bluuebunny/crossref_metadata_2025 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

Repair 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.

Highlights

DNA-PKcs structurally blocks Ku sliding into chromatin in human &Xenopus

A neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin

In S-phase, ATM/CtIP overcomes Ku accumulation

In absence of DNA-PKcs, transcription at the DNA end vicinity is inhibited

eTOC blurb

The 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

Aberrant 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

SARS-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

Leishmaniasis 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

The 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.

Highlights

Identification of > 350 largely dimerization-dependent interactors of p65 / RELA by miniTurboID

The interactome is dominated by transcription factors and epigenetic regulator complexes

Functional validation of 38 high confidence interactors by targeted siRNA screen

Identification 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

In 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 Statement

This 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

Primordial 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

Alternative 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

For 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

Auxin 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

The 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

There 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

PEGylated 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.

Highlights

Impurity identification of PEGylated lipid raw materials based on MS data

Determination of PEG polydispersity using high resolution mass spectrometry

Use of CAD with inverse gradient for impurity quantification
Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons
Authors: Yu Qiguo , Tungsuchat-Huang Tarinee , Ioannou Alexander , Barkan Alice , Maliga Pal
Published: January 2024

Achieving balanced gene expression within synthetic operons requires a spectrum of expression levels. Here we investigate the expression ofgfpreporter gene in tobacco chloroplasts, guided by variants of the plastidatpH5’ UTR, which harbors a binding site for PPR10, a protein that activatesatpHat the post-transcriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maizeatpH5’ UTRs in different design contexts. Notably, high GFP expression was not coupled to stabilization of monocistronicgfptranscripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishesgfpmRNA (and GFP protein), resulting in a substantial reduction in GFP accumulation. When combined with a mutantatpH5’ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning reporter gene expression across a wide range, spanning from 0.02% to 25% of the total soluble cellular protein (TSP). These findings highlight the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels.
Cell envelope structural and functional contributions to antibiotic resistance inBurkholderia cenocepacia
Authors: Hogan Andrew M , Motnenko Anna , Rahman A S M Zisanur , Cardona Silvia T
Published: January 2024

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of theBurkholderia cepaciacomplex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants inB. cenocepaciaK56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, the polymeric O-antigen was important for resistance to cationic antibiotics, while defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase. Susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump inB. cenocepacia. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and β-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.

Importance

The Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. TheBurkholderiacell envelope is known to contain additional modifications that reduce permeability. We investigatedBurkholderiacell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.
Hippocampus oxytocin signaling promotes prosocial eating in rats
Authors: Rea Jessica J. , Liu Clarissa M. , Hayes Anna M.R. , Bashaw Alexander G. , Schwartz Grace , Ohan Rita , Décarie-Spain Léa , Kao Alicia E. , Klug Molly E. , Phung Kenneth J. , Waldow Alice I. , Wood Ruth I. , Kanoski Scott E.
Published: January 2024

The hypothalamic neuropeptide oxytocin (OT) influences both food intake and social behavior. Given that food preference and consumption are heavily affected by social factors in mammals, it is critical to understand the extent that OT’s role in regulating these two fundamental behaviors is interconnected. Here we evaluated the role of OT signaling in the dentate gyrus of the dorsal hippocampus (HPCd), a brain region recently linked with eating and social memory, on food preference and consumption in rats under conditions that vary with regards to social presence and conspecific familiarity. Results from neuropharmacological and virogenetic knockdown approaches reveal that HPCd OT signaling promotes eating in the presence of a familiar but not an unfamiliar conspecific. Additionally, HPCd OT receptor signaling is required for the social transmission of food preference. These findings collectively identify the HPCd as a novel substrate where oxytocin synergistically influences eating and social behaviors.
Single-cell transcriptional dynamics in a living vertebrate
Authors: Eck Elizabeth , Moretti Bruno , Schlomann Brandon H. , Bragantini Jordão , Lange Merlin , Zhao Xiang , VijayKumar Shruthi , Valentin Guillaume , Loureiro Cristina , Soroldoni Daniele , Royer Loïc A. , Oates Andrew C. , Garcia Hernan G.
Published: January 2024

The ability to quantify transcriptional dynamics in individual cells via live imaging has revolutionized our understanding of gene regulation. However, such measurements are lacking in the context of vertebrate embryos. We addressed this deficit by applying MS2-MCP mRNA labeling to the quantification of transcription in zebrafish, a model vertebrate. We developed a platform of transgenic organisms, light sheet fluorescence microscopy, and optimized image analysis that enables visualization and quantification of MS2 reporters. We used these tools to obtain the first single-cell, real-time measurements of transcriptional dynamics of the segmentation clock. Our measurements challenge the traditional view of smooth clock oscillations and instead suggest a model of discrete transcriptional bursts that are organized in space and time. Together, these results highlight how measuring single-cell transcriptional activity can reveal unexpected features of gene regulation and how this data can fuel the dialogue between theory and experiment.
Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly
Authors: Butt Benjamin G. , Fischer Daniela , Rep Alison R. , Schauflinger Martin , Read Clarissa , Böck Thomas , Hirner Manuel , Graham Stephen C. , von Einem Jens
Published: January 2024

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across β-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.
CDC5L surveils cellular stress responses and stress granule formation through transcriptional repression
Authors: Qian Beituo , Li Shunyi , Duan Yongjia , Qiu Feng , Hu Rirong , Yue Wenkai , Cui Jihong , Wang Qiangqiang , Li Wanjin , Fang Yanshan
Published: January 2024

Cells have evolved a variety of mechanisms to respond to stress, such as activating the PERK– eIF2α pathway and forming stress granules (SGs). It is important that these mechanisms are inducted only when necessary and exerted at appropriate levels, to prevent spontaneous or excessive activation of stress responses. However, the mechanisms by which cells keep the stress response programs in check are elusive. In this study, we discovered that downregulation ofCell Division Cycle 5 Like(CDC5L) causes spontaneous SG formation in the absence of any stress, which is independent of its known functions in the cell cycle or the PRP19 complex. Instead, we found that CDC5L binds to thePERKpromoter through its DNA-binding domains and repressesPERKmRNA transcription. As a result, it negatively regulates the abundance of PERK protein and the phosphorylation levels of eIF2α, thereby suppressing the PERK–eIF2α signaling pathway and preventing undesirable SG assembly. Further RNA-sequencing (seq) and chromatin immunoprecipitation (ChIP)-seq analyses reveal a dual function of CDC5L in gene transcription: it acts as a transcriptional activator in cell cycle control but as a repressor in cellular stress responses. Finally, we show that the loss ofCDC5Ldecreases cell viability and fly survival under mild stress conditions. Together, our findings demonstrate a previously unknown role and mechanism of CDC5L in the surveillance of cellular stress through transcriptional repression, which serves as a gatekeeper for the stress response programs such as the PERK–eIF2α pathway and SG formation.

Significance statement

Cells need to respond to stress promptly for survival. Meanwhile, it is equally important to prevent spontaneous or excessive activation of stress response programs when no stress or only minor stress is present. Here, we reveal that the DNA/RNA-binding protein CDC5L represses the transcription of a cluster of stress response genes includingPERK. In doing so, CDC5L suppresses the PERK-eIF2α pathway and prevents spontaneous SG assembly. Downregulation ofCDC5Lreleases the restraint on these genes, resulting in an exaggerated response to stress and decreased viability in both cell and fly models. Taken together, this study demonstrates the existence of a gatekeeper mechanism that surveils the stress response programs and highlights the crucial role of CDC5L-mediated transcriptional repression in this regulation.
Age differences in functional connectivity track dedifferentiation of category representations
Authors: Pauley Claire , Zeithamova Dagmar , Sander Myriam C.
Published: January 2024

With advancing age, the distinctiveness of neural representations of information declines. While the finding of this so-called ‘age-related neural dedifferentiation’ in category-selective neural regions is well-described, the contribution of age-related changes in network organization to dedifferentiation is unknown. Here, we asked whether age differences in a) whole-brain network segregation (i.e., network dedifferentiation) and b) functional connectivity to category-selective neural regions are related to regional dedifferentiation of categorical representations. Younger and older adults viewed blocks of face and house stimuli in the fMRI scanner. We found an age-related decline in neural distinctiveness for faces in the fusiform gyrus (FG) and for houses in the parahippocampal gyrus (PHG). Functional connectivity analyses revealed age-related dedifferentiation of global network structure as well as age differences in connectivity between the FG and early visual cortices. Interindividual correlations demonstrated that regional distinctiveness was related to network segregation. Together, our findings suggest that dedifferentiation of categorical representations may be linked to age-related reorganization of functional networks.

Highlights

Category representations are less distinctive, or dedifferentiated, in older adults

Here, we linked age differences in functional connectivity to dedifferentiation

Age-related declines in network segregation were linked to dedifferentiation
NeoCOMM: A Neocortical Neuroinspired Computational Model for the Reconstruction and Simulation of Epileptiform Events
Authors: Al Harrach Mariam , Yochum Maxime , Ruffini Giulio , Bartolomei Fabrice , Benquet Pascal , Wendling Fabrice
Published: January 2024

Understanding the pathophysiological dynamics which underline interictal epileptiform events (IEEs) such as epileptic spikes, spike-and-waves or High-frequency oscillations (HFOs) is of major importance in the context of neocortical refractory epilepsy, as it paves the way for the development of novel therapies. Typically, these events are detected in local field potential (LFP) recordings obtained through depth electrodes during pre-surgical investigations. Although essential, the underlying pathophysiological mechanisms for the generation of these epileptic neuromarkers remain unclear. The aim of this paper is to propose a novel neurophysiologically relevant reconstruction of the neocortical microcircuitry in the context of epilepsy. This reconstruction intends to facilitate the analysis of a comprehensive set of parameters encompassing physiological, morphological, and biophysical aspects that directly impact the generation and recording of different IEEs. Accordingly, a novel microscale computational model of an epileptic neocortical column was introduced. This model incorporates the intricate multilayered structure of the cortex and allows for the simulation of realistic interictal epileptic signals. The proposed model was validated through comparisons with real IEEs recorded using intracranial stereo-electroencephalography (SEEG) signals from both humans and animals. Using the model, the user can recreate epileptiform patterns observed in different species (human, rodent, and mouse) and study the intracellular activity associated with these patterns. Our model allowed us to unravel the relationship between glutamatergic and GABAergic synaptic transmission of the epileptic neural network and the type of generated IEE. Moreover, sensitivity analyses allowed for the exploration of the pathophysiological parameters responsible for the transitions between these events. Finally, the presented modeling framework also provides an Electrode Tissue Model (ETI) that adds realism to the simulated signals and offers the possibility of studying their sensitivity to the electrode characteristics. The model (NeoCoMM) presented in this work can be of great use in different applications since it offers anin silicoframework for sensitivity analysis and hypothesis testing. It can also be used as a starting point for more complex studies.
The intrinsically disordered N-terminus of SUMO1 is an intramolecular inhibitor of SUMO1 interactions
Authors: Richter Sebastian M. , Jin Fan , Ritterhoff Tobias , Fergin Aleksandra , Maurer Eric , Frank Andrea , Daube Michael , Hajnal Alex , Klevit Rachel , Gräter Frauke , Flotho Annette , Melchior Frauke
Published: January 2024

Ubiquitin-related proteins of the SUMO family are reversibly attached to thousands of proteins in eukaryotic cells. Many SUMO substrates, effectors and enzymes carry short motifs (SIMs) that mediate low affinity interactions with SUMO proteins. How specificity is achieved in target selection, SUMO paralogue choice and SUMO-dependent interactions is largely unknown. A unique but poorly understood feature of SUMO proteins is their intrinsically disordered N-terminus. We reveal a function for N-termini of human,C. elegans,and yeast SUMO proteins as intramolecular inhibitors of SUMO- SIM interactions. Mutational analyses, NMR spectroscopy, and Molecular Dynamics simulations indicate that SUMO’s N-terminus can inhibit SIM binding by fast and fuzzy interactions with SUMO‘s core. Deletion of theC. elegansSUMO1 N-terminus leads to p53-dependent apoptosis during germline development, indicating an important role in DNA damage repair. Our findings reveal a mechanism of disorder-based autoinhibition that contributes to the specificity of SUMOylation and SUMO-dependent interactions.
Behavioural Investigations of Psilocybin in Animals 1962-2021: A Scoping Review
Authors: Shore Ron , Dobson Kat , Thomson Nina , Barnim Nigel , Bergman Hailey , Rideout Katie , McKeown Sandra , Olmstead Mary C. , Goldie Craig , Dumont Eric
Published: January 2024

Background and Aims

Psilocybin is a psychedelic drug that may hold promise for a wide range of human health conditions, yet the identification of therapeutic processes and mechanisms of action remains exploratory. We conducted a scoping review on pre-clinical behavioural investigations of psilocybin in non-human animals to help determine the behavioural effects of psilocybin in non-human animals, to identify studies completed, behavioural tests employed, and what dosing modalities had been studied.

Methods

A librarian-conducted literature search was performed using predefined key terms and search criteria and additional searching was conducted by reviewers, using electronic databases, grey literature sources, and reference lists of relevant articles or reviews. The final search updated occurred in October, 2021. Studies were reviewed, screened and selected against an a priori protocol using Covidence software by multiple reviewers with results plotted across the Research Domains Criteria construct.

Results

From 4124 records identified by database searching, 260 publications were subjected to full-text review with 77 studies included in this scoping review, published between 1962-2021. The preponderance of studies (n=64) investigated behavioural outcomes in rodents. Only 43 studies (55.8%) reported on housing conditions, and seventeen studies (22.1%) failed to report sample size. All studies reported behavioural outcomes following drug administration, with fifty-one studies (66.2%) using psilocybin, thirty studies (42.9%) psilocin, four studies (5.2%) administering whole mushroom extracts (WME), and a further eight studies investigating both psilocybin and psilocin and one study reporting the effects of both psilocin and WME. One hundred and thirty distinct behavioural investigations using fifty different behavioral paradigms were identified. Few adverse events were reported, and even exceedingly high doses were apparently well tolerated.

Conclusion

With seventy-seven publications spanning close to sixty years, there is huge variation in study design and quality. Overall psilocybin presents a unique and strong safety profile with no evidence of biological toxicity, is characterized by unique time and dose-dependent effects, and its pattern of drug action is significantly context and training-sensitive. Data suggest putative effects of psilocybin include acute arousal, dose-dependent sedation, reductions in fear conditioning at low doses, reduced aggression, improved valence, acute disruption of working memory, the rescuing of deficits from chronic stress, and improved learning when combined with repeated environmental exposure after resolution of drug effect.
Revisiting soil fungal biomarkers and conversion factors: Interspecific variability in phospholipid fatty acids, ergosterol and rDNA copy numbers
Authors: Camenzind T , Haslwimmer H , Rillig MC , Ruess L , Finn DR , Tebbe CC , Hempel S , Marhan S
Published: January 2024

The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates, combined with available references, we propose average conversion factors of 95.3 g fungal C g−1ergosterol, 32.0 mg fungal C µmol−1PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful.
Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments
Authors: Begnis Martina , Duc Julien , Offner Sandra , Grun Delphine , Sheppard Shaoline , Rosspopoff Olga , Trono Didier
Published: January 2024

Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping 3D genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods – encompassing, among others, protocadherin and KZFP-encoding genes – with loss of repressive chromatin marks, altered 3D genome architecture andde novoCTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NADs organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.
Species Tree Topology Impacts the Inference of Ancient Whole-Genome Duplications Across the Angiosperm Phylogeny
Authors: McKibben Michael T. W. , Finch Geoffrey , Barker Michael S.
Published: January 2024

Premise

The history of angiosperms is marked by repeated rounds of ancient whole-genome duplications (WGDs). Here we use state of the art methods to provide an up-to-date view of the distribution of WGDs in the history of angiosperms that considers both the uncertainty introduced by inference methods and alternative phylogenetic hypotheses.

Methods

Transcriptomic and genomic data were used to infer and place WGDs across two hypothesized angiosperm phylogenies. Initial WGD hypotheses were made using rate corrections to the distribution of synonymous divergences (Ks) of paralogs and orthologs. WGD hypotheses were tested using syntenic inferences and Bayesian models of duplicate gene gain and loss across the phylogeny.

Key results

The number of ancient WGDs in the history of angiosperms (∼170) is largely similar across different inference methods, but there is often variation in the precise placement of WGDs on the phylogeny. Ksbased methods often yield alternative hypothesized WGD placements largely due to variation in substitution rates among lineages. Phylogenetic models of duplicate gene gain and loss are more robust to topological variation, allowing for post hoc testing of WGD hypotheses. However, errors in species tree inference can still produce spurious WGD hypotheses regardless of method used.

Conclusions

Here we show that different WGD inference methods largely agree on an average of 3.5 WGD in the history of angiosperm species. However, the precise placement of WGDs on the phylogeny is subject to the inference method and tree topology. As researchers continue to test hypotheses regarding the impacts ancient WGDs have on angiosperm evolution, it is important to consider the uncertainty of the phylogeny as well as WGD inference methods.