bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Hindbrain Motion in Chiari Malformation I Patients Measured Through 3D Amplified MRI (3D aMRI)
Authors: Abderezaei Javid , Pionteck Aymeric , Chuang Ya-Chen , Carrasquilla Alejandro , Bilgili Gizem , Lu Tse An , Terem Itamar , Scadeng Miriam , Fillingham Patrick , Morgenstern Peter , Levitt Michael , Ellenbogen Richard G. , Yang Yang , Holdsworth Samantha J. , Shrivastava Raj , Kurt Mehmet
Published: October 2022

Chiari Malformation type 1 (CM-I) is a neurological disorder characterized by morphological defects such as excessive cerebral tonsils herniation and vast associated symptomatology. Given that these structural defects cannot explain the underlying symptomatology, and might result in misdiagnosis, in this work, we studied the brain’s intrinsic motion to better understand the mechanisms of CM-I. We acquired 3D cine MRI of 14 healthy and 14 CM-I subjects and used 3D amplified MRI (3D aMRI) to visualize and measure the brain’s intrinsic motion during the cardiac cycle. We observed that the regional brain motion in CM-I was significantly higher than the healthy subjects, with anterior-posterior (AP) and superior-inferior (SI) displacements in cerebral tonsils and medulla having the highest differences between the healthy and CM-I (∽45% ∽73% increased motion in the CM-I group). The motion of the cerebellum, and brainstem in AP directions (∽42% and ∽31% increased motion in the CM-I group, respectively), followed by the motion of the cerebral tonsils and medulla in medial-lateral (ML) directions were other significant differences found between the two groups (∽16% increased motion in the CM-I group). Additionally, for the CM-I subjects, we measured morphological parameters including the tonsil herniation, ratio of neural tissue in the foramen magnum, and 4thventricle volume. We then used the morphometrics and brain’s intrinsic motion to analyze the symptomatology of the CM-I patients and their surgical outcomes. Interestingly, we found the ratio of neural tissue in the foramen to be directly correlated with the SI motion of the tonsils (r= 0.58). We also found the tonsil herniation to be directly correlated with the AP motion of the tonsils (r= 0.61), and AP and ML motions of the medulla (r= 0.66, andr= 0.57). Additionally, we found the ML motion of the tonsils to be the only indicator of the surgical outcome (AUC= 0.95), in which subjects with higher motion had an improved outcome. Although we did not observe a significant correlation between the brains motion and morphometrics on the CM-I symptoms due to our small sample size, illustrative cases increase our hope for the development of a future tool based on the brain biomechanics.
License to cut: Smart RNA guides for conditional control of CRISPR-Cas9
Authors: Baccouche Alexandre , Adel Arman , Yachie Nozomu , Fujii Teruo , Genot Anthony J.
Published: October 2022

The Cas9 enzyme is a programmable endonuclease, whose target sequence is directed by a companion RNA guide. Cas9 and RNA guides have revolutionized biology, enabling facile editing of the genome in almost all organisms. Controlling where and when Cas9 and the guide operate is indispensable for many fields ranging from developmental biology to therapeutics, but it remains a challenge. Most methods focus on controlling Cas9 with physico-chemical means (which lack finesse, precision or multiplexing), or transcriptional tools (which are slow and difficult to design). Rather than directly engineering Cas9, engineering the RNA guide itself has emerged as a more general and potent way to manage the activity of Cas9. Here we report smart RNA guides that are conditionally activated by the presence of a specific RNA opener. Contrary to most previous approaches, the design affords ample freedom as spacer and the opener are independent. We demonstrate this flexibility by operating SmartGuides activated by a panel of miRNA relevant for human health, and by composing SmartGuides in Boolean logic circuits. Lastly, we test the SmartGuides in mammalian cells - validating the basics tenets of the design, but also highlighting the challenges that remain to be lifted for in-vivo operation.
Cauliflower mosaic virusprotein P6 forms a microenvironment for RNA granule proteins and interferes with stress granule responses
Authors: Hoffmann Gesa , Lopéz-Gonzaléz Silvia , Mahboubi Amir , Hanson Johannes , Hafrén Anders
Published: October 2022

Biomolecular condensation is a multipurpose cellular process that viruses use ubiquitously in their multiplication. CaMV replication complexes are condensates that differ from most viruses in being non-membranous assemblies and consist of RNA and protein, mainly viral protein P6. Despite description of these viral factories already half a century ago with many observations that followed since, functional details of the condensation process, their properties and relevance has remained enigmatic. Our main findings include a large dynamic mobility range of host proteins within viral factories, while the viral matrix protein P6 is immobile in accordance with representing the central node of these condensates. As novel components of VFs we identify stress granule (SG) nucleating factors G3BP7 and the UBP1 family. Similarly, as SG components localize in VFs during infection, ectopic P6 localizes to SGs and reduces their assembly after stress. Intriguingly, it appears that soluble rather than condensed P6 suppresses SGs and mediates also other essential P6 functions, suggesting that the increased condensation over the infection time-course may accompany a progressive shift in selected P6 functions. Together, this study highlights VFs as dynamic condensates and P6 as a complex modulator of SG responses.
Prefrontal control of actions in freely moving macaques
Authors: Voloh Benjamin , Maisson David , Cervera Roberto Lopez , Conover Indirah , Zambre Mrunal , Hayden Benjamin , Zimmermann Jan
Published: October 2022

Our natural behavioral repertoires include complex coordinated actions of characteristic types. To better understand the organization of action and its neural underpinnings, we examined behavior and neural activity in rhesus macaques performing a freely moving foraging task in an open environment. We developed a novel analysis pipeline that can identify meaningful units of behavior, corresponding to recognizable actions such as sitting, walking, jumping, and climbing. On the basis of action transition probabilities, we found that behavior was organized in a modular and hierarchical fashion. We found that, after regressing out many potential confounders, actions are associated with specific patterns of firing in each of six prefrontal brain regions and that, overall, representation of actions is progressively stronger in more dorsal and more caudal prefrontal regions. Conversely, we found that switching between actions resulted in changed firing rates, with more rostral and more ventral regions showing stronger effects. Together, these results establish a link between control of action state and neuronal activity in prefrontal regions in the primate brain.
Antibody-lectin chimeras for glyco-immune checkpoint blockade
Authors: Stark Jessica C. , Gray Melissa A. , Wisnovsky Simon , Ibarlucea-Benitez Itziar , Lustig Marta , Riley Nicholas M. , Ribi Mikaela K. , Errington Wesley J. , Bruncsics Bence , Sarkar Casim A. , Valerius Thomas , Ravetch Jeffrey V. , Bertozzi Carolyn R.
Published: October 2022

Despite the curative potential of checkpoint blockade immunotherapy, most patients remain unresponsive to existing treatments. Glyco-immune checkpoints – interactions of cell-surface glycans with lectin, or glycan-binding, immunoreceptors – have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer. Here, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a cell-targeting antibody domain and a lectin “decoy receptor” domain that directly binds glycans and blocks their ability to engage inhibitory lectin receptors. AbLecs potentiate anticancer immune responses including phagocytosis and cytotoxicity, outperforming most existing therapies and combinations tested. By targeting a distinct axis of immunological regulation, AbLecs synergize with blockade of established immune checkpoints. AbLecs can be readily designed to target numerous tumor and immune cell subsets as well as glyco-immune checkpoints, and therefore represent a new modality for cancer immunotherapy.
CRF receptor type 1 modulates the nigrostriatal dopamine projection and facilitates cognitive flexibility after acute and chronic stress
Authors: Becchi S , Burton CL , Tsoukalas M , Bowring J , Balleine BW , Mor D
Published: October 2022

Chronic unpredictable stress (CUS) impairs cognitive flexibility in rats, particularly when faced with additional mild acute stress (AS). We tested the hypothesis that this impairment is associated with alterations in dopamine activity in the dorsal striatum driven by corticotropin-releasing-factor receptor type 1 (CRFR1) in the substantia nigra pars compacta (SNpc). In experiment 1, rats received CUS or handling for 14 days, before learning two action-outcome associations (lever presses and food rewards). Learning was assessed using outcome devaluation. Cognitive flexibility was then assessed by reversing the outcome identities followed by a second outcome devaluation test, with half of the rats in each group receiving AS prior to reversal training. Dopamine and its metabolite were quantified in the dorsal striatum and CRFR1 mRNA was quantified in the SNpc. Increased dopaminergic activity in the left dorsal striatum and CRFR1 expression in the left SNpc were associated with resilience to AS in naïve rats but with impairment in CUS+AS rats, suggesting a transition in hemispheric control from left to right as a protective mechanism following CUS. This suggestion was tested in experiment 2, where SNpc CRFR1 was blocked unilaterally prior to AS and reversal training. Blocking CRFR1 in the left medial SNpc impaired cognitive flexibility following AS in naïve rats but restored it in CUS rats. Blocking CRFR1 in the left, but not right, lateral SNpc also impaired cognitive flexibility following AS in naïve rats but had no effect in CUS rats.
On an Analogy Between Ephaptic Coupling in Large Tracts and Cross-Modal Integration During Illusory Percept
Authors: Chawla Aman , Morgera Salvatore Domenic
Published: October 2022

In this paper, the authors report simulations analogous to cross-modal integration which can be modeled by thinking of large (in terms of numbers of axons) axon tracts with distinctly demarcated subsets of axons. In such a tract, when the demarcated regions are separately stimulated they fire in response to the stimulation. Due to extracellular current-based coupling, at a later time, unstimulated axons also begin to cross-talk. The simulation graphical result, though on a faster time scale, is analogous to the multi-channel MEG recordings of “AV-(A+V)” obtained by Shams et. al. When the coupling parameter is weakened, this cross-talk disappears. In Shams et. al., the appearance and disappearance of cross-talk are related to the perception of an illusory flash by the subject. Our extrapolated conclusion is that ephaptic coupling might be a part of the mechanism for cross-talk between brain regions mediating, say, audio and visual-processing. We also study noisy ephaptic coupling and additionally do a comparative study with the Kuramoto model, which yields insight into the exact and detailed mechanism of ephaptic synchronization.
Endothelial cells release microvesicles that harbor multivesicular bodies and secrete exosomes
Authors: Petersen Jennifer D. , Mekhedov Elena , Kaur Sukhbir , Roberts David D. , Zimmerberg Joshua
Published: October 2022

Extracellular vesicles (EVs) released by resting endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, we examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy. Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface. The protrusions contained membrane-bound organelles, including multivesicular bodies (MVBs), and appeared to be on the verge of pinching off to form microvesicles. Beyond cell peripheries, membrane-bound vesicles with internal MVBs were observed, and serial sections confirmed that they were not connected to cells. These observations are consistent with the notion that these multi-compartmented microvesicles (MCMVs) pinch-off from protrusions. Remarkably, omega figures formed by fusion of MVBs with the MCMV limiting membrane were directly observed, apparently caught in the act of releasing exosomes from the MCMV. In summary, MCMVs are a novel form of EV that bud from membrane protrusions on the HUVEC surface, contain MVBs and release exosomes. These observations suggest that exosomes can be harbored within and released from transiting microvesicles after departure from the parent cell, constituting a new site of exosome biogenesis occurring from endothelial and potentially additional cell types.
Spatially coordinated collective phosphorylation filters spatiotemporal noises for precise circadian timekeeping
Authors: Chae Seok Joo , Kim Dae Wook , Lee Seunggyu , Kim Jae Kyoung
Published: October 2022

The circadian (∼24h) clock is based on a negative feedback loop centered around the PERIOD protein (PER), translated in the cytoplasm and then enters the nucleus to repress its own transcription at the right time of day. Such precise nucleus entry is mysterious because thousands of PER molecules transit through crowded cytoplasm and arrive at the perinucleus across several hours. To understand this, we developed a mathematical model describing the complex spatiotemporal dynamics of PER as a single random time delay. We find that the spatially coordinated bistable phosphoswitch of PER, which triggers the phosphorylation of accumulated PER at the perinucleus, leads to the synchronous and precise nuclear entry of PER. This leads to robust circadian rhythms even when PER arrival times are heterogenous and perturbed due to changes in cell crowdedness, cell size, and transcriptional activator levels. This shows how the circadian clock compensates for spatiotemporal noise.

Graphical Abstract

Highlights

The time window when PER protein arrives at the perinucleus is wide and keeps changing.

A bistable phosphoswitch enables precise nuclear entry of PER protein.

This leads to robust circadian rhythms when cell congestion level and size change.

This describes how the circadian clock compensates for spatiotemporal noise.
Translesion DNA synthesis polymerase κ is essential to a carcinogen-induced nucleolar stress response
Authors: Paul Shilpi , Rebok Abbey , Cifani Paolo , Paul Anirban , Pappin Darryl , Huang Tony T. , Spratt Thomas E.
Published: October 2022

DNA polymerase kappa (Polκ) has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences and nucleotide excision repair. However, the mechanisms regulating Polκ’s cellular activities are unknown. Since all polymerases insert the canonical deoxynucleotide triphosphates, it is difficult to determine which polymerase is active at a particular genomic site. To counter this difficulty, we utilized the selective Polκ substrate,N2-(4-ethynylbenyl)-2’-deoxyguanosine (EBndG), as bait to capture proteins associated with Polκ synthesized DNA. Here we show that, Polκ is active in the nucleolus and Polκ synthesized DNA are enriched with nucleolar proteins. Exposure of cells to benzo[a]pyrene diol epoxide (BPDE) induced hallmarks of nucleolar stress, increased Polκ stability and nucleolar activity. Other agents that induce nucleolar stress such as mitomycin C, cisplatin and actinomycin D also increased Polκ’s nucleolar activity. The Polκ activity was cell-cycle independent and dependent on PCNA ubiquitination. In addition, we identified that the expression and activity of Polκ is regulated by the polycomb complex protein Ring Finger Protein 2 (RNF2) and by poly(ADP)-ribose polymerase 1 (PARP1) catalyzed PARylation. This study provides insight into the novel role of Polκ in ribosomal RNA synthesis, in maintaining ribosomal DNA integrity after DNA damage thus protecting the cells from nucleolar stress.
Consistent differences in eggshell phenotypes select for bluer eggs in an avian host-parasite system
Authors: Villa Juliana , Wisocki Phillip , Dela Cruz Jacob E. , Hanley Daniel
Published: October 2022

Avian brood parasitism is a well-recognized model for studying coevolution. In this model, hosts adapt the ability to recognize and remove parasitic young to avoid the costs of rearing foreign offspring, while parasites counter-adapt phenotypes to evade detection. A classic example of host-parasite coevolutionary arms race is the great reed warbler and its parasite the common cuckoo (hereafter ‘warbler’ and ‘cuckoo’, respectively). Recent work has found that cuckoo eggs are more likely to be accepted by warblers when those are bluer than their own eggs. Therefore, hosts would select for bluer cuckoo eggs; in turn, this provides directional selection for hosts eggs to remain bluer than cuckoo eggs. Here, we tested whether there were consistent differences in eggshell appearance between cuckoo and warbler eggs using a dataset which provides eggshell coloration of both warblers and cuckoos from the same nest. Despite being a textbook example of mimicry, we found that the cuckoos have significantly browner eggs than the warblers. These findings most likely suggest that this warbler-cuckoo system is experiencing negative frequency-dependent selection, as expected under red queen dynamics. Future research comparing warbler-cuckoo interactions over time would advance our understanding of the coevolution between avian brood parasites and their hosts.
Direct activation of the Ras-Akt network mediates polarity and organizes protrusions in human neutrophil migration
Authors: Pal Dhiman Sankar , Banerjee Tatsat , Lin Yiyan , Borleis Jane , Devreotes Peter N.
Published: October 2022

Functions of Ras oncogenes and their downstream effectors are typically associated with cell proliferation and growth control while their role in immune cell migration has been largely unexplored. Although Ras-mediated signaling cascades have been implicated in immune response, there is no conclusive evidence to show local activation of these pathways on the plasma membrane directly regulates cell motility or polarity. Using spatiotemporally precise, cryptochrome-based optogenetic systems in human neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant-sensing receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or the guanine nucleotide exchange factor, RasGRP4, immediately increased spreading and random motility in neutrophils. Second, creating Ras activity at the cell rear generated new protrusions at the site and reversed pre-existing polarity, similar to the effects of steep chemoattractant gradients. Third, recruiting GTPase activating protein, RASAL3, at cell fronts abrogated existing protrusions and changed the direction of motility whereas dynamically inhibiting nascent fronts stopped migration completely. Fourth, combining pharmacological inhibition studies with optogenetics revealed that mTorC2 is more important than PI3K for Ras-mediated polarity and migration. Finally, local recruitment of Ras-mTorC2 effector, Akt, also generated new protrusions, rearranged pre-existing polarity, and triggered migration, even in absence of PI3K signaling. We propose that actin assembly, cell shape, and migration modes in immune cells are promptly controlled by rapid, local activities of established components of classical growth-control pathways independently of receptor activation.
Gut microbiota assemblages of generalist predators are driven by local- and landscape-scale factors
Authors: Saqib Hafiz Sohaib Ahmed , Sun Linyang , Pozsgai Gabor , Liang Pingping , Goraya Mohsan Ullah , Akutse Komivi Senyo , You Minsheng , Gurr Geoff M. , You Shijun
Published: October 2022

The gut microbiomes of arthropods are reported to have significant impact on key physiological functions such as nutrition, reproduction, behavior, and health. Spiders are diverse and numerically dominant predators in crop fields where they are potentially important regulators of pests. The taxonomic structure of spider gut microbiomes, and environmental drivers of composition are unknown. Harnessing spiders to support agricultural productivity is likely to be supported by an understanding of the gut microbiomes of these predators. This study aimed to deciphering the gut microbiome assembly of predators as well as elucidating the potential implications of key environmental constraints in this process. Here, we used high-throughput sequencing to examine for the first time how the assemblages of bacteria in the gut of spiders are shaped by diverse environmental variables. A total of 27 bacterial phyla were detected with Proteobacteria and Firmicutes dominant. The core bacterial communities included the families Enterobacteriaceae, Chloroplast, Lactobacillaceae, Pseudomonadaceae, Lachnospiraceae, Leuconostocaceae and Ruminococcaceae. Local drivers of microbiome composition were the globally-relevant input use system (organic production versus conventional practice), and crop identity (Chinese cabbage versus cauliflower). Landscape-scale factors, proportion of forest and grassland, compositional diversity, and habitat edge density, also strongly affected gut microbiota. Specific bacterial taxa were enriched in the gut of spiders sampled from different settings and seasons. These findings provide a comprehensive insight into the composition and plasticity of spider gut microbiota. Understanding the temporal responses of specific microbiota could lead to innovative strategies development for boosting biological control services of predators.
MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via Protein Kinase C
Authors: Segui-Perez Celia , Stapels Daphne A.C. , Ma Ziliang , Su Jinyi , Passchier Elsemieke , Westendorp Bart , Wu Wei , van Putten Jos P.M. , Strijbis Karin
Published: October 2022

Regulation and adaptation of intestinal epithelial barrier function is essential for human health. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in intestinal epithelial monolayers MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not result in increased TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC, which is in line with a predicted PKC motif in the MUC13 cytoplasmic tail. The responsible PKC member might be PKCδ based on elevated protein levels in the absence of MUC13. Our results identify MUC13 as a central player in TJ complex stability and intestinal barrier permeability.
Lipid homeostasis is essential for a maximal ER stress response
Authors: Garcia Gilberto , Zhang Hanlin , Moreno Sophia , Kimberly Tsui C. , Webster Brant Michael , Higuchi-Sanabria Ryo , Dillin Andrew
Published: October 2022

Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identitylet-767, a putative hydroxysteroid dehydrogenase, as an essential gene for both lipid and ER protein homeostasis. Knockdown oflet-767reduces lipid stores, alters ER morphology in a lipid-dependent manner, and also blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRERinduction in animals with reducedlet-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream oflet-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell’s response to protein-induced stress.
Carryover effects of tire wear particle leachate threats reproduction across multiple generations
Authors: Chai Yanchao , Wang Haiqing , Lv Mengru , Yang Jiaxin
Published: October 2022

Toxic additives leached from tire wear particle (TWP) have been linked to some collective death events of fish, also impose impacts on zooplankton as secondary consumer in aquatic food web. In addition to direct impacts of TWP leachate at the current generation, potential delayed carryover from past exposure across multi-generational lineage may augment impacts on individual reproduction, then population maintenance. We investigated the carryover effects from persistent exposure and past TWP leachate exposure along generation passaging on the individual reproduction of rotifer,Brachionus calyciflorus, a typical zooplankton. For rotifer treated with TWP leachate across continuous 7 generations, their offspring were divided into parental exposure or no exposure in each generation. And rotifer transferred into no exposure were maintained for 3 generations to eliminate indirect exposure through their parental germ. The similar response of reproduction, via carryover effects from parental exposure, also emerged in offspring without exposure. Persistent exposure across multiple generations additively impaired individual reproduction performance by transferring from its hermetic effects in original generation, even caused final population collapse. TWP leachate could impose cascading toxicity on population persistence of zooplankton via carryover and cumulative effects on reproduction for long term, which must be considered in risk assessment and management policy to alleviate the effects of TWP.
Tell me who your neighbors are: The role of spatial location and tree species identity in determining the ectomycorrhizal community composition of saplings and mature trees in a mixed conifer forest
Authors: Livne-Luzon Stav , Avidar Mor , Herol Lior , Rog Ido , Klein Tamir , Shemesh Hagai
Published: October 2022

The mutualistic interaction between trees and ectomycorrhizal fungi (EMF) can have a major effect on forest dynamics and specifically in seedling establishment. Both intrinsic (i.e., identity of the sapling) and extrinsic (i.e., the identity of mature trees in the vicinity of the sapling) factors can affect the EMF community composition of young saplings.

Here, we compared the EMF community composition associated with the roots of young saplings and mature trees of two co-habiting Pinaceae:Pinus halepensisandCedrus deodaragrowing together in a planted forest plot, using fungal ITS metabarcoding.

We found that the differences between the two sapling groups were mostly attributed to changes in the relative abundance of specific fungal species. Moreover, we found that physical proximity to a specific host species had a significant effect on the community composition of young saplings. However, while no significant differences in sapling size were apparent, the sapling shoot structure was affected by the identity of the nearest mature tree and its unique EMF community composition.

Synthesis: These results suggest that the dynamics of the EMF community are greatly determined by extrinsic factors such as the small-scale distribution of mature trees in the forest, with possible cascading effects on the development of young trees.
RIG-I activation primes and trains innate antiviral immune memory
Authors: Adamson Maike S , Nesic Svetozar , Buness Andreas , Bayrak Kübra , Schmitz Saskia , Soler Sofía , Zillinger Thomas , Marx Samira , Lambing Silke , Andryka-Cegielski Katarzyna , Khan Fawad , Schlee-Guimarães Thais M. , Herberhold Stephan , Proietti Michele , Placek Katarzyna , Reading Patrick , Cicin-Sain Luca , Schlee Martin , van den Boorn Jasper , Hartmann Evelyn , Hartmann Gunther , Bartok Eva
Published: October 2022

Adaptive processes of the innate immune system, known as trained immunity (TI), are critical to human health and disease, yet they have not been systematically investigated downstream of antiviral sensing. Here, we elucidate the potential of the antiviral cytosolic RNA receptor retinoic acid-inducible gene I (RIG-I) to train, prime and tolerize the innate immune system. Using a specific RIG-I agonist, we observed that repetitive stimulation enhanced interferon-stimulated gene (ISG) and pro-inflammatory cytokine induction in human primary monocytes, epithelial cells and fibroblasts and afforded non-specific antiviral protection. RNA sequencing revealed broad, cell type-specific transcriptional changes, indicative of priming of ISGs and training of the NFκB pathway, without measurable tolerization, while ATAC sequencing in monocytes demonstrated chromatin remodeling and enhanced accessibility of key transcription factor-binding motifs such as STAT1. Moreover, while STAT1 signaling was critically required, it was not sufficient to recapitulate RIG-I induced TI. Altogether, our data demonstrate that RIG-I-mediated TI promotes an immunologically alert state with important implications for host defense and the application of RIG-I ligands in anti-infective and anti-tumoral therapies.

One Sentence Summary

RIG-I activation trains and primes innate immune response at the cellular level, affording non-specific immune protection by immune and non-immune cells.
Changes in protein dynamical parameters derived from quasi-elastic neutron scattering spectra induced by coherent scattering: A molecular dynamics simulation study
Authors: Matsuo Tatsuhito
Published: October 2022

Protein dynamics at the sub-nanosecond timescale and the Å length-scale has widely been studied using quasi-elastic neutron scattering (QENS). In almost all QENS studies on hydrogenated proteins in D2O buffers, analysis of the spectra after buffer subtraction is carried out under the assumption that the remaining spectra arise from incoherent scattering of proteins while the contribution of coherent scattering is negligible. On the contrary, a study using polarization analysis has shown that the coherent scattering accounts for more than 10% of the total scattering intensity of hydrogenated proteins (Gaspar et al., Biochim. Biophys. Acta 1804:76–82 (2010)). In addition, the effects of coherent scattering on the values of dynamical parameters of proteins obtained by analysis of QENS spectra remain unclear. Here, molecular dynamics (MD) simulation on hen egg white lysozyme was used to investigate this issue. QENS spectra containing only incoherent scattering and those containing both incoherent and coherent scattering were calculated from the MD trajectory. Dynamical parameters were then extracted from the two simulated QENS spectra. Comparison of the resultant dynamical parameters has shown that the error in the values of the dynamical parameters induced by coherent scattering is at most 6%. This error is unlikely to significantly affect the results of QENS studies that investigate the relative changes in protein dynamics caused by different physicochemical conditions such as temperature unless dynamical parameters need to be determined with high precision at the absolute scale.
Randomly incorporated genomic 6mA delays zygotic transcription initiation
Authors: Febrimarsa  , Gornik Sebastian G , Barreira Sofia N , Salinas-Saavedra Miguel , Schnitzler Christine E , Baxevanis Andreas D , Frank Uri
Published: October 2022

N6-methyldeoxyadenosine (6mA) is a chemical alteration of DNA, observed across all realms of life. The functions of 6mA are well understood in bacteria but its roles in animal genomes have been controversial. We show that 6mA randomly accumulates in early embryos of the cnidarianHydractinia symbiolongicarpus, with a peak at the 16-cell stage followed by clearance to background levels two cell cycles later, at the 64-cell stage – the embryonic stage at which zygotic genome activation occurs in this animal. Knocking downAlkbh1, a putative initiator of animal 6mA clearance, resulted in higher levels of 6mA at the 64-cell stage and a delay in the commencement of zygotic transcription. Our data are consistent with 6mA originating from recycled nucleotides of degraded m6A-marked maternal RNA post-fertilization. Therefore, while 6mA does not function as an epigenetic mark inHydractinia, its random incorporation into the early embryonic genome inhibits transcription. Alkbh1 functions as a genomic 6mA ‘cleaner’, facilitating timely zygotic genome activation. Given the random nature of genomic 6mA accumulation and its ability to interfere with gene expression, defects in 6mA clearance may represent a hitherto unknown cause of various pathologies.
Benchmarking solutions to the T-cell receptor epitope prediction problem: IMMREP22 workshop report
Authors: Meysman Pieter , Barton Justin , Bravi Barbara , Cohen-Lavi Liel , Karnaukhov Vadim , Lilleskov Elias , Montemurro Alessandro , Nielsen Morten , Mora Thierry , Pereira Paul , Postovskaya Anna , Martínez María Rodríguez , Fernandez-de-Cossio-Diaz Jorge , Vujkovic Alexandra , Walczak Aleksandra M. , Weber Anna , Yin Rose , Eugster Anne , Sharma Virag
Published: October 2022

Many different solutions to predicting the cognate epitope target of a T-cell receptor (TCR) have been proposed. However several questions on the advantages and disadvantages of these different approaches remain unresolved, as most methods have only been evaluated within the context of their initial publications and data sets. Here, we report the findings of the first public TCR-epitope prediction benchmark performed on 23 prediction models in the context of the ImmRep 2022 TCR-epitope specificity workshop. This benchmark revealed that the use of paired-chain alpha-beta, as well as CDR1/2 or V/J information, when available, improves classification obtained with CDR3 data, independent of the underlying approach. In addition, we found that straight-forward distance-based approaches can achieve a respectable performance when compared to more complex machine-learning models. Finally, we highlight the need for a truly independent follow-up benchmark and provide recommendations for the design of such a next benchmark.
An exploratory study of a simple approach for evaluating drug solubility in milk related vehicles
Authors: Li Sean , Gabriel Justin J. , Martinez Marilyn N. , Longstaff David G. , Coffey Martin J. , Zhao Fang
Published: October 2022

Milk related materials are frequently used as a vehicle for drug product administration. Therefore, drug solubility information in milk related vehicles is desirable for prediction of how they may influencein vivodrug release and bioavailability. However, there are very limited data published on this topic. This study explored a practical method to address the key challenges associated with solubility assessment in milk, including the sample equilibration time and cleanup procedures. Amitriptyline, acetaminophen, dexamethasone, nifedipine, piroxicam, and prednisolone were selected as model drugs to represent a wide range of physicochemical properties. Their solubilities were determined at room temperature in pH 6.8 phosphate buffer, skim milk, whole milk, reconstituted whole milk powder, and unprocessed raw milk. The overall results confirmed that milk greatly improves the solubility of poorly water-soluble drugs. However, the extent of improvement and mechanism of solubilization appeared unique for each drug, highlighting the importance of evaluating milk solubility experimentally. The method used in this exploratory study can be applied in future investigations of a broader range of drugs and milk-related vehicles.
Optogenetic closed-loop feedback control of the unfolded protein response optimizes protein production
Authors: Benisch Moritz , Benzinger Dirk , Kumar Sant , Hu Hanrong , Khammash Mustafa
Published: October 2022

In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show thatin silicoclosed-loop optogenetic feedback control of the unfolded protein response (UPR) clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of a hard-to-fold protein based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.
Chromosome-scale genome assemblies of five differentBrassica oleraceamorphotypes provide insights in intraspecific diversification
Authors: Cai Chengcheng , Bucher Johan , Finkers Richard , Bonnema Guusje
Published: October 2022

Brassica oleraceais an economically important vegetable and fodder crop species that includes many morphotypes exhibiting enormous phenotypic variations. Previously, a pan-genome study based on short reads mapping approach has shown extensive structural variations betweenB. oleraceamorphotypes. Here, to capture more complete genome sequences ofB. oleracea, we report new chromosome-scale genome assemblies for five different morphotypes, namely broccoli, cauliflower, kale, kohlrabi and white cabbage, which were created by combining long-read sequencing data and Bionano DLS optical maps. The five assemblies are the most continuous and completeB. oleraceagenomes to date (contig N50 > 10 Mb). Comparative analysis revealed both highly syntenic relationships and extensive structural variants among the five genomes. Dispensable and specific gene clusters accounted for ~38.19% of total gene clusters based on a pan-genome analysis including our five newly assembled genomes and four previously reported genomes. Using the pan-genome ofB. oleraceaandB. rapa, we revealed their different evolutionary dynamics of LTR-RTs. Furthermore, we inferred the ancestral genome ofB. oleraceaand the common ancestral genome ofB. oleraceaandB. rapavia a pan-genome approach. We observed faster WGT-derived gene loss inB. rapathan inB. oleraceabefore intraspecific diversification. We also revealed continuing gene loss bias during intraspecific diversification of the two species and a strong bias towards losing only one copy among the three paralogous genes. This study provides valuable genomic resources forB. oleraceaimprovement and insights towards understanding genome evolution during the intraspecific diversification ofB. oleraceaandB. rapa.
Repurposing the CRISPR-Cas9 System for Targeted Chromatin O-linked β-N-acetylglucosamine Editing
Authors: Parker Matthew P. , Dias Wagner B. , Brautman Will , Lowe Nick , Fedosyuk Halyna , Peterson Kenneth R. , Slawson Chad
Published: October 2022

Eukaryotic gene transcription is controlled by many proteins, including the basal transcription machinery, epigenetic chromatin remodeling complexes, and transcription cofactors. Chromatin and genome-mapping consortia identifiedO-linked β-N-acetylglucosamine (O-GlcNAc) as an abundant chromatin post-translational modification involved in numerous transcriptional processes, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. Thus, O-GlcNAc regulation ofcis-regulatory elements is essential for proper gene expression. O-GlcNAc is a single N-acetylglucosamine sugar attached to serine or threonine residues in nuclear, cytoplasmic, or mitochondrial proteins. Two enzymes cycle O-GlcNAc on or off protein; O-GlcNAc transferase (OGT) adds the modification, and O-GlcNAcase (OGA) removes it. O-GlcNAcylation responds to inputs from multiple metabolic and stress pathways including glucose, amino acid, fatty acid, and nucleotide metabolism. Therefore, O-GlcNAc acts as a sensor of cellular homeostasis able to link environmental conditions with gene transcription; however, decoding the precise function of millions of O-GlcNAc regulated elements remains challenging. Technologies to readily manipulate O-GlcNAcylation at specificcis-regulatory elements for functional analysis without pleiotropic consequences are lacking. We have employed novel CRISPR-based gene targeting tools to probe the function of O-GlcNAc regulatedcis-elements. First, we developed a programmable CRISPR-Cas9-based targeting system. This was accomplished by fusing a catalytically dead Cas9 (dCas9) to O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA), which allows for highly specific O-GlcNAc manipulation at chromatincis-regulatory elements. Previously, we demonstrated that O-GlcNAc plays a role in regulating humanAγ-globin gene expression by regulating CHD4 function and the formation of the NuRD (Nucleosome Remodeling and Deacetylase) complex at the -566 GATA repressor-binding site. Thus, as a proof of principle and to further explore the function of O-GlcNAc inγ-globin gene transcription, we targeted both dCas9-OGT and -OGA fusion proteins to theAγ-globin gene promoter. When dCas9-OGT or dCas9-OGA was targeted to the -566 GATA silencer site of theAγ-globin promoter, gene expression decreased or increased, respectively. This data strongly correlates with our previous findings and implicates O-GlcNAc cycling inγ-globin gene regulation. Importantly, this method can be employed to investigate O-GlcNAc events known to exist within the eukaryotic genome in a highly specific manner. Together, this tool will be fundamental in elucidating the function of O-GlcNAc in gene transcription.
Cell-specific expression of key mitochondrial enzymes precludes OXPHOS in astrocytes of the adult human neocortex and hippocampal formation
Authors: Dobolyi Arpad , Bago Attila G , Stepanova Anna , Paal Krisztina , Lee Jeonghyoun , Palkovits Miklos , Chinopoulos Christos
Published: October 2022

The astrocyte-to-neuron lactate shuttle hypothesis entails that glycolytically derived pyruvate in astrocytes is converted to lactate instead of being catabolized in mitochondria. The mechanism of this metabolic rewiring is unclear. Here we show that astrocytes of the adult human neocortex and hippocampal formation do not express mitochondrial proteins critical for performing oxidative phosphorylation (OXPHOS) to a detectable degree, including cytochrome c and complex IV. Without OXPHOS, human brain astrocytes are bound to produce lactate to avoid interruption of glycolysis.
The circadian clock conveys thermal and photoperiodic cues to modulate EYES ABSENT via the neuropeptide PDF to regulate seasonal physiology
Authors: Hidalgo Sergio , Anguiano Maribel , Tabuloc Christine A. , Chiu Joanna C.
Published: October 2022

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain are poorly understood. We previously reported that EYES ABSENT (EYA) inDrosophilashows higher levels in cold temperature or short photoperiod, and genetic ablation ofeyain the fly brain inhibits reproductive dormancy, suggesting that EYA promotes winter physiology. Nevertheless, the mechanisms by which EYA senses seasonal cues are unclear. Pigment-Dispersing Factor (PDF) is a neuropeptide important for photoentrainment and regulation of circadian output rhythms. Interestingly, PDF also regulates reproductive dormancy, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of PDF signaling in mediating the impact of EYA on seasonal biology. First, we subjected flies to different photoperiodic and temperature regimes and observed that PDF abundance is lower in cold and short days, compared to warm and long days. Interestingly, the response of PDF to seasonal cues is opposite of what was observed for EYA. We then determined the potential for PDF to convey seasonal cues and modulate EYA function in seasonality by assessing coexpression of EYA and PDF receptor. Our results indicated that PDF receptor (PDFR) is indeed coexpressed with EYA in the fly brain, including in the circadian clock neuronal network and neurons in thepars intercerebralis. We then manipulated PDF signaling ineya+ cells to show that PDF modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and independent mechanisms. At the molecular level, manipulating PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA and promotes its degradation. This explains the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.
Equalizing epigenetically imprinted centromeres in early mammalian embryos
Authors: Manske Gabriel , Jorgensen Kelsey , Ma Binbin , Aboelenain Mansour , Tower Catherine , Chakraborty Saikat , Ranjan Rajesh , Das Arunika , Lampson Michael A. , Black Ben E. , Schindler Karen , Chen Xin , Hammoud Saher Sue
Published: October 2022

The CENP-A histone variant epigenetically defines centromeres, where its levels and locations are precisely maintained through mitotic cell divisions. However, differences in centromere CENP-A propagation in soma versus female/male germline remains poorly understood. Here, we generated CenpamScarletmice and followed CENP-A dynamics in gametes, zygotes, and embryos. We found that, unlike somatic cells, progenitor female and male germ cells carry high centromeric CENP-A levels that decrease upon terminal differentiation. The reduction in CENP-A is differentially regulated between sexes, resulting in a ten-fold higher level in oocytes compared to sperm. In the zygote, the parent-of-origin CENP-A asymmetry is equalized prior to initial S-phase by redistribution of nuclear CENP-A from maternal to paternal chromosomes. Redistribution of CENP-A requires both CDK1/2 and PLK1 centromeric machinery. These experiments provide direct evidence for resetting of epigenetically imprinted centromeres in early pronuclear stage embryos and imply a mechanism to sense the non-equivalency of parental chromosomes.

Highlights

Increased CENP-A density at centromeres is a conserved property of germline stem cells while CENP-A reduction is coincident with germ cell differentiation

Paternal and maternal CENP-A containing nucleosomes are intergenerationally inherited

CENP-A density at centromeres differs between male and female mature gametes

Upon fertilization, maternal nuclear CENP-A is redistributed to equalize with parental CENP-A

CENP-C and MIS18BP1 are asymmetrically enriched in the parental pronuclei in accordance with CENP-A asymmetry.

Licensing for centromere equalization begins before zygotic DNA replication