bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exon-Skipping Antisense Oligonucleotides for Cystic Fibrosis Therapy
Authors: Kim Young Jin , Sivetz Nicole , Layne Jessica , Voss Dillon , Yang Lucia , Zhang Qian , Krainer Adrian R. 
Published: August 2021

AbstractMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and theCFTR-W1282Xnonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity, but is expressed at a very low level due to nonsense-mediated mRNA decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched forCFTRnonsense mutants. NMD suppression could increase the mutantCFTRmRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable; and read-through drugs are inefficient, partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression ofCFTRmRNA without the PTC-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.
A single shot of a hybrid hAdV5-based anti-COVID-19 vaccine induces a long-lasting immune response and broad coverage against VOC
Authors: López M. Verónica , Vinzón Sabrina E. , Cafferata Eduardo G. A. , Nuñez Felipe J. , Soto Ariadna , Sanchez-Lamas Maximiliano , Afonso Jimena , Aguilar-Cortes Diana , Ríos Gregorio D. , Maricato Juliana T. , Torres-Braconi Carla , Silveira Vanessa Barbosa-da , Montes-de Andrade Tatiane , Carvalho-de Souza Bonetti Tatiana , Ramos Janini Luiz M. , Castello Girão Manoel J. B. , Llera Andrea S. , Gomez Karina , Ortega Hugo H. , Berguer Paula M. , Podhajcer Osvaldo L. 
Published: August 2021

ABSTRACTMost approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric hAdV5 vector. The vaccine (named CoroVaxG.3) is based on three pillars: i) high expression of Spike to enhance its immunodominance by using a potent promoter and a mRNA stabilizer; ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; iii) use of Spike stabilized in a prefusion conformation. Transduction with CoroVaxG.3 expressing Spike (D614G) dramatically enhanced Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3 vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha) and P.1 (gamma) Spikes, as well as an authentic WT and P.1 SARS-CoV-2 isolates. Neutralizing antibodies did not wane even after 5 months making this kind of vaccine a likely candidate to enter clinical trials
A CCaMK/Cyclops response element in the promoter ofL. japonicus Calcium-Binding Protein 1(CBP1) mediates transcriptional activation in root symbioses
Authors: Gong Xiaoyun , Jensen Elaine , Bucerius Simone , Parniske Martin 
Published: August 2021

SummaryEarly gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. TheLotus japonicusT-DNA insertion line T90, carrying a promoterlessuidA(GUS) gene in the promoter ofCalcium Binding Protein1(CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsiblecis-andtrans-acting factors, we subjected deletion/modification series ofCBP1promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrantGUSexpression. We identified onecis-regulatory element required forGUSexpression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack ofGUSexpression in T90whitemutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promotersNIN(RNS),RAM1(AM), andCBP1(AM and RNS), calling for yet-to-be identified specificity-conferring factors.
Human Deep Cortical Neurons Promote Regeneration and Recovery After Cervical Spinal Cord Injury
Authors: Doulames Vanessa M. , Weimann James , Plant Giles W. 
Published: August 2021

AbstractCervical spinal cord injuries (SCI) sever and permanently disrupt sensorimotor neural circuitry. Restoring connectivity within the damaged circuitry is critical to improving function. Herein we report robust regeneration of severed neural circuitry in a rat SCI model following transplantation of human induced pluripotent cells differentiated towards a deep cortical neuron lineage (iPSC-DCNs).In vivo, iPSC-DCNs: (1) integrated within the damaged cord and extended axons to caudal targets, (2) reversed SCI pathophysiology, (3) promoted robust regeneration of severed host supraspinal neural tracts, (4) and improved sensorimotor function. The results herein represent a significant paradigm shift in anatomical and functional outcomes over current preclinical/clinical models and demonstrates the survival and efficacy of human stem cell-derived cortical neurons in a SCI.
A gap-junction mutation in the mouse cochlea reveals cochlear amplification is driven by outer hair cell extracellular receptor potentials
Authors: Levic Snezana , Lukashkina Victoria A. , Simões Patricio , Lukashkin Andrei N. , Russell Ian J. 
Published: August 2021

SummaryCochlear amplification, whereby cochlear responses to low-to-moderate sound levels are amplified and compressed to loud sounds, is attributed to outer hair cell (OHC) electromotility driven by voltage changes across the OHC basolateral membranes due to sound-induced receptor-current modulation. Cochlear operation at high acoustic frequencies is enigmatic because the OHC intracellular receptor potential (RP) is severely attenuated at these frequencies. Clues to understanding the voltage control of OHC electromotility at different frequencies was provided by measurements from CD-1 mice with an A88V mutation of the gap-junction (GJ) protein connexin 30 (Cx30), which with Cx26, form heterogeneous GJs between supporting cells in the organ of Corti (OoC) and stria vascularis. The A88V mutation results in a smaller GJ conductance which may explain why the resistance across the OoC in CD-1Cx30A88V/A88Vmutants is higher compared with wild-type mice. The endocochlear potential, which drives the OHC receptor current and, consequently, the OHC RPs, is smaller in CD-1Cx30A88V/A88Vmutants. Even so, their high-frequency hearing sensitivity equals that of wild-type mice. Preservation of high-frequency hearing correlates with similar amplitude of extracellular receptor potentials (ERPs), measured immediately adjacent to the OHCs. ERPs are generated through OHC receptor current flow across the OoC electrical resistance, which is larger in CD-1Cx30A88V/A88Vthan in wild-type mice. Thus, smaller OHC receptor currents flowing across a larger OoC resistance in CD-1Cx30A88V/A88Vmice may explain why their ERP magnitudes are similar to wild-type mice. It is proposed that the ERPs, which are not subject to low-pass electrical filtering, drive high-frequency cochlear amplification.Significance StatementCochlear amplification, whereby responses to low-to-moderate sound levels are amplified and those to loud sounds are compressed, is attributed to outer hair cell (OHC) electromotility. Electromotility is driven by voltage changes across the OHC basolateral membranes due to modulation of receptor current flow during sound-induced sensory hair bundle displacement. Mechanisms of high-frequency cochlear amplification remain to be elucidated. A mutation of the gap-junction protein connexin 30 decreases OHC intracellular receptor potentials in CD-1 mice. Instead of decreasing auditory sensitivity, the mutation rescues high-frequency hearing by causing OHC extracellular receptor potentials to be similar in amplitude to those of sensitive wild-type mice. It is proposed extracellular, not intracellular, potentials drive high-frequency OHC motility and cochlear amplification at high acoustic frequencies.
Circadian Clock Core Component Bmal1 Dictates Cell Cycle Rhythm of Proliferating Hepatocytes during Liver Regeneration
Authors: Jiang Huaizhou , Garcia Veronica , Yanum Jennifer Abla , Lee Joonyong , Dai Guoli 
Published: August 2021

AbstractFollowing partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH onBmal1+/+and hepatocyte-specificBmal1knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, inBmal1hep-/-hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+hepatocytes underwent three major waves of mitosis, whereasBmal1hep-/-hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators, and dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover,Bmal1+/+hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited inBmal1hep-/-hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, and eventually determines the redox state of regenerated livers.New and NoteworthyWe demonstrated that Bmal1 centrally controls the synchronicity and rhythmicity of the cell cycle and lipid accumulation in replicating hepatocytes during liver regeneration. Bmal1 plays these roles, at least in part, by ensuring formation of the expression peaks of cell cycle components and regulators, as well as the timing and levels of activation of mitogenic signaling molecules.Graphical Abstract
cDNA-detector: Detection and removal of cDNA contamination in DNA sequencing libraries
Authors: Qi Meifang , Nayar Utthara , Ludwig Leif S. , Wagle Nikhil , Rheinbay Esther 
Published: August 2021

SummaryExogenous cDNA introduced into an experimental system, either intentionally or accidentally, can appear as added read coverage over that gene in next-generation sequencing libraries derived from this system. If not properly recognized and managed, this cross-contamination with exogenous signal can lead to incorrect interpretation of research results. Yet, this problem is not routinely addressed in current sequence processing pipelines. Here, we present cDNA-detector, a computational tool to identify and remove exogenous cDNA contamination in DNA sequencing experiments. We apply cDNA-detector to several highly-cited public databases (TCGA, ENCODE, NCBI SRA) and show that contaminant genes appear in sequencing experiments where they lead to incorrect coverage peak calls. Our findings highlight the importance of sensitive detection and removal of contaminant cDNA from NGS libraries before downstream analysis.
RNA sequencing on muscle biopsy from a 5-week bedrest study reveals the effect of exercise and potential interactions with dorsal root ganglion neurons
Authors: McFarland Amelia J. , Ray Pradipta R. , Bhai Salman , Levine Benjamin , Price Theodore J. 
Published: August 2021

AbstractLack of physical activity is a predictor of poor health outcomes that can be prevented or reversed by exercise. Sedentary lifestyle, chronic disease or microgravity can cause muscle deconditioning that then has an impact on other physiological systems. An example is the nervous system, which is adversely affected by decreased physical activity resulting in increased incidence of neurological problems such as chronic pain. We sought to better understand how this might occur by conducting RNA sequencing experiments on muscle biopsies from human volunteers in a 5-week bed-rest study with an exercise intervention arm. We also used a computational method for examining ligand-receptor interactions between muscle and human dorsal root ganglion (DRG) neurons, the latter of which play a key role in nociception and are generators of signals responsible for chronic pain. We identified 1352 differentially expressed genes (DEGs) in bed rest subjects without an exercise intervention but only 132 DEGs in subjects with the intervention. Thirty-six genes were shared between the exercise and no intervention groups. Among 591 upregulated muscle genes in the no intervention arm, 26 of these were ligands that have receptors that are expressed by human DRG neurons. We detected a specific splice variant of one of these ligands, placental growth factor (PGF), in deconditioned muscle that binds to neuropilin 1, a receptor that is highly expressed in DRG neurons and known to promote neuropathic pain. We conclude that exercise intervention protects muscle from deconditioning transcriptomic changes, and prevents changes in expression of ligands that might sensitize DRG neurons that promote pain. Our work creates a set of actionable hypotheses to better understand how deconditioned muscle may influence the function of sensory neurons that innervate the entire body.
Structural basis of adenylyl cyclase 9 activation
Authors: Qi Chao , Lavriha Pia , Mehta Ved , Khanppnavar Basavraj , Mohammed Inayathulla , Li Yong , Lazaratos Michalis , Schaefer Jonas V. , Dreier Birgit , Plückthun Andreas , Bondar Ana-Nicoleta , Dessauer Carmen W. , Korkhov Volodymyr M. 
Published: August 2021

AbstractAdenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.One Sentence SummaryCryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9
Recording gene expression order in DNA by CRISPR addition of retron barcodes
Authors: Bhattarai-Kline Santi , Lear Sierra K. , Fishman Chloe B. , Lopez Santiago C. , Lockshin Elana R. , Schubert Max G. , Nivala Jeff , Church George , Shipman Seth L. 
Published: August 2021

ABSTRACTBiological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons1, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record via simple, logical rules rather than relying on pre-trained classifiers or post-hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.
Experimental evidence that chronic outgroup conflict reduces reproductive success in a cooperatively breeding fish
Authors: Goncalves Ines Braga , Radford Andrew N. 
Published: August 2021

AbstractConflicts with conspecific outsiders are common in group-living species, from ants to primates, and are argued to be an important selective force in social evolution. However, whilst an extensive empirical literature exists on the behaviour exhibited during and immediately after interactions with rivals, only very few observational studies have considered the cumulative fitness consequences of outgroup conflict. Using a cooperatively breeding fish, the daffodil cichlid (Neolamprologus pulcher), we conducted the first experimental test of the effects of chronic outgroup conflict on reproductive investment and output. ‘Intruded’ groups received long-term simulated territorial intrusions by neighbours that generated consistent group-defence behaviour; matched ‘Control’ groups (each the same size and with the same neighbours as an Intruded group) received no intrusions in the same period. Intruded groups had longer inter-clutch intervals and produced eggs with less protein than Control groups. Despite this lower egg investment, Intruded groups provided more parental care, achieving similar hatching success to Control groups. Ultimately, however, Intruded groups had fewer and smaller surviving offspring than Control groups at 1-month post-hatching. We therefore provide experimental evidence that outgroup conflict can decrease fitness via cumulative effects on reproductive success, confirming the selective potential of this empirically neglected aspect of sociality.
The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids
Authors: Monteleone-Cassiano Ana Carolina , Dernowsek Janaina A. , Mascarenhas Romario S. , Assis Amanda Freire , Pitol Dimitrius , Mardegan Issa João Paulo , Donadi Eduardo A. , Passos Geraldo Aleixo 
Published: August 2021

AbstractBesides controlling the expression of peripheral tissue antigens, the autoimmune regulator (AIRE) gene also regulates the expression of adhesion genes in medullary thymic epithelial cells (mTECs), an essential process for mTEC-thymocyte interaction for triggering the negative selection in the thymus. For these processes to occur, it is necessary that the medulla compartment forms an adequate three-dimensional (3D) architecture, preserving the thymic medulla. Previous studies have shown thatAIREknockout (KO) mice have a small and disorganized thymic medulla; however, whetherAireinfluences the mTEC-mTEC interaction in the maintenance of the 3D structure has been little explored. Considering that AIRE controls cell adhesion genes, we hypothesized that this gene affects 3D mTEC-mTEC interaction. To test this, we constructed anin vitromodel system for mTEC spheroid formation, in which cells adhere to each other, establishing a 3D structure. The effect ofAireon mTEC-mTEC adhesion was evaluated by comparingAIREwild type (AIREWT)versus Aire KO (AIRE-/-)mTECs. Considering the 3D spheroid model evaluated, we reported that the absence ofAIREdisorganizes the 3D structure of mTEC spheroids, promotes a differential regulation of mTEC classical surface markers, and modulates genes encoding adhesion and other molecules.
Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen
Authors: Priest Shelby J. , Yadav Vikas , Roth Cullen , Dahlmann Tim A. , Kück Ulrich , Magwene Paul M. , Heitman Joseph 
Published: August 2021

AbstractMicroorganisms survive and compete by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes experiencing strong selective pressures. Infections caused by Cryptococcus neoformans are responsible for ∼15% of AIDS-related deaths and associated with high mortality rates, attributable to a dearth of antifungal drugs and drug resistance. We identified two hypermutator C. neoformans clinical isolates in which Cnl1 transposon insertions were responsible for drug resistance. Whole-genome sequencing revealed both hypermutator genomes harbor a nonsense mutation in the RNAi component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on every chromosome. QTL mapping identified a significant locus associated with hypermutation that included znf3. CRISPR-mediated editing of the znf3 nonsense mutation abolished hypermutation and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from RNAi loss and a significant burden of Cnl1 elements.