bluuebunny/crossref_metadata_2025_split huggingface dataset:
Horizontal Transfer of Histone H3 by Mammalian Cells
Authors: Sulkowski Parker L. , Nicholson Hilary E. , Li Matthew Z. , Perrimon Norbert , Liu James Zhe , Kaelin William G. 
Published: July 2021

The authors have withdrawn their manuscript because many of the experiments described in this paper have not been reproducible, or at least are not robust, in the hands of other members of the Kaelin Laboratory who were not initially involved in this work. While we do see apparent secretion of histone H3 under some conditions, it is usually accompanied by secretion of histone H4. In this regard, the Halo tagged-histone H3 and Halo-tagged H4 constructs used for the single molecule imaging studies we reported, which seemingly confirmed specific secretion and transfer of histone H3, were purported to be sequence validated. Upon resequencing these constructs we discovered a non-synonymous mutation in the Halo tag of the H4 construct. We then redid the imaging experiments with the corrected Halo-H4 together with Halo-H3 and, in contrast to our earlier study, unfused Halo. These experiments were difficult to interpret because of the background signal seen with the unfused Halo but did not support specific secretion and transfer of histones (let alone specific secretion and transfer of histone H3).We have, in some experiments, observed transfer of H3-Cre into reporter cells ex vivo and in vivo, but 1) the transfer is not specific for H3-Cre versus H4-Cre, 2) the transfer efficiency is highly variable, and 3) the transfer efficiency is typically much less than reported in our original paper. We do not yet know whether this lack of reproducibility and robustness reflects technical and biological variables that we do not yet understand and hence were not captured in our experimental protocols.Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
Early detection of SARS-CoV-2 in circulating immune cells in a mouse model
Authors: Geng Tingting , Keilich Spencer , Tafas Triantafyllos , Wang Penghua 
Published: July 2021

ABSTRACTSARS-CoV-2 infects the respiratory tract, lung and then other organs. However, its pathogenesis remains largely unknown. We used RareScope™ Fluorescence Light Sheet Microscopy (FLSM) and fluorescent in situ hybridization of RNA (RNA-FISH) to detect SARS-CoV-2 RNA and dissemination kinetics in mouse blood circulation. By RNA-FISH, we found that SARS-CoV-2 RNA-positive leukocytes, including CD11c cells, appeared as early as one day after infection and continued through day 10 post infection. Our data suggest that SARS-CoV-2-permissive leukocytes contribute to systemic viral dissemination, and RNA-FISH combined with FLSM can be utilized as a sensitive tool for SARS-CoV-2 detection in blood specimens.
A supergene in seaweed flies modulates male traits and female perception
Authors: Enge Swantje , Mérot Claire , Mozūraitis Raimondas , Apšegaitė Violeta , Bernatchez Louis , Martens Gerrit A. , Radžiutė Sandra , Pavia Henrik , Berdan Emma L. 
Published: July 2021

SummarySupergenes, tightly linked allelic combinations that underlie complex adaptive phenotypes represent a critical mechanism protecting intra-specific polymorphism [1, 2]. Supergenes represent some of the best examples of balancing selection in nature and there is increasing evidence that disassortative mating, when individuals preferentially mate with dissimilar phenotypes, is a key force stabilizing supergene polymorphisms. Yet, the underlying biological mechanisms and genetic basis of disassortative mating remain poorly known. Here, we examine a possible mechanism of disassortative mating driven by female mate choice in relation to the overdominant Cf-Inv(1) supergene in the seaweed fly Coelopa frigida by investigating chemical communication and its genomic architecture. We show that Cf-Inv(1) strongly affects chemical signaling; cuticular hydrocarbon (CHC) composition differed between genotypes in males but not females across two continents. In tandem, Cf-Inv(1) affected female perception of these compounds; females are able to sense 36 compounds from the male CHC cocktail but show differential perception between genotypes for almost half of them. This indicates that the genetic underpinnings of male traits and female perceptions are tightly linked within Cf-Inv(1) which likely facilitates disassortative mating [3]. A differential expression approach based on candidate genes for CHC biosynthesis and odorant detection revealed differential expression for CHC biosynthesis in males alone but broad changes in odorant receptors across both sexes. Furthermore, odorant genes clustered together within Cf-Inv(1), with some of them differing between arrangements by 8.3% at the protein level, suggesting evolution via tandem duplication then divergence. We propose that the tight linkage between overdominant loci, male traits, and female perception has helped to maintain the Cf-Inv(1) polymorphism across its range in the face of supergene degeneration.
Optogenetic stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsin variants yield opposite behavioral outcomes
Authors: Baleisyte Aiste , Schneggenburger Ralf , Kochubey Olexiy 
Published: July 2021

SummaryOptogenetic manipulation of genetically-specified neuron populations has become a key tool in circuit neuroscience. The medial amygdala (MeA) receives pheromone information about conspecifics and has crucial functions in social behaviors; interestingly, this amygdalar structure contains a majority of GABAergic projection neurons. A previous study showed that optogenetic activation of MeA GABA neurons with channelrhodopsin-2H134R (ChR2) strongly enhanced inter-male aggression (Hong et al. 2014, Cell). When we attempted to reproduce these findings, accidentally using a faster channelrhodopsin variant (channelrhodopsin-2H134R,E123T or ChETA), we found the opposite results. We therefore systematically compared the behavioral outcome of optogenetic stimulation of MeApd GABA neurons with ChETA versus ChR2, employing two widely used AAV serotypes. This revealed that optogenetic stimulation with ChETA suppressed aggression, whereas optogenetic stimulation with ChR2 increased aggression. Recordings of membrane potential changes following optogenetic stimulation with ChETA versus ChR2 revealed larger plateau depolarizations, smaller action potential amplitudes, and larger local inhibition of neighboring inhibitory neurons with ChR2 as compared to ChETA. Our study shows that channelrhodopsin variants have to be chosen with care for in-vivo optogenetic experiments. Furthermore, the role of MeApd GABA neurons in aggression control should be re- evaluated.
Combination and competition between path integration and landmark navigation in the estimation of heading direction
Authors: Harootonian Sevan K. , Ekstrom Arne D. , Wilson Robert C. 
Published: July 2021

AbstractSuccessful navigation requires the ability to compute one’s location and heading from incoming multisensory information. Previous work has shown that this multisensory input comes in two forms: body-based idiothetic cues, from one’s own rotations and translations, and visual allothetic cues, from the environment (usually visual landmarks). However, exactly how these two streams of information are integrated is unclear, with some models suggesting the body-based idiothetic and visual allothetic cues are combined, while others suggest they compete. In this paper we investigated the integration of body-based idiothetic and visual allothetic cues in the computation of heading using virtual reality. In our experiment, participants performed a series of body turns of up to 360 degrees in the dark with only a brief flash (300ms) of visual feedback en route. Because the environment was virtual, we had full control over the visual feedback and were able to vary the offset between this feedback and the true heading angle. By measuring the effect of the feedback offset on the angle participants turned, we were able to determine the extent to which they incorporated visual feedback as a function of the offset error. By further modeling this behavior we were able to quantify the computations people used. While there were considerable individual differences in performance on our task, with some participants mostly ignoring the visual feedback and others relying on it almost entirely, our modeling results suggest that almost all participants used the same strategy in which idiothetic and allothetic cues are combined when the mismatch between them is small, but compete when the mismatch is large. These findings suggest that participants update their estimate of heading using a hybrid strategy that mixes the combination and competition of cues.Author summarySuccessful navigation requires us to combine visual information about our environment with body-based cues about our own rotations and translations. In this work we investigated how these disparate sources of information work together to compute an estimate of heading. Using a novel virtual reality task we measured how humans integrate visual and body-based cues when there is mismatch between them — that is, when the estimate of heading from visual information is different from body-based cues. By building computational models of different strategies, we reveal that humans use a hybrid strategy for integrating visual and body-based cues — combining them when the mismatch between them is small and picking one or the other when the mismatch is large.
Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging
Authors: Huang Manning Y. , Joshi Meenakshi B. , Boucher Michael J. , Lee Sujin , Loza Liza C. , Gaylord Elizabeth A. , Doering Tamara L. , Madhani Hiten D. 
Published: July 2021

ABSTRACTCryptococcus neoformans, the most common cause of fungal meningitis, is a basidiomycete haploid budding yeast with a complete sexual cycle. Genome modification by homologous recombination is feasible using biolistic transformation and long homology arms, but the method is arduous and unreliable. Recently, multiple groups have reported the use of CRISPR-Cas9 as an alternative to biolistics, but long homology arms are still necessary, limiting the utility of this method. Since the S. pyogenes Cas9 derivatives used in prior studies were not optimized for expression in C. neoformans, we designed, synthesized, and tested a fully C. neoformans-optimized Cas9. We found that a Cas9 harboring only common C. neoformans codons and a consensus C. neoformans intron together with a TEF1 promoter and terminator and a nuclear localization signal (C. neoformans-optimized CAS9 or “CnoCAS9”) reliably enabled genome editing in the widely-used KN99α C. neoformans strain. Furthermore, editing was accomplished using donors harboring short (50 bp) homology arms attached to marker DNAs produced with synthetic oligonucleotides and PCR amplification. We also demonstrated that prior stable integration of CnoCAS9 further enhances both transformation and homologous recombination efficiency; importantly, this manipulation does not impact virulence in animals. We also implemented a universal tagging module harboring a codon-optimized fluorescent protein (mNeonGreen) and a tandem Calmodulin Binding Peptide-2X FLAG Tag that allows for both localization and purification studies of proteins for which the corresponding genes are modified by short homology-directed recombination. These tools enable short-homology genome engineering in C. neoformans.
Network-Based Discovery of Opioid Use Vulnerability in Rats Using the Bayesian Stochastic Block Model
Authors: Allen Carter , Kuhn Brittany N. , Cannella Nazzareno , Crow Ayteria D. , Roberts Analyse T. , Lunerti Veronica , Ubaldi Massimo , Hardiman Gary , Solberg Woods Leah C. , Ciccocioppo Roberto , Kalivas Peter W. , Chung Dongjun 
Published: July 2021

AbstractOpioid use disorder is a psychological condition that affects over 200,000 people per year in the U.S., causing the Centers for Disease Control and Prevention to label the crisis as a rapidly spreading public health epidemic. It has been found that the behavioral relationship between opioid exposure and development of opioid use disorder varies greatly between individuals, implying existence of sup-populations with varying degrees of opioid vulnerability. In this study, we assessed several behavioral variables across heroin taking, refraining and seeking to establish how these factors interact with one another resulting in a heroin dependent, resilient, or vulnerable behavioral phenotype. Over 400 (male and female) heterogeneous stock rats were used in these two studies, and data were collected from two geographically distinct locations. Rats underwent heroin self-administration training, followed by a progressive ratio and heroin-primed reinstatement test. Next, rats underwent extinction training and a cue-induced reinstatement test. To assess how these variables contribute to heroin addiction vulnerability, we developed a network-based data analysis workflow. Specifically, we integrated different cohorts of rats, remove possible batch effects, and constructed a rat-rat similarity network based on their behavioral patterns. We then implemented community detection on this similarity network using a Bayesian degree-corrected stochastic block model to uncover sub-populations of rats with differing levels of opioid vulnerability. We discovered three distinct behavioral sub-populations, each with significantly different behavioral outcomes that allowed for unique characterization of each cluster in terms of vulnerability to opioid use and seeking. We implement this analysis workflow as an open source R package, named mlsbm.
Rapid synaptic plasticity contributes to a learned conjunctive code of position and choice-related information in the hippocampus
Authors: Zhao Xinyu , Hsu Ching-Lung , Spruston Nelson 
Published: July 2021

AbstractTo successfully perform goal-directed navigation, animals must know where they are and what they are doing—e.g., looking for water, bringing food back to the nest, or escaping from a predator. Hippocampal neurons code for these critical variables conjunctively, but little is known about how this where/what code is formed or flexibly routed to other brain regions. To address these questions, we performed intracellular whole-cell recordings in mouse CA1 during a cued, two-choice virtual navigation task. We demonstrate that plateau potentials in CA1 pyramidal neurons rapidly strengthen synaptic inputs carrying conjunctive information about position and choice. Plasticity-induced response fields were modulated by cues only in animals previously trained to collect rewards based on these cues. Thus, we reveal that gradual learning is required for the formation of a conjunctive population code, upstream of CA1, while plateau-potential-induced synaptic plasticity in CA1 enables flexible routing of the code to downstream brain regions.
Integrative analysis of the shikonin metabolic network identifies new gene connections and reveals evolutionary insight into shikonin biosynthesis
Authors: Suttiyut Thiti , Auber Robert P. , Ghaste Manoj , Kane Cade N. , McAdam Scott A. M. , Wisecaver Jennifer H. , Widhalm Joshua R. 
Published: July 2021

SummaryPlant specialized 1,4-naphthoquinones present a remarkable case of convergent evolution. Species across multiple discrete orders of vascular plants produce diverse 1,4-naphthoquinones via one of several pathways using different metabolic precursors. Evolution of these pathways was preceded by events of metabolic innovation and many appear to share connections with biosynthesis of photosynthetic or respiratory quinones. Here, we sought to shed light on the metabolic connections linking shikonin biosynthesis with its precursor pathways and on the origins of shiknoin metabolic genes. Downregulation of Lithospermum erythrorhizon geranyl diphosphate synthase (LeGPPS), recently shown to have been recruited from a cytoplasmic farnesyl diphosphate synthase (FPPS), resulted in reduced shikonin production and a decrease in expression of mevalonic acid and phenylpropanoid pathway genes. Next, we used LeGPPS and other known shikonin pathway genes to build a coexpression network model for identifying new gene connections to shikonin metabolism. Integrative in silico analyses of network genes revealed candidates for biochemical steps in the shikonin pathway arising from Boraginales-specific gene family expansion. Multiple genes in the shikonin coexpression network were also discovered to have originated from duplication of ubiquinone pathway genes. Taken together, our study provides evidence for transcriptional crosstalk between shikonin biosynthesis and its precursor pathways, identifies several shikonin pathway gene candidates and their evolutionary histories, and establishes additional evolutionary links between shikonin and ubiquinone metabolism. Moreover, we demonstrate that global coexpression analysis using limited transcriptomic data obtained from targeted experiments is effective for identifying gene connections within a defined metabolic network.
Structural studies on cell wall biogenesis-I: A method to produce high-quality protoplasts from alga Micrasterias denticulate, an emerging model in plant biology
Authors: Selvaraj M. 
Published: July 2021

AbstractMicrasterias denticulate is a freshwater unicellular green alga emerging as a model system in plant cell biology. This is an algae that has been examined in the context of cell wall research from early 1970’s. Protoplast production from such a model system is important for many downstream physiological and cell biological studies. The algae produce intact protoplast in a straight two-step protocol involving 5% mannitol, 2% cellulysin, 4mM calcium chloride under a ‘temperature ramping’ strategy. The process of protoplast induction and behavior of protoplast was examined by light microscopy and reported in this study.
TMEM120A/TACAN inhibits mechanically activated Piezo2 channels
Authors: Del Rosario John Smith , Gabrielle Matthew , Yudin Yevgen , Rohacs Tibor 
Published: July 2021

ABSTRACTMechanically activated Piezo2 channels are key mediators of light touch and proprioception in mice and humans. Relatively little is known about what other proteins regulate Piezo2 activity in a cellular context. TACAN (TMEM120A) was proposed to act as a high threshold mechanically activated ion channel in nociceptive dorsal root ganglion (DRG) neurons. Here we find that TACAN co-expression robustly reduced mechanically activated Piezo2 currents, but did not inhibit mechanically activated Piezo1 and TREK1 currents. TACAN co-expression did not affect cell surface expression of either Piezo1 or Piezo2 and did not have major effects on the cortical actin or tubulin cytoskeleton. TACAN expression alone did not result in the appearance of mechanically activated currents above background. In addition, TACAN and Piezo2 expression in DRG neurons overlapped, and siRNA mediated knockdown of TACAN did not decrease the proportion of slowly adapting mechanically activated currents, but resulted in an increased proportion of rapidly adapting currents. Our data do not support TACAN being a mechanically activated ion channel, and identify it as a negative modulator of Piezo2 channel activity.
daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71
Authors: Wirick Matthew J. , Cale Allison R. , Smith Isaac T. , Alessi Amelia F. , Starostik Margaret R. , Cuko Liberta , Lalk Kyal , Schmidt Mikayla N. , Olson Benjamin S. , Salomon Payton M. , Santos Alexis , Schmitter-Sánchez Axel , Galagali Himani , Ranke Kevin J. , Wolbert Payton A. , Knoblock Macy L. , Kim John K. , Karp Xantha 
Published: July 2021

AbstractMany tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.
Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease
Authors: Pattabhi Sowmya , Lotti Samantha N. , Berger Mason P. , Singh Swati , Rawlings David J. 
Published: July 2021

AbstractSickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.