bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring the promoter activity of long noncoding RNAs and stem cell differentiation through knock-in of sgRNA flanked by tRNA in an intron
Authors: Zhao Yu-Ting , Wang Yangming 
Published: January 2021

AbstractMammalian genomes encode thousands of long noncoding RNAs (lncRNAs) that are often expressed in a tissue and cell specific manner. Therefore, a reporter that can faithfully reflect the expression or activity of lncRNAs can provide tools useful not only for uncovering the regulators of lncRNAs, but also for tracking cell fate and disease status. Here, we design a sgRNA precursor in anintron (GRIT) strategy that can monitor the promoter activity of lncRNAs. We used this strategy to report the expression of Lncenc1 and Neat1 in mouse embryonic stem cells (ESCs). Furthermore, we show that GRIT may be used to track differentiation status of stem cells. We anticipate that GRIT will be applicable in dissecting regulatory mechanisms underlying the transcription of lncRNAs, tracking cell fate switch during differentiation or disease progression and integrating the promoter activity of various RNAs for synthetic biology applications.
Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing inDrosophila
Authors: Eastwood Evelyn L. , Jara Kayla A. , Bornelöv Susanne , Munafò Marzia , Frantzis Vasileios , Kneuss Emma , Barbar Elisar J. , Czech Benjamin , Hannon Gregory J. 
Published: January 2021

AbstractIn animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. InDrosophilaovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through thePanoramix-inducedco-transcriptionalsilencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.
Sex and origin-specific inbreeding effects on flower attractiveness to specialised pollinators
Authors: Schrieber Karin , Paul Sarah Catherine , Höche Levke Valena , Salas Andrea Cecilia , Didszun Rabi , Mößnang Jakob , Müller Caroline , Erfmeier Alexandra , Eilers Elisabeth Johanna 
Published: January 2021

AbstractWe investigate whether inbreeding has particularly fatal consequences for dioecious plants by diminishing their floral attractiveness and the associated pollinator visitation rates disproportionally in females. We also test whether the magnitude of such effects depends on the evolutionary histories of plant populations. We recorded spatial, olfactory, colour and rewarding flower attractiveness traits as well as pollinator visitation rates in experimentally inbred and outbred, male and femaleSilene latifoliaplants from European and North American populations differing in their evolutionary histories. We found that inbreeding specifically impairs spatial and olfactory attractiveness. Our results support that sex-specific selection and gene expression partially magnified these inbreeding costs for females, and that divergent evolutionary histories altered the genetic architecture underlying inbreeding effects across population origins. Moreover, they highlight that inbreeding effects on olfactory attractiveness have a huge potential to disrupt interactions among plants and specialist moth pollinators, which are mediated by elaborate chemical communication.
Somatic mutations in CTCF zinc fingers produce cellular phenotypes explained by structure-function relationships
Authors: Bailey Charles G , Gupta Shailendra , Metierre Cynthia , Amarasekera Punkaja MS , O’Young Patrick , Kyaw Wunna , Laletin Tatyana , Francis Habib , Semaan Crystal , Singh Krishna P , Mullighan Charles G , Wolkenhauer Olaf , Schmitz Ulf , Rasko John EJ 
Published: January 2021

AbstractBackgroundHuman cancers commonly contain mutations in transcription factors that lead to aberrant DNA binding or altered effector function at target sites. One such factor significantly mutated in cancer is the evolutionarily-conserved CCCTC-binding factor (CTCF), which has fundamental roles in maintaining chromatin architecture and transcriptional regulation. Numerous cancer genome sequencing and functional studies have revealed CTCF’s role as a haploinsufficient tumour suppressor gene. However, to date, structure-function relationships of somatic CTCF mutations have not been examined.MethodsWe collated somatic CTCF mutations from cancer genome portals and published studies to determine their nature, frequency, distribution and potential functional impact. We undertook an in-depth examination of 5 CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. We performed functional analyses including cell growth, colony-formation, chromatin immunoprecipitation and transcriptional reporter assays. Based on their homology, each ZF mutation was then modelled on CTCF’s ZF domain crystal structure and its structural impact analysed using molecular dynamics simulations.ResultsWe observed an enrichment of somatic missense mutations occurring in the ZF region of CTCF, compared to the unstructured N- and C-termini. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated.In silicomutagenesis revealed that L309P had the highest mutation energy and thus most severe impact on protein stability. Molecular docking and molecular dynamics simulations confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding (R339Q, R339W, R377H). Remarkably, R339Q and G420D were stabilised by the formation of new primary DNA bonds. All mutations exhibited some loss or gain of bonds at neighbouring residues, often in adjacent zinc fingers.ConclusionsOur data confirm the significant negative impact haploinsufficient CTCF ZF mutations have on its tumour suppressor function. A spectrum of loss-, change- and gain-of-function impacts in CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. We have established that diverse cellular phenotypes in CTCF are explained by examining structure-function relationships.
RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zeamays
Authors: Guo Wei , Wang Dafang , Lisch Damon 
Published: January 2021

AbstractIn large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent onModifer of Paramutation 1(Mop1), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silencedMuDRtransposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K27 trimethylation, a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in amop1mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even inmop1wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in amop1mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.Author SummaryMost plant genomes are mostly transposable elements (TEs), most of which are held in check by modifications of both DNA and histones. The bulk of silenced TEs are associated with methylated DNA and histone H3 lysine 9 demethylation (H3K9me2). In contrast, epigenetically silenced genes are often associated with histone lysine 27 trimethylation (H3K27me3). Although stress can affect each of these modifications, plants are generally competent to rapidly reset them following that stress. Here we demonstrate that although DNA methylation is not required to maintain silencing of theMuDRelement, it is essential for preventing heat-induced, stable and heritable changes in both H3K9me2 and H3K27me3 at this element, and for concomitant changes in transcriptional activity. These finding suggest that RdDM acts to buffer the effects of heat on silenced transposable elements, and that a loss of DNA methylation under conditions of stress can have profound and long lasting effects on epigenetic silencing in maize.
Cep55 regulation of PI3K/Akt signaling is required for neocortical development and ciliogenesis
Authors: Rashidieh Behnam , Shohayeb Belal , Bain Amanda Louise , Fortuna Patrick R. J. , Sinha Debottam , Burgess Andrew , Mills Richard , Adams Rachael C. , Lopez J. Alejandro , Blumbergs Peter , Finnie John , Kalimutho Murugan , Piper Michael , Hudson James Edward , Ng Dominic , Khanna Kum Kum 
Published: January 2021

AbstractHomozygous nonsense mutations in CEP55 are associated with several congenital malformations that lead to perinatal lethality suggesting that it plays a critical role in regulation of embryonic development. CEP55 has previously been studied as a critical regulator of cytokinesis predominantly in transformed cells and its deregulation is linked to carcinogenesis. However, its molecular functions during embryonic development in mammals have not been clearly defined. We have generated a Cep55 knockout (Cep55-/-) mouse model which demonstrated perinatal lethality associated with a wide range of neural defects. Focusing our analysis on the neocortex, we show that Cep55-/- embryos exhibited depleted neural stem/progenitor cells in the ventricular zone as a result of significantly increased cellular apoptosis. Mechanistically, we demonstrated that Cep55-loss downregulates the pGsk3β/β-Catenin/Myc axis in an Akt-dependent manner. The phenotype was recapitulated using human cerebral organoids and we could rescue the phenotype by inhibiting active Gsk3β. Additionally, we show that Cep55-loss leads to a significant reduction of ciliated cells, highlighting its novel role in regulating ciliogenesis. Collectively, our findings demonstrate a critical role of Cep55 during brain development and provide mechanistic insights that may have important implications for genetic syndromes associated with Cep55-loss.
Family cluster comparisons to detect infection byMycobacterium lepraein at-risk populations of six endemic regions in Colombia
Authors: Serrano-Coll Héctor , Osorio-Leal Yuliana , Escobar-Builes María Victoria , Cardona-Castro Nora 
Published: January 2021

AbstractIntroductionLeprosy is a chronic infectious disease, caused byMycobacterium leprae, which is endemic in some tropical countries. It is necessary to implement strategies for its detection and elimination. We propose a strategy useful could be identifying risk factors associated with a seropositive test in leprosy.ObjectiveWe aimed to quantify the infection rates and identify risk factors for M. leprae infection using the serological evaluations against NDO-LID in family clusters of leprosy patients, from regions with a high burden of leprosy in Colombia.ResultsWe observed that belong to a low socioeconomic condition (OR 5.6 [95% IC 1.1-29]) and living in geographic area of residing such as Chocó and Atlántico (OR 2 [95% IC 1.1-3.7]) could be risk factors related to infection byM. lepraebetween the members of a family cluster.ConclusionsLeprosy is a persistent disease that affects vulnerable and large family clusters, in which the detection of antibodies against NDO-LID can be a useful tool for early detection ofM. lepraein family clusters with high risk for this infection.
Lamina-associated polypeptide 2α is required for intranuclear MRTF-A activity
Authors: Sidorenko Ekaterina , Sokolova Maria , Pennanen Antti , Kyheröinen Salla , Posern Guido , Foisner Roland , Vartiainen Maria K. 
Published: January 2021

ABSTRACTMyocardin-related transcription factor A (MRTF-A), a coactivator of serum response factor (SRF), regulates the expression of many cytoskeletal genes in response to cytoplasmic and nuclear actin dynamics. Here we describe a novel mechanism to regulate MRTF-A activity within the nucleus by showing that lamina-associated polypeptide 2α (Lap2α), the nucleoplasmic isoform of Lap2, is a direct binding partner of MRTF-A, and required for the efficient expression of MRTF-A/SRF target genes. Mechanistically, Lap2α is not required for MRTF-A nuclear localization, unlike most other MRTF-A regulators, but is required for binding of MRTF-A to its target genes. This regulatory step takes place prior to MRTF-A chromatin binding, because Lap2α neither interacts with, nor specifically influences active histone marks on MRTF-A/SRF target genes. Phenotypically, Lap2α is required for serum-induced cell migration, and deregulated MRTF-A activity may also contribute to muscle and proliferation phenotypes associated with loss of Lap2α. Our studies therefore add another regulatory layer to the control of MRTF-A-SRF-mediated gene expression, and broaden the role of Lap2α in transcriptional regulation.
Pin Electrode Reactor: A novel cold atmospheric plasma device and its potential in glioblastoma treatment
Authors: de Carvalho Andressa Maria Aguiar , Behan Sean , Scally Laurence , Sarangapani Chaitanya , Malone Renee , Cullen Patrick J. , Tiwari Brijesh , Curtin James F. 
Published: January 2021

AbstractGlioblastoma multiforme (GBM) is the most common and biologically aggressive brain tumour. The current standard therapy for GBM consists in surgical resection, followed by radiotherapy and chemotherapy. Yet, the treatment is limited due to the area for the surgical resection and for the inability of some drugs to cross the brain blood barrier, leading to a general prognostic of no more than a year. Cold atmospheric plasma (CAP) is a new approach in the treatment of this challenging disease. CAP interaction with cells is dependent on physical and chemical factors, with different plasma discharges, cell type, and culture conditions leading to different CAP activity. Considering the plasma self-adaptation that different plasma discharge modes can undergo, which leads to different interaction plasma/cells, the characterization of a new device is essential. In this study we analysed the effect of a novel large pin-to-plate non-thermal atmospheric plasma on U-251 MG cells under different conditions. The analysis of reactive oxygen and nitrogen species (RONS) on plasma, media and cells were also assessed. We were able to demonstrate that the pin-to-plate device is cytotoxic to GBM cells in a dose, time and ROS dependent manner. The measurements of RONS on plasma/media also give us an insight on the chemical effect of this novelty device, and the possibility to better understand the use of this device as a promising GBM therapy.
Caspase inhibition mitigates tau cleavage and neurotoxicity in iPSC-induced neurons with the V337MMAPTmutation
Authors: Theofilas Panos , Wang Chao , Butler David , Morales Dulce O. , Petersen Cathrine , Chin Brian , Yang Teddy , Khan Shireen , Ng Raymond , Kayed Rakez , Karch Celeste M. , Miller Bruce L. , Gestwicki Jason E. , Gan Li , Temple Sally , Arkin Michelle R. , Grinberg Lea T. 
Published: January 2021

AbstractTau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD). Proteolytic cleavage of tau by active caspases, including caspase-6, represents an underexplored tau PTM implicated in tau pathology. Caspase-cleaved tau is toxic and prone to self-aggregation in experimental models. To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two neoepitope monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites and cortical neurons from induced pluripotent stem cells (iPSCs) with the frontotemporal dementia (FTD)-causing V337MMAPTmutation. FTLD V337MMAPTand AD postmortem brains showed positivity for both cleaved tau mAbs as well as active caspase-6. Relative to isogenic wild-typeMAPTcontrols, V337MMAPTneurons showed a time-dependent increase in pathogenic tau in the form of tau oligomers, caspase-cleaved tau, and p-tau. Accumulation of toxic tau species in 3-month V337MMAPTneurons also increased vulnerability to stress, which was pharmacologically rescued by caspase inhibition. We propose a model in which time-dependent accumulation of caspase-cleaved tau in V337MMAPTneurons promotes neurotoxicity that is reversed by caspase-6 inhibition. Caspase-cleaved tau may be a biomarker of tauopathy, and caspases could be viable targets for therapeutic intervention against tau pathogenesis in FTLD and other tauopathies.SignificanceThe mechanisms leading to tau pathology in frontotemporal dementia (FTD) and Alzheimer’s disease (AD) remain elusive. Experimental studies in AD demonstrate that tau cleavage by active caspase-6 contributes to tau pathology since cleaved tau may be toxic and prone to self-aggregation. Yet, the role of caspase-cleaved tau in promoting toxicity and cell death is unclear. Here, we generated two neoepitope monoclonal antibodies against caspase-6 tau and evaluated tau cleavage in postmortem human brains, iPSC-induced cortical neurons with the FTD-causing V337MMAPTmutation, and isogenic wild-typeMAPTcontrols. Our results demonstrate a time-dependent accumulation of caspase-cleaved tau and increased neurotoxicity in the mutant iNs that is reversed by caspase-6 inhibition. Caspases could be viable therapeutic targets against tau pathology in tauopathies.
Ad26.COV2.S-elicited immunity protects against G614 spike variant SARS-CoV-2 infection in Syrian hamsters and does not enhance respiratory disease in challenged animals with breakthrough infection after sub-optimal vaccine dosing
Authors: van der Lubbe Joan E.M. , Rosendahl Huber Sietske K. , Vijayan Aneesh , Dekking Liesbeth , van Huizen Ella , Vreugdenhil Jessica , Choi Ying , Baert Miranda R.M. , Feddes-de Boer Karin , Gil Ana Izquierdo , van Heerden Marjolein , Dalebout Tim J. , Myeni Sebenzile K. , Kikkert Marjolein , Snijder Eric J. , de Waal Leon , Stittelaar Koert J. , Tolboom Jeroen T.B.M. , Serroyen Jan , Muchene Leacky , van der Fits Leslie , Rutten Lucy , Langedijk Johannes P.M. , Barouch Dan H. , Schuitemaker Hanneke , Zahn Roland C. , Wegmann Frank 
Published: January 2021

ABSTRACTPreviously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1×109vp and 1×1010vp elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers which was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these pre-clinical data confirm efficacy of a 1-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.
Clr4SUV39H1ubiquitination and non-coding RNA mediate transcriptional silencing via heterochromatic phase transitions
Authors: Kim Hyun-Soo , Roche Benjamin , Bhattacharjee Sonali , Todeschini Leila , Chang An-Yun , Hammell Christopher , Verdel André , Martienssen Robert A. 
Published: January 2021

AbstractTranscriptional silencing by RNAi paradoxically relies on transcription, but how the transition from transcription to silencing is achieved has remained unclear. The Cryptic Loci Regulator complex (CLRC) inSchizosaccharomyces pombeis a cullin-ring E3 ligase required for silencing that is recruited by RNAi. We found that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and mono-ubiquitinates the histone H3K9 methyltransferase Clr4SUV39H1, promoting the transition from co-transcriptional gene silencing (H3K9me2) to transcriptional gene silencing (H3K9me3). Ubiquitination of Clr4 occurs in an intrinsically disordered region (IDR), which undergoes robust liquid-liquid phase separation (LLPS), along with Swi6HP1the effector of transcriptional gene silencing. Phase separation of Clr4 and Swi6 is exquisitely sensitive to non-coding RNA (ncRNA), which promotes dimerization, chromatin association, and di-, but not tri-methylation instead. Ubc4-CLRC also targets the transcriptional co-activator Bdf2BRD4, down-regulating centromeric transcription and small RNA production. The deubiquitinase Ubp3 counteracts both activities.