bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidermal growth factor receptors in vascular endothelial cells contribute to functional hyperemia in the brain
Authors: Ferris Hannah R. , Hill-Eubanks David C. , Nelson Mark T. , Wellman George C. , Koide Masayo
Published: September 2023

Functional hyperemia – activity-dependent increases in local blood perfusion – underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by exogenous administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs depletes PIP2and attenuates functional hyperemia, underscoring the central role of the endothelial EGFR signaling in cerebral blood flow regulation.
Pyruvate Kinase Activity Regulates Cystine Starvation Induced Ferroptosis through Malic Enzyme 1 in Pancreatic Cancer Cells
Authors: Ensink Elliot , Jordan Tessa , D Medeiros Hyllana C , Thurston Galloway , Pardal Anmol , Yu Lei , Lunt Sophia Y.
Published: September 2023

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high mortality and limited efficacious therapeutic options. PDAC cells undergo metabolic alterations to survive within a nutrient-depleted tumor microenvironment. One critical metabolic shift in PDAC cells occurs through altered isoform expression of the glycolytic enzyme, pyruvate kinase (PK). Pancreatic cancer cells preferentially upregulate pyruvate kinase muscle isoform 2 isoform (PKM2). PKM2 expression reprograms many metabolic pathways, but little is known about its impact on cystine metabolism. Cystine metabolism is critical for supporting survival through its role in defense against ferroptosis, a non-apoptotic iron-dependent form of cell death characterized by unchecked lipid peroxidation. To improve our understanding of the role of PKM2 in cystine metabolism and ferroptosis in PDAC, we generated PKM2 knockout (KO) human PDAC cells. Fascinatingly, PKM2KO cells demonstrate a remarkable resistance to cystine starvation mediated ferroptosis. This resistance to ferroptosis is caused by decreased PK activity, rather than an isoform-specific effect. We further utilized stable isotope tracing to evaluate the impact of glucose and glutamine reprogramming in PKM2KO cells. PKM2KO cells depend on glutamine metabolism to support antioxidant defenses against lipid peroxidation, primarily by increased glutamine flux through the malate aspartate shuttle and utilization of ME1 to produce NADPH. Ferroptosis can be synergistically induced by the combination of PKM2 activation and inhibition of the cystine/glutamate antiporterin vitro. Proof-of-conceptin vivoexperiments demonstrate the efficacy of this mechanism as a novel treatment strategy for PDAC.

Graphical Abstract

Highlights

PKM2KO in pancreatic ductal adenocarcinoma (PDAC) cells produces enhanced defense against cystine starvation induced ferroptosis.

Pharmacologic activation of pyruvate kinase (PK) activity promotes ferroptosis under cystine starvation, while inhibition promotes ferroptosis survival in PDAC cells.

Decrease in PK activity reprograms glutamine metabolism to increase use of malic enzyme 1 and promote survival under cystine starvation in PDAC cells.

Cystine starvation and activation of pyruvate kinase synergistically decreases progression of pancreatic cancerin vivo.
A three-genome ultraconserved element phylogeny of Cryptophytes
Authors: Greenwold Matthew J. , Merritt Kristiaän , Richardson Tammi L. , Dudycha Jeffry L.
Published: September 2023

Cryptophytes are single celled protists found in all aquatic environments. They are composed of a heterotrophic genus,Goniomonas, and a largely autotrophic group comprising many genera. Cryptophytes evolved through secondary endosymbiosis between a host eukaryotic heterotroph and a symbiont red alga. This merger resulted in a four-genome system that includes the nuclear and mitochondrial genomes from the host and a second nuclear genome (nucleomorph) and plastid genome inherited from the symbiont. Here, we make use of different genomes (with potentially distinct evolutionary histories) to perform a phylogenomic study of the early history of cryptophytes. Using ultraconserved elements from the host nuclear genome and symbiont nucleomorph and plastid genomes, we produce a three-genome phylogeny of 91 strains of cryptophytes. Our phylogenetic analyses find that that there are three major cryptophyte clades: Clade 1 comprisesChroomonasandHemiselmisspecies, Clade 2, a taxonomically rich clade, comprises at least twelve genera, and Clade 3, comprises the heterotrophicGoniomonasspecies. Each of these major clades include both freshwater and marine species, but subclades within these clades differ in degrees of niche conservatism. Finally, we discuss priorities for taxonomic revision to Cryptophyceae based on previous studies and in light of these phylogenomic analyses.
Effects of Polyphosphate metabolism Mutations on Biofilm, Capsule Formation and Virulence Traits in Hypervirulent ST23Klebsiella pneumoniaeSGH10
Authors: Rojas Diego , Marcoleta Andrés E. , Gálvez Matías , Varas Macarena A. , Díaz Mauricio , Hernández Mauricio , Vargas Cristian , Nourdin Guillermo , Koch Elard , Saldivia Pablo , Vielma Jorge , Gan Yunn-Hwen , Chen Yahua , Guiliani Nicolás , Chávez Francisco P.
Published: September 2023

The emergence of hypervirulentKlebsiella pneumoniae(hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP usingDictyostelium discoideumamoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence inD. discoideumcompared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulentK. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence inK. pneumoniaeand provides insights into potential therapeutic targets for treatingK. pneumoniaeinfections.
Cone-Opponent Ganglion Cells in the Primate Fovea Tuned to Non-Cardinal Color Directions
Authors: Godat Tyler , Kohout Kendall , Yang Qiang , Parkins Keith , McGregor Juliette E. , Merigan William H. , Williams David R. , Patterson Sara S.
Published: September 2023

A long-standing question in vision science is how the three cone photoreceptor types – long (L), medium (M) and short (S) wavelength sensitive – combine to generate our perception of color. Hue perception can be described along two opponent axes: red-green and blue-yellow. Psychophysical measurements of color appearance indicate that the cone inputs to the red-green and blue-yellow opponent axes are M vs. L+S and L vs. M+S, respectively. However, the “cardinal directions of color space” revealed by psychophysical measurements of color detection thresholds are L vs. M and S vs. L+M. The cardinal directions match the most common cone-opponent retinal ganglion cells (RGCs) in the primate retina. Accordingly, the cone opponency necessary for color appearance is thought to be established in cortex. However, small populations with the appropriate M vs. L+S and L vs. M+S cone-opponency have been reported in large surveys of cone inputs to primate RGCs and their projections to the lateral geniculate nucleus (LGN) yet their existence continues to be debated. Resolving this long-standing open question is needed as a complete account of the cone-opponency in the retinal output is critical for efforts to understand how downstream neural circuits process color. Here, we performed adaptive optics calcium imaging to longitudinally and noninvasively measurements of the foveal RGC light responses in the living macaque eye. We confirm the presence of L vs. M+S and M vs. L+S neurons with non-cardinal cone-opponency and demonstrate that cone-opponent signals in the retinal output are substantially more diverse than classically thought.
Androgen blockade primes NLRP3 in macrophages to induce tumor phagocytosis
Authors: Chaudagar Kiranj , Rameshbabu Srikrishnan , Mei Shenglin , Hirz Taghreed , Hu Ya-Mei , Argulian Anna , Labadie Brian , Desai Kunal , Grimaldo Sam , Kahramangil Doga , Nair Rishikesh , DSouza Sabina , Zhou Dylan , Li Mingyang , Doughan Farah , Chen Raymond , Shafran Jordan , Loyd Mayme , Xia Zheng , Sykes David B. , Moran Amy , Patnaik Akash
Published: September 2023

Immune-based therapies induce durable remissions in subsets of patients across multiple malignancies. However, there is limited efficacy of immunotherapy in metastatic castrate-resistant prostate cancer (mCRPC), manifested by an enrichment of immunosuppressive (M2) tumor- associated macrophages (TAM) in the tumor immune microenvironment (TME). Therefore, therapeutic strategies to overcome TAM-mediated immunosuppression are critically needed in mCRPC. Here we discovered that NLR family pyrin domain containing 3 (NLRP3), an innate immune sensing protein, is highly expressed in TAM from metastatic PC patients treated with standard-of-care androgen deprivation therapy (ADT). Importantly,ex vivostudies revealed that androgen receptor (AR) blockade in TAM upregulates NLRP3 expression, but not inflammasome activity, and concurrent AR blockade/NLRP3 agonist (NLRP3a) treatment promotes cancer cell phagocytosis by immunosuppressive M2 TAM. In contrast, NLRP3a monotherapy was sufficient to enhance phagocytosis of cancer cells in anti-tumor (M1) TAM, which exhibit highde novoNLRP3 expression. Critically, combinatorial treatment with ADT/NLRP3a in a murine model of advanced PC resulted in significant tumor control, with tumor clearance in 55% of mice via TAM phagocytosis. Collectively, our results demonstrate NLRP3 as an AR-regulated “macrophage phagocytic checkpoint”, inducibly expressed in TAM by ADT and activated by NLRP3a treatment, the combination resulting in TAM-mediated phagocytosis and tumor control.
Imprinting but not cytonuclear interactions affects parent-of-origin effect on seed size inArabidopsishybrids
Authors: June Viviana , Song Xiaoya , Chen Z. Jeffrey
Published: September 2023

The parent-of-origin effect on seed size can result from imprinting or a combinational effect between cytoplasmic and nuclear genomes, but their relative contributions remain unknown. To discern these confounding effects, we generated cytoplasmic-nuclear substitution (CNS) lines using recurrent backcrossing in theArabidopsis thalianaecotypes Col-0 and C24. These CNS lines differ only in the nuclear genome (imprinting) or in the cytoplasm. The CNS reciprocal hybrids with the same cytoplasm display a ∼20% seed size difference as observed in the conventional hybrids. However, seed size is similar between the reciprocal cybrids with fixed imprinting. Transcriptome analyses in the endosperm of CNS hybrids using laser-capture microdissection have identified 104 maternally expressed genes (MEGs) and 90 paternally-expressed genes (PEGs). These imprinted genes are involved in pectin catabolism and cell wall modification in the endosperm.HDG9, an epiallele and one of 11 cross-specific imprinted genes, controls seed size. In the embryo, a handful of imprinted genes is found in the CNS hybrids but only one is expressed higher in the embryo than endosperm.AT4G13495encodes a long-noncoding RNA (lncRNA), but no obvious seed phenotype is observed in the lncRNA knockout lines.NRPD1, encoding the largest subunit of RNA Pol IV, is involved in the biogenesis of small interfering RNAs. Seed size and embryo is larger in the cross usingnrpd1as the maternal parent than in the reciprocal cross. In spite of limited ecotypes tested, these results suggest potential roles of imprinting andNRPD1-mediated small RNA pathway in seed size variation in hybrids.
Phosphate starvation regulates cellulose synthesis to modify root growth
Authors: Khan Ghazanfar Abbas , Dutta Arka , Van de Meene Allison , Frandsen Kristian EH. , Ogden Michael , Whelan James , Persson Staffan
Published: September 2023

In the model plantArabidopsis thaliana, the absence of the essential macro-nutrient phosphate reduces primary root growth through decreased cell division and elongation, requiring alterations to the polysaccharide-rich cell wall surrounding the cells. Despite its significance, the regulation of cell wall synthesis in response to low phosphate levels is not well understood. In this study, we show that plants increase cellulose synthesis in roots under limiting phosphate conditions, which leads to changes in the thickness and structure of the cell wall. These changes contribute to the reduced growth of primary roots in low phosphate conditions. Furthermore, we found that the cellulose synthase complex activity at the plasma membrane increases during phosphate deficiency. Moreover, we show that this increase in the activity of the cellulose synthase complex is likely due to alterations in the phosphorylation status of cellulose synthases in low phosphate conditions. Specifically, phosphorylation of CELLULOSE SYNTHASE 1 at the S688 site decreases in low phosphate conditions. Phosphomimic versions of CELLULOSE SYNTHASE 1 with an S688E mutation showed significantly reduced cellulose induction and primary root length changes in low phosphate conditions. Protein structure modelling suggests that the phosphorylation status of S688 in CELLULOSE SYNTHASE 1 could play a role in stabilizing and activating the cellulose synthase complex. This mechanistic understanding of root growth regulation under limiting phosphate conditions provides potential strategies for changing root responses to soil phosphate content.
Comparison of image registration techniques in functional ultrasound imaging
Authors: Zhong Shan , Agyeman Kofi , Syed Shanze , Tobing Richard , Choi Wooseong , Liu Charles , Lee Darrin , Christopoulos Vassilios
Published: September 2023

Functional Ultrasound Imaging (fUSI) is an emerging hemodynamic-based functional neuroimaging technique that combines high spatiotemporal resolution and sensitivity, as well as extensive brain coverage, enabling a range of applications in both control and disease animal models. Based on power Doppler (pD) imaging, fUSI measures changes in cerebral blood volume (CBV) by detecting the back-scattered echoes from red blood cells moving within its field of view (FOV). However, the expansion of fUSI technology is partly limited by the challenge to co-register pD vascular maps acquired across different sessions or animals to one reference; an approach that could widen the scope of experimental paradigms and enable advanced data analysis tools. In this study, we seek to address this critical limitation. We evaluate six image registration techniques, predominantly used in other neuroimaging studies, using 2D sagittal whole-brain fUSI data from 82 anesthetized mice, and tested the quality of registration using multiple metrics. Our findings indicate a substantial enhancement in the alignment of fUSI images post registration. Among the tested techniques, the non-rigid registration algorithmImregdeformyielded superior performance. We offer the first comparative study of image registration techniques for a 2D fUSI brain dataset, paving a way for improved utilization of fUSI in future pre-clinical research applications.
Evaluating batch correction methods for image-based cell profiling
Authors: Arevalo John , Su Ellen , van Dijk Robert , Carpenter Anne E. , Singh Shantanu
Published: September 2023

High-throughput image-based profiling platforms are powerful technologies capable of collecting data from billions of cells exposed to thousands of perturbations in a time- and cost-effective manner. Therefore, image-based profiling data has been increasingly used for diverse biological applications, such as predicting drug mechanism of action or gene function. However, batch effects pose severe limitations to community-wide efforts to integrate and interpret image-based profiling data collected across different laboratories and equipment. To address this problem, we benchmarked seven high-performing scRNA-seq batch correction techniques, representing diverse approaches, using a newly released Cell Painting dataset, the largest publicly accessible image-based dataset. We focused on five different scenarios with varying complexity, and we found that Harmony, a mixture-model based method, consistently outperformed the other tested methods. Our proposed framework, benchmark, and metrics can additionally be used to assess new batch correction methods in the future. Overall, this work paves the way for improvements that allow the community to make best use of public Cell Painting data for scientific discovery.
Ontogeny of Projections from the Motor Cortex Governing Whisker and Neck Movements to the Striatum in the Rat
Authors: Gu Albert T , Han Victor Z , Jiang Yanfen
Published: September 2023

Corticostrial or cortico-basal ganglia circuitry plays an important role in integrating sensory and motor information, developing appropriate goal-directed behavior, promoting the maturation of GABAergic interneurons in the striatum, and regulating the nigrostriatal pathways. Dysfunction of this circuitry has been seen in some movement disorders. However, the dynamic changes of this circuitry in early life are not fully elucidated. Previous studies demonstrated that projections from motor cortices of caudal forelimb and jaw-lip-tongue areas to the striatum developed postnatally with terminal-like fibers evident at postnatal day 7. Here we report the development of the projections from the motor cortex governing whisker and neck movements to the striatum. Corticostriatal projections from this area were mainly ipsilateral and also underwent a progressive, postnatal development. The pyramidal tract and its collaterals to the dorsal striatum appeared on the day of birth (postanatal day 0 (P0)), peaked on P6 in density, and continued to be tuned until P36. The intertelencephalic projections in the dorsolateral striatum were established between P6 and P12 and continued to be refined between P20 and P36. Neurons in this motor cortex sent their axons to the contralateral motor cortex via the corpus callosum at the age between P6 and P12. Our results suggest that the time window between P6 and P12 is critical for the development of the projections from the motor cortex governing whisker and neck movement to the striatum in the rat. The overall process of the development of this circuitry appears to correspond to the functional development of whisker movement and locomotor activities.
Improving the performance of supervised deep learning for regulatory genomics using phylogenetic augmentation
Authors: Duncan Andrew G , Mitchell Jennifer A , Moses Alan M
Published: September 2023

Structured abstract

Motivation

Supervised deep learning is used to model the complex relationship between genomic sequence and regulatory function. Understanding how these models make predictions can provide biological insight into regulatory functions. Given the complexity of the sequence to regulatory function mapping (the cis-regulatory code), it has been suggested that the genome contains insufficient sequence variation to train models with suitable complexity. Data augmentation is a widely used approach to increase the data variation available for model training, however current data augmentation methods for genomic sequence data are limited.

Results

Inspired by the success of comparative genomics, we show that augmenting genomic sequences with evolutionarily related sequences from other species, which we term phylogenetic augmentation, improves the performance of deep learning models trained on regulatory genomic sequences to predict high-throughput functional assay measurements. Additionally, we show that phylogenetic augmentation can rescue model performance when the training set is down-sampled and permits deep learning on a real-world small dataset, demonstrating that this approach improves experimental data efficiency. Overall, this data augmentation method represents a solution for improving model performance that is applicable to many supervised deep learning problems in genomics.

Availability and implementation

The open-source GitHub repository agduncan94/phylogenetic_augmentation_paper includes the code for rerunning the analyses here and recreating the figures.

Contact

alan.moses@utoronto.ca
A robust knock-in approach using a minimal promoter and a minicircle
Authors: Keating Margaret , Hagle Ryan , Osorio-Mendez Daniel , Rodriguez-Parks Anjelica , Almutawa Sarah I , Kang Junsu
Published: September 2023

Knock-in reporter (KI) animals are essential tools in biomedical research to study gene expression impacting diverse biological events. While CRISPR/Cas9-mediated genome editing allows for the successful generation of KI animals, several factors should be considered, such as low expression of the target gene, prevention of bacterial DNA integration, and in-frame editing. To circumvent these challenges, we developed a new strategy that utilizes minicircle technology and introduces a minimal promoter. We demonstrated that minicircles serve as an efficient donor DNA in zebrafish, significantly enhancing KI events compared to plasmids containing bacterial backbones. In an attempt to generate a KI reporter forscn8ab,we precisely integrated a fluorescence gene at the start codon. However, the seamlessly integrated reporter was unable to direct expression that recapitulates endogenousscn8abexpression. To overcome this obstacle, we introduced thehsp70minimal promoter to provide an ectopic transcription initiation site and succeeded in establishing stable KI transgenic reporters forscn8ab. This strategy also created afgf20bKI reporter line with a high success rate. Furthermore, our data revealed that an unexpectedly edited genome can inappropriately influence the integrated reporter gene expression, highlighting the importance of selecting a proper KI line. Overall, our approach utilizing a minicircle and an ectopic promoter establishes a robust and efficient strategy for KI generation, expanding our capacity to create KI animals.
Development of an undergraduate cell biology laboratory to assess pigmentation and cell size in a zebrafish model of uveal melanoma
Authors: Henle Andrea M.
Published: September 2023

This study outlines a two-week laboratory module for an authentic cell biology undergraduate research experience that uses zebrafish (Danio rerio), a popular model organism for research. Previous research has indicated that course-based undergraduate research experiences such as this one increase student confidence, active learning, and retention. During this research experience, students investigate variations in pigmentation in the caudal fins of wild type and transgenic fish (Tg(mitfa:GNAQQ209L)). The transgenic fish express a hyperactive Gα protein, GNAQQ209L, under the melanocyte-specific mitfa promoter, offering insights into uveal melanoma, a common eye cancer. Students specifically analyze the black pigmented cells, melanophores, within the caudal fin. We determined that the transgenic zebrafish have increased pigmentation in their caudal fins but smaller melanophores. These results suggest there are more melanophores in theTg(mitfa:GNAQQ209L)fish compared to the wild type. Future undergraduate research could investigate these cellular differences. This research experience imparts microscopy and image analysis skills and instills the ability to grapple with large datasets, statistical tests, and data interpretation in alignment with biology education principles. Post-lab surveys reveal students attain confidence in the above skills and in handling animals, along with a deeper appreciation for model organism research and its relevance to cancer cell biology.
Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry
Authors: Wancewicz Benjamin , Pergande Melissa R. , Zhu Yanlong , Gao Zhan , Shi Zhuoxin , Plouff Kylie , Ge Ying
Published: September 2023

The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates such as fatty acids, carbohydrates, lipids, and amino acids to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic extractions) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection - direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF MS/MS). Using DI-FTICR MS, 9,521 metabolic features were detected where 7,699 were assigned a chemical formula and 1,756 were assigned an annotated by accurate mass assignment. Using LC-Q-TOF MS/MS, 21,428 metabolic features were detected where 626 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 2276 heart metabolites were identified in this study which span a wide range of polarities including polar (benzenoids, alkaloids and derivatives and nucleosides) as well as non-polar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results of this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites.

Table of Contents Graphical Abstract
Single-cell nascent RNA sequencing using click-chemistry unveils coordinated transcription
Authors: Mahat Dig B. , Tippens Nathaniel D. , Martin-Rufino Jorge D. , Waterton Sean K. , Fu Jiayu , Blatt Sarah E. , Sharp Phillip A.
Published: September 2023

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers1–5. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations6–9. However, fundamental questions in the temporal regulation of transcription and enhancer-gene synchrony remain unanswered primarily due to the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq - a novel single-cell nascent RNA sequencing assay using click-chemistry - and unveil the coordinated transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription, and estimates burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells. It reveals the co-transcription of functionally related genes and leverages the replication-dependent non-polyadenylated histone genes transcription to elucidate cell-cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq identifies networks of enhancers and genes and indicates that the bursting of transcription at super-enhancers precedes the burst from associated genes. By imparting insights into the dynamic nature of transcription and the origin and propagation of transcription signals, scGRO-seq demonstrates its unique ability to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression.
Computational Insights on Xenobiotic Nuclear Receptor-Air Pollutant Interactions Through Molecular Docking and Dynamics Simulations
Authors: Ullah Samee , Khan Nasir Ali
Published: September 2023

Air pollution is a major global health risk, contributing to millions of deaths each year. Exposure to air pollutants can lead to a variety of adverse health effects, including oxidative stress, inflammation, and DNA damage. The nuclear receptor pregnane X receptor (PXR) is a xenobiotic sensor that plays a key role in the detoxification of environmental chemicals and air pollutants can activate these receptors, leading to the expression of genes involved in drug metabolism and detoxification. Elucidating the structural interactions of air pollutants with PXR provides insights into the mechanisms by which these pollutants exert their toxic effects. Using computational techniques, we screened 124 air pollutants toxic compounds from (APDB) against PXR and identified priority compounds for molecular dynamics simulation that revealed the dynamic binding mechanisms and structural conformations involved in PXR activation. Furthermore, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analyses characterized the stability and flexibility of PXR-pollutant complexes. Together, these computational approaches provide unprecedented insights into the molecular basis of PXR activation by air pollutants. By enabling high-throughput screening and multi-faceted biophysical characterization of PXR interactions, this work elucidates the structural mechanisms linking air pollution to impaired xenobiotic metabolism. Integration of virtual screening, molecular dynamics, and rigorous biophysical analyses represents a powerful computational systems pharmacology pipeline for assessing health risks posed by ubiquitous environmental toxicants.
Tailpiece Heating device for effective prevention of biofilm growth in sink plumbing
Authors: Kotay Shireen M , Guilford William , Pettygrove Brian A , Komisarcik Alan J , Hughes Samantha A. , Mathers Amy J
Published: September 2023

In recent years, numerous hospitals have linked patient infections toKlebsiella pneumoniaeproducing Enterobacterales (KPCE) and other resistant bacterial species in their wastewater systems and handwashing sinks. Wastewater plumbing provides a reservoir for bacteria, making them incredibly difficult to eliminate through traditional disinfection methods. Data suggests that patients become infected when bacteria grow or migrate up the proximal wastewater plumbing and into the sink basin, and are subsequently dispersed onto surrounding surfaces. Therefore, a novel electronic device was developed that acts at the highest risk area, just below the sink drain, to heat and dry out the biofilm and creating a biofilm barrier and prevent upward growth from the sink trap. The efficacy of the first prototype of a tailpiece heater (TPH) in preventing drain colonization was tested using GFP-expressingEscherichia coli(GFP-E. coli) as the challenge organism. In control sinks without the TPH, GFP-E. colibiofilm grew from the p-trap upwards to colonize the drain within 7 days. Sinks with a TPH set to 75°C were found to prevent sink drain colonization. In contrast, 65°C was not adequate to prevent drain colonization. Using KPCE the TPH was more effective than no heat control in preventing drain colonization in sinks over time. Lastly, when challenged with seeding from above, the TPH also effectively prevented KPCE colonization at the drain level. Heating of the tailpiece may offer a safe, effective, and economically attractive approach to preventing the spread of resistant bacterial species from contaminated drain biofilm to patients.
Evidence for individual vocal recognition in a pair bonding poison frog? Insights from no-choice and two-choice paradigms
Authors: Podraza Molly E. , Moss Jeanette B. , Fischer Eva K.
Published: September 2023

Individually distinctive vocalizations are widespread in nature, although the ability of receivers to discriminate these signals has only been studied through limited taxonomic and social lenses. Here we ask whether anuran advertisement calls, typically studied for their role in territory defense and mate attraction, facilitate recognition and preferential association with partners in a pair bonding poison frog. Combining no- and two-stimulus choice playback experiments, we evaluated behavioral and physiological responses of females to male acoustic stimuli. Virgin females oriented to and approached speakers broadcasting male calls independent of caller identity, implying that females are generally attracted to male acoustic stimuli outside the context of a pair bond. When pair bonded females were presented with calls of a mate and a stranger, they showed a slight preference for calls of their mate. Moreover, behavioral responses varied with breeding status: females with eggs were faster to approach stimuli and spent more time in the mate arm than females that were pair bonded but did not currently have eggs. Our study suggests a potential role for individual vocal recognition in the formation and maintenance of pair bonds in a poison frog and raises new questions about how acoustic signals are perceived in the context of monogamy and biparental care.
Dietary Availability Acutely Governs Puberty Onset via Hypothalamic Neural Circuit
Authors: Goto Teppei , Hagihara Mitsue , Irie Satsuki , Abe Takaya , Kiyonari Hiroshi , Miyamichi Kazunari
Published: September 2023

Reproduction poses a substantial burden, especially for mammalian females. Puberty onset serves as a vital checkpoint, regulated based on the body’s energy state, to prevent inappropriate reproductive activity under malnutrition. However, the neural basis of this puberty checkpoint remains poorly understood. Here, we demonstrate that peripubertal malnutrition in female mice reduces the synchronous activity episodes of arcuate kisspeptin neurons (SEskiss), which are critical regulators of the gonadotropin axis. Improved dietary availability boosts SEskissfrequency, facilitating puberty onset. Using a viral-genetic approach, we show that the activating agouti-related protein neurons in the arcuate nucleus (ARCAgrpneurons), a hunger center, suppresses SEskiss, even with enough food. Conversely, loss-of-function of ARCAgrpneurons enhances SEskissduring malnutrition, partly promoting irregular sexual maturation. Collectively, a neural circuit connecting feeding to reproductive centers is responsible for disinhibiting SEskissfrequency based on dietary availability, which sheds light on the neural basis of puberty checkpoint.

Highlights

The pulsatile activity of arcuate kisspeptin neurons emerges and grows before vaginal opening.

Pubertal impairment resulting from malnutrition is associated with a reduction in the pulsatile activity of kisspeptin neurons.

Pubertal recovery by food availability follows the elevated pulsatile activity of kisspeptin neurons during catch-up growth.

The arcuate Agrp neurons suppress the frequency of pulsatile activity of kisspeptin neurons under negative energy balance.
bamSliceR: a Bioconductor package for rapid, cross-cohort variant and allelic bias analysis
Authors: Huang Yizhou Peter , Harmon Lauren , Deering-Gardner Eve , Ma Xiaotu , Harsh Josiah , Xue Zhaoyu , Wen Hong , Ramos Marcel , Davis Sean , Triche Timothy J.
Published: September 2023

The NCI Genomic Data Commons (GDC) provides controlled access to sequencing data from thousands of subjects, enabling large-scale study of impactful genetic alterations such as simple and complex germline and structural variants. However, efficient analysis requires significant computational resources and expertise, especially when recalling variants from raw sequence reads. We thus developedbamSliceR, an R/Bioconductor package that builds upon theGenomicDataCommonspackage to extract aligned sequence reads from cross-GDC meta-cohorts, followed by targeted analysis of variants and effects (including transcript-aware variant annotation from transcriptome-aligned GDC RNA data). Here we demonstrate population-scale genomic & transcriptomic analyses with minimal compute burden viabamSliceR, identifying recurrent, clinically relevant sequence and structural variants in the TARGET AML and BEAT-AML cohorts. We then validate results in the (non-GDC) Leucegene cohort, demonstrating how thebamSliceRpipeline can be seamlessly applied to replicate findings in non-GDC cohorts. These variants directly yield clinically impactful and biologically testable hypotheses for mechanistic investigation.bamSliceRhas been submitted to the Bioconductor project, where it is presently under review, and is available on GitHub athttps://github.com/trichelab/bamSliceR.
Carvedilol suppresses ryanodine receptor-dependent Ca2+bursts in human neurons bearingPSEN1variants found in early onset Alzheimer’s disease
Authors: Hori Atsushi , Ai Tomohiko , Hato Takashi , Inaba Haruka , Tanaka Kimie , Sato Shoichi , Okamoto Mizuho , Horiuchi Yuna , Paran Faith Jessica , Tabe Yoko , Rosales Corina , Akamatsu Wado , Murayama Takashi , Kurebayashi Nagomi , Sakurai Takashi , Miida Takashi
Published: September 2023

Seizures are increasingly being recognized as the hallmark of Alzheimer’s disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid β (Aß) depositions. However, it can also be a cell-autonomous phenomenon causing AD by Aß-independent mechanisms. Indeed, various studies using animal models showed that Ca2+releases from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+signaling in neural cells derived from three early-onset AD patients harboring variants of Presenilin-1 (PSEN1p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal Aß. We found that allPSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, anRyR2 inhibitor, and VK-II-86, an analog of carvedilol without the β-blocking effects, sufficiently eliminated the abnormal Ca2+bursts. In contrast, Dantrolene, a RyR1 inhibitor, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246Ecells compared to the WT neurons. Thus, we propose that aberrant Ca2+signaling is a key link between human pathogenicPSEN1variants and cell-intrinsic hyperactivity prior to deposition of abnormal Aß, offering prospects for the development of targeted prevention strategies for at-risk individuals.

One Sentence Summary

Aberrant Ca2+-signaling causesPSEN1-related early onset Alzheimer’s disease.
Mathematical Analysis of Left Ventricular Elastance with respect to Afterload Change During Ejection Phase
Authors: Kato Shiro , Himeno Yukiko , Amano Akira
Published: September 2023

Since the left ventricle (LV) has pressure (Pιυ) and volume (Vιυ), we can define LV elastance from the ratio betweenPιυandVιυ, termed as “instantaneous elastance.” On the other hand, end-systolic elastance (Emax) is known to be a good index of LV contractility, which is measured by the slope of several end-systolicPιυ-Vιυpoints obtained by using different loads. The wordEmaxoriginates from the assumption that LV elastance increases during the ejection phase and attains its maximum at the end-systole. From this concept, we can define another elastance determined by the slopes of isochronousPιυ-Vιυpoints, that isPιυ-Vιυpoints at a certain time after the ejection onset time by using different loads. We refer to this elastance as “load-dependent elastance.”

To reveal the relation between these two elastances, we used a hemodynamic model that included a detailed ventricular myocyte contraction model. From the simulation results, we found that the isochronousPιυ-Vιυpoints lay in one line and that the line slope corresponding to the load-dependent elastance slightly decreased during the ejection phase, which is quite different from the instantaneous elastance.

Subsequently, we analyzed the mechanism determining these elastances from the model equations. We found that instantaneous elastance is directly related to contraction force generated by the ventricular myocyte, but the load-dependent elastance is determined by two factors: one is the transient characteristics of the cardiac cell, i.e., the velocity–dependent force drops characteristics in instantaneous shortening. The other is the force–velocity relationship of the cardiac cell. We also found that the linear isochronous pressure–volume relation is based on the approximately linear relation between the temporal differential of the cellular contraction force and the cellular shortening velocity that results from the combined characteristics of LV and aortic compliances.
Enhancing KCC2 function reduces interictal activity and prevents seizures in mesial temporal lobe epilepsy
Authors: Donneger Florian , Zanin Adrien , Besson Jeremy , Kadiri Yoness , Pagan Carla , David Nicolas , Russeau Marion , Bielle Franck , Devaux Bertrand , Mathon Bertrand , Navarro Vincent , Chassoux Francine , Poncer Jean Christophe
Published: September 2023

The neuronal K/Cl cotransporter KCC2 controls intraneuronal chloride and subsequently the efficiency of GABA signaling. In many neurological disorders, including mesial temporal lobe epilepsy (mTLE), reduced KCC2 expression or function may lead to depolarizing GABA signaling, which may then contribute to pathological activity and seizures. Compensating for the dysregulation of chloride transport in the pathology therefore appears to be a promising therapeutic strategy. Two small molecules - prochlorperazine (PCPZ) and CLP-257 - recently identified from library screening as candidate KCC2 enhancers, appear to improve symptoms in animal models of disorders associated with KCC2 extinction. However, their mode of action in cortical neurons and their therapeutic potential in epilepsy remain elusive and even controversial. Here, we show in rat hippocampal neurons that PCPZ and CLP-257 act by increasing KCC2 function and clustering while decreasing its membrane diffusion, independent of phosphorylation of canonical regulatory residues. Consistently, both compounds suppress spontaneous interictal-like discharges in postoperative tissue from patients with mTLE. Chronic administration of PCPZ also reduces seizure frequency and interictal activity in a mouse model of mTLE. These results unravel the mechanism of action of two KCC2 enhancers and validate their therapeutic potential in mesial temporal lobe epilepsy.
A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria
Authors: Vento Justin M. , Durmusoglu Deniz , Li Tianyu , Sullivan Sean , Ttofali Fani , Schaik John van , Yu Yanying , Barquist Lars , Crook Nathan , Beisel Chase L.
Published: September 2023

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic-biology chasses. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here we report IMPRINT, a generalized, rapid and scalable approach based on cell-free transcription-translation (TXTL) systems to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from the bacterial host’s restriction-modification systems. The expressed methyltransferases then methylate DNAin vitroto match the host DNA’s methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also developed a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to facilitate a library screen for translational rules in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling use of increasingly sophisticated genetic manipulation tools across the bacterial world.
HLA-Based Banking of Human Induced Pluripotent Stem Cells in Saudi Arabia
Authors: Alowaysi Maryam , Lehmann Robert , Al-Shehri Mohammad , Baadheim Moayad , Alzahrani Hajar , Aboalola Doaa , Zia Asima , Malibari Dalal , Daghestani Mustafa , Alghamdi Khaled , Haneef Ali , Jawdat Dunia , Hakami Fahad , Gomez-Cabrero David , Tegner Jesper , Alsayegh Khaled
Published: September 2023

Human iPSCs’ derivation and use in clinical studies are transforming medicine. Yet, there is a high cost and long waiting time for autologous iPS-based cellular therapy, and the genetic engineering of hypo-immunogenic iPS cell lines is hampered with numerous hurdles. Therefore, it is increasingly interesting to create cell stocks based on HLA haplotype distribution in a given population. In this study, we assessed the potential of HLA-based iPS banking for the Saudi population. First, we analyzed the HLA database of the Saudi Stem Cell Donor Registry (SSCDR), which contains high-resolution HLA genotype data of 64,315 registered Saudi donors at the time of analysis. We found that only 13 iPS lines would be required to cover 30% of the Saudi population, 39 iPS lines would offer 50% coverage and 596 for more than 90% coverage.

Next, As a proof-of-concept, we launched the first HLA-based banking of iPSCs in Saudi Arabia. Using clinically relevant methods, we generated the first iPSC line from a homozygous donor for the most common HLA haplotype in Saudi. The two generated clones expressed pluripotency markers, could be differentiated into all three germ layers, beating cardiomyocytes and neuronal progenitors. To ensure that our reprogramming method generates genetically stable iPSCs, we assessed the mutational burden in the generated clones and the original blood sample from which the iPSCs were derived using whole-genome sequencing. All detected variants were found in the original donor sample and were classified as benign according to current guidelines of the American College of Medical Genetics and Genomics (ACMG).

This study sets a road map for introducing iPS-based cell therapy in the Kingdom of Saudi Arabia.