bluuebunny/crossref_metadata_2025 huggingface dataset:
Precision-Cut Liver Slices as anex vivomodel to evaluate antifibrotic therapies for liver fibrosis and cirrhosis
Authors: Wang Yongtao , Leaker Ben , Qiao Guoliang , Sojoodi Mozhdeh , Eissa Ibrahim Ragab , Epstein Eliana T. , Eddy Jonathan , Dimowo Oizoshimoshiofu , Lauer Georg M. , Chung Raymond T. , Qadan Motaz , Lanuti Michael , Fuchs Bryan C. , Tanabe Kenneth K.
Published: November 2023

Background

Precision-Cut Liver Slices (PCLS) are anex vivoculture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximatesin vivopathology thanin vitromethods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS.

Methods

PCLS of 250 μm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl4). PCLS were treated with 5 μM Erlotinib for 72 hours. Histology and gene expression were then compared within vivomurine experiments and TGF-β1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver.

Results

PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genesIl6,Col1a1andTimp1in PCLS from CDAHFD-induced cirrhotic mice, andIl6,Col1a1andTgfb1in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression ofCol1a1,Timp1,Tgfb1andIl6, which was consistent with the impact of erlotinib onCol1a1andTgfb1expression inin vivoDEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl4-induced cirrhosis caused reduced expression ofTimp1,Col1a1andTgfb1, which was consistent with the effect of erlotinib inin vivoCCl4-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-β1 treatment upregulatedActa2(αSMA), while treatment with erlotinib inhibited the expression ofActa2. Similar expression results were observed in TGF-β1 treatedin vitroHSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples.

Conclusion

The responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observedin vivoandin vitro. Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).
High Frequency Oscillations (>250Hz) Outnumber Interictal Spikes in Preclinical Studies of Alzheimer’s Disease
Authors: Lisgaras Christos Panagiotis , Scharfman Helen E.
Published: November 2023

Interictal spikes (IIS) and seizures are well-documented in Alzheimer’s disease (AD). IIS typically outnumber seizures, supporting their role as a prominent EEG biomarker in AD. In preclinical models, we showed that high frequency oscillations (HFOs>250Hz) also occur, but it is currently unknown how HFOs compare to IIS. Therefore, we asked whether the incidence of HFOs and IIS differed and if they are differentially affected by behavioral state.

We used three mouse lines that simulate aspects of AD: Tg2576, presenilin 2 knockout, and Ts65Dn mice. We recorded and quantified HFOs and IIS in the hippocampus during wakefulness, slow-wave sleep, and rapid eye movement sleep.

In all three mouse lines, HFOs were more frequent than IIS. High numbers of HFOs correlated with fewer IIS, suggesting for the first time possible competing dynamics among them in AD. Notably, HFOs occurred in more behavioral states than IIS.

In summary, HFOs were the most abundant EEG abnormality when compared to IIS, and occurred in all behavioral states, suggesting they are a better biomarker than IIS. These findings pertained to three mouse lines, which is important because they simulate different aspects of AD. We also show that HFOs may inhibit IIS.

SHORT SUMMARY

Interictal spikes (IIS) and seizures are common in Alzheimer’s disease (AD). IIS are more frequent than seizures and occur during earlier disease stages. In preclinical models, we showed that high frequency oscillations (HFOs>250Hz) occur, but a comparison between IIS and HFOs is lacking. Here we used 3 mouse lines with AD features and local field potential recordings to quantify IIS and HFOs. We found that HFOs outnumbered IIS and that their total numbers were inversely correlated with IIS. HFOs occurred during more behavioral states than IIS. Therefore, HFOs were the most abundant EEG abnormality, and this was generalizable across 3 types of preclinical AD.
Lightway access to AlphaMissense data that demonstrates a balanced performance of this missense mutation predictor
Authors: Tordai H. , Torres O. , Csepi M. , Padányi R. , Lukács G. L. , Hegedűs T.
Published: November 2023

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function, leading to potential pathological consequences. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of single nucleotide polymorphism variants. In our study, we assessed the efficacy of AlphaMissense across several protein groups, such as mitochondrial, housekeeping, transmembrane proteins, and specific proteins like CFTR, using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on the CFTR and disordered, membrane-interacting MemMoRF datasets. However, an enhanced performance with CFTR was shown when benchmarked against the CFTR2 database. Our results also emphasize that quality of AlphaFold’s predictions can seriously influence AlphaMissense predictions. Most importantly, AlphaMissense’s consistent capability in predicting pathogenicity across diverse protein groups, spanning both transmembrane and soluble domains was found. Moreover, the prediction of likely-pathogenic labels for IBS and CFTR coupling helix residues emphasizes AlphaMissense’s potential as a tool for pinpointing functionally significant sites. Additionally, to make AlphaMissense predictions more accessible, we have introduced a user-friendly web resource (https://alphamissense.hegelab.org) to enhance the utility of this valuable tool. Our insights into AlphaMissense’s capability, along with this online resource, underscore its potential to significantly aid both research and clinical applications.
Characterization of alternative splicing in high-risk Wilms’ tumors
Authors: Trink Yaron , Urbach Achia , Dekel Benjamin , Hohenstein Peter , Goldberger Jacob , Kalisky Tomer
Published: November 2023

The significant heterogeneity of Wilms’ tumors between different patients is thought to arise from genetic and epigenetic distortions that occur during various stages of fetal kidney development in a way that is poorly understood. To address this, we characterized the heterogeneity of alternative mRNA splicing in Wilms’ tumors using a publicly available RNAseq dataset of high-risk Wilms’ tumors and normal kidney samples. Through Pareto task inference and cell deconvolution, we found that the tumors and normal kidney samples are organized according to progressive stages of kidney development within a triangle-shaped region in latent space, whose vertices, or “archetypes,” resemble the cap mesenchyme, the nephrogenic stroma, and epithelial tubular structures of the fetal kidney. We identified a set of genes that are alternatively spliced between tumors located in different regions of latent space and found that many of these genes are associated with the Epithelial to Mesenchymal Transition (EMT) and muscle development. Using motif enrichment analysis, we identified putative splicing regulators, some of which are associated with kidney development. Our findings provide new insights into the etiology of Wilms’ tumors and suggest that specific splicing mechanisms in early stages of development may contribute to tumor development in different patients.
Local Delivery of SBRT and IL12 by mRNA Technology Overcomes Immunosuppressive Barriers to Eliminate Pancreatic Cancer
Authors: Hughson Angela L. , Hannon Gary , Salama Noah A. , Vrooman Tara G. , Stockwell Caroline A. , Mills Bradley N. , Garrett-Larsen Jesse , Qiu Haoming , Katerji Roula , Benoodt Lauren , Johnston Carl J. , Murphy Joseph D. , Kruger Emma , Ye Jian , Gavras Nicholas W. , Keeley David C. , Qin Shuyang S. , Lesch Maggie L. , Muhitch Jason B. , Love Tanzy M.T. , Calvi Laura M. , Lord Edith M. , Luheshi Nadia , Elyes Jim , Linehan David C. , Gerber Scott A.
Published: November 2023

The immunosuppressive milieu in pancreatic cancer (PC) is a significant hurdle to treatments, resulting in survival statistics that have barely changed in 5 decades. Here we present a combination treatment consisting of stereotactic body radiation therapy (SBRT) and IL-12 mRNA lipid nanoparticles delivered directly to pancreatic murine tumors. This treatment was effective against primary and metastatic models, achieving cures in both settings. IL-12 protein concentrations were transient and localized primarily to the tumor. Depleting CD4 and CD8 T cells abrogated treatment efficacy, confirming they were essential to treatment response. Single cell RNA sequencing from SBRT/IL-12 mRNA treated tumors demonstrated not only a complete loss of T cell exhaustion, but also an abundance of highly proliferative and effector T cell subtypes. SBRT elicited T cell receptor clonal expansion, whereas IL-12 licensed these cells with effector function. This is the first report demonstrating the utility of SBRT and IL-12 mRNA in PC.

Statement of significance

This study demonstrates the use of a novel combination treatment consisting of radiation and immunotherapy in murine pancreatic tumors. This treatment could effectively treat local and metastatic disease, suggesting it may have the potential to treat a cancer that has not seen a meaningful increase in survival in 5 decades.
Hsp90 shapes adaptation by controlling the fitness consequences of regulatory variation
Authors: Jakobson Christopher M. , Aguilar-Rodríguez José , Jarosz Daniel F.
Published: November 2023

The essential stress-responsive chaperone Hsp90 impacts development and adaptation from microbes to humans. Yet despite evidence of its role in evolution, pathogenesis, and oncogenic transformation, the molecular mechanisms by which Hsp90 alters the consequences of mutations remain vigorously debated. Here we exploit the power of nucleotide-resolution genetic mapping inSaccharomyces cerevisiaeto uncover more than 1,000 natural variant-to-phenotype associations governed by this molecular chaperone. Strikingly, Hsp90 more frequently modified the phenotypic effects ofcis-regulatory variation than variants that altered protein sequence. Moreover, these interactions made the largest contribution to Hsp90-dependent heredity. Nearly all interacting variants—both regulatory and protein-coding—fell within clients of Hsp90 or targets of its direct binding partners. Hsp90 activity affected mutations in evolutionarily young genes, segmental deletions, and heterozygotes, highlighting its influence on variation central to evolutionary novelty. Reconciling the diverse epistatic effects of this chaperone, synthetic transcriptional regulation and reconstructions of natural alleles by genome editing revealed a central role for Hsp90 in regulating the fundamental relationship between activity and phenotype. Our findings establish that non-coding variation is a core driver of Hsp90’s influence on heredity, offering a mechanistic explanation for the chaperone’s strong effects on evolution and development across species.
Critical role of CD206+ macrophages in promoting a cDC1-NK-CD8 T cell anti-tumor immune axis
Authors: Ray Arja , Hu Kenneth H. , Kersten Kelly , Courau Tristan , Kuhn Nicholas F. , Zaleta-Linares Itzia , Samad Bushra , Combes Alexis J. , Krummel Matthew F.
Published: November 2023

Tumor-associated macrophages (TAMs) are frequently categorized as being ‘M1’ or ‘M2’ polarized, even as substantial data challenges this binary modeling of macrophage cell state. One molecule consistently referenced as a delineator of a putative immunosuppressive ‘M2’ state is the surface protein CD206. We thus made a novel conditional CD206 (Mrc1) knock-in mouse to specifically visualize and/or deplete CD206+ ‘M2-like’ TAMs and assess their correspondence with pro-tumoral immunity. Early, but not late depletion of CD206+ macrophages and monocytes (here, ‘Mono/Macs’) led to an indirect loss of a key anti-tumor network of NK cells, conventional type I dendritic cells (cDC1) and CD8 T cells. Among myeloid cells, we found that the CD206+ TAMs are the primary producers of CXCL9, and able to differentially attract activated CD8 T cells. In contrast, a population of stress-responsive TAMs (“Hypoxic” orSpp1+) and immature monocytes, which lack CD206 expression and become prominent following early depletion, expressed markedly diminished levels of CXCL9. Those NK and CD8 T cells which enter CD206- depleted tumors express vastly reduced levels of the corresponding receptorCxcr3,the cDC1- attracting chemokineXcl1and cDC1 growth factorFlt3ltranscripts. Consistent with the loss of this critical network, early CD206+ TAM depletion decreased tumor control by antigen specific CD8 T cells in mice. Likewise, in humans, the CD206Replete, but not the CD206DepletedMono/Mac gene signature correlated robustly with CD8 T cell, NK cell and stimulatory cDC1 gene signatures and transcriptomic signatures skewed towards CD206RepleteMono/Macs associated with better survival. Together, these findings negate the unqualified classification of CD206+ ‘M2-like’ macrophages as immunosuppressive by illuminating contexts for their role in organizing a critical tumor-reactive archetype of immunity.
A proteogenomic pipeline for the analysis of protein biosynthesis errors in the human pathogenCandida albicans
Authors: Correia Inês , Oliveira Carla , Reis Andreia , Guimarães Ana Rita , Aveiro Susana , Domingues Pedro , Bezerra Ana Rita , Vitorino Rui , Moura Gabriela , Santos Manuel A. S.
Published: November 2023

Candida albicansis a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals, but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37°C, the ability to switch between yeast and hyphal forms and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. The latter leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex proteomic samples.

To address this question, we have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying low-level amino acid misincorporation events at the proteome level. We have also analysed the proteomic profile of an engineeredC. albicansstrain that exhibits high level of leucine misincorporation at protein CUG sites and employed anin vivoquantitative gain-of-function fluorescence reporter system to validate our MS/MS data. The data show thatC. albicansmisincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG sites, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins. The study also demonstrates that increasing misincorporation of Leucine at CUG sites does not alter the translational fidelity of the other codons. These findings advance existing knowledge on amino acid misincorporations inC. albicansand add a new dimension to the remarkable capacity of this fungus to diversify its proteome.
Anticipatory smooth pursuit eye movements scale with the probability of visual motion: the role of target speed and acceleration
Authors: Morita Vanessa Carneiro , Souto David , Masson Guillaume S. , Montagnini Anna
Published: November 2023

Sensory-motor systems can extract statistical regularities in dynamic uncertain environments, enabling quicker responses and anticipatory behavior for expected events. Anticipatory smooth pursuit eye movements (aSP) have been observed in primates when the temporal and kinematic properties of a forthcoming visual moving target are fully or partially predictable. To investigate the nature of the internal model of target kinematics underlying aSP, we tested the effect of varying the target kinematics and its predictability. Participants tracked a small visual target in a constant direction with either constant, accelerating or decelerating speed. Across experimental blocks, we manipulated the probability of each kinematic condition varying either speed or acceleration across trials; with either one kinematic condition (providing certainty) or with a mixture of conditions with a fixed probability within a block. We show that aSP is robustly modulated by target kinematics. With constant-velocity targets, aSP velocity scales linearly with target velocity in blocked sessions, and matches the probability-weighted average in the mixture sessions. Predictable target acceleration does also have an influence on aSP, suggesting that the internal model of motion which drives anticipation contains some information about the changing target kinematics, beyond the initial target speed. However, there is a large variability across participants in the precision and consistency with which this information is taken into account in order to control anticipatory behavior.
Decreased fucosylation impacts epithelial integrity and increases risk for COPD
Authors: Swaby Carter , Yeung-Luk Bonnie , Thapa Shreeti , Nishida Kristine , Wally Arabelis , Ghosh Baishakhi , Niederkofler Austin , Luk Sean , Girgis Mirit , Keller Allison , Cortez Cecilia , Ramaswamy Sahana , Wilmsen Kai , Bouché Laura , Dell Anne , Drummond M. Bradley , Putcha Nirupama , Haslam Stuart M. , Mathias Rasika , Hansel Nadia N. , Sheng Jian , Sidhaye Venkataramana
Published: November 2023

COPD causes significant morbidity and mortality worldwide. Epithelial damage is fundamental to disease pathogenesis, although the mechanisms driving disease remain undefined. Published evidence from a COPD cohort (SPIROMICS) and confirmed in a second cohort (COPDgene) demonstrate a polymorphism inFucosyltransferese-2 (FUT2)is a trans-pQTL for E-cadherin, which is critical in COPD pathogenesis. We found by MALDI-TOF analysis thatFUT2increased terminal fucosylation of E-cadherin. Using atomic force microscopy, we found that FUT2-dependent fucosylation enhanced E-cadherin-E-cadherin bond strength, mediating the improvement in monolayer integrity. Tracheal epithelial cells fromFut2-/-mice have reduced epithelial integrity, which is recovered with reconstitution ofFut2. Overexpression ofFUT2in COPD derived epithelia rescues barrier function.Fut2-/-mice show increased susceptibility in an elastase model of disease developing both emphysema and fibrosis. We propose this is due to the role ofFUT2in proliferation and cell differentiation. Overexpression of FUT2 significantly increased proliferation. Loss ofFut2results in accumulation of Spc+ cells suggesting a failure of alveolar type 2 cells to undergo transdifferentiation to alveolar type 1. Using a combination of population data, genetically manipulated mouse models, and patient-derived cells, we present a novel mechanism by which post-translational modifications modulate tissue pathology and serve as a proof of concept for the development of a disease-modifying target in COPD.
Scalable gradients enable Hamiltonian Monte Carlo sampling for phylodynamic inference under episodic birth-death-sampling models
Authors: Shao Yucai , Magee Andrew F. , Vasylyeva Tetyana I. , Suchard Marc A.
Published: November 2023

Birth-death models play a key role in phylodynamic analysis for their interpretation in terms of key epidemiological parameters. In particular, models with piecewiseconstant rates varying at different epochs in time, to which we refer as episodic birthdeath-sampling (EBDS) models, are valuable for their reflection of changing transmission dynamics over time. A challenge, however, that persists with current time-varying model inference procedures is their lack of computational efficiency. This limitation hinders the full utilization of these models in large-scale phylodynamic analyses, especially when dealing with high-dimensional parameter vectors that exhibit strong correlations. We present here a linear-time algorithm to compute the gradient of the birth-death model sampling density with respect to all time-varying parameters, and we implement this algorithm within a gradient-based Hamiltonian Monte Carlo (HMC) sampler to alleviate the computational burden of conducting inference under a wide variety of structures of, as well as priors for, EBDS processes. We assess this approach using three different real world data examples, including the HIV epidemic in Odesa, Ukraine, seasonal influenza A/H3N2 virus dynamics in New York state, America, and Ebola outbreak in West Africa. HMC sampling exhibits a substantial efficiency boost, delivering a 10to 200-fold increase in minimum effective sample size per unit-time, in comparison to a Metropolis-Hastings-based approach. Additionally, we show the robustness of our implementation in both allowing for flexible prior choices and in modeling the transmission dynamics of various pathogens by accurately capturing the changing trend of viral effective reproductive number.
Sensory Feedback and Central Neuronal Interactions in Mouse Locomotion
Authors: Molkov Yaroslav I. , Yu Guoning , Ausborn Jessica , Bouvier Julien , Danner Simon M. , Rybak Ilya A.
Published: November 2023

Locomotion is a complex process involving specific interactions between the central neural controller and the mechanical components of the system. The basic rhythmic activity generated by locomotor circuits in the spinal cord defines rhythmic limb movements and their central coordination. The operation of these circuits is modulated by sensory feedback from the limbs providing information about the state of the limbs and the body. However, the specific role and contribution of central interactions and sensory feedback in the control of locomotor gait and posture remain poorly understood. We use biomechanical data on quadrupedal locomotion in mice and recent findings on the organization of neural interactions within the spinal locomotor circuitry to create and analyze a tractable mathematical model of mouse locomotion. The model includes a simplified mechanical model of the mouse body with four limbs and a central controller composed of four rhythm generators, each operating as a state machine controlling the state of one limb. Feedback signals characterize the load and extension of each limb as well as postural stability (balance). We systematically investigate and compare several model versions and compare their behavior to existing experimental data on mouse locomotion. Our results highlight the specific roles of sensory feedback and some central propriospinal interactions between circuits controlling fore and hind limbs for speed-dependent gait expression. Our models suggest that postural imbalance feedback may be critically involved in the control of swing-to-stance transitions in each limb and the stabilization of walking direction.
Multi-spectral Fluorescent Reporter Influenza A Viruses Allow forin vivoStudies of Innate Immune Function in Zebrafish
Authors: Soos Brandy-Lee , Ballinger Alec , Weinstein Mykayla , Foreman Haley , Grampone Julianna , Weafer Samuel , Aylesworth Connor , King Benjamin L.
Published: November 2023

Influenza virus infection can cause severe respiratory disease and is estimated to cause millions of illnesses annually. Studies of the contribution of the innate immune response to influenza A virus (IAV) to viral pathogenesis may yield new antiviral strategies. Zebrafish larvae are useful models to study the innate immune response to pathogens, including IAV,in vivo. Here, we demonstrate how Color-flu, four fluorescent IAV strains originally developed for mice, can be used to study host-virus interactions by simultaneously monitoring virus particles, neutrophils, and macrophagesin vivo. Using this model, we show how the angiotensin-converting enzyme inhibitor, ramipril, and mitophagy inhibitor, MDIVI-1, improved survival, decreased viral burden, and improved the respiratory burst response to IAV infection. The Color-flu zebrafish model of IAV infection is complementary to other models as it is the only model where interactions between virus particles and host cells in an intact vertebrate can be visualizedin vivo.
Transcriptome and Mutant Analysis of Neuronal Genes for Memory Formation and Retrieval inCaenorhabditis elegans
Authors: Chen Yen-Ju , Wu Shyang-Jen , Tang Yu-Cheng , Wu Yu-Chun , Chiang Yueh-Chen , Pan Chun-Liang
Published: November 2023

Although it is generally believed that the formation and retrieval of memory require distinct sets of genes, little is known about the identity and temporal specificity of genes that act at different memory stages. Here we characterize the transcriptomes for the formation and retrieval of stress-induced aversive memory in the nematodeCaenorhabditis elegans. Our RNA-sequencing analysis show that neuronal genes for calcium homeostasis, membrane excitability, synaptic function and signaling are progressively activated during the formation of aversive memory. Stage-specific transcriptomes further reveal that memory formation and retrieval display distinct gene activation patterns. We carried out a candidate screen of mutants for these memory genes and verified the temporal requirement of memory function for several of them. Further in-depth characterization ofcasy-1/calsyntenin show that while the short CASY-1B isoform acts in memory retrieval, the long CASY-1A isoform functions during memory formation, and its shed extracellular N-terminus fragment is likely a critical signal for memory regulation. Our work uncovers gene expression patterns associated with distinct memory stages and provides a foundation for future mechanistic interrogation of memory functions.
Predicting the subjective intensity of imagined sensory experiences from electrophysiological measures of oscillatory brain activity
Authors: Arnold Derek H. , Saurels Blake W. , Anderson Natasha , Andresen Isabella , Schwarzkopf Dietrich S.
Published: November 2023

Most people can conjure images and sounds that they experience in their minds. There are, however, marked individual differences. Some people report that they cannot generate imagined sensory experiences at all (aphantasics) and others report that they have unusually intense imagined experiences (hyper-phantasics). These individual differences have been linked to activity in sensory brain regions, driven by feedback. We would therefore expect imagined experiences to be associated with specific frequencies of oscillatory brain activity, as these can be a hallmark of neural interactions within and across regions of the brain. Replicating a number of other studies, relative to meditation we find that the act of engaging in imagining auditory or visual sensations is linked to reductions in the power of oscillatory brain activity across a broad range of frequencies, with prominent peaks in the alpha band (8-12 Hz). This oscillatory activity, however, did not predict individual differences in the subjective intensity of imagined experiences. For imagined audio experiences, these were rather predicted by reductions within the theta (6 – 9 Hz) and gamma (33 – 38 Hz) bands, and by increases in beta (15 – 17Hz) band activity. For imagined visual experiences these were predicted by reductions in lower (14 – 16Hz) and upper (29 – 32 Hz) beta band activity, and by an increase in mid-beta band (24 – 26 Hz) activity. Our data suggest that there is sufficient ground truth to the subjective reports that people use to describe the intensity of their imagined sensory experiences such that these can be predicted by the power of distinct rhythms of brain activity.
Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes
Authors: Schafer Christopher T. , Pauszek Raymond F. , Gustavsson Martin , Handel Tracy M. , Millar David P.
Published: November 2023

The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.
A distinctive family of L,D-transpeptidases catalyzing L-Ala-mDAP crosslinks in Alpha and Betaproteobacteria
Authors: Espaillat Akbar , Alvarez Laura , Torrens Gabriel , Beek Josy ter , Miguel-Ruano Vega , Irazoki Oihane , Gago Federico , Hermoso Juan A. , Berntsson Ronnie Per-Arne , Cava Felipe
Published: November 2023

Most bacteria are surrounded by an essential protective mesh-like structure called peptidoglycan, made of glycan chains crosslinked through short peptides by enzymes known as transpeptidases. Of these, penicillin-binding DD-transpeptidases connect adjacent peptide stems between their 4thand 3rdamino acids (4,3-type), D-alanine and a meso-diaminopimelic acid (mDAP) in Gram negatives, whereas LD-transpeptidases make the 3,3-type between mDAP3residues. While these two processes explain the formation of crosslinks in most bacteria, recent investigations involving non-model species have brought to light novel crosslinking mechanisms that point to the existence of less-explored groups of peptidoglycan crosslinking enzymes. Here, we present the identification and characterization of a novel LD-transpeptidase found in the acetic acid bacterialGluconobacter oxydans, named LDTGo, which performs 1,3-type crosslinks between L-Ala1and mDAP3. LDTGo-like proteins are conserved among Alpha and Betaproteobacteria species that do not encode LD3,3-transpeptidases. Using a highly active ortholog, we demonstratedin vitrothat this enzyme can work with non-terminal peptide bonds in the crosslinking process. This property is different from the strict specificity of typical LD- and DD-transpeptidases, which only deal with terminal peptide bonds. The high-resolution crystal structure of LDTGorevealed significant distinctions when compared to 3,3-type LD-transpeptidases. These include a proline-rich region near the N-terminus that restricts substrate access to the active site, and an unprecedented cavity designed to accommodate both the glycan chain and the peptide stem from donor muropeptides, a feature that exhibits broad conservation among LD1,3-transpeptidases. Finally, we demonstrated the involvement of DD-crosslinking turnover in supplying the necessary substrate for LD1,3-transpeptidation. This phenomenon underscores the interplay between structurally distinct crosslinking mechanisms in maintaining cell wall integrity inG. oxydans.
Fast and robust 3D MINFLUX excitation with a variable phase plate
Authors: Deguchi Takahiro , Ries Jonas
Published: November 2023

MINFLUX has achieved record resolution in superresolution imaging and single fluorophore tracking. It is based on localizing single fluorophores by rapid probing with a patterned beam that features a local intensity minimum. Current implementations, however, are complex and expensive and are limited in speed and robustness.

Here, we show that a combination of an electro-optical modulator with a segmented birefringent element such as a spatial light modulator produces a variable phase plate for which the phase can be scanned on the MHz timescale. Bisected or top-hat phase patterns generate high-contrast compact excitation point-spread functions for MINFLUX localization in the x,y, and z-direction, respectively, which can be scanned around a fluorophore within a microsecond and alternated among different excitation wavelengths.

We discuss how to compensate for non-optimal performance of the components and present a robust 3D and multi-color MINFLUX excitation module with record speed, which we envision as an integral component of a high-performance and cost-effective open-source MINFLUX.
Genes acquired by horizontal gene transfer share common regulatory patterns inPhotorhabdus laumondii
Authors: Rili Lydia , Simoes Marcus , Ali Maythem , Findlay Brandon L.
Published: November 2023

Biosynthetic gene clusters are readily transferred between microbes through horizontal gene transfer, but to provide benefit to their host they must be induced to high levels during times of stress. Working with the insect pathogenPhotorhabdus laumondii, here we study the effect of plasmid-induced stress on natural product biosynthesis. We find that the expression plasmid pARO190 alone was sufficient to induce production of an array of natural products, and that the induction could be increased with heterologous expression of either bacterial enhancer binding proteins or a random DNA sequence. Overall, this suggests that generalized stress may be an effective way to increase production of natural products inP. laumondii.
Synanthropic Culicidae (Diptera) in three fragments of Atlantic Forest in northern Parana, Brazil
Authors: Grossi Luiz Eduardo , da Silva Leticia Bernardete , Novaes Vinicius Martins , Golias Halison Correia , da Silva Mario Antonio Navarro , Zequi João Antonio Cyrino
Published: November 2023

Synanthropic Culicidae were sampled using natural and artificial traps in three forest fragments (Daher Wood, Botanical Garden, and Mata dos Godoy State Park) in Londrina, Paraná, Brazil. To collect Culicidae, six traps were distributed in two separate transects of 70 m each, parallel, with a distance between them of 30 m. Three traps were natural (bamboo) and the other three were artificial (tires). In total, we collected 15,048 specimens distributed in eight species. The peri-urban fragments showed the highest richness. Tires were preferred as breeding sites. The seasons of highest and lowest sampling were summer and winter. The Daher Wood and Botanical Garden showed high similarity, and the Godoy Wood, an intact environment, showed the greatest dominance. The mosquitoes demonstrated varying degrees of synanthropy. Correlations were positive between temperature (0.53) and precipitation (0.40) with Culicid abundance. The Principal Component Analysis indicated that tires were most influenced by temperature, relative humidity, and bamboos by Total Dissolved Solids. Four of the collected species showed potential to be vectors of etiological agents. Abiotic factors directly influence the biology of mosquitoes, which is reflected in higher diversity in warmer and rainy periods. Urban and peri-urban areas showed more synanthropic mosquitoes due to their preference for modified sites. Vector species in these areas are of concern because they can modify disease cycles, and for this reason, it is essential to monitor these areas.
Brain-derived neurotrophic factor contributes to activity-induced muscle pain in male but not female mice
Authors: Hayashi Kazuhiro , Lesnak Joseph B. , Plumb Ashley N. , Janowski Adam J. , Smith Angela F. , Hill Joslyn K. , Sluka Kathleen A.
Published: November 2023

Activity-induced muscle pain increases release of interleukin-1β (IL-1β) in muscle macrophages and the development of pain is prevented by blockade of IL-1β. Brain derived neurotrophic factor (BDNF) is released from sensory neurons in response to IL-1β and mediates both inflammatory and neuropathic pain. Thus, we hypothesized that metabolites released during fatiguing muscle contractions activate macrophages to release IL-1β, which subsequently activate sensory neurons to secrete BDNF. To test this hypothesis, we used an animal model of activity-induced pain induced by repeated intramuscular acidic saline injections combined with fatiguing muscle contractions. Intrathecal or intramuscular injection of inhibitors of BDNF-Tropomyosin receptor kinase B (TrkB) signaling, ANA-12 or TrkB-Fc, reduced the decrease in muscle withdrawal thresholds in male, but not in female, mice when given before or 24hr after, but not 1 week after induction of the model. BDNF messenger ribonucleic acid (mRNA) was significantly increased in L4–L6 dorsal root ganglion (DRG), but not the spinal dorsal horn or gastrocnemius muscle, 24hr after induction of the model in either male or female mice. No changes in TrkB mRNA or p75 neurotrophin receptor mRNA were observed. BDNF protein expression via immunohistochemistry was significantly increased in L4–L6 spinal dorsal horn and retrogradely labelled muscle afferent DRG neurons, at 24hr after induction of the model in both sexes. In cultured DRG, fatigue metabolites combined with IL-1β significantly increased BDNF expression in both sexes. In summary, fatigue metabolites release, combined with IL-1β, BDNF from primary DRG neurons and contribute to activity-induced muscle pain only in males, while there were no sex differences in the changes in expression observed in BDNF.
Environment-organism feedbacks drive changes in ecological interactions
Authors: Meacock Oliver J. , Mitri Sara
Published: November 2023

Ecological interactions, the impact of one organism on the growth and death of another, underpin our understanding of the long-term composition and the functional properties of communities. In recent years, the context-dependency of interactions – their tendency to change values in different environments, locations and at different times – has become an increasingly important theme in ecological research. However, an overarching theoretical assumption has been that external environmental factors are responsible for driving these changes. Here, we derive a theoretical interaction framework which teases apart the separate roles played by these extrinsic environmental inputs and the intrinsic environmental changes driven by organisms within the environment itself. At the heart of our theory is the ‘instantaneous interaction’, a quantity that captures the feedback between environmental composition and the growth of organisms within it. In the limit that intrinsic, organismdriven environmental change dominates over external drivers, we find that this feedback can give rise to temporal and spatial context-dependencies as organisms modify the environment over time and/or space. We use small synthetic microbial communities as model ecosystems to demonstrate the power of this framework, using it to predict time-dependent intra-specific interactions in a toxin degradation system and to relate time and spatial dependencies in crossfeeding communities. Our framework helps to explain the ubiquity of interaction context-dependencies in systems where population changes are driven by environmental changes – such as microbial communities – by placing the environment on an equal theoretical footing as the organisms that inhabit it.
From average transient transporter currents to microscopic mechanism – A Bayesian analysis
Authors: George August , Zuckerman Daniel M.
Published: November 2023

Electrophysiology studies of secondary active transporters have revealed quantitative, mechanistic insights over many decades of research. However, the emergence of new experimental and analysis approaches calls for investigation of the capabilities and limitations of the newer methods. We examine the ability of solid-supported membrane electrophysiology (SSME) to characterize discrete-state kinetic models with>10 rate constants. We use a Bayesian framework applied to synthetic data for three tasks: to quantify and check (i) the precision of parameter estimates under different assumptions, (ii) the ability of computation to guide selection of experimental conditions, and (iii) the ability of SSME data to distinguish among mechanisms. When the general mechanism – event order – is known in advance, we show that a subset of kinetic parameters can be “practically identified” within∼1 order of magnitude, based on SSME current traces that visually appear to exhibit simple exponential behavior. This remains true even when accounting for systematic measurement bias and realistic uncertainties in experimental inputs (concentrations) are incorporated into the analysis. When experimental conditions are optimized or different experiments are combined, the number of practically identifiable parameters can be increased substantially. Some parameters remain intrinsically difficult to estimate through SSME data alone, suggesting additional experiments are required to fully characterize parameters. We additionally demonstrate the ability to perform model selection and determine the order of events when that is not known in advance, comparing Bayesian and maximum-likelihood approaches. Finally, our studies elucidate good practices for the increasingly popular, but subtly challenging, Bayesian calculations for structural and systems biology.
An immunohistochemical atlas of necroptotic pathway expression
Authors: Chiou Shene , Al-Ani Aysha H. , Pan Yi , Patel Komal M. , Kong Isabella Y. , Whitehead Lachlan W. , Light Amanda , Young Samuel N. , Barrios Marilou , Sargeant Callum , Rajasekhar Pradeep , Zhu Leah , Hempel Anne , Lin Ann , Rickard James A. , Hall Cathrine , Gangatirkar Pradnya , Yip Raymond K.H. , Cawthorne Wayne , Jacobsen Annette V. , Horne Christopher R. , Martin Katherine R. , Ioannidis Lisa J. , Hansen Diana S. , Day Jessica , Wicks Ian P. , Law Charity , Ritchie Matthew E. , Bowden Rory , Hildebrand Joanne M. , O’Reilly Lorraine A. , Silke John , Giulino-Roth Lisa , Tsui Ellen , Rogers Kelly L. , Hawkins Edwin D. , Christensen Britt , Murphy James M. , Samson André L.
Published: November 2023

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell deathin vivohas proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators – Caspase-8, RIPK1, RIPK3 and MLKL – in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signalingin vivo.

Highlights

13 automated immunohistochemistry protocols for detecting the necroptotic pathway

Necroptotic pathway expression is confined to fast-cycling immune barriers

Necroptotic pathway expression changes at sites of immunoinflammatory challenge

Immunodetection of necrosomes in IBD patients is a putative new diagnostic tool
The curse of zombie dispersal in discrete-time models of spatial population dynamics
Authors: Neubert Michael G. , van Daalen Silke F.
Published: November 2023

In many metapopulation and metacommunity models, individuals disperse between discrete habitat patches. When those models treat time as a discrete variable, the formulation of the dispersal term must be handled with care. A commonly made mistake is to model dispersal with terms identical to those found in continuous-time models. Such terms can inadvertently resurrect dead individuals, effectively creating “zombie dispersers.” Zombie dispersal, in turn, can have dramatic, but spurious, effects on model dynamics. In this manuscript, we illustrate the misleading effects generated by zombie dispersal in a published model used to investigate how dispersal mediates synchrony in population dynamics.