bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus
Authors: Kong Yvonne X , Rehan Rajan , Moreno Cesar L , Madsen Søren , Zhang Yunwei , Zhao Huiwen , Houlahan Callum , Cartland Siân P , Robertshaw Declan , Trang Vincent , Liang Ong Frederick Jun , Cielesh Michelle , Cooke Kristen C , Potter Meg , Stӧckli Jacqueline , Morahan Grant , Kalev-Zylinska Maggie , Rondina Matthew T , Schulman Sol , Yang Jean , Neely G Gregory , James David , Kavurma Mary M , Hocking Samantha , Twigg Stephen M , Weaver James , Larance Mark , Passam Freda H
Published: February 2024

High platelet reactivity is associated with adverse clinical events and is more frequent in people with diabetes mellitus (DM). To better understand platelet dysfunction in DM, we performed a proteomic analysis in platelets from a matched cohort of 34 people without, and 42 people with type 2 DM. The cohorts were matched by clinical characteristics including age, sex, and coronary artery disease burden. Using high sensitivity unbiased proteomics, we consistently identified over 2,400 intracellular proteins, and detected proteins that are differentially released by platelets from people with diabetes in response to low dose thrombin. Importantly, we identified the endoplasmic reticulum (ER) protein SEC61 translocon subunit beta (SEC61B) was increased in platelets from humans and mice within vivohyperglycemia. SEC61B was increased in megakaryocytes in mouse models of diabetes, in association with megakaryocyte ER stress. A rise in cytosolic calcium is a key aspect in platelet activation, and the SEC61 translocon is known to act as a channel for ER calcium leak. We demonstrate that cultured cells overexpressing SEC61B have increased calcium flux and decreased protein synthesis. In accordance, hyperglycemic mouse platelets mobilized more calcium to the cytosol and had lower protein synthesis compared with normoglycemic platelets. Independently,in vitroinduction of ER stress increased platelet SEC61B expression and markers of platelet activation. We propose a mechanism whereby ER stress-induced upregulation of platelet SEC61B leads to increased cytosolic calcium, potentially contributing to platelet hyperactivity in people with diabetes.

Key Points

Platelet SEC61B is increased in hyperglycemia and contributes to increased endoplasmic reticulum (ER) calcium leak

Increased ER calcium leak is associated with ER stress and platelet hyperactivity
Local Delivery of Soluble Fractalkine (CX3CL1) Peptide Restore Ribbon Synapses After Noise-Induced Cochlear Synaptopathy
Authors: Manickam Vijayprakash , Maity Sibaprasad , Murali Sree Varshini , Gawande Dinesh Y. , Stothert Andrew R. , Batalkina Lyudamila , Cardona Astrid , Kaur Tejbeer
Published: February 2024

Efficacy of chemokine fractalkine isoforms was evaluated for restoration of loss of inner hair cell ribbon synapses and hearing after noise-induced cochlear synaptopathy (NICS). Previously, we have demonstrated a critical role for fractalkine signaling axis (CX3CL1-CX3CR1) in synaptic repair where in the presence of fractalkine receptor (CX3CR1) expressed by cochlear macrophages, the damaged synapses are spontaneously repaired. Here, we examined whether overexpression of fractalkine ligand (CX3CL1 or FKN) in the form of a peptide is effective in restoring the lost synapses and hearing after NICS. Remarkably, single transtympanic (TT) injection of soluble isoform of FKN (sFKN) peptide at 1 day after synaptopathic noise trauma showed significant recovery of ABR thresholds, ABR peak I amplitudes and ribbon synapses in both FKN-wildtype and knockout mice when compared to mice injected with full length membrane-bound FKN peptide (mFKN). Mechanistically, sFKN peptide treatment increased macrophage numbers in the cochlea and in the absence of those macrophages, sFKN failed to restore loss of synapses and hearing after NICS. Furthermore, sFKN treatment attenuated cochlear inflammation after noise overexposure without altering the expression of CX3CR1. Finally, sFKN peptide was detectable inside the cochlea localized to the sensory epithelium for 24 hours after TT injection. These data provide a robust proof-of-principle that local delivery of an immune factor, sFKN is effective in restoring lost ribbon synapses and hearing after NICS in a macrophage-dependent manner and highlights the potential of sFKN as an immunotherapy for cochlear synaptopathy due to noise or aging.

Transtympanic delivery of soluble FKN peptide is effective in restoring lost inner hair cell ribbon synapses and hearing after noise-induced cochlear synaptopathy in a macrophage-dependent manner.
Uncovering the Interleukin-12 Pharmacokinetic Desensitization Mechanism and Its Consequences with Mathematical Modeling
Authors: DeBonis Jonathon , Veiseh Omid , Igoshin Oleg A.
Published: February 2024

The cytokine interleukin-12 (IL-12) is a potential immunotherapy because of its ability to induce a Th1 immune response. However, success in the clinic has been limited due to a phenomenon called IL-12 desensitization – the trend where repeated exposure to IL-12 leads to reduced IL-12 concentrations (pharmacokinetics) and biological effects (pharmacodynamics). Here, we investigated IL-12 pharmacokinetic desensitization via a modeling approach to (i) validate proposed mechanisms in literature and (ii) develop a mathematical model capable of predicting IL-12 pharmacokinetic desensitization. Two potential causes of IL-12 pharmacokinetic desensitization were identified: increased clearance or reduced bioavailability of IL-12 following repeated doses. Increased IL-12 clearance was previously proposed to occur due to the upregulation of IL-12 receptor on T-cells that causes increased receptor-mediated clearance in the serum. However, our model with this mechanism, the accelerated-clearance model, failed to capture trends in clinical trial data. Alternatively, our novel reduced-bioavailability model assumed that upregulation of IL-12 receptor on T-cells in the lymphatic system leads to IL-12 sequestration, inhibiting the transport to the blood. This model accurately fits IL-12 pharmacokinetic data from three clinical trials, supporting its biological relevance. Using this model, we analyzed the model parameter space to illustrate that IL-12 desensitization occurs over a robust range of parameter values and to identify the conditions required for desensitization. We next simulated local, continuous IL-12 delivery and identified several methods to mitigate systemic IL-12 exposure. Ultimately, our results provide quantitative validation of our proposed mechanism and allow for accurate prediction of IL-12 pharmacokinetics over repeated doses.
The evolutionary dynamics of alternative splicing during primate neuronal differentiation
Authors: Ritter Alex , Wallace Andrew , Ronaghi Neda , Sanford Jeremy R
Published: February 2024

Alternative splicing (AS) is emerging as an important regulatory process for complex biological processes such as neuronal differentiation. To uncover the functional consequences of AS during neuronal differentiation we performed a comparative transcriptomic analysis using human, rhesus, chimpanzee and orangutan pluripotent stem cells. Transcriptomic studies commonly involve the identification and quantification of alternative processing events, but the need for predicting the functional consequences of changes to the relative inclusion of alternative events remains largely unaddressed. Many tools exist for the former task, albeit often limited to rudimentary event types. Few tools exist for the latter task; each with significant limitations. To address these issues we developed junctionCounts, which captures both simple and complex pairwise AS events and quantifies them with straightforward exon-exon and exon-intron junction reads in RNA-seq data, performing competently among similar tools in terms of sensitivity, false discovery and quantification accuracy. Its partner utility, cdsInsertion identifies transcript coding sequence information, including the presence of premature termination codons, gathered viain silicotranslation from annotated start codons. It then couples transcript-level information to AS events to predict functional effects, i.e. nonsense-mediated decay (NMD). We used junctionCounts and related tools to discover both conserved and species-specific splicing dynamics as well as regulation of NMD during differentiation. Our work demonstrates this tool’s capacity to robustly characterize AS and bridge the gap of predicting its potential effect on mRNA isoform fate.

GRAPHICAL ABSTRACT

junctionCounts is an alternative splicing analysis tool that identifies both simple and complex splicing events from a gene annotation and then measures their percent spliced-in from mapped RNA-seq junction reads.
The Dorsal Column Nuclei Scale Mechanical Sensitivity in Naive and Neuropathic Pain States
Authors: Upadhyay Aman , Gradwell Mark A. , Vajtay Thomas J. , Conner James , Sanyal Arnab A. , Azadegan Chloe , Patel Komal R. , Thackray Joshua K. , Bohic Manon , Imai Fumiyasu , Ogundare Simon O. , Yoshida Yutaka , Abdus-Saboor Ishmail , Azim Eiman , Abraira Victoria E.
Published: February 2024

Tactile perception relies on reliable transmission and modulation of low-threshold information as it travels from the periphery to the brain. During pathological conditions, tactile stimuli can aberrantly engage nociceptive pathways leading to the perception of touch as pain, known as mechanical allodynia. Two main drivers of peripheral tactile information, low-threshold mechanoreceptors (LTMRs) and postsynaptic dorsal column neurons (PSDCs), terminate in the brainstem dorsal column nuclei (DCN). Activity within the DRG, spinal cord, and DCN have all been implicated in mediating allodynia, yet the DCN remains understudied at the cellular, circuit, and functional levels compared to the other two. Here, we show that the gracile nucleus (Gr) of the DCN mediates tactile sensitivity for low-threshold stimuli and contributes to mechanical allodynia during neuropathic pain in mice. We found that the Gr contains local inhibitory interneurons in addition to thalamus-projecting neurons, which are differentially innervated by primary afferents and spinal inputs. Functional manipulations of these distinct Gr neuronal populations resulted in bidirectional changes to tactile sensitivity, but did not affect noxious mechanical or thermal sensitivity. During neuropathic pain, silencing Gr projection neurons or activating Gr inhibitory neurons was able to reduce tactile hypersensitivity, and enhancing inhibition was able to ameliorate paw withdrawal signatures of neuropathic pain, like shaking. Collectively, these results suggest that the Gr plays a specific role in mediating hypersensitivity to low-threshold, innocuous mechanical stimuli during neuropathic pain, and that Gr activity contributes to affective, pain-associated phenotypes of mechanical allodynia. Therefore, these brainstem circuits work in tandem with traditional spinal circuits underlying allodynia, resulting in enhanced signaling of tactile stimuli in the brain during neuropathic pain.
Subunit Communication within Dimeric SF1 DNA Helicases
Authors: Nguyen Binh , Hsieh John , Fischer Christopher J. , Lohman Timothy M.
Published: February 2024

Monomers of the Superfamily (SF) 1 helicases,E. coliRep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with lower processivity than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results withE. coliUvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.
Computed Tomography of Polymeric Biomedical Implants from Bench to Bedside
Authors: Pawelec Kendell M , Schoborg Todd A , Shapiro Erik M
Published: February 2024

Implanted biomedical devices require porosity to encourage tissue regeneration. However, characterizing porosity, which affects many functional device properties, is non-trivial. Computed tomography (CT) is a quick, versatile, and non-destructive way to gain 3D structural information. While optimization of CT for polymeric devices has been investigated at the bench on high-resolution micro-CT (μCT) scanners, pre-clinical and clinical systems cannot be tuned the same way, given an overriding objective to minimize ionizing radiation exposure to living tissues. Therefore, in this study we tested feasibility of obtaining structural information in pre-clinical systems and μCT under physiological conditions. The size of resolved features in porous structures is highly dependent on the resolution (voxel size) of the scan. Lower resolution underestimated porosity and overestimated pore size. With the homogeneous introduction of radiopaque nanoparticle contrast agent into both biopolymers and synthetic polymers, devices could be imaged in the hydrated state, even at high-resolution. Biopolymers had significant structural changes at the micro-scale post-hydration, including a mean increase of 130% in pore wall thickness that could potentially impact biological response. Through optimizing devices for medical imaging, CT has the potential to be a facile way to monitor devices from initial design stages through to clinical translation.
Engineering a three-dimensional multilayer multicellular model of endometrial cancer for high throughput drug screening and novel treatment methods
Authors: Cadena Ines A , Rowlands Claire , Buchanan Mina R , Jenne Molly A , Keefe Bailey , Almer Alyssa , Obasi Ndubuisi , Harris Conor G , Rochefort Willie E , Givens Brittany E. , Fogg Kaitlin C
Published: February 2024

Endometrial cancer is one of the most common gynecological cancers in the world, with an estimated 382,000 new cases and 90,000 deaths each year. There is no specific treatment, as the underlying causes of endometrial cancer neoplasia are poorly understood. This study focuses on the development and validation of a three-dimensional (3D)in vitromultilayer, multicellularhydrogel that facilitates drug screening analysis. We hypothesized that a specific combination of natural (collagen type I and IV, fibrinogen, fibronectin, Laminin) and synthetic (GELMA, PEGDA) polymers would maximize microvessel formation and cell invasion. The 3D model incorporates human microvascular endothelial cells (hMVEC) and endometrial cancer cells (HEC-1A) atop hydrogel formulations mimicking cell-specific extracellular matrix components. Using a D-optimal experimental design, 45 hydrogel combinations were generated. The predicted hydrogel formulation to maximize all cell responses enhanced higher microvessel formation and cancer invasion compared to the gold standard Matrigel. Subsequent validation emphasizes the importance of a disease-specific model and cell crosstalk in maximizing microvessel formation and cancer invasion. The optimized 3D model adeptly captures variances in cell responses among endometrial cancer cell lines from distinct stages. Finally, the platform is employed to compare cell viability, microvessel formation, and cancer invasion across Ishikawa, KLE, and HEC-1A cells after Paclitaxel exposure, delivered both as a free drug and loaded in poly(caprolactone) (PCL) nanoparticles. Overall, this study provides a valuable tool for exploring intricate interactions within the tumor microenvironment, offering a holistic understanding of cell responses and fostering the development of targeted therapeutic strategies for endometrial cancer.
Label free metabolic imaging to enhance the efficacy of Chimeric Antigen Receptor T cell therapy
Authors: Pham Dan L. , Cappabianca Daniel , Forsberg Matthew H. , Weaver Cole , Mueller Katherine P. , Tommasi Anna , Vidugiriene Jolanta , Lauer Anthony , Sylvester Kayla , Bugel Madison , Capitini Christian M. , Saha Krishanu , Skala Melissa C.
Published: February 2024

Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and the immunosuppressive tumor microenvironment. The manufacturing condition directly impacts CAR T cell yield, phenotype, and metabolism, which correlate within vivopotency and persistence. Optical metabolic imaging (OMI) is a non-invasive, label-free method to evaluate single cell metabolism based on autofluorescent metabolic coenzymes NAD(P)H and FAD. Using OMI, we identified the dominating impacts of media composition over the selection of antibody stimulation and/or cytokines on anti-GD2 CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrated that OMI parameters were indicative of cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. Notably, OMI accurately predicted oxidative metabolic phenotype of virus-free CRISPR-edited anti-GD2 CAR T cells that correlated to higherin vivopotency against neuroblastoma. Our data supports OMI’s potential as a robust, sensitive analytical tool that enables dynamic and optimal manufacturing conditions for increased CAR T cell yield and metabolic fitness.

One sentence summary

Autofluorescence imaging informs manufacturing conditions that enhance yield and metabolic fitness of CAR T cells for neuroblastoma.
Estimating distances to desertification points from dryland ecosystem images
Authors: Pichon Benoît , Donnet Sophie , Gounand Isabelle , Kéfi Sonia
Published: February 2024

Resource-limited ecosystems, such as drylands, can exhibit self-organized spatial patterns. Theory suggests that these patterns can reflect increasing degradation levels as ecosystems approach possible tipping points to degradation. However, we still lack ways of estimating a distance to degradation points that is comparable across sites. Here, we present an approach to do just that from images of ecosystem landscapes’. After validating the approach on simulated landscapes, we applied it to a global dryland dataset, estimated the distance of each of the sites to their degradation point and investigated the drivers of that distance. Crossing this distance with aridity projections makes it possible to pinpoint the most fragile sites among those studied. Our approach paves the way for a risk assessment method for spatially-organized ecosystems.
Manipulating syntax without taxing working memory: MEG correlates of syntactic dependencies in a Verb-Second language
Authors: Krogh Simone , Pylkkänen Liina
Published: February 2024

The neural basis of syntax is notoriously difficult to study without working memory and lexico-semantic confounds. To tackle these challenges, we presented dependencies in minimal Danish two-word sentences using Rapid Parallel Visual Presentation (RPVP), which eliminated the tem-poral delay between a filler and gap. Our stimuli involved dependencies high and low in the tree as a function of manipulating syntactic frame (declarative, yes/no question) and verb argument structure (unergative, unaccusative, alternating unaccusative). Neural magnetoencephalography signals were increased and behaviour facilitated for sentences compared to list controls, replicating the Sentence Superiority Effect (SSE) of prior RPVP studies. Our left-lateral neural SSE at 231– 407ms co-occurred with a right-lateral fronto-medial argument structure effect, while syntactic frame affected neural signals at 500–723ms. Thus hierarchically lower dependencies elicited faster neurophysiological correlates, raising the possibility that RPVP may offer a unique window into the brain’s detection of syntax when removing temporally extended parsing decisions.
Ndufs4knockout induces transcriptomic signatures of Alzheimer’s Diseases that are partially reversed by mitochondrial complex I inhibitor
Authors: Gao Huanyao , Jensen Kate , Nesbit Jarred , Ostroot Mark , Baloni Priyanka , Funk Cory , Trushina Eugenia
Published: February 2024

Mitochondrial dysfunction is well documented in Alzheimer’s Disease (AD). However, whether it instigates the onset of AD remains unclear. We demonstrate that a reduction of complex I activity in wild type (WT) mice caused by a global knockout ofNdufs4, an accessory mitochondrial complex I subunit, was sufficient to induce transcriptomic changes in the brain reminiscent of those observed in AD patients and familial mouse models of AD. Reduced complex I activity affected expression of genes in the networks related to mitochondrial homeostasis, neuronal and synaptic function. Transcriptomic signatures in male and femaleNdufs4-/-mice reflected a different severity of AD phenotype. Unexpectedly, these changes were partially rescued by a neuroprotective small molecule mild complex I inhibitor CP2. Consistent with studies in AD mice, CP2 treatment inNdufs4-/-mice augmented the expression of genes associated with mitochondrial biogenesis and turnover, synaptic activity, autophagy, redox balance, and reduced expression of genes related to inflammation. FemaleNdufs4-/-mice demonstrated a greater reversal of gene expression toward WT mice. These studies provide further support for mitochondria as a causative factor of AD pathophysiology and complex I as a putative therapeutic target.
Targeted NAD+Delivery for Intimal Hyperplasia and Re-endothelialization: A Novel Anti-restenotic Therapy Approach
Authors: Yin Li , Tong Yao , Islam Zain Husain , Zhang Kaijie , Xie Ruosen , Burger Jacobus , Hoyt Nicholas , Kent Eric William , Marcum William Aaron , Johnston Campbell , Kanchetty Rohan , Tetz Zoe , Stanisic Sophia , Huang Yitao , Guo Lian-Wang , Gong Shaoqin , Wang Bowen
Published: February 2024

Endovascular interventions often fail due to restenosis, primarily caused by smooth muscle cell (SMC) proliferation, leading to intimal hyperplasia (IH). Current strategies to prevent restenosis are far from perfect and impose significant collateral damage on the fragile endothelial cell (EC), causing profound thrombotic risks. Nicotinamide adenine dinucleotide (NAD+) is a co-enzyme and signaling substrate implicated in redox and metabolic homeostasis, with a pleiotropic role in protecting against cardiovascular diseases. However, a functional link between NAD+repletion and the delicate duo of IH and EC regeneration has yet to be established. NAD+repletion has been historically challenging due to its poor cellular uptake and low bioavailability. We have recently invented the first nanocarrier that enables direct intracellular delivery of NAD+in vivo. Combining the merits of this prototypic NAD+-loaded calcium phosphate (CaP) nanoparticle (NP) and biomimetic surface functionalization, we created a biomimetic P-NAD+-NP with platelet membrane coating, which enabled an injectable modality that targets IH with excellent biocompatibility. Using human cell primary culture, we demonstrated the benefits of NP-assisted NAD+repletion in selectively inhibiting the excessive proliferation of aortic SMC, while differentially protecting aortic EC from apoptosis. Moreover, in a rat balloon angioplasty model, a single-dose treatment with intravenously injected P-NAD+-NP immediately post angioplasty not only mitigated IH, but also accelerated the regeneration of EC (re-endothelialization)in vivoin comparison to control groups (i.e., saline, free NAD+solution, empty CaP-NP). Collectively, our current study provides proof-of-concept evidence supporting the role of targeted NAD+repletion nanotherapy in managing restenosis and improving re-endothelialization.
Contribution of Urea to Nitrite Production in Southern Ocean Waters with Contrasting Nitrifying Communities
Authors: Hollibaugh J T. , Okotie-Oyekan A. , Damashek J. , Ducklow H. , Popp B. N. , Wallsgrove N. , Allen T.
Published: February 2024

We compared the contribution of ammonia and urea to nitrite production in >100 samples of Southern Ocean waters with abundant and diverse ammonia-oxidizing archaeal (AOA) communities. Ammonia (AO) and urea (UO) oxidation rates were distributed uniformly within a water mass across coastal and slope waters west of the Antarctic Peninsula; however, rates and AOA community composition displayed strong vertical gradients. Rates in most samples from Antarctic surface and slope water were at or below the limit of detection. Highest mean rates of both processes were in the Winter Water (WW, epipelagic, 21.2 and 1.6 nmol N L-1d-1), and the Circumpolar Deep Water (CDW, mesopelagic, 7.9 and 2.5 nmol N L-1d-1), for AO and UO, respectively. However, we also found that the response of AO and UO to substrate amendments varied by water mass. AO rates in WW samples increased by ∼200% with 44 vs 6 nM amendments, but decreased (down to 7%) in CDW samples. UO rates responded similarly, but to a lesser degree. This response suggests that even low NH4+amendments may inhibit AO by mesopelagic Thaumarchaeota populations. AO and UO rates were not correlated, nor were they correlated with the abundance or ratios of abundance of marker genes, or with the concentrations of ammonium or urea. Our data suggest that while ammonium is the primary substrate, urea-N is responsible for a significant fraction (∼25% of that from AO alone) of nitrite production in the Southern Ocean, comparable to its contribution at lower latitudes.

IMPORTANCE

Southern Ocean nitrification fuels denitrification in oxygen depleted zones at higher latitudes, one of the controls of N:P ratios in the global ocean. N2O, a powerful greenhouse gas, is by-product of nitrification. We contrast the contributions of ammonium and urea-N to nitrification in the Southern Ocean. Our work constrains rates and demonstrates that the contribution of urea-N to nitrite production in polar waters is comparable to that in temperate oceans. Correlations between activity and the abundance or ratios of Thaumarchaeota marker genes were weak, questioning their use as indicators of activity. We document differential responses of activity to substrate amendments by water mass: enhanced in epipelagic but inhibited in mesopelagic samples. We interpret this difference in the context of community composition and the production of reactive oxygen species. Our insights into environmental controls of nitrification are relevant to microbial ecologists studying Thaumarchaeota and to modeling the global nitrogen cycle.
The genome of Przewalski’s horse (Equus ferus przewalskii)
Authors: Flack Nicole , Hughes Lauren , Cassens Jacob , Enriquez Maya , Gebeyehu Samrawit , Alshagawi Mohammed , Hatfield Jason , Kauffman Anna , Brown Baylor , Klaeui Caitlin , Mabrouk Islam F. , Walls Carrie , Yeater Taylor , Rivas Anne , Faulk Christopher
Published: February 2024

The Przewalski’s horse (Equus ferus przewalskii) is an endangered equid native to the steppes of central Asia. After becoming extinct in the wild, multiple conservation efforts convened to preserve the species including captive breeding programs, reintroduction and monitoring systems, protected lands, and cloning. Availability of a highly contiguous reference genome is essential to support these continued efforts. We used Oxford Nanopore sequencing to produce a scaffold-level 2.5 Gb nuclear assembly and 16,002 bp mitogenome from a captive Przewalski’s mare. All assembly drafts were generated from 111 Gb of sequence from a single PromethION R10.4.1 flow cell. The mitogenome contained 37 genes in the standard mammalian configuration and was 99.63% identical to the domestic horse (Equus caballus). The nuclear assembly, EquPr2, contained 2,146 scaffolds with an N50 of 85.1 Mb, 43X mean depth, and BUSCO quality score of 98.92%. EquPr2 successfully improves upon the existing Przewalski’s horse reference genome (Burgud), with 25-fold fewer scaffolds, a 166-fold larger N50, and phased pseudohaplotypes. Modified basecalls revealed 79.5% DNA methylation and 2.1% hydroxymethylation globally. Allele-specific methylation analysis between pseudohaplotypes revealed 226 differentially methylated regions (DMRs) in known imprinted genes and loci not previously reported as imprinted. The heterozygosity rate of 0.165% matches previous estimates for the species and compares favorably to other endangered animals. This improved Przewalski’s horse assembly will serve as a valuable resource for conservation efforts and comparative genomics investigations.
Modulation of stress granule dynamics by phosphorylation and ubiquitination in plants
Authors: He Siou-Luan , Wang Xiling , Kim Sungil , Kong Liang , Wang Lei , He Ping , Shan Libo , Wang Ying , Jang Jyan-Chyun
Published: February 2024

The Arabidopsis tandem CCCH zinc finger 1 (TZF1) is an RNA-binding protein that plays a crucial role in plant growth and stress response. TZF1 can localize to ribonucleoprotein (RNP) granules in response to various abiotic stresses. However, very little is known about the composition, function, and assembly mechanism of plant RNP granules. In this report, we show that TZF1 contains two intrinsically disordered regions (IDRs) necessary for its localization to stress granules (SGs), a subclass of RNP granules. TZF1 recruits mitogen-activated protein kinase (MAPK) signaling components and an E3 ubiquitin ligase KEEP-ON-GOING (KEG) to SGs. TZF1 is phosphorylated by MPKs and ubiquitinated by KEG. The phosphorylation sites of TZF1 were mapped by mass spectrometry. Mutant studies revealed that phosphorylation and ubiquitination of specific residues played differential roles in enhancing or reducing TZF1 SG assembly and protein-protein interaction with mitogen-activated kinase kinase 5 (MKK5) in SGs. TZF1 is extremely unstable, and its accumulation can be enhanced by proteosome inhibitor MG132. We showed that TZF1 was ubiquitinated in vivo and in vitro by KEG and TZF1 accumulated at a much lower level in gain-of-function mutantkeg-4, compared to the WT. Ubiquitination appeared to play a positive role in TZF1 SG assembly, because either single or higher order mutations caused reduced number of SGs per cell, while enhanced the coalescence of small SGs into a large nucleus-like SG encompassing the nucleus. Together, our results demonstrate that the assembly of TZF1 SGs is distinctively regulated by ubiquitination and phosphorylation.
Human Alveolar and Monocyte-derived Human Macrophage Responses to Mycobacterium tuberculosis
Authors: Campo Monica , Dill-McFarland Kimberly A. , Peterson Glenna J. , Benson Basilin , Skerrett Shawn J. , Hawn Thomas R.
Published: February 2024

Rationale

Alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) mediate early lung immune responses toMycobacterium tuberculosis(Mtb). Differences in the response of these distinct cell types is poorly understood and may provide insight into mechanisms of TB pathogenesis.

Objectives

To determine whether Mtb induces unique and essential anti-microbial pathways in human AMs compared to MDMs.

Methods

Using paired human AMs and 5-day MCSF-derived MDMs from 6 healthy volunteers, we infected cells with Mtb H37Rv for 6 hours, isolated RNA, and analyzed transcriptomic profiles with RNASeq.

Main Results

We found 681 genes that were Mtb-dependent in AMs compared to MDMs and 4538 that were Mtb-dependent in MDMs but not AMs (FDR < 0.05). Using hypergeometric enrichment of DEGs in Broad Hallmark gene sets, we found that Type I and II IFN Response were the only gene sets selectively induced in Mtb-infected AM (FDR <0.05). In contrast, MYC targets, unfolded protein response and MTORC1 signaling, were selectively enriched in MDMs (FDR < 0.05). IFNA1, IFNA8, IFNE, and IFNL1 were specifically and highly upregulated in AMs compared to MDMs at baseline and/or after Mtb infection. IFNA8 modulated Mtb-induced pro-inflammatory cytokines and, compared to other interferons, stimulated unique transcriptomes. Several DNA sensors and Interferon Regulatory Factors had higher expression at baseline and/or after Mtb infection in AMs compared to MDMs.

Conclusions

These findings demonstrate that Mtb infection induced unique transcriptional responses in human AMs compared to MDMs, including upregulation of the IFN response pathway and specific DNA sensors.
Adverse structural and mechanical remodelling of main pulmonary artery in experimental pulmonary arterial hypertension is associated with impaired right ventricle-pulmonary artery coupling and function
Authors: Mirani Bahram , Dauz John D. , Yazaki Kana , Latifi Neda , Santerre J Paul , Bendeck Michelle P. , Simmons Craig A. , Friedberg Mark K.
Published: February 2024

Rationale

Coupling between right ventricular function and the pulmonary vasculature determines outcomes in pulmonary arterial hypertension. The mechanics of the main pulmonary artery is an important but understudied determinant of right ventricular-pulmonary artery coupling.

Objectives

To investigate the histology and mechanics of the pulmonary artery in relationship to right ventricular remodeling, mechanics, hemodynamics and coupling in experimental pulmonary arterial hypertension.

Methods

In a sugen+hypoxia rat model of pulmonary arterial hypertension, right ventricular hemodynamics were assessed by conductance catheters. Active tension-strain curves were generated using echocardiography. Main pulmonary artery and right ventricle free-wall were harvested to determine their macro- and micro-structure, composition, and mechanical properties. Comprehensive multivariate analyses elucidated relationships between pulmonary artery and right ventricle mechanics, structure and coupling.

Measurements and Main Results

Pulmonary hypertensive main pulmonary arteries developed fibrosis relative to healthy controls, as did right ventricles, which also hypertrophied, with re-orientation of muscle fibres toward a tri-layer architecture reminiscent of normal left ventricular architecture. Increased glycosaminoglycan deposition and increased collagen-to-elastin ratio in the pulmonary artery; and increased collagen, as well as hypertrophy and reorganization of myofibers in the right ventricle, led to increased stiffness. This increase in stiffness was more pronounced in the longitudinal direction in the high- and low-strain regime for the pulmonary artery and right ventricle, respectively, causing increased mechanical anisotropy. Main pulmonary artery stiffening correlated significantly with right ventricular tissue mechanical remodelling and reduced systolic performance, cardiac output and right ventricle-pulmonary artery coupling.

Conclusions

Compositional, structural, and mechanical changes in the main pulmonary artery correlate with adverse right ventricular remodeling, mechanics, function and coupling in pulmonary arterial hypertension. Therefore, increasing mechanical compliance of the large pulmonary arteries may be an important and novel therapeutic strategy for mitigating right ventricular failure.
Responses of neurons in macaque V4 to object and texture images
Authors: Lieber Justin D. , Oleskiw Timothy D. , Simoncelli Eero P. , Movshon J. Anthony
Published: February 2024

Humans and monkeys can effortlessly recognize objects in everyday scenes. This ability relies on neural computations in the ventral stream of visual cortex. The intermediate computations that lead to object selectivity are not well understood, but previous studies implicate V4 as an early site of selectivity for object shape. To explore the mechanisms of this selectivity, we generated a continuum of images between “scrambled” textures and photographic images of both natural and manmade environments, using techniques that preserve the local statistics of the original image while discarding information about scene and shape. We measured the responses of single units in awake macaque V4 to these images. On average, V4 neurons were slightly more active in response to photographic images than to their scrambled counterparts. However, responses in V4 varied widely both across different cells and different sets of images. An important determinant of this variation was the effectiveness of image families at driving strong neural responses. Across the full V4 population, a cell’s average evoked firing rate for a family reliably predicted that family’s preference for photographic over scrambled images. Accordingly, the cells that respond most strongly to each image family showed a much stronger difference between photographic and scrambled images and a graded level of modulation for images scrambled at intermediate levels. This preference for photographic images was not evident until ∼50 ms after the onset of neuronal activity and did not peak in strength until 140 ms after activity onset. Finally, V4 neural responses seemed to categorically separate photographic images from all of their scrambled counterparts, despite the fact that the least scrambled images in our set appear similar to the originals. When these same images were analyzed with DISTS (Deep Image Structure and Texture Similarity), an image-computable similarity metric that predicts human judgements of image degradation, this same pattern emerged. This suggests that V4 responses are highly sensitive to small deviations from photographic image structure.
Transcriptional perturbation of LINE-1 elements reveals theircis-regulatory potential
Authors: Pérez-Rico Yuvia A , Bousard Aurélie , Misikova Lenka Henao , Mulugeta Eskeatnaf , de Almeida Sérgio F , Muotri Alysson R , Heard Edith , Gendrel Anne-Valerie
Published: February 2024

Long interspersed element-1 (LINE-1 or L1) retrotransposons constitute the largest transposable element (TE) family in mammalian genomes and contribute prominently to inter- and intra-individual genetic variation. Although most L1 elements are inactive, some evolutionary younger elements remain intact and genetically competent for transcription and occasionally retrotransposition. Despite being generally more abundant in gene-poor regions, intact or full-length L1s (FL-L1) are also enriched around specific classes of genes and on the eutherian X chromosome. How proximal FL-L1 may affect nearby gene expression remains unclear. In this study, we aim to examine this in a systematic manner using engineered mouse embryonic stem cells (ESCs) where the expression of one representative active L1 subfamily is specifically perturbed. We found that ∼1,024 genes are misregulated following FL-L1 activation and to a lesser extent (∼81 genes), following their repression. In most cases (68%), misexpressed genes contain an intronic FL-L1 or lie near a FL-L1 (<260 kb). Gene ontology analysis shows that upon L1 activation, up-regulated genes are enriched for neuronal function-related terms, suggesting that some L1 elements may have evolved to control neuronal gene networks. These results illustrate thecis-regulatory impact of FL-L1 elements and suggest a broader role for L1s than originally anticipated.
Long read subcellular fractionation and sequencing reveals the translational fate of full length mRNA isoforms during neuronal differentiation
Authors: Ritter Alexander J , Draper Jolene M , Vollmers Chris , Sanford Jeremy R
Published: February 2024

Alternative splicing (AS) alters the cis-regulatory landscape of mRNA isoforms leading to transcripts with distinct localization, stability and translational efficiency. To rigorously investigate mRNA isoform-specific ribosome association, we generated subcellular fractionation and sequencing (Frac-seq) libraries using both conventional short reads and long reads from human embryonic stem cells (ESC) and neural progenitor cells (NPC) derived from the same ESC. We performedde novotranscriptome assembly from high-confidence long reads from cytosolic, monosomal, light and heavy polyribosomal fractions and quantified their abundance using short reads from their respective subcellular fractions. Almost half of all transcripts exhibited association with particular subcellular fractions relative to the cytosol. Of the multi-isoform genes, 27% and 18% exhibited significant differential isoform sedimentation in ESC and NPC, respectively. Alternative promoter usage and internal exon skipping accounted for the majority of differences between isoforms from the same gene. Random forest classifiers implicated 3’ and 5’ untranslated region (UTR) GC-content and coding sequence (CDS) and UTR lengths as important determinants of isoform-specific sedimentation profiles. Taken together our data demonstrate that alternative mRNA processing within the CDS and UTRs impacts the translational control of mRNA isoforms during stem cell differentiation, and highlights the utility of using a novel long read sequencing-based method to study translational control.
Gene expression differences associated with intrinsic hindfoot muscle loss in the jerboa,Jaculus jaculus
Authors: Tran Mai P. , Ochoa Reyes Daniel , Weitzel Alexander J. , Saxena Aditya , Hiller Michael , Cooper Kimberly L.
Published: February 2024

Vertebrate animals that run or jump across sparsely vegetated habitats, such as horses and jerboas, have reduced the number of distal limb bones, and many have lost most or all distal limb muscle. We previously showed that nascent muscles are present in the jerboa hindfoot at birth and that these myofibers are rapidly and completely lost soon after by a process that shares features with pathological skeletal muscle atrophy. Here, we apply an intra- and inter-species approach, comparing jerboa and mouse muscles, to identify gene expression differences associated with the initiation and progression of jerboa hindfoot muscle loss. We show evidence for reduced Hepatocyte Growth Factor (HGF) and Fibroblast Growth Factor (FGF) signaling and an imbalance in nitric oxide signaling; all are pathways that are necessary for skeletal muscle development and regeneration. We also find evidence for phagosome formation, which hints at how myofibers may be removed by autophagy or by non-professional phagocytes without evidence for cell death or immune cell activation. Last, we show significant overlap between genes associated with jerboa hindfoot muscle loss and genes that are differentially expressed in a variety of human muscle pathologies and rodent models of muscle loss disorders. All together, these data provide molecular insight into the mechanism of evolutionary and developmental muscle loss in jerboa hindfeet.
Large-pore connexin hemichannels function as molecule transporters independently of ion conduction
Authors: Gaete Pablo S. , Kumar Deepak , Fernandez Cynthia I. , Valdez-Capuccino Juan Manuel , Bhatt Aashish , Jiang Wenjuan , Lin Yi-Chun , Liu Yu , Harris Andrew L. , Luo Yun L. , Contreras Jorge E.
Published: February 2024

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore — a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.
Diaminopurine in Nonenzymatic RNA Template Copying
Authors: Jia Xiwen , Fang Ziyuan , Kim Seohyun Chris , Ding Dian , Zhou Lijun , Szostak Jack W.
Published: February 2024

In the primordial RNA World, before the advent of ribozymes, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides are heavily biased towards the incorporation of G and C. Diaminopurine (D) can form a D:U base pair that is almost as strong as a G:C base pair. We therefore asked whether replacing A with D might lead to more efficient and less biased nonenzymatic template copying. As expected, primer extension substrates containing D bind to U in the template more tightly than substrates containing A. However, primer extension with D exhibited elevated reaction rates on a C template, leading to concerns about fidelity. To investigate the nature of the D:C mismatch, we solved the crystal structure of RNA duplexes containing D:C mismatches, and showed that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease mismatched primer extension. We performed nonenzymatic primer extension with all four activated nucleotides on randomized RNA templates containing all four letters, and used deep sequencing to analyze the products. We found that the DUCG genetic system exhibited a more even product distribution and a lower mismatch frequency than the canonical AUCG system. Furthermore, primer extension is greatly reduced following all mismatches, including the D:C mismatch. Our study suggests that diaminopurine deserves further attention for its possible role in the RNA World, and as a potentially useful component of artificial nonenzymatic RNA replication systems.
Fast and accurate short read alignment with hybrid hash-tree data structure
Authors: Makino Junichiro , Ebisuzaki Toshikazu , Himeno Ryutaro , Hayashizaki Yoshihide
Published: February 2024

Rapidly increasing amount of short read data generated by NGSs (new-generation sequencers) calls for the development of fast and accurate read alignment programs. The programs based on hash table (BLAST) and Burrows-Wheeler transform (bwa-mem) are used, and the latter is known to give superior performance. We here present a new algorithm, a hybrid of hash table and suffix tree, which we designed to speed up the alignment of short reads against large reference sequences such as human genome. The total turnaround time for processing one human genome sample (read depth of 30) is just 31 minutes with our system while that was more than 25 hours with bwa-mem/gatk. The time for aligner alone is 28 minutes for our system but around 2 hours for bwa-mem. Our new algorithm is 4.4 times faster than bwa-mem while achieving similar accuracy. Variant calling and other downstream analyses after the alignment can be done with open-source tools such as SAMtools and Genome Analysis Toolkit (gatk) packages, as well as our own fast variant caller, which is well parallelized and much faster than gatk.