bluuebunny/crossref_metadata_2025 huggingface dataset:
Common roles for serotonin in rats and humans for computations underlying flexible decision-making
Authors: Luo Qiang , Kanen Jonathan W. , Bari Andrea , Skandali Nikolina , Langley Christelle , Knudsen Gitte Moos , Alsiö Johan , Phillips Benjamin U. , Sahakian Barbara J. , Cardinal Rudolf N. , Robbins Trevor W.
Published: February 2023

Serotonin is critical for adapting behavior flexibly to meet changing environmental demands. Cognitive flexibility is important both for successful attainment of goals, as well as for social interactions, and is frequently impaired in neuropsychiatric disorders, including obsessive-compulsive disorder (OCD). However, a unifying mechanistic framework accounting for the role of serotonin in behavioral flexibility has remained elusive. Here, we demonstrate common effects of manipulating serotonin function across two species (rats and humans) on latent processes supporting choice behavior during probabilistic reversal learning using computational modelling. The findings support a role of serotonin in behavioral flexibility and plasticity, indicated, respectively, by increases or decreases in choice repetition (‘stickiness’) or reinforcement learning rates depending upon manipulations intended to increase or decrease serotonin function. More specifically, the rate at which expected value increased following reward and decreased following punishment (reward and punishment ‘learning rates’) was greatest after sub-chronic administration of the selective serotonin reuptake (SSRI) citalopram (5 mg/kg for 7 days followed by 10 mg/kg twice a day for 5 days) in rats. Conversely, humans given a single dose of an SSRI (20mg escitalopram), which can decrease post-synaptic serotonin signalling, and rats that received the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which destroys forebrain serotonergic neurons, exhibited decreased reward learning rates. A basic perseverative tendency (‘stickiness’), or choice repetition irrespective of the outcome produced, was likewise increased in rats after the 12-day SSRI regimen and decreased after single dose SSRI in humans and 5,7-DHT in rats. These common effects of serotonergic manipulations on rats and humans – identified via computational modelling – suggest an evolutionarily conserved role for serotonin in plasticity and behavioral flexibility and have clinical relevance transdiagnostically for neuropsychiatric disorders.
Fine-tuning spatial-temporal dynamics and surface receptor expression support plasma cell-intrinsic longevity
Authors: Jing Zhixin , Galbo Phillip , Ovando Luis , Demouth Megan , Welte Skylar , Park Rosa , Chandran Kartik , Wu Yinghao , MacCarthy Thomas , Zheng Deyou , Fooksman David
Published: February 2023

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intra-vital two-photon imaging, we find that in contrast to most plasma cells in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44 and CD48, important for adhesion and homing. Conditional deletion ofCxcr4in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

HIGHLIGHTS

LLPCs have reduced motility and increased clustering in the BM

LLPCs accumulate in the BM PC pool, with mouse age

LLPCs have unique surfaceome, transcriptome, and BCR clonality

CXCR4 controls maintenance of PCs and antibody titers
Inter-laboratory comparison of plant volatile analyses in the light of intra-specific chemodiversity
Authors: Eckert Silvia , Eilers Elisabeth J. , Jakobs Ruth , Anaia Redouan Adam , Aragam Kruthika Sen , Bloss Tanja , Popp Moritz , Sasidharan Rohit , Schnitzler Jörg-Peter , Stein Florian , Steppuhn Anke , Unsicker Sybille B. , van Dam Nicole M. , Yepes Sol , Ziaja Dominik , Müller Caroline
Published: February 2023

Introduction

Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses.

Objectives

In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we usedTanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps.

Methods

Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures.

Results

Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles.

Conclusion

Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.
The Development of Synapses in Mouse and Macaque Primary Sensory Cortices
Authors: Wildenberg Gregg , Li Hanyu , Kasthuri Narayanan
Published: February 2023

We report that the rate of synapse development in primary sensory cortices of mice and macaques is unrelated to lifespan, as was previously thought. We analyzed 28,084 synapses over multiple developmental time points in both species and find, instead, that net excitatory synapse development of mouse and macaque neurons primarily increased at similar rates in the first few postnatal months, and then decreased over a span of 1-1.5 years of age. The development of inhibitory synapses differed qualitatively across species. In macaques, net inhibitory synapses first increase and then decrease on excitatory soma at similar ages as excitatory synapses. In mice, however, such synapses are added throughout life. These findings contradict the long-held belief that the cycle of synapse formation and pruning occurs earlier in shorter-lived animals. Instead, our results suggest more nuanced rules, with the development of different types of synapses following different timing rules or different trajectories across species.
Strengthening connectivity between premotor and motor cortex increases inter-areal communication in the human brain
Authors: Trajkovic Jelena , Romei Vincenzo , Rushworth Matthew F. S. , Sel Alejandra
Published: February 2023

The ventral premotor cortex (PMv) is an important component of cortico-cortical pathways mediating prefrontal control over primary motor cortex (M1) function. Paired associative stimulation (ccPAS) is known to change PMv influence over M1 in humans, which manifests differently depending on the behavioural context. Here we show that these changes in influence are functionally linked to PMv-M1 phase synchrony changes induced by repeated paired stimulation of the two areas. PMv-to-M1 ccPAS leads to increased phase synchrony in alpha and beta bands while reversed order M1-to-PMv ccPAS leads to decreased theta phase synchrony. These changes are visible at rest but they are predictive of changes in oscillatory power in the same frequencies during movement execution and inhibition, respectively. The results unveil a link between the physiology of the motor network and the resonant frequencies mediating its interactions, and provide a putative mechanism underpinning the relationship between synaptic efficacy and brain oscillations.
Systematic analysis of disease-linked rare germline variants reveals new classes of cancer predisposing genes
Authors: Song Seulki , Koh Youngil , Kim Seokhyeon , Lee Sang Mi , Kim Hyun Uk , Ko Jung Min , Lee Se-Hoon , Yoon Sung-Soo , Park Solip
Published: February 2023

Despite recent insights into cancer predisposition genes (CPGs), the contribution of rare germline variants to carcinogenesis remains unclear. Here, we examine the enrichment of rare germline variants in Mendelian disease-associated genes using PCAWG and the 1,000 Genomes Project. Using an integrative approach that considers disease classes, cellular pathways, and tissue expression profiles, we find that subsets of Online Mendelian Inheritance in Man (OMIM) genes are strong candidate CPGs. These genes were classified into five clusters suggesting diverse mechanisms underlie tumor progression. We further analyzedPAHgene which shows the strongest prevalence of rare germline variants in cancers (2.2%) compared to controls (0.7%). This enrichment was validated in two independent cancer cohorts and present the possibility of tumorigenesis by contributing immune-associated pathways. Our data argue that rare germline variants of Mendelian disease-associated genes contribute to cancer progression and suggest new CPG classifications that may underlie diverse tumorigenesis mechanisms.
A biologically realistic model to predict wildlife-vehicle collision risks
Authors: Bénard Annaëlle , Lengagne Thierry , Bonenfant Christophe
Published: February 2023

Road networks have major ecological impacts on living organisms consequent to habitat loss and fragmentation, chemical and acoustic pollution, and direct mortality when wildlife-vehicle collisions are involved (WVC). The many past empirical studies revealed biological traits shared by species most vulnerable to roadkills (e.g. population density). Similarly, spatial locations of WVC hot-spots are associated to landscape features at large spatial scales, and to road characteristics at small spatial scale. We currently lack a comprehensive theoretical framework for WVC. Animal movement in relation to habitats is an essential driver of encounters with roads, but this remains largely ignored in studies, even when movement ecology provides the necessary tools to investigate the impact of animal movement on WVC. We built a general individual-based model incorporating recent knowledge in movement ecology (movement typology: roaming, migratory route crossing a road, active attraction and active repulsion of roads) to estimate WVC risks. We explored the relative effects of animal and vehicle movement parameters (speed, abundance, road sinuosity and animal movement pattern) on collision probability. We show that animal behaviour toward roads has major impacts on the number and risks of WVC, but also modulate the effects of other factors (animal traveling speed, species local abundance, road traffic volume) on WVC. Sensitivity analyses show that the movement and behaviour of the animal has more influence on WVC risks than any of the characteristics of roads and vehicles we tested. Our results suggest that (1) effective roadkill mitigation should be species-specific and could vary in efficiency depending on the target’s movement pattern (mating and migratory seasons, foraging habits…) and (2) empirical studies of WVC should incorporate knowledge about the behavioural habits of the focal species in relation to roads.
A Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling
Authors: George Amy L. , Sidgwick Frances R. , Watt Jessica E. , Martin Mathew P. , Trost Matthias , Marín-Rubio José Luis , Dueñas Maria Emilia
Published: February 2023

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMT) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification (LFQ) approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukaemia (AML) cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable or better than TMT-DDA in their ability to reproducibly detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labelled quantitation in the TPP pipeline.
High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy
Authors: Lee Moosung , Jeong Hyuntae , Lee Chaeyeon , Lee Mahn Jae , Delmo Benedict Reve , Heo Won Do , Shin Jennifer H. , Park YongKeun
Published: February 2023

A critical requirement for studying cell mechanics is three-dimensional (3D) assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy (TFM) with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow 3D acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), a high-speed volumetric technique that simultaneously quantifies the 3D morphology and traction force of cells. RI-TFM reconstructs a 3D tomogram of single cells on a planar hydrogel elastomer using a high-speed illumination scheme with 0.5-Hz temporal resolution. This allows for quantitative analyses of 3D dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix without labelling. When combined with a constrained total variation-based deconvolution algorithm, the proposed method provides 0.55-Pa shear (in-plane) and 1.59-Pa normal (out-of-plane) traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate the utility of RI-TFM by assessing the effects of compromised intracellular stress due to actin disruption or low temperature and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.
EEG decoding reveals neural predictions for naturalistic material behaviors
Authors: Kaiser Daniel , Stecher Rico , Doerschner Katja
Published: February 2023

Material properties like softness or stickiness determine how an object can be used. Based on our real-life experience, we form strong expectations about how objects should behave under force, given their typical material properties. Such expectations have been shown to modulate perceptual processes, but we currently do not know how expectation influences the temporal dynamics of the cortical visual analysis for objects and their materials. Here, we tracked the neural representations of expected and unexpected material behaviors using time-resolved EEG decoding in a violation-of-expectation paradigm, where objects fell to the ground and deformed in expected or unexpected ways. Participants were 25 men and women. Our study yielded three key results: First, both objects and materials were represented rapidly and in a temporally sustained fashion. Second, objects exhibiting unexpected material behaviors were more successfully decoded than objects exhibiting expected behaviors within 190ms after the impact, which might indicate additional processing demands when expectations are unmet. Third, general signals of expectation fulfillment that generalize across specific objects and materials were found within the first 150ms after the impact. Together, our results provide new insights into the temporal neural processing cascade that underlies the analysis of real-world material behaviors. They reveal a sequence of predictions, with cortical signals progressing from a general signature of expectation fulfillment towards increased processing of unexpected material behaviors.

Significance Statement

In the real world, we can make accurate predictions about how an object’s material shapes its behavior: For instance, we know that cups are typically made of porcelain and shatter when we accidentally drop them. Here, we use EEG to experimentally test how expectations about material behaviors impact neural processing. We showed our participants videos of objects that exhibited expected material behaviors (such as a glass shattering when falling to the ground) or unexpected material behaviors (such as a glass melting upon impact). Our results reveal a hierarchy of predictions in cortex: The visual system rapidly generates signals that index whether expectations about material behaviors are met. These signals are followed by increased processing of objects displaying unexpected material behaviors.
Cochlear transcriptome analysis of an outbred mouse population (CFW)
Authors: Boussaty Ely Cheikh , Tedeschi Neil , Novotny Mark , Ninoyu Yuzuru , Du Eric , Draf Clara , Zhang Yun , Manor Uri , Scheuermann Richard H. , Friedman Rick
Published: February 2023

Age-related hearing loss (ARHL) is the most common cause of hearing loss and one of the most prevalent conditions affecting the elderly worldwide. Despite evidence from our lab and others about its polygenic nature, little is known about the specific genes, cell types and pathways involved in ARHL, impeding the development of therapeutic interventions. In this manuscript, we describe, for the first time, the complete cell-type specific transcriptome of the aging mouse cochlea using snRNA-seq in an outbred mouse model in relation to auditory threshold variation. Cochlear cell types were identified using unsupervised clustering and annotated via a three-tiered approach - first by linking to expression of known marker genes, then using the NS-Forest algorithm to select minimum cluster-specific marker genes and reduce dimensional feature space for statistical comparison of our clusters with existing publicly-available data sets on the gEAR website (https://umgear.org/), and finally, by validating and refining the annotations using Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH) and the cluster-specific marker genes as probes. We report on 60 unique cell-types expanding the number of defined cochlear cell types by more than two times. Importantly, we show significant specific cell type increases and decreases associated with loss of hearing acuity implicating specific subsets of hair cell subtypes, ganglion cell subtypes, and cell subtypes withing the stria vascularis in this model of ARHL. These results provide a view into the cellular and molecular mechanisms responsible for age-related hearing loss and pathways for therapeutic targeting.
Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex
Authors: Deng Chengyu , Whalen Sean , Steyert Marilyn , Ziffra Ryan , Przytycki Pawel F. , Inoue Fumitaka , Pereira Daniela A. , Capauto Davide , Norton Scott , Vaccarino Flora M. , Pollen Alex , Nowakowski Tomasz J. , Ahituv Nadav , Pollard Katherine S.
Published: February 2023

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated thecis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

One Sentence Summary

We identify 46,802 enhancers and 164 psychiatric disorder variants with regulatory effects in the developing cortex and organoids.
Colour polymorphism and conspicuousness do not increase speciation rates in Lacertids
Authors: de Solan Thomas , Sinervo Barry , Geniez Philippe , David Patrice , Crochet Pierre-André
Published: February 2023

Conspicuous body colours and colour polymorphism have been hypothesized to increase rates of speciation. Conspicuous colours are evolutionary labile, and often involved in intraspecific sexual signalling and thus may provide a raw material from which reproductive isolation can easily evolve, while polymorphism could favour rapid evolution of new lineages through morphic speciation. Here, we investigated the influence of the presence/absence of conspicuous colourations, and of colour polymorphism on the speciation of Lacertids. We used several state-dependent diversification models, and showed that, regardless of the methods, conspicuous colourations and colour polymorphism were not related to species speciation. While the lack of correlation between conspicuous colourations and speciation rates is in line with most of the literature testing this hypothesis, the results for colour polymorphism contradict previous studies, and question the generality of the morphic speciation hypothesis.
STUN: forward-time Simulation on TUnable fitNess landscapes in recombining populations
Authors: Amado André , Li Juan , Bank Claudia
Published: February 2023

Understanding the population genetics of complex polygenic traits during adaptation is challenging. Here, we implement a forward-in-time population-genetic simulator (STUN) based on Wright-Fisher dynamics. STUN is a flexible and user-friendly software package for simulating the polygenic adaptation of recombining haploid populations using either new mutations or standing genetic variation. STUN assumes that populations adapt to sudden environmental changes by undergoing selection on a new fitness landscape. With pre-implemented fitness landscape models like Rough Mount Fuji, NK, Block, additive, and House-of-Cards, users can explore the effect of different levels of epistasis (ruggedness of the fitness landscape). Custom fitness landscapes and recombination maps can also be defined. STUN empowers both experimentalists and advanced programmers to study the evolution of complex polygenic traits and to dissect the adaptation process.

Availability and implementation

STUN is implemented in Rust. Its source code is available athttps://github.com/banklab/STUN, which also includes a link to the software’s manual and binary files for Linux, macOS and Windows. Upon publication, the code will also be archived on Zenodo and a doi will be provided here.
The Zinc-BED transcription factor Bedwarfed promotes proportional dendritic growth and branching through transcriptional and translational regulation inDrosophila
Authors: Bhattacharjee Shatabdi , Iyer Eswar Prasad R. , Iyer Srividya Chandramouli , Nanda Sumit , Rubaharan Myurajan , Ascoli Giorgio A. , Cox Daniel N.
Published: February 2023

Dendrites are the primary points of sensory or synaptic inputs to a neuron and play an essential role in synaptic integration and neural function. Despite the functional importance of dendrites, relatively less is known about the underlying mechanisms regulating cell-type specific dendritic patterning. Herein, we have dissected functional roles of a previously uncharacterized gene,CG3995, in cell-type specific dendritic development inDrosophila melanogaster.CG3995, which we have namedbedwarfed(bdwf), encodes a zinc-finger BED-type protein which is required for proportional growth and branching of dendritic arbors, exhibits nucleocytoplasmic expression, and functions in both transcriptional and translational cellular pathways. At the transcriptional level, we demonstrate a reciprocal regulatory relationship between Bdwf and the homeodomain transcription factor (TF) Cut. We show that Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins produced phenotypically similar dendritic hypotrophy defects as observed inbdwfmutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, our findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. Taken together, these results provide new insights into the complex, combinatorial and multi-functional roles of transcription factors (TFs) in directing diversification of cell-type specific dendritic development.
A novel rotavirus reverse genetics platform supports flexible insertion of exogenous genes and enables rapid development of a high-throughput neutralization assay
Authors: Wei Jiajie , Radcliffe Scott , Lu Meiqing , Li Yuan , Cassaday Jason , Newhard William , Heidecker Gwendolyn J , Rose William A , He Xi , Freed Daniel , Citron Michael , Espeseth Amy , Wang Dai
Published: February 2023

Despite the success of rotavirus vaccines, rotaviruses are still one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality especially in low- and middle-income countries. Reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins such as vaccine antigens and therapeutic payloads. Here, we identified three positions in rotavirus genome, the C terminus of NSP1, NSP3 and NSP5, for reporter gene insertion. By using rotavirus expressing GFP, we developed a high-throughput neutralization assay and revealed the pre-existing immunity against rotavirus in human and other animal species. Our work shows the plasticity of rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.
Virotest: a bioinformatics pipeline for virus identification in plants
Authors: Gonzalez-Ibeas Daniel
Published: February 2023

High-throughput DNA sequencing is being introduced as an emerging technology for viral diagnosis. Despite several tools being available, lack of standardized bioinformatic pipelines and price are major limitations for its implementation. Many extant tools focus on the assembly of virus-derived small RNAs into contigs and their subsequent annotation. Better sensitivity has been reported by several studies when this approach is complemented with read mapping on known viral genome sequences as an ancillary step, requiring less number of reads and allowing price optimization during DNA sequencing. This publication introducesvirotest, a bioinformatics pipeline for the identification of viruses in plants from small RNA data. The tool prioritizes read mapping over sequence assembly for the purpose of obtaining better sensitivities and requiring lower sequencing depths.
Functional significance of PUF partnerships inC. elegansgermline stem cells
Authors: Ferdous Ahlan S. , Costa Dos Santos Stephany J. , Kanzler Charlotte R. , Shin Heaji , Carrick Brian H. , Crittenden Sarah L. , Wickens Marvin , Kimble Judith
Published: February 2023

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal ofC. elegansgermline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy. Here, we investigate LST-1–PUF and SYGL-1–PUF partnerships and their molecular activities in their natural context – nematode stem cells. We confirm LST-1–PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(AmBm) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(AmBm) is used to explore the functional significance of the LST-1–PUF partnership. Tethered LST-1 requires the partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs. Comparison of PUF-LST-1 and Pumilio–Nanos reveals fundamental molecular differences, making PUF–LST-1 a distinct paradigm for PUF partnerships.

Summary statement

Partnerships between PUF RNA-binding proteins and intrinsically disordered proteins are essential for stem cell maintenance and RNA repression.
Assessing the performance of the Cell Painting assay across different imaging systems
Authors: Tromans-Coia Callum , Jamali Nasim , Abbasi Hamdah Shafqat , Giuliano Kenneth A. , Hagimoto Mai , Jan Kevin , Kaneko Erika , Letzsch Stefan , Schreiner Alexander , Sexton Jonathan Z. , Suzuki Mahomi , Trask O. Joseph , Yamaguchi Mitsunari , Yanagawa Fumiki , Yang Michael , Carpenter Anne E. , Cimini Beth A.
Published: February 2023

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments. Here, we examine the performance of the Cell Painting assay across multiple high-throughput microscope systems and find that all are compatible with this assay. Furthermore, we determine independently for each microscope system the best performing settings, providing those who wish to adopt this assay an ideal starting point for their own assays. We also explore the impact of microscopy setting changes in the Cell Painting assay and find that few dramatically reduce the quality of a Cell Painting profile, regardless of the microscope used.
Finite Element Modeling of Residual Hearing after Cochlear Implant Surgery in Chinchillas
Authors: Castle Nicholas , Liang Junfeng , Smith Matthew , Petersen Brett , Matson Cayman , Eldridge Tara , Zhang Ke , Lee Chung-Hao , Liu Yingtao , Dai Chenkai
Published: February 2023

Cochlear implant (CI) surgery is one of the most utilized treatments for severe hearing loss. However, the effects of a successful scala tympani insertion on the mechanics of hearing are not yet fully understood. This paper presents a finite element (FE) model of the chinchilla inner ear for studying the interrelationship between the mechanical function and the insertion angle of a CI electrode. This FE model includes a three-chambered cochlea and full vestibular system, accomplished using μ-MRI and μ-CT scanning technology. This model’s first application found minimal loss of residual hearing due to insertion angle after CI surgery and indicates that it is a reliable and helpful tool for future application in CI design, surgical planning, and stimuli setup.
Integrated single cell and unsupervised spatial transcriptomic analysis defines molecular anatomy of the human dorsolateral prefrontal cortex
Authors: Huuki-Myers Louise , Spangler Abby , Eagles Nick , Montgomery Kelsey D. , Kwon Sang Ho , Guo Boyi , Grant-Peters Melissa , Divecha Heena R. , Tippani Madhavi , Sriworarat Chaichontat , Nguyen Annie B. , Ravichandran Prashanthi , Tran Matthew N. , Seyedian Arta ,   PsychENCODE consortium, Hyde Thomas M. , Kleinman Joel E. , Battle Alexis , Page Stephanie C. , Ryten Mina , Hicks Stephanie C. , Martinowich Keri , Collado-Torres Leonardo , Maynard Kristen R.
Published: February 2023

The molecular organization of the human neocortex has been historically studied in the context of its histological layers. However, emerging spatial transcriptomic technologies have enabled unbiased identification of transcriptionally-defined spatial domains that move beyond classic cytoarchitecture. Here we used the Visium spatial gene expression platform to generate a data-driven molecular neuroanatomical atlas across the anterior-posterior axis of the human dorsolateral prefrontal cortex (DLPFC). Integration with paired single nucleus RNA-sequencing data revealed distinct cell type compositions and cell-cell interactions across spatial domains. Using PsychENCODE and publicly available data, we map the enrichment of cell types and genes associated with neuropsychiatric disorders to discrete spatial domains. Finally, we provide resources for the scientific community to explore these integrated spatial and single cell datasets atresearch.libd.org/spatialDLPFC/.

Generation of a molecular neuroanatomical map of the human prefrontal cortex reveals novel spatial domains and cell-cell interactions relevant for psychiatric disease.
Mixed IgG Fc immune complexes exhibit blended binding profiles and refine FcR affinity estimates
Authors: Tan Zhixin Cyrillus , Lux Anja , Biburger Markus , Varghese Prabha , Lees Stephen , Nimmerjahn Falk , Meyer Aaron S.
Published: February 2023

Immunoglobulin (Ig)G antibodies coordinate immune effector responses by selectively binding to target antigens and then interacting with various effector cells via the Fcγ receptors. The Fc domain of IgG can promote or inhibit distinct effector responses across several different immune cell types through variation based on subclass and Fc domain glycosylation. Extensive characterization of these interactions has revealed how the inclusion of certain Fc subclasses or glycans results in distinct immune responses. During an immune response, however, IgG is produced with mixtures of Fc domain properties, so antigen-IgG immune complexes are likely to almost always be comprised of a combination of Fc forms. Whether and how this mixed composition influences immune effector responses has not been examined. Here, we measured Fcγ receptor binding to immune complexes of mixed Fc domain composition. We found that the binding properties of the mixed-composition immune complexes fell along a continuum between those of the corresponding pure cases. Binding quantitatively matched a mechanistic binding model, except for several low-affinity interactions mostly involving IgG2. We found that the affinities of these interactions are different than previously reported, and that the binding model could be used to provide refined estimates of these affinities. Finally, we demonstrated that the binding model can predict effector-cell elicited platelet depletion in humanized mice, with the model inferring the relevant effector cell populations. Contrary to the previous view in which IgG2 poorly engages with effector populations, we observe appreciable binding through avidity, but insufficient amounts to observe immune effector responses. Overall, this work demonstrates a quantitative framework for reasoning about effector response regulation arising from IgG of mixed Fc composition.

Summary points

The binding behavior of mixed Fc immune complexes is a blend of the binding properties for each constituent IgG species.

An equilibrium, multivalent binding model can be generalized to incorporate immune complexes of mixed Fc composition.

Particularly for low-affinity IgG-Fcγ receptor interactions, immune complexes provide better estimates of affinities.

The FcγR binding model predicts effector-elicited cell clearance in humanized mice.
Cancer cell sedimentation in 3D cultures reveals active migration regulated by self-generated gradients and adhesion sites
Authors: Dimitriou Nikolaos M. , Flores-Torres Salvador , Kyriakidou Maria , Kinsella Joseph Matthew , Mitsis Georgios D.
Published: February 2023

Cell sedimentation in 3D hydrogel cultures refers to the vertical migration of cells towards the bottom of the space. To explain this poorly understood phenomenon, we conducted a multiscale experimental and mathematical examination of 3D cancer growth in triple negative breast cancer cells. Migration was examined in the presence and absence of Paclitaxel, in high and low adhesion environments and in the presence of fibroblasts. The observed behaviour was modeled by hypothesizing active migration due to self-generated chemotactic gradients. Our results confirmed this hypothesis, whereby migration was regulated by the MAPK and TGF-β pathways. The mathematical model enabled us to describe the experimental data in absence (normalized error< 40%) and presence of Paclitaxel (normalized error< 10%), suggesting inhibition of random motion and advection in the latter case. Inhibition of sedimentation in low adhesion and co-culture experiments further supported the conclusion that cells actively migrated downwards due to the presence of signals produced by cells already attached to the adhesive glass surface.
Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis
Authors: Rosenberger George , Li Wenxue , Turunen Mikko , He Jing , Subramaniam Prem S , Pampou Sergey , Griffin Aaron T , Karan Charles , Kerwin Patrick , Murray Diana , Honig Barry , Liu Yansheng , Califano Andrea
Published: February 2023

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies, over the past decade, we developed VESPA—an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations—and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.
Highly sensitivein vivodetection of dynamic changes in enkephalins following acute stress
Authors: Mikati Marwa O. , Erdmann-Gilmore Petra , Connors Rose , Conway Sineadh M. , Malone Jim , Woods Justin , Sprung Robert W. , Townsend R. Reid , Al-Hasani Ream
Published: February 2023

Enkephalins are opioid peptides that modulate analgesia, reward, and stress.In vivodetection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani, 2018, eLife). Here we show improved detection resolution and stabilization of enkephalin detection, which allowed us to investigate enkephalin release during acute stress. We present an analytical method for real-time, simultaneous detection of Met- and Leu-Enkephalin (Met-Enk & Leu-Enk) in the mouse Nucleus Accumbens shell (NAcSh) after acute stress. We confirm that acute stress activates enkephalinergic neurons in the NAcSh using fiber photometry and that this leads to the release of Met- and Leu-Enk. We also demonstrate the dynamics of Met- and Leu-Enk release as well as how they correlate to one another in the ventral NAc shell, which was previously difficult due to the use of approaches that relied on mRNA transcript levels rather than post-translational products. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enkephalin through methionine oxidation, and provides novel insight into the relationship between Met- and Leu-Enkephalin following stress.
Mitochondrial metabolism inDrosophilamacrophage-like cells regulates body growth via modulation of cytokine and insulin signaling
Authors: Sriskanthadevan-Pirahas Shrivani , Tinwala Abdul Qadeer , Turingan Michael J , Khan Shahoon , Grewal Savraj S
Published: February 2023

Macrophages play key roles in regulating and maintaining tissue and whole-body metabolism in both normal and disease states. While the cell-cell signaling pathway that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. Here we investigate this by examining Drosophila macrophage-like cells called hemocytes. We used knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. We find that this reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. We also find that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, we show that genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size but without altering hemocyte numbers. Our data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that mitochondrial metabolism can be controlled by nutrient availability, our findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis.
Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation
Authors: Evans Christopher R. , Smiley Marina K. , Thio Sean Asahara , Wei Mian , Price-Whelan Alexa , Min Wei , Dietrich Lars E.P.
Published: February 2023

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacteriumPseudomonas aeruginosaproduces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. Here, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone ofP. aeruginosabiofilms. Deletion ofrpoSorcrctherefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity—an apparent beneficial effect—in hypoxic/anoxic subpopulations within biofilms. However, we also find that biofilms lacking Crc show increased sensitivity to an exogenously added methylated phenazine, indicating that the increased metabolic activity in this mutant comes at a cost. Together, these results suggest that complex regulation of PhzM allowsP. aeruginosato simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.

Significance Statement

P. aeruginosacauses biofilm-based infections and is known for its production of colorful phenazine derivatives. Among these the methylated phenazines are the most toxic and can cause condition-dependent damage to their producer. In this study, we show that methylated phenazines also have a beneficial effect in that they specifically support metabolic activity at depth inP. aeruginosabiofilms, where oxygen limitation would otherwise stall metabolism. We describe a new link betweenP. aeruginosaglobal regulators that control methylated phenazine production in a manner that limits their toxicity while simultaneously enabling their contribution to metabolism. These results expand our understanding of the strategies that enableP. aeruginosasurvival in multicellular structures, which is key to its success during chronic host colonization.