bluuebunny/crossref_metadata_2025_split huggingface dataset:
Promoters constrain evolution of expression levels of essential genes inEscherichia coli
Authors: Tsuru Saburo , Hatanaka Naoki , Furusawa Chikara 
Published: May 2024

AbstractVariability in expression levels in response to random genomic mutations varies among genes, influencing both the facilitation and constraint of phenotypic evolution in organisms. Despite its importance, both the underlying mechanisms and evolutionary origins of this variability remain largely unknown due to the mixed contributions ofcis- andtrans-acting elements. To address this issue, we focused on the mutational variability ofcis-acting elements, that is, promoter regions, inEscherichia coli. Random mutations were introduced into the natural and synthetic promoters to generate mutant promoter libraries. By comparing the variance in promoter activity of these mutant libraries, we found no significant difference in mutational variability in promoter activity between promoter groups, suggesting the absence of a signature of natural selection for mutational robustness. In contrast, the promoters controlling essential genes exhibited a remarkable bias in mutational variability, with mutants displaying higher activities than the wild types being relatively rare compared to those with lower activities. Our evolutionary simulation on a rugged fitness landscape provided a rationale for this vulnerability. These findings suggest that past selection created non-uniform mutational variability in promoters biased toward lower activities of random mutants now constrain future evolution of downstream essential genes toward higher expression levels.
Combined forces of hydrostatic pressure and actin polymerization drive endothelial tip cell migration and sprouting angiogenesis
Authors: Kondrychyn Igor , He Liqun , Wint Haymar , Betsholtz Christer , Phng Li-Kun 
Published: May 2024

SUMMARYCell migration is a key process in the shaping and formation of tissues. During sprouting angiogenesis, endothelial tip cells invade avascular tissues by generating actomyosin-dependent forces that drive cell migration and vascular expansion. Surprisingly, ECs can still invade if actin polymerization is inhibited. In this study, we show that endothelial tip cells employ an alternative mechanism of cell migration that is dependent on Aquaporin (Aqp)-mediated water inflow and increase in hydrostatic pressure. In the zebrafish, ECs expressaqp1a.1andaqp8a.1in newly formed vascular sprouts in a VEGFR2-dependent manner. Aqp1a.1 and Aqp8a.1 loss-of-function studies show an impairment in intersegmental vessels formation because of a decreased capacity of tip cells to increase their cytoplasmic volume and generate membrane protrusions, leading to delayed tip cell emergence from the dorsal aorta and slower migration. Further inhibition of actin polymerization resulted in a greater decrease in sprouting angiogenesis, indicating that ECs employ two mechanisms for robust cell migrationin vivo. Our study thus highlights an important role of hydrostatic pressure in tissue morphogenesis.
NPD1/GPR37 axis protects painful traumatic brain injury and its complications
Authors: Zhao Junli , Chandra Sharat , Wang Yuqing , Zhang Vivian , Wang Haichen , Ji Ru-Rong 
Published: May 2024

AbstractPatients with traumatic brain injury (TBI) frequently exhibit heightened pain and associated complications such as cognitive decline, depression, and anxiety. GPR37 is widely expressed in various brain regions, but its function remains largely unclear. We recently discovered neuroprotectin D1 (NPD1) as a novel GPR37 ligand. In this study, we examined the protective role of the NPD1/GPR37 signaling pathway in TBI-induced neuropathic pain and its complications. TBI was induced by closed-head impact and resulted in transient neuropathic pain for less than two weeks, showing periorbital and cutaneous mechanical allodynia/hyperalgesia, as well as motor deficiency and cognitive impairment. We found that peri-surgical treatment with NPD1, effectively prevented TBI-induced mechanical hypersensitivity, motor deficiency, and cognitive impairment. NPD1 treatment also substantially inhibited TBI-induced microgliosis, astrogliosis (including A1 astrocyte markers), and neuroinflammation in the sensory cortex and hippocampus. RNA sequencing and GO enrichment analysis revealed downregulations of genes related to “calcium ion homeostasis,” and “GPCR signaling pathway” in the TBI-affected brain. These downregulations were restored by NPD1 treatment. RNAscopein situhybridization revealed predominantGpr37mRNA expression in oligodendrocytes. TBI resulted in rapid and remarkable demyelination and downregulation ofGpr37mRNA expression in oligodendrocytes, and both were protected by NPD1 treatment. NPD1’s inhibition of periorbital and cutaneous mechanical pain was abolished inGpr37-/-mice. Moreover, TBI-induced neuropathic pain was prolonged by swimming stress, and NPD1 treatment prevented the stress-induced transition from acute to chronic pain in wild-type mice but notGpr37-/-mice. Finally, chronic pain was associated with depression and anxiety, and NPD1 treatment mitigated these chronic pain complications through GPR37. Thus, through modulation of demyelination, diverse responses of glial cells, and neuroinflammation, the NPD1/GPR37 axis serves as a protective mechanism and a therapeutic target against painful traumatic brain injury and its complications.
N-cadherin facilitates trigeminal sensory neuron outgrowth and target tissue innervation
Authors: Halmi Caroline A. , Leonard Carrie E. , McIntosh Alec T. , Taneyhill Lisa A. 
Published: May 2024

ABSTRACTDuring embryonic development, diverse cell types coordinate to form functionally complex tissues. Exemplifying this process, the trigeminal ganglion emerges from the condensation of two distinct precursor cell populations, cranial placodes and neural crest, with neuronal differentiation of the former preceding the latter. While its dual cellular origin has been understood for decades, the molecules orchestrating trigeminal ganglion formation remain relatively obscure. Initial assembly of the trigeminal ganglion is mediated by cell adhesion molecules, including neural cadherin (N-cadherin), which is first expressed by placodal neurons and is required for their proper condensation with other neurons and neural crest cells. Axon outgrowth first occurs from placodal neurons, but as gangliogenesis proceeds, neural crest cells also differentiate into N-cadherin-expressing neurons, and together both extend axons toward target tissues. However, a role for N-cadherin in regulating axon outgrowth and innervation of target tissues by trigeminal neurons has not been explored. To this end, we depleted N-cadherin from chick trigeminal placode cells and observed decreases in trigeminal ganglion size, nerve growth, and target innervationin vivo, phenotypes that could only partially be attributed to increased apoptosis early in gangliogenesis. Accordingly, neurite number and branching of neural crest-derived neurons was decreasedin vitroin response to N-cadherin knockdown in placode cells, providing a novel non-cell autonomous explanation for these morphological changes. Inhibiting N-cadherin-mediated adhesion with a function-blocking antibody prevented axon extension in most, but not all, placode-derived trigeminal neuronsin vitro, indicating potential unique requirements for N-cadherin in various neuronal subtypes. Collectively, these findings reveal persistent cell autonomous and non-cell autonomous functions for N-cadherin, thus highlighting the critical role of N-cadherin in mediating reciprocal interactions between neural crest and placode neuronal derivatives during trigeminal ganglion development.
Sphingosine-1-phosphate signaling inhibition suppresses Th1-like Treg generation by reversing mitochondrial uncoupling
Authors: Coulombeau Rachel , Selck Claudia , Giang Nicolas , Al-Mohammad Abdulrahman , Ng Natalie , Maher Allison K , Argüello Rafael , Scalfari Antonio , Varley James , Nicholas Richard , Dominguez-Villar Margarita 
Published: May 2024

AbstractInflammatory environments induce the generation of dysfunctional IFNγ+T-bet+FOXP3+Th1-like Tregs, which show defective function and are found in autoimmune conditions including multiple sclerosis (MS). The pathways that control the generation of Th1-like Tregs are not well understood. Sphingosine-1-phosphate (S1P) signaling molecules are upregulated in Th1-like Tregs, andin vivoS1P inhibition with Fingolimod (FTY720) inhibits the expression of genes responsible for Treg plasticity in MS patients. However, the underlying mechanisms are unknown. Here we show that S1P signaling inhibition by FTY720 inhibits the generation of Th1-like Tregs and rescues their suppressive function. These effects are mediated by a decrease in mTORC1 signaling and reversal of the mitochondrial uncoupling that Tregs undergo during their reprogramming into Th1-like Tregsin vitro. Finally, these results are validated inin vivogenerated Th1-like Tregs, as the Tregs from MS patients treated with FTY720 display decreased Th1-like Treg frequency, increased suppressive function, and mitochondrial metabolism rebalance. These results highlight the involvement of mitochondrial uncoupling in Treg reprogramming and identify S1P signaling inhibition as a target to suppress the generation of dysfunctional Th1-like Tregs.
The insulin / IGF axis is critically important controlling gene transcription in the podocyte
Authors: Hurcombe Jennifer A. , Dayalan Lusyan , Barrington Fern , Burdet Frédéric , Ni Lan , Coward Joseph T. , Brinkkoetter Paul T. , Holzenberger Martin , Jeffries Aaron , Oltean Sebastian , Welsh Gavin I. , Coward Richard J.M. 
Published: May 2024

SUMMARYSignalling to the podocyte via the structurally related insulin receptor (IR) or insulin-like growth factor 1 receptor (IGF1) is important for podocyte function. This study sought to elucidate the compound role of the insulin/IGF1 axis in podocytes using transgenic mice and cell culture models deficient in both receptors.Podocyte specific IR/IGF1R knockdown mice developed a severe kidney phenotype with albuminuria, glomerulosclerosis and renal failure with death occurring in some mice between 4 and 24 weeks. Simultaneous knockdown of both receptors in cultured podocytes resulted in >50% cell death by 7 days.Proteomic analysis revealed a striking downregulation of spliceosome-related proteins in IR/IGF1R knockdown podocytes with long-read RNA sequence data indicating an increased fraction of transcripts with intron retention/premature termination codons in these cells.This work underlines the critical importance of podocyte insulin/IGF signalling revealing a novel role for this extrinsic hormonal signalling axis in regulating gene transcription.
Cigarette smoke impairs pulmonary vascular function through nAChR activation
Authors: Munar-Rubert O , Andreu-Martínez R , Rodríguez-Pérez J , López N , Barreira B , Fernández-Malavé E , Peces-Barba G , Muñoz-Calleja C , Cogolludo A , Calzada MJ 
Published: May 2024

ABSTRACTTobacco smoke is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), a major health concern worldwide. Despite current therapies alleviate symptoms; there remain some limitations in the efficacy of treatments to curb COPD and its cardiovascular morbidities, particularly pulmonary hypertension. Our previous studies demonstrate that cigarette smoke (CS) has direct effects on pulmonary vascular tone homeostasis and contribute to pulmonary arterial dysfunction. This is in part due to altered activity of the voltage-dependent K+channel, and to an exacerbated oxidative stress promoting a switch in the sGCs redox state. However, further characterization of the molecular basis of CS-mediated PA dysfunction is needed for more effective targeted treatment and prevention. Our current studies explored these molecular pathways and specifically addressed their contribution to the cellular contractile apparatus within pulmonary arteries. Our results proved deleterious effects on the contractile machinery of pulmonary artery smooth muscle cells. Increased oxidative stress and calcium dysregulation resulting from the activation of acetylcholine receptors (nAChR) in the pulmonary artery led to the manifestation of these effects. This groundbreaking discovery unveiled, for the first time, the expression of these receptors in human pulmonary arteries. Furthermore, we proved that inhibitors directed at these receptors demonstrate efficacy in alleviating various harmful effects of smoking and safeguarding pulmonary artery function from damage. These discoveries hold significant clinical implications, as they suggest that treatment with nAChR-targeted inhibitors could constitute a viable therapeutic option for COPD-related pulmonary hypertension in patients who do not respond to conventional medication.
Cell type-specific network analysis in Diversity Outbred mice identifies genes potentially responsible for human bone mineral density GWAS associations
Authors: Dillard Luke J , Calabrese Gina M , Mesner Larry D , Farber Charles R 
Published: May 2024

AbstractGenome-wide association studies (GWASs) have identified many sources of genetic variation associated with bone mineral density (BMD), a clinical predictor of fracture risk and osteoporosis. Aside from the identification of causal genes, other difficult challenges to informing GWAS include characterizing the roles of predicted causal genes in disease and providing additional functional context, such as the cell type predictions or biological pathways in which causal genes operate. Leveraging single-cell transcriptomics (scRNA-seq) can assist in informing BMD GWAS by linking disease-associated variants to genes and providing a cell type context for which these causal genes drive disease. Here, we use large-scale scRNA-seq data from bone marrow–derived stromal cells cultured under osteogenic conditions (BMSC-OBs) from Diversity Outbred (DO) mice to generate cell type-specific networks and contextualize BMD GWAS-implicated genes. Using trajectories inferred from the scRNA-seq data, we identify networks enriched with genes that exhibit the most dynamic changes in expression across trajectories. We discover 21 network driver genes, which are likely to be causal for human BMD GWAS associations that colocalize with expression/splicing quantitative trait loci (eQTL/sQTL). These driver genes, includingFgfrl1andTpx2,along with their associated networks, are predicted to be novel regulators of BMD via their roles in the differentiation of mesenchymal lineage cells. In this work, we showcase the use of single-cell transcriptomics from mouse bone-relevant cells to inform human BMD GWAS and prioritize genetic targets with potential causal roles in the development of osteoporosis.
Sequential deactivation across the thalamus-hippocampus-mPFC pathway during loss of consciousness
Authors: Chen Xiaoai , Cramer Samuel R. , Chan Dennis C.Y. , Han Xu , Zhang Nanyin 
Published: May 2024

AbstractHow consciousness is lost in states such as sleep or anesthesia remains a mystery. To gain insight into this phenomenon, we conducted concurrent recordings of electrophysiology signals in the anterior cingulate cortex and whole-brain functional magnetic resonance imaging (fMRI) in rats exposed to graded propofol, undergoing the transition from consciousness to unconsciousness. Our results reveal that upon the loss of consciousness (LOC), as indicated by the loss of righting reflex, there is a sharp increase in low-frequency power of the electrophysiological signal. Additionally, simultaneously measured fMRI signals exhibit a cascade of deactivation across a pathway including the hippocampus, thalamus, and medial prefrontal cortex (mPFC) surrounding the moment of LOC, followed by a broader increase in brain activity across the cortex during sustained unconsciousness. Furthermore, sliding window analysis demonstrates a temporary increase in synchrony of fMRI signals across the hippocampus-thalamus-mPFC pathway preceding LOC. These data suggest that LOC might be triggered by sequential activities in the hippocampus, thalamus and mPFC, while wide-spread activity increases in other cortical regions commonly observed during anesthesia-induced unconsciousness might be a consequence, rather than a cause of LOC. Taken together, our study identifies a cascade of neural events unfolding as the brain transitions into unconsciousness, offering critical insight into the systems-level neural mechanisms underpinning LOC.
Determination of metal ion transport rate of human ZIP4 using stable zinc isotopes
Authors: Jiang Yuhan , MacRenaris Keith , O’Halloran Thomas V. , Hu Jian 
Published: May 2024

AbstractThe essential microelement zinc is absorbed in the small intestine mainly by the zinc transporter ZIP4, a representative member of the Zrt/Irt-like protein (ZIP) family. ZIP4 is reportedly upregulated in many cancers, making it a promising oncology drug target. To date, there have been no reports on the turnover number of ZIP4, which is a crucial missing piece of information needed to better understand the transport mechanism. In this work, we used a non-radioactive zinc isotope,70Zn, and inductively coupled plasma mass spectrometry (ICP-MS) to study human ZIP4 (hZIP4) expressed in HEK293 cells. Our data showed that70Zn can replace the radioactive65Zn as a tracer in kinetic evaluation of hZIP4 activity. This approach, combined with the quantification of the cell surface expression of hZIP4 using biotinylation or surface-bound antibody, allowed us to estimate the apparent turnover number of hZIP4 to be in the range of 0.08-0.2 s-1. The turnover numbers of the truncated hZIP4 variants are significantly smaller than that of the full-length hZIP4, confirming a crucial role for the extracellular domain in zinc transport. Using64Zn and70Zn, we measured zinc efflux during the cell-based transport assay and found that it has little effect on the zinc import analysis under these conditions. Finally, we demonstrated that use of laser ablation (LA) ICP-TOF-MS on samples applied to a solid substrate significantly increased the throughput of the transport assay. We envision that the approach reported here can be applied to the studies of metal transporters beyond the ZIP family.
Polar flagellin glycan metaheterogeneity ofAeromonas hydrophilastrain ATCC 7966T
Authors: Fulton Kelly M. , Mendoza-Barberà Elena , Tomás Juan M. , Twine Susan M. , Smith Jeffrey C. , Merino Susana 
Published: May 2024

ABSTRACTMotile pathogens often rely upon flagellar motility as an essential virulence factor and in many species the structural flagellin protein is glycosylated. This post-translational modification has been shown to be necessary for proper folding of the flagellin structural proteins and proper function of the flagellar filament in a number of bacterial species.Aeromonas hydrophilais a ubiquitous aquatic pathogen with a constitutively expressed polar flagellum. Using a suite of mass spectrometry techniques, the flagellin FlaA and FlaB structural proteins ofA. hydrophilastrain ATCC 7966Twere shown to be glycosylated with significant metaheterogeneity: heterologous glycans were observed with variable site occupancy. The penta- and hexa-saccharide glycan chains contained a previously unreported pseudaminic acid derivative with a mass of 422 Da as the linking sugar, followed in sequence by two hexoses, anN-acetylglucosamine derivative, a deoxyN-acetylglucosamine derivative, and sometimes an additionalN-acetylglucosamine.
Decoding Secondary Motor Cortex Neuronal Activity during Cocaine Self-Administration: Insights from Longitudinalin vivoCalcium Imaging
Authors: Chen Yingying , Fu Haoying , Korada Amith , Lange Michal A. , Rayanki Chandrashekar , Lu Tao , Lai Dongbing , Fang Shiaofen , Guo Changyong , Ma Yao-Ying 
Published: May 2024

ABSTRACTBackgroundWe recently reported that the risk of cocaine relapse is linked to hyperexcitability in the secondary motor cortex (M2) after prolonged withdrawal following intravenous self-administration (IVSA). However, the neuronal mechanisms underlying drug-taking behaviors and the response of M2 neurons to contingent drug delivery remain poorly understood.MethodsMice received cocaine as reinforcement (RNF) following active lever presses (ALP) but not inactive lever presses (ILP). Usingin vivocalcium imaging during cocaine IVSA, we tracked M2 neuronal activity with single-cell resolution. We then analyzed Ca2+transients in M2 at the earlyvs. late stages during the 1-hr daily sessions on IVSA Day 1 and Day 5.ResultsM2 neurons adapted to both operant behaviors and drug exposure history. Specifically, saline mice showed a reduction in both saline taking behaviors and Ca2+transient frequency with the 1-hr session. In contrast, cocaine mice maintained high ALP and RNF counts, with increased Ca2+transient frequency and amplitude on Day 1, persisting through Day 5. Compared to saline controls, cocaine mice exhibited a lower % of positively responsive neurons (Pos) and higher % of negatively responsive neurons (Neg) before ALP and after RNF, a difference not seen before ILP. Furthermore, as drug-taking behaviors progressed during the daily session, cocaine mice showed greater neuronal engagement with a larger population, particularly linked to ALP and RNF, with reduced overlap in neurons associated with ILP.ConclusionThe M2 undergoes dynamic neuronal adaptations during early drug-taking behaviors, indicating its role as a potential substrate mediating the persistence of drug-seeking behaviors in cocaine relapse.
High-density CRISPRi screens reveal adaptive transcriptional gradients in cyanobacteria
Authors: Hren Andrew , Lollini Nicole , Carper Dana L. , Abraham Paul , Cameron Jeffrey C. , Fox Jerome M. , Eckert Carrie 
Published: May 2024

ABSTRACTCyanobacteria are the oldest form of photosynthetic life on Earth and contribute to primary production in nearly every habitat, from permafrost to hot springs. Despite longstanding interest in the biochemical basis of environmental adaptation in these microbes, it remains poorly understood and challenging to re-wire. This study uses a high-density, genome-wide CRISPR interference (CRISPRi) screen to examine the influence of gene-specific transcriptional variation on the growth ofSynechococcussp. PCC 7002 under environmental extrema. Surprisingly, many partial knockdowns enhanced fitness under cold monochromatic conditions. Notably, transcriptional repression of a gene for a core subunit of the NDH-1 complex, which is important for photosynthesis and carbon uptake, improved growth rates under both red and blue light but at distinct, color-specific optima. In general, most genes with fitness-improving knockdowns were distinct to each light color, evidencing unique stress responses and alleviation mechanisms. Multi-target transcriptional repression produced nonadditive effects. Findings reveal diverse mechanisms of environmental adaptation in cyanobacteria and provide a new approach for using gradients in sgRNA activity to pinpoint biochemically influential transcriptional changes in cells.SIGNIFICANCE STATEMENTCyanobacteria are the most abundant photosynthetic organisms on Earth, where they endure a striking variety of environmental fluctuations. This study examines the biochemical basis of environmental adaptation inSynechococcussp. PCC 7002, an important model strain, by modulating the expression of every gene in its genome. Results show that partial, but not complete, reduction in the expression of a subset of influential genes can improve growth under cold monochromatic conditions. Optimal expression levels differ between red and blue light and shift with multi-gene adjustments. Findings show how minor transcriptional adjustments can yield major improvements in growth under environmental extrema and provide a powerful systems-level approach for studying—and fine-tuning—the adaptive capacity of microbes.