bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sample preparation and data collection for Serial Block Face Scanning Electron Microscopy of Mammalian Cell Monolayers
Authors: Antao Noelle V. , Sall Joseph , Petzold Christopher , Ekiert Damian C. , Bhabha Gira , Liang Feng-Xia 
Published: September 2023

ABSTRACTVolume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focussed ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted toin vitrotissue culture samples. Here we describe detailed protocols for two sample embedding methods forin vitrotissue culture cells intended to be studied using SBF-SEM. The first protocol focuses on cell pellet embedding and the second onen faceembedding.En faceembedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events in a light microscope, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables that will facilitate the acquisition of high quality datasets. Users experienced with electron microscopy sample preparation methodology will be able to complete this protocol in 10-11 days from initial seeding of cells in tissue culture to image acquisition.
Contribution of image statistics and semantics in local vs. distributed EEG decoding of rapid serial visual presentation
Authors: Holm Eric Lützow , Slezak Diego Fernández , Tagliazucchi Enzo 
Published: September 2023

AbstractSpatio-temporal patterns of evoked brain activity contain information that can be used to decode and categorize the semantic content of visual stimuli. This procedure can be biased by statistical regularities which can be independent from the concepts that are represented in the stimuli, prompting the need to dissociate between the contributions of image statistics and semantics to decoding accuracy. We trained machine learning models to distinguish between concepts included in the THINGS-EEG dataset using electroencephalography (EEG) data acquired during a rapid serial visual presentation protocol. After systematic univariate feature selection in the temporal and spatial domains, we constructed simple models based on local signals which superseded the accuracy of more complex classifiers based on distributed patterns of information. Simpler models were characterized by their sensitivity to biases in the statistics of visual stimuli, with some of them preserving their accuracy after random replacement of the training dataset while maintaining the overall statistics of the images. We conclude that model complexity impacts on the sensitivity to confounding factors regardless of performance; therefore, the choice of EEG features for semantic decoding should ideally be informed by the underlying neurobiological mechanisms.
CYP79B2 and CYP79B3 contribute to root branching through production of the auxin precursor indole-3-acetonitrile
Authors: van Zelm Eva , Koevoets Iko T. , Meyer A. Jessica , van der Velde Kyra , de Zeeuw Thijs A. J. , Verstappen Francel , Holmer Rens , Kohlen Wouter , Willemsen Viola , Gommers Charlotte M.M. , Testerink Christa 
Published: September 2023

AbstractLateral root placement, outgrowth and density are influenced by environmental changes, including salinity stress. CYP79B2 and B3 are two cytochrome P450 enzymes previously identified as required for root architecture remodeling in salt. They produce iAOx, a metabolite that can be converted into indole glucosinolates (IGs), camalexin and indole-3-acetic acid (IAA), a type of auxin. We report here that lateral root appearance, induced by an auxin maximum in the bending zone after gravistimulation, is delayed in the absence of CYP79B2/B3. This delay traces back to a decrease in early lateral root growth after emergence, taking place before lateral roots are macroscopically visible. We measured gene transcripts and abundance of metabolites in the iAOx pathway in root segments that are forming lateral roots. Genes involved in tryptophane and IG biosynthesis were upregulated incyp79b2/b3mutants, suggesting a transcriptional feedback-loop. Salt stress was found to increase the expression of genes involved in IAN biosynthesis, a precursor of both IAA and camalexin, in the root during lateral root formation. Moreover, salt increases the concentration of IAN in tissue forming lateral roots in a CYP79B2/B3 dependent manner, but these changes in IAN did not coincide with altered IAA levels. Both the reduction in lateral root density under salt and the delayed lateral root appearance incyp79b2/b3knock-out mutants can be complemented by exogenous application of IAN. Our results reveal a role for the iAOx pathway in regulating the timing of lateral root appearance, allowing the modulation of lateral root density under salt stress.
Bed Nucleus of the Stria Terminalis (BNST) neurons containing the serotonin 5HT2creceptor modulate operant alcohol self-administration behavior in mice
Authors: Flanigan Meghan E. , Gianessi Carol , Castle Megan , Dorlean Winifred , Sides Tori , Kash Thomas L. 
Published: September 2023

AbstractThe serotonin 5HT2creceptor has been widely implicated in the pathophysiology of alcohol use disorder (AUD), particularly alcohol seeking and the affective consequences of chronic alcohol consumption. However, little is known about the brain sites in which 5HT2cexerts its effects on specific alcohol-related behaviors, especially in females. Here, we investigated the effects of site-specific manipulation of the 5HT2creceptor system in the BNST on operant alcohol self-administration behaviors in adult mice of both sexes, including the acquisition and maintenance of fixed-ratio responding, motivation for alcohol (progressive ratio), and quinine-adulterated responding for alcohol on a fixed-ratio schedule (punished alcohol seeking). Knockdown of 5HT2cin the BNST did not affect the acquisition or maintenance of operant alcohol self-administration, nor did it affect progressive ratio responding for alcohol. This manipulation had only a subtle effect on responding for quinine alcohol selectively in females. On the other hand, chemogenetic inhibition of BNST 5HT2c-containing neurons (BNST5HT2c) increased operant alcohol self-administration behavior in both sexes on day 2, but not day 9, of testing. It also increased operant responding for 1000 μM quinine-adulterated alcohol selectively in males. Importantly, chemogenetic inhibition of BNST5HT2cdid not alter operant sucrose responding or motivation for sucrose in either sex. We then performed cell-type specific anterograde tracing, which revealed that BNST5HT2cproject to similar regions in males and females, many of which have been previously implicated in AUD. We next used chemogenetics and quantification of the immediate early gene cFos to characterize the functional influence of BNST5HT2cinhibition on vlPAG activity. We show that chemogenetic inhibition of BNST5HT2creduces vlPAG cFos in both sexes, but that this reduction is more robust in males. Together these findings suggest that BNST5HT2cneurons, and to a small extent the BNST 5HT2creceptor, serve to promote aversive responses to alcohol consumption, potentially through sex-dependent disinhibition of vlPAG neurons.
The Use of Electronic Health Record Embedded MRC-ICU as a Metric for Critical Care Pharmacist Workload
Authors: Webb Andrew J , Carver Bayleigh , Rowe Sandra , Sikora Andrea 
Published: September 2023

AbstractObjectiveA lack of pharmacist-specific risk-stratification scores in the electronic health record (EHR) may limit resource optimization. The medication regimen complexity-intensive care unit (MRC-ICU) score was implemented into our center’s EHR for use by clinical pharmacists. The purpose of this evaluation was to evaluate MRC-ICU as a predictor of pharmacist workload and to assess its potential as an additional dimension to traditional workload measures.MaterialsData were abstracted from the EHR on adult ICU patients, including MRC-ICU scores and two traditional measures of pharmacist workload: numbers of medication orders verified and interventions logged.MethodsThis was a single-center study of an EHR-integrated MRC-ICU tool. The primary outcome was the association of MRC-ICU with institutional metrics of pharmacist workload. Associations were assessed using the initial 24-hour maximum MRC-ICU score’s Pearson’s correlation with overall admission workload and the day-to-day association using generalized linear mixed-effects modeling.ResultsA total of 1,205 patients over 5,083 patient-days were evaluated. Baseline MRC-ICU was correlated with both cumulative order volume (Spearman’s rho 0.41, p < 0.001) and cumulative interventions placed (Spearman’s rho 0.27, p < 0.001). A one-point increase in maximum daily MRC-ICU was associated with 31% increase in order volume (95% CI 24-38%) and 4% increase in interventions (95% CI 2-5%).DiscussionThe MRC-ICU is a validated score that has been previously correlated with important patient-centered outcomes. Here, MRC-ICU was modestly associated with two traditional objective measures of pharmacist workload, including orders verified and interventions placed, which is an important step for its use as a tool for resource utilization needs.Lay SummaryMeasuring critical care clinical pharmacist workload is challenging because currently available metrics, including number of medication orders verified or medication interventions logged, do not capture the full breadth of work critical care pharmacists do. The medication regimen complexity-intensive care unit (MRC-ICU) score is a tool designed to quantify the complexity of an ICU patient’s medication regimen and may serve as an alternative measure of overall critical care pharmacist workload. In this study, we assessed whether MRC-ICU scores from 1,205 ICU patients admitted to a single academic medical center were correlated with traditional metrics used to assess pharmacist workload, including medication orders and documented interventions. MRC-ICU was correlated with both workflow measures and traditional measures of patient acuity and also was predictive of the next day’s workload, suggesting MRC-ICU could be explored as an additional tool to optimize critical care pharmacist resource utilization. Further studies should assess how MRC-ICU can be utilized to optimize critical care pharmacist workload.
Notch signalling plays a critical role in patterning the ventral mesoderm during early embryogenesis inDrosophila melanogaster
Authors: Megaly Marvel , Foran Gregory , Ali Arsala , Turgambayeva Anel , Hallam Ryan D. , Necakov Aleksandar 
Published: September 2023

AbstractNotch signaling is a critical regulator of multiple developmental processes through its ability to control gene expression, and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Although Notch signalling has been implicated in myogenesis during late embryogenesis, the role of Notch signalling in early mesoderm development in largely unexplored. Endocytosis of the ligand and receptor extracellular domain is observed in the mesoderm and mesectoderm and therefore indicative of signalling activity. Here we present evidence that critical mesodermal genes require and are responsive to Notch signalling activity. We also developed a novel light-inducible variant of NICD to allow precise control of ectopic activation of Notch signalling. Using a combination of high-resolution fluorescent mRNAin situhybridizations and qPCR, we identified a set of mesodermal genes that are directly regulated by Notch signalling. We also show evidence that Notch signalling indirectly regulates the dorsal-ventral patterning program mediated by the Toll signalling pathway through the Dorsal/ Twist/ Snail network. Our findings in this paper show that Notch signalling regulates mesoderm patterning and boundary formation between it and the ectoderm by regulating the expression of critical genes and providing negative feedback on the upstream patterning network.
Temporal profiling ofSalmonellatranscriptional dynamics during macrophage infection using a comprehensive reporter library
Authors: Nguyen Taylor H. , Diaz Oscar R. , Rajendram Manohary , Butler Daniel S.C. , Wang Benjamin X. , Hinton Jay C. D. , Monack Denise , Huang Kerwyn Casey 
Published: September 2023

AbstractThe transcriptome ofSalmonella entericaserovar Typhimurium (S. Tm) dynamically responds to the rapid environmental shifts intrinsic toS.Tm lifestyle, exemplified by entry into theSalmonella-containing vacuole (SCV) within macrophages. IntracellularS. Tm must respond to the acidity of the SCV, accumulation of reactive oxygen/nitrogen species, and fluctuations in nutrient availability. Despite thorough RNA-seq-based investigations, the precise transcriptional timing of the expression of many secretion systems, metabolic pathways, and virulence effectors involved in infection has yet to be elucidated. Here, we construct a comprehensive library of GFP-reporter strains representing ∼3,000 computationally identifiedS.Tm promoter regions to study the dynamics of transcriptional regulation. We quantified promoter activity duringin vitrogrowth in defined and complex media and throughout the timeline of intracellular infection of RAW 246.7 macrophages. Using bulk measurements and single-cell imaging, we uncovered condition-specific transcriptional regulation and population-level heterogeneity in the activity of virulence-related promoters, including SPI2 genes such asssaRandssaG. We discovered previously unidentified transcriptional activity from 234 genes, including ones with novel activity during infection that are associated with pathogenecity islands and are involved in metabolism and metal homeostasis. Our library and data sets should provide powerful resources for systems-level interrogation ofSalmonellatranscriptional dynamics.
Development of genetically-encoded fluorescent KSR1-based probes to track ceramides during phagocytosis
Authors: Girik Vladimir , van Ek Larissa , Quadri Isabelle Dentand , Azam Maral , Cobo Maria Cruz , Mandavit Marion , Riezman Isabelle , Riezman Howard , Gavin Anne-Claude , Nunes-Hasler Paula 
Published: September 2023

AbstractCeramides regulate phagocytosis, however their exact function remains poorly understood. Here we sought 1) to develop genetically encoded fluorescent tools for imaging ceramide, and 2) to use them to examine ceramide dynamics during phagocytosis. Fourteen EGFP fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of KSR1 localized to plasma membrane or endolysosomes. C-terminally-tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR preferentially binds to glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial lysosomal degradation, C-KSR robustly accumulated at the plasma membrane during phagocytosis, whereas N-KSR becomes cytoplasmic at later timepoints. Moreover, weak recruitment of C-KSR-GS to endoplasmic reticulum and phagosomes was enhanced by overexpression of the endoplasmic reticulum proteins STIM1 and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.
Molecular signatures of heat tolerance and sensitivity in the alpine plantWahlenbergia ceraceaduring moderate warming
Authors: Notarnicola Rocco F. , Arnold Pieter A. , Feng Zhi-Ping , Hamilton Jemimah , Jones Ashley W. , Loke Stella , Schwessinger Benjamin , Nicotra Adrienne B. , Ganguly Diep R. 
Published: September 2023

AbstractUnderstanding the molecular basis of plant heat tolerance helps to predict the consequences of climate warming on plant performance, particularly in vulnerable environments. Our current understanding comes primarily from studies inArabidopsis thaliana(Arabidopsis) and select crops exposed to short and intense periods of heat stress. In this study we sought to characterise the transcriptomic responses ofWahlenbergia ceracea(W. ceracea), an Australian alpine herb, to growth under sustained moderate warming. We compared responses of pre-defined tolerant and sensitive lines, based on measures of photosynthetic thermostability, to growth under cool (24/15 °C day/night) or warm (30/20 °C day/night) temperatures to identify the pathways involved in heat tolerance and acclimation. Under warmer growth temperatures,W. ceraceaup-regulated genes involved in RNA metabolism, while down-regulating those involved in photosynthesis and pigment metabolism. In tolerant lines, genes related to photosystem II and light-dependent photosynthetic reactions were more strongly down- regulated. This suggests that the regulation of electron transport and its components may be involved in thermal acclimation. Our results also highlight the importance of hormonal gene networks, particularly those responsive to ethylene, during longer-term moderate warming. In conclusion, our results point to post-transcriptional processes and the stabilisation of the electron transport chain as candidate mechanisms for thermal acclimation in heat-tolerantW. ceracea. The study also revealed many temperature-responsive genes with no detectable ortholog in Arabidopsis, whose characterization may enhance our understanding of physiological acclimation and have relevance for the biotechnological improvement of threatened species and crops.
Proteolytic activation of fatty acid synthase signals pan-stress resolution
Authors: Wei Hai , Weaver Yi M. , Yang Chendong , Zhang Yuan , Hu Guoli , Karner Courtney M. , DeBerardinis Ralph J. , Weaver Benjamin P. 
Published: September 2023

AbstractChronic stress and inflammation are not only outcomes of pathological states but rather major drivers of many human diseases1-4. Ideally, a given stress program is downregulated to basal levels upon restoration of homeostasis. Chronic responsiveness despite stress mitigation suggests a failure to sense the resolution of the initiating stressor. Here we show that a proteolytic cleavage event of fatty acid synthase (FASN) activates a global cue for stress resolution. FASN is well-established as the multifunctional enzyme catalyzingde novobiosynthesis of saturated fatty acid5, 6. Surprisingly, our results demonstrate FASN functioning as a signaling molecule promoting an anti-inflammatory profile apart from fatty acid synthesis. Redox-dependent proteolytic cleavage of FASN by caspase activates a truncated C-terminal enzymatic fragment (FASN-CTF) that is sufficient to down-regulate multiple aspects of stress-responsiveness including gene expression and metabolic programs. Only a fraction of FASN is cleaved allowing for continued fat synthesis. FASN-CTF can signal stress resolution across tissues in a cell non-autonomous manner. Consistent with these findings, FASN processing is also seen in well-fed but not fasted mouse liver. As down-regulation of stress responsiveness is critical to health, our findings provide a potential pathway to control the magnitude for diverse aspects of stress responses.
Engineered Lactiplantibacillus plantarum as a Biosensor Probe for the Lungs
Authors: Brasino Michael , Wagnell Eli , Manalo Elise C , Drennan Samuel , Fischer Jared M , Merritt Justin 
Published: September 2023

AbstractLungs are a frequent site for disease but are difficult to image and probe, potentially exacerbating lung disease, delaying diagnoses and impacting survival. Here, we demonstrate a low cost, minimally invasive method to probe the lungs for disease, using genetically engineeredLactiplantibacillus plantarumstrain WCFS1. Genetically modified WCFS1 was delivered specifically to the lungs of mice, where it was shown to remain transcriptionally active for several hours and then be cleared without further colonization. WCFS1 was further modified to secrete nanoluciferase as a synthetic biomarker, which traveled from the bacteria in the lung to the urine where it was easily detected. The administered bacteria also secreted nano-luciferase upon detecting a specific peptide secreted by a mouse lung cancer cell line in vitro or in vivo from syngeneic tumors.
Trait level somatic arousal modulates fMRI neural synchrony to naturalistic stimuli
Authors: Klamer K. , Craig J. , Sullivan K. , Haines C. , Ekstrand C. 
Published: September 2023

AbstractSomatic arousal refers to the physiological and bodily responses that occur in reaction to different emotional and psychological stimuli and is a crucial component of the fight or flight response. Symptoms associated with higher levels of somatic arousal such as higher heart and respiration rates have been shown to impact the blood oxygenation level dependent (BOLD) signal during functional magnetic resonance imaging (fMRI) studies. Differences in baseline levels of somatic arousal may therefore modulate the brain’s response to incoming stimuli during fMRI. Previous studies typically investigate somatic arousal as a state, rather than as a trait, in which some individuals are more likely to have heightened physiological responses to psychological stimuli, causing the neurological mechanisms behind baseline somatic arousal levels to remain poorly understood. The current study seeks to identify how differing levels of baseline somatic arousal modulate neural synchrony in response to an audiovisual film. We hypothesize that individuals with higher levels of somatic arousal will show overall heightened neural synchrony in response to a complex audiovisual stimulus. We identified that higher levels of somatic arousal are associated with widespread neural synchrony across the brain, including frontal gyri, parietal and temporo-occipital cortices. Taken together, this research suggests that baseline somatic arousal levels should be measured during naturalistic fMRI paradigms, as baseline somatic arousal levels may have a profound influence on synchronous neural activity.
A map of the rubisco biochemical landscape
Authors: Prywes Noam , Philips Naiya R. , Oltrogge Luke M. , Lindner Sebastian , Tsai Yi-Chin Candace , de Pins Benoit , Cowan Aidan E. , Taylor-Kearney Leah J. , Chang Hana A. , Hall Laina N. , Bellieny-Rabelo Daniel , Nisonoff Hunter M. , Weissman Rachel F. , Flamholz Avi I. , Ding David , Bhatt Abhishek Y. , Shih Patrick M. , Mueller-Cajar Oliver , Milo Ron , Savage David F. 
Published: September 2023

AbstractRubisco is the primary CO2fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineeredE. coliwhere enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2concentrations, we inferred enzyme velocity and CO2affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.