bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the main barriers to Ku accumulation in chromatin
Authors: Bossaert Madeleine , Moreno Andrew , Peixoto Antonio , Pillaire Marie-Jeanne , Chanut Pauline , Frit Philippe , Calsou Patrick , Loparo Joseph John , Britton Sébastien 
Published: January 2024

SummaryRepair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.HighlightsDNA-PKcs structurally blocks Ku sliding into chromatin in human &XenopusA neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatinIn S-phase, ATM/CtIP overcomes Ku accumulationIn absence of DNA-PKcs, transcription at the DNA end vicinity is inhibitedeTOC blurbThe DNA end binding protein Ku can slide onto naked DNA but this is limited in cells. Using human cells andXenopusegg extracts, DNA-PKcs is identified as the main structural barrier to Ku entry into chromatin, along with two active mechanisms which limit Ku accumulation in absence of DNA-PKcs.Graphical abstract
RBM7 deficiency promotes breast cancer metastasis by coordinating MFGE8 splicing switch and NF-kB pathway
Authors: Huang Fang , Dai Zhenwei , Yu Jinmiao , Wang Kainan , Chen Chaoqun , Chen Dan , Zhang Jinrui , Zhao Jinyao , Li Mei , Zhang Wenjing , Li Xiaojie , Qi Yangfan , Wang Yang 
Published: January 2024

AbstractAberrant alternative splicing is well-known to be closely associated with tumorigenesis of various cancers. However, the intricate mechanisms underlying breast cancer metastasis driven by deregulated splicing events remain largely unexplored. Here, we unveiled that RBM7 is decreased in lymph node and distant organ metastases of breast cancer as compared to primary lesions and low expression of RBM7 is correlated with the reduced disease-free survival of breast cancer patients. Breast cancer cells with RBM7 depletion exhibited an increased potential for lung metastasis compared to scramble control cells. The absence of RBM7 stimulated breast cancer cell migration, invasion, and angiogenesis. Mechanistically, RBM7 controlled the splicing switch of MFGE8, favoring the production of the predominant isoform of MFGE8, MFGE8-L. This resulted in the attenuation of STAT1 phosphorylation and alterations in cell adhesion molecules. MFGE8-L exerted an inhibitory effect on the migratory and invasive capability of breast cancer cells, while the truncated isoform MFGE8-S, which lack the second F5/8 type C domain had the opposite effect. In addition, RBM7 negatively regulates the NF-κB cascade and an NF-κB inhibitor could obstruct the increase in HUVEC tube formation caused by RBM7 silencing. Clinically, we noticed a positive correlation between RBM7 expression and MFGE8 exon7 inclusion in breast cancer tissues, providing new mechanistic insights for molecular-targeted therapy in combating breast cancer.
An infection and pathogenesis mouse model of SARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR)
Authors: Wei Lai , Liu Shuiqing , Lu Shanshan , Luo Shengdong , An Xiaoping , Fan Huahao , Chen Weiwei , Li Erguang , Tong Yigang , Song Lihua 
Published: January 2024

ABSTRACTSARS-CoV-2-related pangolin coronavirus GX_P2V(short_3UTR) is highly attenuated, but can cause mortality in a specifically designed human ACE2-transgenic mouse model, making it an invaluable surrogate model for evaluating the efficacy of drugs and vaccines against SARS-CoV-2.
Assessment of autophagy inLeishmaniaparasites
Authors: Onwah Somtochukwu S. , Uzonna Jude E. , Ghavami Saeid 
Published: January 2024

AbstractLeishmaniasis is a neglected tropical disease caused by numerous species ofLeishmaniaparasites, includingLeishmania major.The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown thatLeishmaniamodulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy inLeishmaniais inconclusive because of the limited tools available to study theLeishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy inLeishmania major. Transmission electron microscopy (TEM) allowed us to visualizeLeishmaniaautophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments inLeishmania majorby detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation inLeishmaniaparasites that could provide insights into developing improved therapeutic targets against leishmaniasis.
The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics
Authors: Leib Lisa , Juli Jana , Jurida Liane , Meier-Soelch Johanna , Mayr-Buro Christin , Schmitz M. Lienhard , Heylmann Daniel , Weber Axel , Papantonis Argyris , Bartkuhn Marek , Wilhelm Jochen , Linne Uwe , Kracht Michael 
Published: January 2024

AbstractThe protein interactome of p65 / RELA, the most active subunit of the transcription factor (TF) NF-κB, has not been previously determined in living cells. Using p65-miniTurbo fusion proteins, we identified by biotin tagging > 350 RELA interactors from untreated and IL-1α-stimulated cells, including many TFs (47 % of all interactors) and > 50 epigenetic regulators belonging to different classes of chromatin remodeling complexes. According to point mutants of p65, the interactions primarily require intact dimerization rather than DNA binding properties. A targeted RNAi screen for 38 interactors and subsequent functional transcriptome and bioinformatics studies identified gene regulatory (sub)networks, each controlled by RELA in combination with one of the TFs ZBTB5, GLIS2, TFE3 / TFEB or S100A8 / A9. The remarkably large, dynamic and versatile high resolution interactome of RELA and its gene-regulatory logics provides a rich resource and a new framework for explaining how RELA cooperativity determines gene expression patterns.HighlightsIdentification of > 350 largely dimerization-dependent interactors of p65 / RELA by miniTurboIDThe interactome is dominated by transcription factors and epigenetic regulator complexesFunctional validation of 38 high confidence interactors by targeted siRNA screenIdentification of genetic networks regulated by RELA and six of its interactors in the IL-1α response
Kernels of Motor Memory Formation: Temporal Generalization in Bimanual Adaptation
Authors: Howard Ian S. , Franklin Sae , Franklin David W. 
Published: January 2024

AbstractIn daily life, we coordinate both simultaneous and sequential bimanual movements to manipulate objects. Our ability to rapidly account for different object dynamics suggests there are neural mechanisms to quickly deal with them. Here we investigate how actions of one arm can serve as a contextual cue for the other arm, and facilitate adaptation. Specifically, we examine the temporal characteristics that underlie motor memory formation and recall, by testing the contextual effects of prior, simultaneous, and post contralateral arm movements in both male and female human participants. To do so, we measure their temporal generalization in three bimanual interference tasks. Importantly, the timing context of the learned action plays a pivotal role in the temporal generalization. While motor memories trained with post adaptation contextual movements generalize broadly, motor memories trained with prior contextual movements exhibit limited generalization, and motor memories trained with simultaneous contextual movements do not generalize to prior or post contextual timings. This highlights temporal tuning in sensorimotor plasticity: different training conditions yield substantially different temporal generalization characteristics. Since these generalizations extend far beyond any variability in training times, we suggest that the observed differences may stem from inherent differences in the use of prior, current and post-adaptation contextual information in the generation of natural behavior. This would imply differences in the underlying neural circuitry involved in learning and executing the corresponding coordinated bimanual movements.Significance StatementThis study addresses a fundamental question in the field of sensorimotor neuroscience of how multiple movements are temporally linked within a single motor memory. We examine the temporal generalization of motor memory formation by varying the timing of contextual movements associated with a learned motor memory across a range of prior, current, and post adaptation movement times using a bimanual motor learning task. We observed distinct patterns of temporal generalization based on whether the contextual movements occurred prior to, simultaneously with, or after the adaptation movement. For the first time, our findings reveal that the timing of contextual movements is crucial in the formation and generalization of motor memories.
Integrated analysis and systematic characterization of the regulatory network for human germline development
Authors: Gu Yashi , Chen Jiayao , Wang Ziqi , Li Zhekai , Xiao Xia , Shao Qizhe , Xiao Yitian , Liu Wenyang , Xie Sisi , Ye Yaxuan , Jiang Jin , Xiao Xiaoying , Yu Ya , Jin Min , Young Robert , Hou Lei , Chen Di 
Published: January 2024

AbstractPrimordial germ cells (PGCs) are the precursors of germline that are specified at embryonic stage. Recent studies reveal that humans employ different mechanisms for PGC specification compared with model organisms such as mice. Moreover, the specific regulatory machinery is still largely unexplored, mainly due to the inaccessible nature of this complex biological process. Here, we collect and integrate multi-omics data, including 581 RNA-seq, 54 ATAC-seq, 45 ChIP-seq, and 69 single-cell RNA-seq samples from different stages of human PGC development to recapitulate the precisely controlled and stepwise process, presenting an atlas in the human PGC database (hPGCdb). With these uniformly processed data and integrated analyses, we characterize the potential key transcription factors and regulatory networks governing human germ cell fate. We validate the important roles of some of the key factors in germ cell development by CRISPRi knockdown. We also identify the soma-germline interaction network and discover the involvement of SDC2 and LAMA4 for PGC development, as well as soma-derived NOTCH2 signaling for germ cell differentiation. Taken together, we have built a database for human PGCs and demonstrate that hPGCdb enables the identification of the missing pieces of mechanisms governing germline development, including both intrinsic and extrinsic regulatory programs.
Alternative splicing plays a nonredundant role in greater amberjack (Seriola dumerili) in the adaptation to ambient salinity fluctuations
Authors: Zhao Chunyu , Liu Yuqi , Zhang Panpan , Xia Xinhui , Yang Yuchen 
Published: January 2024

AbstractAlternative splicing (AS) is an important post-transcriptional mechanism for adaptation of fish to environmental stress. Here, we performed a genome-wide investigation to explore the biological importance of AS dynamics in greater amberjack (Seriola dumerili), an economical marine teleost species, in response to hypo- (10 ppt) and hyper-salinity (40 ppt) stresses. The results revealed high level of differential splicing in both gills and kidney upon the exposure to undesired salinity regimes. In gills, genes involved in energy metabolism, stimulus response and epithelial cell differentiation were differentially spliced in response to the deviation of normal water salinity, while sodium ion transport, erythrocyte homeostasis and cellular amide metabolism were enhanced in kidney to combat the adverse impacts of salinity changes. More importantly, the majority of the differentially spliced genes were not differentially expressed, and AS was found to regulate different biological processes from differential gene expression, indicative of the functionally nonredundant role of AS in modulating salinity acclimation in greater amberjack. Together, our study highlights the important contribution of post-transcriptional mechanisms to the adaptation of fish to ambient salinity fluctuations, and provides a theoretical guidance to the conservation of marine fishery resources under the increasingly extreme environmental challenges.
Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade
Authors: Powers Jordan , Zhang Xing , Reyes Andres V. , Zavaliev Raul , Xu Shou-Ling , Dong Xinnian 
Published: January 2024

SUMMARYFor over 60 years, salicylic acid (SA) has been known as a plant immune signal required for both basal and systemic acquired resistance (SAR). SA activates these immune responses by reprogramming up to 20% of the transcriptome through the function of NPR1. However, components in the NPR1-signaling hub, which appears as nuclear condensates, and the NPR1-signaling cascade remained elusive due to difficulties in studying transcriptional cofactors whose chromatin associations are often indirect and transient. To overcome this challenge, we applied TurboID to divulge the NPR1-proxiome, which detected almost all known NPR1-interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, NPR1-proxiome shares a striking similarity to GBPL3-proxiome involved in SA synthesis, except associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise greenCUT&RUN analyses showed that, upon SA-induction, NPR1 initiates the transcriptional cascade primarily through association with TGA TFs to induce expression of secondary TFs, predominantly WRKYs. WRKY54 and WRKY70 then play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, a loss of NPR1 condensate formation decreases its chromatin-association and transcriptional activity, indicating the importance of condensates in organizing the NPR1-signaling hub and initiating the transcriptional cascade. This study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades.
Light and temperature-dependent developmental role of Auxin Binding Protein 1 (ABP1) in Arabidopsis thaliana
Authors: Patnaik Alena , Behera Anshuman , Kumar Aman , Dalai Aadishakti , Mukundan S , Priyadarshini Nibedita , Panigrahy Madhusmita , Panigrahi Kishore CS 
Published: January 2024

AbstractAuxin Binding Protein 1 (ABP1) is a small glycoprotein of about 22 kDa that has long been debated as the auxin receptor, and has been put into question for its unclear functions. Despite its conservancy during land plant evolution, its precise role in plant development is still elusive. Historically, it has been implicated in various rapid responses such as membrane polarization, calcium fluxes, TMK1-based cell-surface signalling, auxin canalization, etc. A relatively recent observation questioning the role of ABP1 in plant development led us to explore its probable functions if any. In the current study, we reinvestigated the plausible function of ABP1 using its CRISPR-based loss-of-function mutants, namelyabp1-C1andabp1-C2. Here we show that, ABP1 acts as a positive regulator for primary root elongation under red and secondary root elongation under blue light in seedlings at 22 °C. Under red light at 18 °C, it has a negative effect on hypocotyl growth inhibition. Furthermore, it is involved in flowering time control at 18 °C irrespective of the photoperiod. We show that the transcript levels of Phytochrome B (phyB) and GIGANTEA (GI) are altered in the mutants of ABP1 under red light and low temperature (18 °C) regimes. Further, ABP1 show a pronounced role in tolerance to dehydration induced due to low temperature (18 °C), which correlates with an increase in endogenous abscisic acid (ABA), salicylic acid (SA), and a decrease in jasmonic acid (JA) content in leaves. The functional roles of ABP1 under red light, low temperature and dehydration tolerance inArabidopsis thalianaonce again frames it to be an important regulator under adverse and varied conditions that the plant can experience, and thus opened up new avenues for further studies.
Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks
Authors: Jacobus Ana P. , Cavassana Stella D. , Oliveira Isabelle I. , Barreto Joneclei A. , Rohwedder Ewerton , Frazzon Jeverson , Basso Thalita P. , Basso Luis C. , Gross Jeferson 
Published: January 2024

AbstractThe design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. We conducted an adaptive laboratory evolution by challenging four populations (P1–P4) of the Brazilian bioethanol yeast,Saccharomyces cerevisiaePE-2_H4, through 68–82 cycles of 2-h ethanol shocks (19%–30% v/v) and outgrowths. Colonies isolated from the final populations (P1c–P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c–P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting fromBUD3disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutationscyr1A1474T/usv1Δconferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. Thecyr1A1474T/usv1Δstrain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. These results demonstrate that some alleles selected for acute stress survival may further confer stress tolerance and optimal performance under industrial conditions.
EPISOMAL VECTORS FOR STABLE PRODUCTION OF RECOMBINANT PROTEINS AND ENGINEERED ANTIBODIES
Authors: Fallahee Ian , Hawiger Daniel 
Published: January 2024

ABSTRACTThere is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance though an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein-split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody-siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications.
Characterization of DSPE-mPEG raw materials from different vendors reveals differences in impurity profiles and polymer chain length polydispersity
Authors: Hackbusch Sven , White Sissi , Du Min 
Published: January 2024

AbstractPEGylated lipids such as 1,2-distearoyl-glycero-3-phosphatidylethanolamine-N-methoxy-polyethyleneglycol-2000 (DSPE-mPEG) are essential components of lipid nanoparticle formulations. They have an impact on the efficacy and quality of the drug products they used in, and as such should be carefully characterized. The present work used liquid chromatography with charged aerosol detection and high resolution mass spectrometry to investigate the polydispersity and purity of several lots of DSPE-mPEG, revealing significant differences in the level and identity of impurities. Additionally, mass spectral data showed that there were variations in the polyethylene glycol chain length between lots of comparable purity, which could not be detected by charged aerosol detection alone. The obtained results may be of importance in the development of new lipid nanoparticle based drugs.HighlightsImpurity identification of PEGylated lipid raw materials based on MS dataDetermination of PEG polydispersity using high resolution mass spectrometryUse of CAD with inverse gradient for impurity quantification