bluuebunny/crossref_metadata_2025 huggingface dataset:
TorchLens: A Python package for extracting and visualizing hidden activations of PyTorch models
Authors: Taylor JohnMark , Kriegeskorte Nikolaus
Published: March 2023

Deep neural network models (DNNs) are essential to modern AI and provide powerful models of information processing in biological neural networks. Researchers in both neuroscience and engineering are pursuing a better understanding of the internal representations and operations that undergird the successes and failures of DNNs. Neuroscientists additionally evaluate DNNs as models of brain computation by comparing their internal representations to those found in brains. It is therefore essential to have a method to easily and exhaustively extract and characterize the results of the internal operations of any DNN. Many models are implemented in PyTorch, the leading framework for building DNN models. Here we introduceTorchLens, a new open-source Python package for extracting and characterizing hidden-layer activations in PyTorch models. Uniquely among existing approaches to this problem,TorchLenshas the following features: (1) it exhaustively extracts the results of all intermediate operations, not just those associated with PyTorch module objects, yielding a full record of every step in the model’s computational graph, (2) it provides an intuitive visualization of the model’s complete computational graph along with metadata about each computational step in a model’s forward pass for further analysis, (3) it contains a built-in validation procedure to algorithmically verify the accuracy of all saved hidden-layer activations, and (4) the approach it uses can be automatically applied to any PyTorch model with no modifications, including models with conditional (if-then) logic in their forward pass, recurrent models, branching models where layer outputs are fed into multiple subsequent layers in parallel, and models with internally generated tensors (e.g., injections of noise). Furthermore, usingTorchLensrequires minimal additional code, making it easy to incorporate into existing pipelines for model development and analysis, and useful as a pedagogical aid when teaching deep learning concepts. We hope this contribution will help researchers in AI and neuroscience understand the internal representations of DNNs.
Reconstructing prehistoric viral genomes from Neanderthal sequencing data
Authors: Ferreira Renata C. , Alves Gustavo V. , Ramon Marcello , Antoneli Fernando , Briones Marcelo R. S.
Published: March 2023

DNA viruses that produce persistent infections have been proposed as potential causes for the extinction of Neanderthals and therefore, the identification of viral genome remnants in Neanderthal sequence reads is an initial step to address this hypothesis. Here, as proof of concept, we searched for viral remnants in sequence reads of Neanderthal genome data by mapping to adenovirus, herpesvirus and papillomavirus, which are double stranded DNA viruses that may establish lifelong latency and can, produce persistent infections. The reconstructed ancient viral genomes of adenovirus, herpesvirus and papillomavirus revealed conserved segments, with nucleotide identity to extant viral genomes, and variable regions in coding regions with substantial divergence to extant close relatives. Sequence reads mapped to extant viral genomes showed deamination patterns of ancient DNA and that these ancient viral genomes showed divergence consistent with the age of these samples (≈50,000 years) and viral evolutionary rates (10-5to 10-8substitutions/site/year). Analysis of random effects shows that the Neanderthal mapping to genomes of extant persistent viruses is above the expected by random similarities of short reads. Also, negative control with a nonpersistent DNA virus does not yield statistically significant assemblies. This work demonstrates the feasibility of identifying viral genome remnants in archaeological samples with signal-to-noise assessment.
Expanding the Characterization of Microbial Ecosystems using DIA-PASEF Metaproteomics
Authors: Gómez-Varela David , Xian Feng , Grundtner Sabrina , Sondermann Julia Regina , Carta Giacomo , Schmidt Manuela
Published: March 2023

Metaproteomics is gaining momentum in microbiome research due to the multi-dimensional information it provides. However, current approaches have reached their detection limits. We present a highly-sensitive metaproteomic workflow using the extra information captured by Parallel Accumulation-Serial Fragmentation (PASEF) technology. The comparison of different acquisition modes and data analysis software packages showed that DIA-PASEF and DIA-NN doubled protein identifications of mouse gut microbiota and, importantly, also of the host proteome. DIA-PASEF significantly improved peptide detection reproducibility and quantification accuracy, which resulted in more than twofold identified taxa, reaching depths comparable to metagenomic studies. Consequently, DIA-PASEF exhibited improved coverage of functional networks revealing 131 additional pathways compared to DDA-PASEF. We applied our optimized workflow to a pre-clinical mouse model of chronic pain, in which we deciphered novel host-microbiome interactions. In summary, we present here a metaproteomic approach that paves the way for increasing the functional characterization of microbiome ecosystems and is applicable to diverse fields of biological research.
Aedes aegyptigut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes
Authors: Hegde Shivanand , Brettell Laura E. , Quek Shannon , Etebari Kayvan , Saldaña Miguel A. , Asgari Sassan , Coon Kerri L. , Heinz Eva , Hughes Grant L.
Published: March 2023

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, far more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities are associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique.
Loss Of function of Male-Specific Lethal 3 (Msl3) Does Not Affect Spermatogenesis in Rodents
Authors: Mitchell T.A. , Lin J.M. , Hicks S.M. , James J.R. , Rangan P. , Forni P.E.
Published: March 2023

Msl3 is a member of the chromatin-associated male-specific lethal MSL complex which is responsible for the transcriptional upregulation of genes on the X chromosome in males Drosophila. Although the dosage complex operates differently in mammals, the Msl3 gene is conserved from flies to humans.

Msl3 is required for meiotic entry during Drosophila oogenesis. Recent reports indicate that also in primates, Msl3 is expressed in undifferentiated germline cells before meiotic entry. However, if Msl3 plays a role in the meiotic entry of mammals has yet to be explored. To study this, we used mouse spermatogenesis as a study model. Analyses of single cells RNA-seq data revealed that, in mice, Msl3 is mostly expressed in meiotic cells.

To test the role of Msl3 in meiosis, we used a male germline-specific Stra8-iCre driver and a newly generated Msl3floxconditional knock-out mouse line. Msl3 conditional loss-of-function in spermatogonia did not cause spermatogenesis defects or changes in the expression of genes related to meiosis. Our data suggest that, in mice, Msl3 exhibits delayed expression compared to Drosophila and primates, and loss-of-function mutations disrupting the chromodomain of Msl3 alone do not impede meiotic entry in rodents.
Simple and highly efficient detection of PSD95 using a nanobody and its recombinant minibody derivative
Authors: Kilisch Markus , Gere-Becker Maja , Wüstefeld Liane , Bonnas Christel , Crauel Alexander , Mechmershausen Maja , Martens Henrik , Götzke Hansjörg , Opazo Felipe , Frey Steffen
Published: March 2023

The post-synaptic density protein 95 (PSD95) is a crucial scaffolding protein participating in the organization and regulation of synapses. PSD95 interacts with numerous molecules, including neurotransmitter receptors and ion channels. The functional dysregulation of PSD95 as well as its abundance and localization has been implicated with several neurological disorders, making it an attractive target to develop strategies able to monitor PSD95 accurately for diagnostics and therapeutics. This study characterizes a novel camelid single-domain antibody (nanobody) that binds strongly and with high specificity to rat, mouse and human PSD95. This nanobody allows for a more precise detection and better quantification of PSD95 in various biological samples. We expect that the flexibility and unique performance of this thoroughly characterized affinity tool will help to further understand the role of PSD95 in normal and diseased neuronal synapses.
Effect of predicting familiar melodies on alpha power
Authors: Ito Shuma , Matsunaga Kazuki , Chanpornpakdi Ingon , Tanaka Toshihisa
Published: March 2023

The processing of music and language share similar characteristics. Previous studies indicated that the similarity between language and melody was observed in event-related potentials to the deviation of a word and tone, respectively. We focused on a language study that demonstrated strong suppression of the alpha power in the presence of easily predictable words. Motivated by the physiological similarity between language and music, this study hypothesized that predictable music might suppress the alpha power. We measured electroencephalogram (EEG) signals while a melody followed by silence was presented for a participant who imagined the melody during the silent part of the music. The participants scored the melody’s familiarity to quantify the ease of prediction and imagination. We observed similarity to language processing. For familiar melodies, alpha power suppression was observed in the left frontal and left central regions. Further, we observed Bereitschaftspotential (a negative slope) in both familiar and unfamiliar conditions before the silent interval. Moreover, a network analysis revealed that information flow from the right sensory-motor cortex to the right auditory cortex in the beta band was stronger for familiar music than for unfamiliar music. Considering the previous findings that motor preparation and execution suppress the alpha power in the left frontal and left central regions, the alpha band suppression under music prediction suggests a motor interaction during music processing in the prediction of melodies.
ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways
Authors: Rubina Anzelika , Patel Mihil , Nightingale Katie , Potts Martin , Fielding Ceri A. , Kollnberger Simon , Lau Betty , Ladell Kristin , Miners Kelly L. , Nichols Jenna , Nobre Luis , Roberts Dawn , Trinca Terrence M. , Twohig Jason P. , Vlahava Virginia-Maria , Davison Andrew J. , Price David A. , Tomasec Peter , Wilkinson Gavin W.G. , Weekes Michael P. , Stanton Richard J. , Wang Eddie C.Y.
Published: March 2023

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defences. In exploring the finding that HCMV infection upregulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory anti-viral cytokine TNFa, we discovered the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype ‘sheddase’, a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in itsUL/b’ region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with a HCMV double deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (p<0.05) in an ADAM17-dependent fashion. These included known substrates of ADAM17 with established immunological functions such as TNFR2 and Jagged1, but also numerous novel host and viral targets, such as Nectin1, UL8 and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

Significance statement

Human cytomegalovirus (HCMV) is an important pathogen, being the commonest infectious cause of brain damage to babies and the primary reason for hospital readmissions in transplant recipients. Even though HCMV induces the strongest immune responses by any human pathogen, it evades host defences and persists for life. This study describes a novel immunoregulatory strategy through which HCMV modulates multiple immune pathways simultaneously, by targeting a single host protein. HCMV UL148 and UL148D impair the maturation of the sheddase, A Disintegrin And Metalloproteinase 17, profoundly altering surface expression of numerous immunoregulatory proteins. This is the first description of viral genes targeting this pathway. Our findings may be relevant for future viral therapies and understanding the impact of HCMV in developmental biology.
Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer
Authors: Traphagen Nicole A. , Schwartz Gary N. , Tau Steven , Jiang Amanda , Hosford Sarah R. , Goen Abigail E. , Roberts Alyssa M. , Romo Bianca A. , Johnson Anneka L. , Duffy Emily-Claire K. , Demidenko Eugene , Heverly Paul , Mosesson Yaron , Soucy Shannon M. , Kolling Fred , Miller Todd W.
Published: March 2023

Purpose

Clinical evidence indicates that treatment with estrogens elicits anti-cancer effects in ∼30% of patients with advanced endocrine-resistant estrogen receptor alpha (ER)-positive breast cancer. Despite the proven efficacy of estrogen therapy, its mechanism of action is unclear and this treatment remains under-utilized. Mechanistic understanding may offer strategies to enhance therapeutic efficacy.

Experimental Design

We performed genome-wide CRISPR/Cas9 screening and transcriptomic profiling in long-term estrogen-deprived (LTED) ER+ breast cancer cells to identify pathways required for therapeutic response to the estrogen 17β-estradiol (E2). We validated findings in cell lines, patient-derived xenografts (PDXs), and patient samples, and developed a novel combination treatment through testing in cell lines and PDX models.

Results

Cells treated with E2 exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops). Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced E2-induced DNA damage. PARP inhibition synergized with E2 to suppress growth and prevent tumor recurrence inBRCA1/2-mutant andBRCA1/2-wild-type cell line and PDX models.

Conclusions

E2-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells. Inhibition of the DNA damage response using drugs such as PARP inhibitors can enhance therapeutic response to E2. These findings warrant clinical exploration of the combination of E2 with DNA damage response inhibitors in advanced ER+ breast cancer, and suggest that PARP inhibitors may synergize with therapeutics that exacerbate transcriptional stress.
Neuron-specific protein expression at striatal excitatory synapses
Authors: Wang Yi-Zhi , Perez-Rosello Tamara , Surmeier D. James , Savas Jeffrey N.
Published: March 2023

Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types1-5. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies6-9. To overcome the limitations associated with bulk measures of synaptic protein abundance6,10, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomesin vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Studies in mice lacking Shank3 gene exons 13-16 revealed a robust postsynaptic proteome alteration in iSPNs. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.
The relationship between object-based spatial ability and virtual navigation performance
Authors: Garg Tanya , Velasco Pablo Fernández , Patai Zita Eva , Malcolm Charlotte P. , Kovalets Victor , Bohbot Veronique D. , Coutrot Antoine , Hegarty Mary , Hornberger Michael , Spiers Hugo J.
Published: March 2023

Spatial navigation is a multi-faceted behaviour drawing on many different aspects of cognition. Visuospatial abilities, such as spatial working memory and mental rotation, in particular, may be key factors. A range of tests have been developed to assess visuospatial processing and memory, but how such tests relate to navigation ability remains unclear. This understanding is important to advance tests of navigation for disease monitoring in Alzheimer’s Disease, where disorientation is an early symptom. Here, we report the use of an established mobile gaming app, Sea Hero Quest, as a measure of navigation ability. We used three separate tests of navigation embedded in the game: wayfinding, path integration and spatial memory in a radial arm maze. In the same participants, we also collected measures of mental rotation (Mental Rotation Test), visuospatial processing (Design Organization Test) and visuospatial working memory (Digital Corsi). We found few strong correlations across our measures. Being good at wayfinding in a virtual navigation test does not mean an individual will rate themself as a confident navigator, be good at path integration, or have a superior memory in a radial arm maze. However, those good at wayfinding tend to perform well on the three visuospatial tasks examined here, and to also use a landmark strategy in the radial maze task. These findings help clarify the inter-relationships between different abilities supporting visuospatial and navigation skills.

Highlights

Three navigation tests embedded in the game Sea Hero Quest were examined in relation to three object-based visuospatial tasks, and self-ratings of navigation ability and stress during navigation.

No associations were observed among performance on wayfinding, path integration and radial arm maze levels of Sea Hero Quest.

Object-based visuospatial abilities were selectively correlated with performance on wayfinding levels of Sea Hero Quest.

Gameplay stress and navigation strategy were not associated with performance on Sea Hero Quest navigation tasks.
Rise of the Cybercrabs: how digital cloning in an integrated taxonomic framework can support deep-sea exploration
Authors: Reynaud Emmanuel G. , Gutierrez-Heredia Luis , Garbett Amy , Horn Esben , Carlsson Jens , Collins Patrick C.
Published: March 2023

Taxonomy has been a labour-intensive field of expertise based on hours of manual work and lengthy comments that are mainly bound to books and publications on a two-dimensional world. But every species described is a three-dimensional organism that needs to be seen, manipulated to be fully understood in its native shape. Nowadays, digital technology allows us to transform everyone in an avatar or a digital clone with ease, but collections do not provide many type specimens in a digital format. Here we present a simple approach used to study a specific deep-sea crabSegonzacia mesatlanticaand provide online digital taxonomy across four repository sites. This offers the possibility to describe, exchange digitally and analyse specimens in its full 3D, establish their taxonomy and share them widely on online databases as well as physically by additive manufacturing to duplicate them in collections and outreach activities. Using an integrated taxonomic approach that included the use of 3D type specimens and molecular barcoding we provide evidence that the genusSegonzaciamay be more diverse than previously understood
Choroid Plexus Modulates Subventricular Zone Adult Neurogenesis and Olfaction Through Secretion of Small Extracellular Vesicles
Authors: Liu Luke L. , Shannahan Jonathan , Zheng Wei
Published: March 2023

The choroid plexus (CP) in brain ventricles secrete cerebrospinal fluid (CSF) that bathes the adjacent subventricular zone (SVZ); the latter is the largest neurogenic region in adult brain harboring neural stem/progenitor cells (NSPCs) and supplies newborn neurons to the olfactory bulb (OB) for normal olfaction. We discovered the presence of a CP-SVZ regulatory (CSR) axis in which the CP, by secreting small extracellular vesicles (sEVs), regulated adult neurogenesis in the SVZ and maintained olfaction. The proposed CSR axis was supported by 1) differential neurogenesis outcomes in the OB when animals treated with intracerebroventricular (ICV) infusion of sEVs collected from the CP of normal or manganese (Mn)-poisoned mice, 2) progressively diminished SVZ adult neurogenesis in mice following CP-targeted knockdown of SMPD3 to suppress CP sEV secretion, and 3) compromised olfactory performance in these CP-SMPD3-knockdown mice. Collectively, our findings demonstrate the biological and physiological presence of this sEV-dependent CSR axis in adult brains.

Highlights

CP-secreted sEVs regulate adult neurogenesis in the SVZ.

CP-secreted sEVs modulate newborn neurons in the OB.

Suppression of sEV secretion from the CP deteriorates olfactory performance.
FLT3 signaling inhibition preserves opioid analgesia while abrogating tolerance and hyperalgesia
Authors: Jouvenel Antoine , Tassou Adrien , Thouaye Maxime , Ruel Jérôme , Antri Myriam , Leyris Jean-Philippe , Paquet Aurore , Mallié Sylvie , Sar Chamroeum , Diouloufet Lucie , Sonrier Corinne , Daubeuf François , Bertin Juliette , Alves Stacy , Ventéo Stéphanie , Frossard Nelly , Carroll Patrick , Mechaly Ilana , Rognan Didier , Sokoloff Pierre , Dallel Radhouane , Delmas Patrick , Valmier Jean , Rivat Cyril
Published: March 2023

Opioid analgesia is counteracted on chronic use by tolerance and hyperalgesia inducing dose escalation and life-threatening overdoses. Mu opiate receptors (MOR) expressed in primary sensory neurons were recently found to control tolerance and hyperalgesia, but the underlying mechanisms remained elusive. Here we show that genetic inactivation offms-like tyrosine kinase receptor 3 (FLT3) receptor in sensory neurons abrogates morphine tolerance and hyperalgesia by preventing MOR-induced hyperactivation of the cAMP signaling pathway and subsequent excitatory adaptive processes. Moreover, the specific FLT3 inhibitor BDT001 potentiates morphine analgesia in acute and chronic pain models, without aggravating morphine adverse effects, and reverses tolerance and hyperalgesia once installed. Thus, FLT3 appears as a key regulator of the MOR signaling pathway and its pharmacological blockade shows promise to enhance chronic opioid analgesic efficacy.
Pangenomes reveal genomic signatures of microbial adaptation to experimental soil warming
Authors: Choudoir Mallory J. , Narayanan Achala , Rodriguez-Ramos Damayanti , Simoes Rachel , Efroni Alon , Sondrini Abigail , DeAngelis Kristen M.
Published: March 2023

Below-ground carbon transformations represent a natural climate change mitigation solution, but newly-acquired traits adaptive to climate stress may alter microbial climate feedback mechanisms. To better define microbial evolutionary responses to long-term climate warming, we study microorganisms from an ongoing in situ soil warming experiment at the Harvard Forest Long-term Ecological Research (LTER) site where, for over three decades, soils are continuously heated 5 °C above ambient temperatures. We hypothesize that across generations of chronic warming, genomic signatures within diverse bacterial lineages reflect trait-based adaptations related to growth and carbon utilization. From our bacterial culture collection isolated from experimental heated and control plots, we sequenced genomes representing taxa dominant in soil communities and sensitive to warming, including lineages of Alphaproteobacteria, Actinobacteria, and Betaproteobacteria. We investigated differences in genomic attributes and patterns of functional gene content to identify genomic signatures of adaptation. Comparative pangenomics revealed accessory gene clusters related to central metabolism, competition, and carbon substrate degradation. Overall, genomes from control plots were relatively enriched in carbon and fatty acid metabolism pathways, while genomes from heated plots were relatively enriched in nitrogen metabolism pathways. We also observed differences in global codon usage bias between heated and control genomes, suggesting potential adaptive traits related to growth or growth efficiency. This effect was more varied for organisms with fewer 16S rrn operons, suggesting that these organisms experience different selective pressures on growth efficiency. Together, these data illustrate the emergence of lineage-specific traits as well as common ecological-evolutionary microbial responses to climate change.
MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states
Authors: Bailey Logan R.J. , Bugg Darrian , Reichardt Isabella M. , Ortaç C. Dessirée , Gunaje Jagadambika , Johnson Richard , MacCoss Michael J. , Sakamoto Tomoya , Kelly Daniel P. , Regnier Michael , Davis Jennifer M.
Published: March 2023

Discovering determinants of cardiomyocyte maturity and the maintenance of differentiated states is critical to both understanding development and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Here, the RNA binding protein Muscleblind-like 1 (MBNL1) was identified as a critical regulator of cardiomyocyte differentiated states and their regenerative potential through transcriptome-wide control of RNA stability. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of the estrogen-related receptor signaling axis was essential for maintaining cardiomyocyte maturity. In accordance with these data, modulating MBNL1 dose tuned the temporal window of cardiac regeneration, where enhanced MBNL1 activity arrested myocyte proliferation, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. Collectively these data suggest MBNL1 acts as a transcriptome-wide switch between regenerative and mature myocyte states postnatally and throughout adulthood.
Curved adhesions mediate cell attachment to soft matrix fibres in 3D
Authors: Zhang Wei , Lu Chih-Hao , Nakamoto Melissa L. , Tsai Ching-Ting , Roy Anish R. , Lee Christina E. , Yang Yang , Jahed Zeinab , Li Xiao , Cui Bianxiao
Published: March 2023

Mammalian cells adhere to the extracellular matrix (ECM) and sense mechanical cues through integrin-mediated adhesions1, 2. Focal adhesions and related structures are the primary architectures that transmit forces between the ECM and the actin cytoskeleton. Although focal adhesions are abundant when cells are cultured on rigid substrates, they are sparse in soft environments that cannot support high mechanical tensions3. Here, we report a new class of integrin-mediated adhesions, curved adhesions, whose formation is regulated by membrane curvature instead of mechanical tension. In soft matrices made of protein fibres, curved adhesions are induced by membrane curvatures imposed by the fibre geometry. Curved adhesions are mediated by integrin ɑVβ5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin β5 and a curvature-sensing protein FCHo2. We find that curved adhesions are prevalent in physiologically relevant environments. Disruption of curved adhesions by knocking down integrin β5 or FCHo2 abolishes the migration of multiple cancer cell lines in 3D matrices. These findings provide a mechanism of cell anchorage to natural protein fibres that are too soft to support the formation of focal adhesions. Given their functional importance for 3D cell migration, curved adhesions may serve as a therapeutic target for future development.
Sex differences in anxiety and threat avoidance in GAD65 knock-out mice
Authors: Ulrich Michelle , Pollali Evangelia , Çalışkan Gürsel , Stork Oliver , Albrecht Anne
Published: March 2023

Anxiety disorders have been linked to a disbalance of excitation and inhibition in a network of brain structures comprising frontal cortical regions, the amygdala and the hippocampus, among others. Recent imaging studies suggest sex differences in the activation of this anxiety network during the processing of emotional information. Rodent models with genetically altered ϒ-amino butyric acid (GABA) neurotransmission allow studying the neuronal basis of such activation shifts and their relation to anxiety endophenotypes, but to date sex effects have rarely been addressed. Using mice with a null mutation of the GABA synthetizing enzyme glutamate decarboxylase 65 (GAD65-/-), we started to compare anxiety-like behavior and avoidance in male vs. female GAD65-/- mice and their wildtype littermates. In an open field, female GAD65-/- mice displayed increased activity, while male GAD65-/- mice showed an increased adaptation of anxiety-like behavior over time. GAD65-/- mice of both sexes had a higher preference for social interaction partners, which was further heightened in male mice. In male mice higher escape responses were observed during an active avoidance task. Together, female mice showed more stable emotional responses despite GAD65 deficiency. To gain insights into interneuron function in network structures controlling anxiety and threat perception, fast oscillations (10-45 Hz) were measured inex vivoslice preparations of the anterior cingulate cortex (ACC). GAD65-/- mice of both sexes displayed increased gamma power in the ACC and a higher density of PV-positive interneurons, which are crucial for generating such rhythmic activity. In addition, GAD65-/- mice had lower numbers of somatostatin-positive interneurons in the basolateral amygdala and in the dorsal dentate gyrus especially in male mice, two key regions important for anxiety and active avoidance responses. Our data suggest sex differences in the configuration of GABAergic interneurons in a cortico-amygdala-hippocampal network controlling network activity patterns, anxiety and threat avoidance behavior.

Highlights

Role of GABA in sex-specific anxiety endophenotypes demonstrated in GAD65-/- mice

Sex- and GAD65-dependent alterations in anxiety, social preference and avoidance

Enhanced in vitro gamma-beta oscillations in ACC slices of GAD65-/- mice

Increased parvalbumin(+) interneuron counts in ACC slices of GAD65-/- mice

Reduced somatostatin(+) interneuron counts in dorsal DG and BLA of male GAD65-/- mice

Graphical Abstract
Transitions between cognitive topographies: contributions of network structure, neuromodulation, and disease
Authors: Luppi Andrea I. , Singleton S. Parker , Hansen Justine Y. , Bzdok Danilo , Kuceyeski Amy , Betzel Richard F. , Misic Bratislav
Published: March 2023

Patterns of neural activity underlie human cognition. Transitions between these patterns are orchestrated by the brain’s network architecture. What are the mechanisms linking network structure to cognitively relevant activation patterns? Here we implement principles of network control to investigate how the architecture of the human connectome shapes transitions between 123 experimentally defined cognitive activation maps (cognitive topographies) from the NeuroSynth meta-analytic engine. We also systematically incorporate neurotransmitter receptor density maps (18 receptors and transporters) and disease-related cortical abnormality maps (11 neurodegenerative, psychiatric and neurodevelopmental diseases;N= 17 000 patients,N= 22 000 controls). Integrating large-scale multimodal neuroimaging data from functional MRI, diffusion tractography, cortical morphometry, and positron emission tomography, we simulate how anatomically-guided transitions between cognitive states can be reshaped by pharmacological or pathological perturbation. Our results provide a comprehensive look-up table charting how brain network organisation and chemoarchitecture interact to manifest different cognitive topographies. This computational framework establishes a principled foundation for systematically identifying novel ways to promote selective transitions between desired cognitive topographies.
Coupling of Slack and NaV1.6 sensitizes Slack to quinidine blockade and guides anti-seizure strategy development
Authors: Yuan Tian , Wang Yifan , Jin Yuchen , Yang Hui , Xu Shuai , Zhang Heng , Chen Qian , Li Na , Ma Xinyue , Song Huifang , Peng Chao , Geng Ze , Dong Jie , Duan Guifang , Sun Qi , Yang Yang , Yang Fan , Huang Zhuo
Published: March 2023

Quinidine has been used as an anticonvulsant to treat patients with KCNT1-related epilepsy by targeting gain-of-function KCNT1 pathogenic mutant variants. However, the detailed mechanism underlying quinidine’s blockade against KCNT1 (Slack) remains elusive. Here, we report a functional and physical coupling of the voltage-gated sodium channel NaV1.6 and Slack. NaV1.6 binds to and highly sensitizes Slack to quinidine blockade. Homozygous knockout of NaV1.6 reduces the sensitivity of native sodium-activated potassium currents to quinidine blockade. NaV1.6-mediated sensitization requires the involvement of NaV1.6’s N-and C-termini binding to Slack’s C-terminus, and is enhanced by transient sodium influx through NaV1.6. Moreover, disrupting the Slack-NaV1.6 interaction by viral expression of Slack’s C-terminus can protect against SlackG269S-induced seizures in mice. These insights about a Slack-NaV1.6 complex challenge the traditional view of “Slack as an isolated target” for anti-epileptic drug discovery efforts, and can guide the development of innovative therapeutic strategies for KCNT1-related epilepsy.

GRAPHICAL ABSTRACT
Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae
Authors: Pelaez Julianne N. , Gloss Andrew D. , Goldman-Huertas Benjamin , Kim Bernard , Lapoint Richard T. , Pimentel-Solorio Giovani , Verster Kirsten I. , Aguilar Jessica M. , Dittrich Anna C. Nelson , Singhal Malvika , Suzuki Hiromu C. , Matsunaga Teruyuki , Armstrong Ellie E. , Charboneau Joseph L.M. , Groen Simon C. , Hembry David H. , Ochoa Christopher J. , O’Connor Timothy K. , Prost Stefan , Zaaijer Sophie , Nabity Paul D. , Wang Jiarui , Rodas Esteban , Liang Irene , Whiteman Noah K.
Published: March 2023

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families – genes directly mediating interactions with plant chemical defenses – underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution acrossScaptomyza,a genus nested withinDrosophilathat includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorousScaptomyzahave among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions inDrosophila.
Bioactive Natural Product Discovery via Deuterium Adduct Bioactivity Screening
Authors: Zill N.A. , Du Y. , Marinkovich S. , Gu D. , Seidel J. , Zhang W.
Published: March 2023

The discovery of bioactive natural products lies at the forefront of human medicine. The continued discovery of these molecules is imperative in the fight against infection and disease. While natural products have historically dominated the drug market, discovery in recent years has slowed significantly, partly due to limitations in current discovery methodologies. This work demonstrates a new workflow, Deuterium Adduct Bioactivity Screening (DABS), which pairs untargeted isotope labeling with whole cell binding assays for bioactive natural product discovery. DABS was validated and led to the discovery of a new isoprenyl guanidine alkaloid, zillamycin, which showed anti-cancer and anti-microbial activities. DABS thus represents a new workflow to accelerate discovery of natural products with a wide range of bioactive potential.
The Ribosome Profiling landscape of yeast reveals a high diversity in pervasive translation
Authors: Papadopoulos Chris , Arbes Hugo , Chevrollier Nicolas , Blanchet Sandra , Cornu David , Roginski Paul , Rabier Camille , Atia Safiya , Lespinet Olivier , Namy Olivier , Lopes Anne
Published: March 2023

Pervasive translation is a widespread phenomenon that plays an important role in de novo gene birth; however, its underlying mechanisms remain unclear. Based on multiple Ribosome Profiling (Ribo-Seq) datasets, we investigated the RiboSeq landscape of coding and noncoding regions of yeast. Therefore, we developed a representation framework which allows the visual representation and rational classification of the entire diversity of Ribo-Seq signals that could be observed in yeast. We show that if coding regions are restricted to specific areas of the Ribo-Seq landscape, noncoding regions are associated with a wide diversity of translation signals and, conversely, populate the entire yeast Ribo-Seq landscape. Specifically, we reveal that noncoding regions are associated with canonical translation signals, but also with non-canonical ones absent from coding regions, and which appear to be a hallmark of pervasive translation. Notably, we report thousands of translated noncoding ORFs among which, 251 led to detectable products with Mass Spectrometry while being characterized by a wide range of translation specificities. Overall, we show that pervasive translation is not random with noncoding ORF translation signals being consistent across Ribo-Seq experiments. Finally, we show that the translation signal of noncoding ORFs is not explained by features related to the emergence of function, but rather determined by the translation start codon and the codon distribution in their two alternative frames. Overall, our results enable us to propose a topology of the pervasive Ribo-Seq landscape of a species, and open the way to future comparative analyses of this translation landscape under different conditions.
Clec12a tempers inflammation while restricting expansion of a colitogenic commensal
Authors: Chiaro Tyson R. , Bauer Kaylyn M. , Ost Kyla S. , Stephen-Victor Emmanuel , Nelson Morgan C. , Hill Jennifer H. , Bell Rickesha , Harwood Morgan , Voth Warren , Jackson Taylor , Klag Kendra A , O’Connell Ryan M. , Zac Stephens W. , Round June L.
Published: March 2023

SUMMARY

Regulation of the microbiota is critical to intestinal health yet the mechanisms employed by innate immunity remain unclear. Here we show that mice deficient in the C-Type-lectin receptor, Clec12a developed severe colitis, which was dependent on the microbiota. Fecal-microbiota-transplantation (FMT) studies into germfree mice revealed a colitogenic microbiota formed within Clec12a-/-mice that was marked by expansion of the gram-positive organism,Faecalibaculum rodentium. Treatment withF. rodentiumwas sufficient to worsen colitis in wild-type mice. Macrophages within the gut express the highest levels of Clec12a. Cytokine and sequencing analysis in Clec12a-/-macrophages revealed heighten inflammation but marked reduction in genes associated with phagocytosis. Indeed, Clec12a-/-macrophages are impaired in their ability to uptakeF. rodentium.Purified Clec12a had higher binding to gram-positive organisms such asF. rodentium. Thus, our data identifies Clec12a as an innate immune surveillance mechanism to control expansion of potentially harmful commensals without overt inflammation.
Alamar Blue assay optimization to minimize drug interference and inter-assay viability
Authors: Dinh Mina N. , Hitomi Masahiro , Al-Turaihi Zahraa A. , Scott Jacob G.
Published: March 2023

Alamar Blue (AB) has become an increasingly popular reagent of choice for cell viability assays. We chose AB over other reagents such as MTT and Cell-Titer Glo due to its cost effectiveness and its ability to be a nondestructive assay. While analyzing the effect of osimertinib, an EGFR inhibitor on the non-small cell lung cancer cell line, PC-9, we noticed unexpected right-shifts of dose response curves as compared to the curve obtained by Cell Titer Glo assay. Here, we describe our modified AB assay method to avoid right shift right shift in dose response curve. Unlike some of the redox drugs that were reported to directly affected AB reading, osimertinib itself did not directly increase AB reading. Yet, the removal of the drug containing medium prior to AB addition eliminated falsely increased reading giving comparable dose response curve as the one determined by Cell Titer Glo assay. When a panel of 11 drugs were assessed, we found that this modified AB assay eliminated unexpected similar right shifts detected in other epidermal growth factor receptor (EGFR) inhibitors. We also found that plate-to-plate variability can be minimized by adding an appropriate concentration of rhodamine B solution to the assay plates to calibrate fluorimeter sensitivity. This calibration method also enables a continuous longitudinal assay to monitor cell growth or recovery from drug toxicity over time. Our new modified AB assay is expected to provide accuratein vitromeasurement of EGFR targeted therapies.
Frequency-dependent ecological interactions increase the prevalence, and shape the distribution, of pre-existing drug resistance
Authors: Maltas Jeff , Tadele Dagim Shiferaw , Durmaz Arda , McFarland Christopher D. , Hinczewski Michael , Scott Jacob G.
Published: March 2023

The evolution of resistance remains one of the primary challenges for modern medicine from infectious diseases to cancers. Many of these resistance-conferring mutations often carry a substantial fitness cost in the absence of treatment. As a result, we would expect these mutants to undergo purifying selection and be rapidly driven to extinction. Nevertheless, pre-existing resistance is frequently observed from drug-resistant malaria to targeted cancer therapies in non-small cell lung cancer (NSCLC) and melanoma. Solutions to this apparent paradox have taken several forms from spatial rescue to simple mutation supply arguments. Recently, in an evolved resistant NSCLC cell line, we found that frequency-dependent ecological interactions between ancestor and resistant mutant ameliorate the cost of resistance in the absence of treatment. Here, we hypothesize that frequency-dependent ecological interactions in general play a major role in the prevalence of pre-existing resistance. We combine numerical simulations with robust analytical approximations to provide a rigorous mathematical framework for studying the effects of frequency-dependent ecological interactions on the evolutionary dynamics of pre-existing resistance. First, we find that ecological interactions significantly expand the parameter regime under which we expect to observe pre-existing resistance. Next, even when positive ecological interactions between mutants and ancestors are rare, these resistant clones provide the primary mode of evolved resistance because even weak positive interaction leads to significantly longer extinction times. We then find that even in the case where mutation supply alone is sufficient to predict pre-existing resistance, frequency-dependent ecological forces still contribute a strong evolutionary pressure that selects for increasingly positive ecological effects (negative frequency-dependent selection). Finally, we genetically engineer several of the most common clinically observed resistance mechanisms to targeted therapies in NSCLC, a treatment notorious for pre-existing resistance. We find that each engineered mutant displays a positive ecological interaction with their ancestor. As a whole, these results suggest that frequency-dependent ecological effects can play a crucial role in shaping the evolutionary dynamics of pre-existing resistance.
A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α–primase
Authors: Mullins Elwood A. , Salay Lauren E. , Durie Clarissa L. , Bradley Noah P. , Jackman Jane E. , Ohi Melanie D. , Chazin Walter J. , Eichman Brandt F.
Published: March 2023

The mechanism by which polymerase α–primase (polα–primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα–primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5′-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα–primase.