile across the remaining visible spectra. Multiple species produce floral odour resembling ripe fruits. The flowers ofMeiogynespecies andPolyalthia xanthocarpaemitted mostly branched-chain esters, while flowers ofUvariareleased mainly straight-chain esters. ThePseuduvariaspecies instead emitted scent reminiscent of rotten fruits, largely consisting of 2,3-butanediol. The inner petal corrugation inMeiogynefunctions as a food reward, and the inner petal growth serves as a nectary gland forPseuduvaria.

Conclusions

Our study identifies the visual and olfactory cues of multiple Annonaceae species and provides insights into how Annonaceae flowers attract different guilds of pollinators.
HiFi long-read genomes for difficult-to-detect clinically relevant variants
Authors: Höps Wolfram , Weiss Marjan M. , Derks Ronny , Galbany Jordi Corominas , den Ouden Amber , van den Heuvel Simone , Timmermans Raoul , Smits Jos , Mokveld Tom , Dolzhenko Egor , Chen Xiao , van den Wijngaard Arthur , Eberle Michael A. , Yntema Helger G. , Hoischen Alexander , Gilissen Christian , Vissers Lisenka E.L.M.
Published: September 2024

Clinical short-read exome and genome sequencing approaches have positively impacted diagnostic testing for rare diseases. Yet, technical limitations associated with short reads challenge their use for detection of disease-associated variation in complex regions of the genome. Long-read sequencing (LRS) technologies may overcome these challenges, potentially qualifying as a first-tier test for all rare diseases. To test this hypothesis, we performed LRS (30x HiFi genomes) for 100 samples with 145 known clinically relevant germline variants that are challenging to detect using short-read sequencing and necessitate a broad range of complementary test modalities in diagnostic laboratories.

We show that relevant variant callers readily re-identify the majority of variants (120/145, 83%), including ∼90% of structural variants, SNVs/InDels in homologous sequences and expansions of short tandem repeats. Another 10% (n=14) was visually apparent in the data but not automatically detected. Our analyses also identified systematic challenges for the remaining 7% (n=11) of variants such as the detection of AG-rich repeat expansions. Titration analysis showed that 89% of all automatically called variants could also be identified using 15-fold coverage.

Thus, long-read genomes identified 93% of pathogenic variants that are most challenging to detect using short-read technologies. Even with reduced coverage, the vast majority of variants remained detectable, possibly enhancing cost-effective diagnostic implementation. Most importantly, we show the potential to use a single technology to accurately identify all types of clinically relevant variants.
Structural basis for immune cell binding ofFusobacterium nucleatumvia the trimeric autotransporter adhesin CbpF
Authors: Marongiu Gian Luca , Fink Uwe , Oder Andreas , von Kries Jens Peter , Roderer Daniel
Published: September 2024

Fusobacterium nucleatum(Fn), a commensal in the human oral cavity, is overrepresented in the colon microbiota of colorectal cancer (CRC) patients and is linked to tumor chemoresistance, metastasis, and a poor therapeutic prognosis. Fn produces numerous adhesins that mediate tumor colonization and downregulation of the host’s anti-tumor immune response. One of these, the trimeric autotransporter adhesin (TAA) CEACAM binding protein ofFusobacterium(CbpF), targets CEACAM1 on T-cells and has been associated with immune evasion of Fn-colonized tumors. Whereas the role of CEACAM1 in homophilic and heterophilic cell interactions and immune evasion is well described, the mechanistic details of its interaction with fusobacterial CbpF remain unknown due to the lack of a high-resolution structure of the adhesin-receptor complex.

Here, we present two structures of CbpF alone and in complex with CEACAM1, obtained by cryogenic electron microscopy and single particle analysis. They reveal that CbpF forms a stable homotrimeric complex whose N-terminal part of the extracellular domain comprises a 64 Å long β roll domain with a unique lateral loop extension. CEACAM1 binds to this loop via its N-terminal IgV-like domain with high affinity with a nanomolar dissociation constant, as obtained by surface plasmon resonance. This study provides the first structural description of a fusobacterial TAA, illustrates a yet undescribed CEACAM1 binding mode, and paves the way for rational drug design targeting Fn in CRC.
Extrachromosomal DNA Associates with Nuclear Condensates and Reorganizes Chromatin Structures to Enhance Oncogenic Transcription
Authors: Taghbalout Aziz , Tung Chia-Hao , Clow Patricia A. , Wang Ping , Tjong Harianto , Wong Chee Hong , Mao Diane D. , Maurya Rahul , Huang Meng-Fan , Ngan Chew Yee , Kim Albert H. , Wei Chia-Lin
Published: September 2024

Extrachromosomal, circular DNA (ecDNA) is a prevalent oncogenic alteration in cancer genomes, often associated with aggressive tumor behavior and poor patient outcome. While previous studies proposed a chromatin-based mobile enhancer model for ecDNA-driven oncogenesis, its precise mechanism and impact remains unclear across diverse cancer types. Our study, utilizing advanced multi-omics profiling, epigenetic editing, and imaging approaches in three cancer models, reveals that ecDNA hubs are an integrated part of nuclear condensates and exhibit cancer-type specific chromatin connectivity. Epigenetic silencing of the ecDNA-specific regulatory modules or chemically disrupting liquid-liquid phase separation breaks down ecDNA hubs, displaces MED1 co-activator binding, inhibits oncogenic transcription, and promotes cell death. These findings substantiate thetrans-activator function of ecDNA and underscore a structural mechanism driving oncogenesis. This refined understanding expands our views of oncogene regulation and opens potential avenues for novel therapeutic strategies in cancer treatment.
SLC39A8.p.(Ala391Thr) is associated with poorer cognitive ability: a cross-sectional study of schizophrenia and the general UK population
Authors: Smart Sophie E , Legge Sophie E. , Fenner Eilidh , Pardiñas Antonio F. , Woolway Grace , Lynham Amy J. , Escott-Price Valentina , Hall Jeremy , Wilkinson Lawrence , Holmans Peter , O’Donovan Michael C. , Owen Michael J. , Walters James T.R.
Published: September 2024

The missense SNP NC_000004.12:g.102267552C>T (SLC39A8.p.(Ala391Thr), rs13107325) inSLC39A8,which encodes a zinc transporter, has been linked to schizophrenia and is the likely causal variant for one of the genome-wide association loci associated with the disorder.

We tested whether the schizophrenia-risk allele at p.(Ala391Thr) was associated with schizophrenia-related phenotypes, including positive, negative, and disorganised symptoms, cognitive ability, educational attainment, and age of psychosis onset, within three schizophrenia cohorts (combined N=1,232) and, with equivalent phenotypes, in a sample of population controls (UK Biobank, N=355,069). We used regression analyses controlling for age, sex, and population stratification.

Within the schizophrenia cohorts, after correction for multiple testing, p.(Ala391Thr) was not significantly associated with any schizophrenia-related phenotypes. In the unaffected participants from the UK Biobank, the schizophrenia-risk allele at p.(Ala391Thr) was associated with significantly poorer cognitive ability and fluid intelligence, a lower probability of obtaining GCSEs or a degree-level qualification, and fewer years in education. There was no association between p.(Ala391Thr) and self-reported psychotic experiences in this cohort.

The schizophrenia-risk allele was associated with poorer cognitive ability, but not psychotic experiences, in a volunteer sample drawn from of the general population. To determine whether p.(Ala391Thr) is associated with cognitive phenotypes in people with schizophrenia, and to understand the role of p.(Ala391Thr) in the aetiology of cognitive impairment in schizophrenia, larger independent samples are required.
Functional connectivity is dominated by aperiodic, rather than oscillatory, coupling
Authors: Monchy N. , Duprez J. , Houvenaghel J-F. , Legros A. , Voytek B. , Modolo J.
Published: September 2024

Functional connectivity has attracted significant interest in the identification of specific circuits underlying brain (dys-)function. Classical analyses to estimate functional connectivity (i.e., filtering electrophysiological signals in canonical frequency bands and using connectivity metrics) assume that these reflect oscillatory networks. However, this approach conflates non-oscillatory, aperiodic neural activity with oscillations; raising the possibility that these functional networks may reflect aperiodic rather than oscillatory activity. Here, we provide the first study quantifying, in two different human electroencephalography (EEG) databases, the contribution of aperiodic activity on reconstructed oscillatory functional networks in resting state. We found that more than 99% of delta, theta, and gamma functional networks, more than 90% of beta functional networks and between 23 and 55% of alpha functional networks were actually driven by aperiodic activity. While there is no universal consensus on how to identify and quantify neural oscillations, our results demonstrate that oscillatory functional networks are drastically sparser than commonly assumed. These findings suggest that most functional connectivity studies focusing on resting state actually reflect aperiodic networks instead of oscillations-based networks. We highly recommend that oscillatory network analyses first check the presence of aperiodicity-unbiased neural oscillations before estimating their statistical coupling to strengthen the robustness, interpretability, and reproducibility of functional connectivity studies.
In-cell processing enables rapid and in-depth proteome analysis of low-inputCaenorhabditis elegans
Authors: Elsayyid Malek , Tanis Jessica E. , Yu Yanbao
Published: September 2024

Caenorhabditis elegansis a widely used genetic model organism, however, the worm cuticle complicates extraction of intracellular proteins, a prerequisite for typical bottom-up proteomics. Conventional physical disruption procedures are not only time-consuming, but can also cause significant sample loss, making it difficult to perform proteomics with low-input samples. Here, for the first time, we present an on-filter in-cell (OFIC) processing approach, which can digestC. elegansproteins directly in the cells of the organism after methanol fixation. With OFIC processing and single-shot LCMS analysis, we identified over 9,400 proteins from a sample of only 200 worms, the largestC. elegansproteome reported to date that did not require fractionation or enrichment. We systematically evaluated the performance of the OFIC approach by comparing it with conventional lysis-based methods. Our data suggest equivalent and unbiased performance of OFIC processing forC. elegansproteome identification and quantitation. We further evaluated the OFIC approach with even lower input samples, then used this method to determine how the proteome is impacted by loss of superoxide dismutasesod-1, the ortholog of humanSOD-1, a gene associated with amyotrophic lateral sclerosis (ALS). Analysis of 8,800 proteins from only 50 worms as the initial input showed that loss ofsod-1affects the abundance of proteins required for stress response, ribosome biogenesis, and metabolism. In conclusion, our streamlined OFIC approach, which can be broadly applied to other systems, minimizes sample loss while offering the simplest workflow reported to date forC. elegansproteomics analysis.
Cleland immunoblotting unmasks unexpected cysteine redox proteoforms
Authors: Cobley James N. , Noble Anna , Guille Matthew
Published: September 2024

Cysteine redox proteoforms are virtually unstudied because they are extremely difficult to detect. They are difficult to detect by immunoblotting when the polyethylene glycol (PEG)-payloads used to mobility-shift proteoforms into distinct bands block antibody binding. Here, we synthesised a novel compound to reversibly crosslink the PEG-payloads to oxidised cysteines using disulfide bonds. To reductively release the PEG-payloads from mobility-shifted proteoforms, we soaked the gel in Cleland’s reagent: 1,4-dithiothreitol (DTT). Hence, Cleland immunoblotting. Cleland immunoblotting unmasked hitherto undetectable cysteine redox proteoforms. InXenopusoocytes, we detected 2 cdc20-specific coordinates in an otherwise abstract space housing 1,024 theoretical proteoforms. The coordinates mapped to the fully, all 10 cysteine residues, 100%-reduced and 100%-oxidised proteoforms. The unexpected absence of partially oxidised molecules along the 10-reaction path to the 100%-oxidised proteoform implies novel biology. By providing the technological means to detect cysteine redox proteoforms, Cleland immunoblotting opens new avenues of discovery.
Herpes Simplex Virus Assembly and Spread in murine skin after infection from the outside
Authors: Richardo Timmy , Liu Xiaokun , Döhner Katinka , Buch Anna , Binz Anne , Pohlmann Anja , de le Roi Madeleine , Baumgärtner Wolfgang , Brand Korbinian , Bauerfeind Rudolf , Förster Reinhold , Sodeik Beate , Halle Stephan
Published: September 2024

Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murineex vivoexplant model using gentle depilation but no further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy studies showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently-tagged capsid protein VP26, and monitored the formation of nuclear capsid assembly sites, nuclear capsid egress, and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinicalex vivoinfection model.

IMPORTANCE

This study describes a novel murineex vivoskin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers growing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.
Re-analysis of CLAP data affirms PRC2 as an RNA binding protein
Authors: Lee YongWoo , Das Priyojit , Rosenberg Michael , Blum Roy , Lee Jeannie
Published: September 2024

Using CLAP methodology, Guo et al. recently concluded that PRC2 is not an RNA binding protein (RBP). They suggest that prior findings were CLIP artifacts and argue against RNAs direct role in PRC2 regulation. Here, we re-analyze their raw datasets and reach contrary conclusions. Through an independent computational pipeline, we observe significant PRC2 enrichment throughout the transcriptome, including XIST. Applying the authors published computational pipeline also reaffirms PRC2 as an RBP. Detailed investigation of the authors pipeline reveals several unconventional practices. First, Guo et al. retained reads from foreign species and other unmappable reads to obtain a normalization factor. Second, they selectively removed read duplicates from the mappable fraction, while retaining them in the unmappable fraction. Finally, the authors applied an arbitrary cutoff for enrichment values in XIST. Their pipeline thereby inflated PRC2s background reads and suppressed mappable signals, creating the impression that PRC2 is not a robust RBP.
Acute increase of protein O-GlcNAcylation in mice leads to transcriptome changes in the brain opposite to what is observed in Alzheimer’s Disease
Authors: Bell Margaret , Kane Mariame S , Ouyang Xiaosen , Young Martin E , Jegga Anil G , Chatham John C. , Darley-Usmar Victor , Zhang Jianhua
Published: September 2024

Enhancing protein O-GlcNAcylation by pharmacological inhibition of the enzyme O-GlcNAcase (OGA) is explored as a strategy to decrease tau and amyloid-beta phosphorylation, aggregation, and pathology in Alzheimer’s disease (AD). There is still more to be learned about the impact of enhancing global protein O-GlcNAcylation, which is important for understanding the mechanistic path of using OGA inhibition to treat AD. In this study, we investigated the acute effect of pharmacologically increasing O-GlcNAc levels, using OGA inhibitor Thiamet G (TG), on normal mouse brains. We hypothesized that the transcritome signature in respones to TG treatment provides a comprehensive view of the effect of OGA inhibition. We sacrificed the mice and dissected their brains after 3 hours of saline or 50 mg/kg TG treatment, and then performed mRNA sequencing using NovaSeq PE 150 (n=5 each group). We identified 1,234 significant differentially expressed genes with TG versus saline treatment. Functional enrichment analysis of the upregulated genes identified several upregulated pathways, including genes normally down in AD. Among the downregulated pathways were the cell adhesion pathway as well as genes normally up in AD and aging. When comparing acute to chronic TG treatment, protein autophosphorylation and kinase activity pathways were upregulated, whereas cell adhesion and astrocyte markers were downregulated in both datasets. Interestingly, mitochondrial genes and genes normally down in AD were up in acute treatment and down in chronic treatment. Data from this analysis will enable the evaluation of the mechanisms underlying the potential benefits of OGA inhibition in the treatment of AD. In particular, although OGA inhibitors are promising to treat AD, their downstream chronic effects related to bioenergetics may be a limiting factor.

Abstract Figure
Labor- and cost-effective long-read amplicon sequencing using a plasmid analysis service: Application to transposon-inserted alleles in Japanese morning glory
Authors: Nakagawa Soya , Hoshino Atsushi , Park Kyeung-Il
Published: September 2024

The sequencing of PCR fragments amplified from specific regions of genomes is a fundamental technique in molecular genetics. Sanger sequencing is commonly used for this analysis; however, amplicon sequencing utilizing next-generation sequencing has become widespread. In addition, long-read amplicon sequencing, using Nanopore or PacBio sequencers to analyze long PCR fragments, has emerged, although it is often more expensive than Sanger sequencing.

Recently, low-cost commercial services for full-length plasmid DNA sequencing using Nanopore sequencers have been launched in several countries, including Japan. This study explored the potential of these services to sequence long PCR fragments without the need for cloning into plasmid DNA. PCR fragments of 4–11 kb, amplified from theDFR-Bgene involved in the biosynthesis of anthocyanin, with or withoutTpn1transposons in Japanese morning glory (Ipomoea nil), were circularized using T4 ligase and analyzed as templates. Although some inaccuracies in the length of homopolymer stretches were observed, the remaining sequences were obtained without significant errors. This method could potentially reduce the labor and costs associated with cloning, primer synthesis, and sequence assembly, thus making it a viable option for the analysis of long PCR fragment sequences. Moreover, this study reconfirmed thatTpn1transposons are major mutagens inI. niland demonstrated their transposition in the Violet line, a long-used standard in plant physiology.
Population-level control of two manganese oxidases expands the niche for bacterial manganese biomineralization
Authors: Gehin Gaitan , Carraro Nicolas , Roelof van der Meer Jan , Peña Jasquelin
Published: September 2024

The enzymatic oxidation of aqueous divalent manganese (Mn) is a widespread microbial trait that produces reactive Mn(III, IV) oxide minerals. These biominerals drive carbon, nutrient, and trace metal cycles, thus playing important environmental and ecological roles. However, the regulatory mechanisms and physiological functions of Mn biomineralization are unknown. This challenge arises from the common occurrence of multiple Mn oxidases within the same organism and the use of Mn oxides as indicators of combined gene activity. Through detection of gene activation in individual cells, we discover that expression ofmnxGandmcoA, two Mn oxidase-encoding genes inPseudomonas putidaGB-1, is confined to subsets of cells within the population, with each gene showing distinct spatiotemporal patterns that reflect local microenvironments. These coordinated intra-population dynamics control Mn biomineralization and illuminate the strategies used by microbial communities to dictate the extent, location and timing of biogeochemical transformations.
Understanding the regulation of chronic wounds by tissue inhibitors of matrix metalloproteinases through mathematical modelling
Authors: Dari Sonia , O’Dea Reuben D. , Fadai Nabil T.
Published: September 2024

Understanding the biochemistry and pharmacodynamics of chronic wounds is of key importance, due to the millions of people in the UK affected and the significant cost to the NHS. Chronic wounds are characterised by elevated concentrations of matrix metalloproteinases (MMPs) that destroy the surrounding extracellular matrix (ECM). However, fibroblasts can produce tissue inhibitors of MMPs (TIMPs) in order to regulate wound healing. Therefore, the role of TIMPs in both acute and chronic wounds needs to be properly understood in order to develop therapeutic treatments. In this work, we propose a reaction-diffusion system of four partial differential equations that describe the interaction of the ECM, fibroblasts, MMPs, and TIMPs in a wound. We observe that, subject to biologically realistic parameter values, this mathematical model gives rise to travelling wave solutions. Using bifurcation analysis, we demonstrate that deregulated apoptosis in the ECM results in the emergence of chronic wounds, and the reversal of these chronic wounds is prohibited for lower TIMP production values. These results are replicated within a simplified model obtained via a parameter sensitivity analysis. This model is further extended to more realistic spatial domains where we demonstrate the effectiveness of a therapeutic hydrogel containing TIMPs as a treatment for chronic wounds.
Cytoophidium complexes resonate with cell fates
Authors: Li Yi-Lan , Liu Ji-Long
Published: September 2024

Metabolism is a fundamental characteristic of life. In 2010, we discovered that the metabolic enzyme CTP synthase (CTPS) can assemble a snake like structure inside cells, which we call the cytoophidium. Including CTPS, an increasing number of metabolic enzymes have been found to form cytoophidia in cells. However, the distribution and relationship among cytoophidia formed by different metabolic enzymes remain elusive. Here we investigate five metabolic enzymes that can form cytoophidia, namely Asn1, Bna5, CTPS (ie. Ura7), Glt1, and Prs5 inSaccharomyces cerevisiae. We find that multiple cytoophidia can be assembled into cytoophidium complexes by docking one after another. Glt1 cytoophidia tend to assemble in non-quiescent cells, while CTPS cytoophidia are more abundant in quiescent cells and form complexes with Prs5 and Asn1 cytoophidia. Blocking CTPS cytoophidium assembly can lead to a non-quiescent phenotype and increase the assembly of Glt1 cytoophidia, Bna5 cytoophidia, and cytoophidium complexes. Blocking CTPS cytoophidium assembly also inhibits the NAD biosynthesis pathway, which includes Bna5 and Sir2. Consistent with this result, the non-quiescent phenotype caused by blocking CTPS cytoophidium assembly can be rescued by blocking Glt1 cytoophidium assembly, supplementing nicotinic acid, or overexpressing Sir2. Our results indicate that the assembly of cytoophidium complexes with different compositions resonates with distinct cell fates.

Abstract Figure

HIGHLIGHTS

Multiple cytoophidia can be assembled into cytoophidium complexes by docking one after another.

CTPS cytoophidia blockage impedes quiescence and boosts Glt1-Bna5 cytoophidium complex.

Glt1 cytoophidium blockage restores quiescence and Bna5 inhibition in NAD pathway.

Activating NAD utilizing SIR2 rescues CTPS cytoophidium blockage induced non-quiescence.
Revealing nonclassicality of multiphoton optical beams via artificial neural networks
Authors: Machulka Radek , Peřina Jan , Michálek Václav , León-Montiel Roberto de J. , Haderka Ondřej
Published: September 2024

The identification of nonclassical features of multiphoton quantum states represents a crucial task in the development of many quantum photonic technologies. Under realistic experimental conditions, a photonic quantum state gets affected by its interaction with several nonideal optoelectronic devices, including those used to guide, detect, or characterize it. The result of such noisy interaction is that the nonclassical features of the original quantum state get considerably reduced or are completely absent in the detected, final state. In this work, the self-learning features of artificial neural networks are exploited to experimentally show that the nonclassicality of multiphoton quantum states can be assessed and fully characterized, even in the cases in which the nonclassical features are concealed by the measuring devices. Our work paves the way toward artificial-intelligence-assisted experimental-setup characterization, as well as quantum state nonclassicality identification.

Published by the American Physical Society
2024
Development of a global landscape of undergraduate physics laboratory courses
Authors: Geschwind Gayle , Alemani Micol , Fox Michael F. J. , Logman P. S. W. M. , Tufino Eugenio , Lewandowski H. J.
Published: September 2024

Physics education research (PER) is a global endeavor, with a wealth of work performed at a variety of institutions worldwide. However, results from research into undergraduate physics laboratory courses are often difficult to compare due to the broad variations in courses. We report here how we developed and validated a survey to classify these courses, as well as compare and contrast them. This will be useful in two key endeavors: comparisons between PER studies and providing useful data for individual instructors hoping to improve their courses. While we are still in the process of collecting sufficient data to create a full taxonomy of laboratory courses, we present here details of the survey creation itself, including its face, construct, and content validation, as well as a first look at the data collected, which includes a broad landscape of lab courses in 41 countries. We used both quantitative and qualitative methods to analyze the data collected. Some of these results include similarities between courses, such as students often using preconstructed apparatuses and instructors hoping for students to learn technical skills. We also find differences in courses, such as in the number and types of goals of the course, as well as the activities students participate in. Thus, this survey and its results can provide information relevant to both researchers and instructors.

Published by the American Physical Society
2024