-
BioRxiv : the Preprint Server For... Jun 2024Matching arousal level to the motor activity of an animal is important for efficiently allocating cognitive resources and metabolic supply in response to behavioral...
Matching arousal level to the motor activity of an animal is important for efficiently allocating cognitive resources and metabolic supply in response to behavioral demands, but how the brain coordinates changes in arousal and wakefulness in response to motor activity remains an unclear phenomenon. We hypothesized that the locus coeruleus (LC), as the primary source of cortical norepinephrine (NE) and promoter of cortical and sympathetic arousal, is well-positioned to mediate movement-arousal coupling. Here, using a combination of physiological recordings, fiber photometry, optogenetics, and behavioral tracking, we show that the LC activation is tightly coupled to the return of organized movements during waking from an anesthetized state. Moreover, in an awake animal, movement initiations are coupled to LC activation, while movement arrests, to LC deactivation. We also report that LC activity covaries with the depth of anesthesia and that LC photoactivation leads to sympathetic activation, consistent with its role in mediating increased arousal. Together, these studies reveal a more nuanced, modulatory role that LC plays in coordinating movement and arousal.
PubMed: 38948871
DOI: 10.1101/2024.06.18.599619 -
BioRxiv : the Preprint Server For... Jun 2024DNA replication is regulated by factors that promote or inhibit initiation. In YabA is a negative regulator of DNA replication initiation while the newly identified...
UNLABELLED
DNA replication is regulated by factors that promote or inhibit initiation. In YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that Δ and several alleles under-initiate DNA replication while ablation of or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA damaging agents. We show that cells under-initiating and over-initiating DNA replication were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA replication, we propose that an increase in the number of replication forks leads to replication fork stress which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled as changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.
IMPORTANCE
The regulation of DNA replication is fundamental to cell growth and cell cycle control. In eukaryotes under-initiation or over-initiation leads to genome instability. For bacteria, it is unclear how changes in replication initiation frequency impact DNA replication status and genome integrity. We show that tight regulation of DNA replication initiation is critical for maintaining genome integrity. Cells over-initiating or under-initiating DNA replication are sensitive to DNA damage. Further, cells over-initiating DNA replication experience replication fork stress at levels that phenocopy cells encountering DNA damage from the crosslinking agent mitomycin C. Our results establish the critical importance of properly regulating DNA replication initiation frequency because an imbalance in initiation results in replication fork perturbations, deficiencies in DNA repair, and genome instability.
PubMed: 38948856
DOI: 10.1101/2024.06.18.599555 -
BioRxiv : the Preprint Server For... Jun 2024Inhibitory control is a crucial cognitive-control ability for behavioral flexibility that has been extensively investigated through action-stopping tasks. Multiple...
UNLABELLED
Inhibitory control is a crucial cognitive-control ability for behavioral flexibility that has been extensively investigated through action-stopping tasks. Multiple neurophysiological features have been proposed to represent 'signatures' of inhibitory control during action-stopping, though the processes signified by these signatures are still controversially discussed. The present study aimed to disentangle these processes by comparing simple stopping situations with those in which additional action revisions were needed. Three experiments in female and male humans were performed to characterize the neurophysiological dynamics involved in action-stopping and - changing, with hypotheses derived from recently developed two-stage 'pause-then-cancel' models of inhibitory control. Both stopping and revising an action triggered an early broad 'pause'-process, marked by frontal EEG β-bursts and non-selective suppression of corticospinal excitability. However, partial-EMG responses showed that motor activity was only partially inhibited by this 'pause', and that this activity can be further modulated during action-revision. In line with two-stage models of inhibitory control, subsequent frontocentral EEG activity after this initial 'pause' selectively scaled depending on the required action revisions, with more activity observed for more complex revisions. This demonstrates the presence of a selective, effector-specific 'retune' phase as the second process involved in action-stopping and -revision. Together, these findings show that inhibitory control is implemented over an extended period of time and in at least two phases. We are further able to align the most commonly proposed neurophysiological signatures to these phases and show that they are differentially modulated by the complexity of action-revision.
SIGNIFICANCE STATEMENT
Inhibitory control is one of the most important control processes by which humans can regulate their behavior. Multiple neurophysiological signatures have been proposed to reflect inhibitory control. However, these play out on different time scales and appear to reflect different aspects of cognitive control, which are controversially debated.Recent two-stage models of inhibitory control have proposed that two phases implement the revisions of actions: 'pause' and 'retune'. Here, we provide the first empirical evidence for this proposition: Action revisions engendered a common initial low-latency 'pause', during which motor activity is broadly suppressed. Later activity, however, distinguishes between simple stopping of actions and more complex action revisions. These findings provide novel insights into the sequential dynamics of human action control.
PubMed: 38948849
DOI: 10.1101/2024.06.18.597172 -
BioRxiv : the Preprint Server For... Jun 2024Bed bugs are blood-feeders that rapidly proliferate into large indoor infestations. Their bites can cause allergies, secondary infections and psychological stress, among...
Bed bugs are blood-feeders that rapidly proliferate into large indoor infestations. Their bites can cause allergies, secondary infections and psychological stress, among other problems. Although several tactics for their management have been used, bed bugs continue to spread worldwide wherever humans reside. This is mainly due to human-mediated transport and their high resistance to several classes of insecticides. New treatment options with novel modes of action are required for their control. In this study, we evaluated the use of nitisinone (NTBC), an FDA-approved drug, for bed bug control in an insecticide-susceptible (HH) and an insecticide-resistant (CIN) population. Although NTBC was lethal to both populations when administered orally or applied topically in very low doses, we observed a slight but significant resistance in the CIN population. Transcriptomic analysis in both populations indicated that NTBC treatment elicited a broad suppression of genes associated with RNA post-transcriptional modifications, translation, endomembrane system, protein post-translational modifications and protein folding. The CIN population exhibited higher ATP production and xenobiotic detoxification. Feeding studies on a mouse model highlight that NTBC could be used as a control method of bed bugs by host treatment. The results demonstrate that NTBC can be used as a new active ingredient for bed bug control by topical or oral treatment and shed light on the molecular mechanisms of suppressed tyrosine metabolism following NTBC treatment.
PubMed: 38948842
DOI: 10.1101/2024.06.18.599347 -
BioRxiv : the Preprint Server For... Jun 2024The multidrug-resistant, nosocomial pathogen is a major threat to human health. A sensor kinase-response regulator system, BfmS-BfmR, is a potential antimicrobial...
The multidrug-resistant, nosocomial pathogen is a major threat to human health. A sensor kinase-response regulator system, BfmS-BfmR, is a potential antimicrobial target in the bacterium due to its essential role in promoting drug resistance and virulence. Important questions remain, however, about how the system controls resistance and pathogenesis. Although knockout of BfmR is known to alter expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls BfmR is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and reactions using a small phosphodonor to study how phosphorylation affects BfmR function. We show that phosphorylation requires the BfmR active site Asp58 and is essential to gene regulation, antibiotic resistance, and virulence in a sepsis model. Consistent with activation of the protein, phosphorylation induces dimerization and increases its affinity for target DNA. Integrated analysis of the genome-wide binding and transcriptional profiles of BfmR led to several key findings: (1) Phosphorylation dramatically expands the number of genomic sites bound by BfmR, from 4 to >250; (2) BfmR recognizes a direct repeat motif widespread across promoters; (3) BfmR directly regulates >300 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (eg, type IV pilus machinery); (4) The regulator also directly controls a set of non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving resistance and pathogenicity and unravel the extensive gene regulatory network under its control.
PubMed: 38948834
DOI: 10.1101/2024.06.16.599214 -
BioRxiv : the Preprint Server For... Jun 2024Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically...
Gas vesicles (GVs) based on acoustic reporter genes have emerged as potent contrast agents for cellular and molecular ultrasound imaging. These air-filled, genetically encoded protein nanostructures can be expressed in a variety of cell types to visualize cell location and activity or injected systemically to label and monitor tissue function. Distinguishing GVs from tissue signal deep inside intact organisms requires imaging approaches such as amplitude modulation (AM) or collapse-based pulse sequences, however they have limitations in sensitivity or require irreversible collapse of the GVs that restricts its scope for imaging dynamic cellular processes. To address these limitations, this study explores the utility of harmonic imaging to enhance the sensitivity of non-destructive imaging of GVs and cellular processes. Traditional fundamental-frequency imaging utilizing cross-wave AM (xAM) sequences has been deemed optimal for GV imaging. Contrary to this, we hypothesize that harmonic imaging, integrated with xAM could significantly elevate GV detection sensitivity. To verify our hypothesis, we conducted imaging on tissue-mimicking phantoms embedded with purified GVs, mammalian cells genetically modified to express GVs, and live mice after systemic GV infusion. Our findings reveal that harmonic xAM (HxAM) imaging markedly surpasses traditional xAM in isolating GVs' nonlinear acoustic signature, showcasing significant enhancements in signal-to-background and contrast-to-background ratios across all tested samples. Further investigation into the backscattered spectra elucidates the efficacy of harmonic imaging in conjunction with xAM. HxAM imaging enables the detection of lower concentrations of GVs and cells with ultrasound and extends the imaging depth by up to 20% and imaging performance metrics by up to 10dB. These advancements bolster the capabilities of ultrasound for molecular and cellular imaging, underscoring the potential of using harmonic signals to amplify GV detection.
PubMed: 38948831
DOI: 10.1101/2024.06.18.599141 -
BioRxiv : the Preprint Server For... Jun 2024Neurotransmiter release is triggered in microseconds by Ca -binding to the Synaptotagmin-1 C domains and by SNARE complexes that form four-helix bundles between...
UNLABELLED
Neurotransmiter release is triggered in microseconds by Ca -binding to the Synaptotagmin-1 C domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca -sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C B domain to SNARE complexes through a 'primary interface' comprising two regions (I and II). The Synaptotagmin-1 Ca -binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca -binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca causes reorientation of the C B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca causes reorientation of the C B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper.
SIGNIFICANCE STATEMENT
Neurotransmiter release requires SNARE complexes that fuse synaptic vesicles with the plasma membrane and the Ca -sensor synaptotagmin-1, which was thought to facilitate membrane fusion directly through its Ca -binding loops. However, binding of Synaptotagmin-1 to SNARE complexes orients these loops away from the fusion site. Using molecular dynamics simulations, we show that placing Synaptotagmin-1 at the fusion site hinders the action of SNARE complexes. Spectroscopic studies show that Ca binding to Synaptotagmin-1 can change its interactions with SNARE complexes and, together with molecular modeling, suggest that Synaptotagmin-1 acts as a lever, pulling SNARE complexes and thus facilitating their action on the membranes to induce fusion. Functional studies described in the accompanying paper support this hypothesis.
PubMed: 38948826
DOI: 10.1101/2024.06.17.599417 -
BioRxiv : the Preprint Server For... Jun 2024Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a...
Cascade is a class 1, type 1 CRISPR-Cas system with a variety of roles in prokaryote defense, specifically against DNA-based viruses. The transposon, Tn6677, encodes a variant of the type 1F Cascade known as type 1F-3. This Cascade variant complexes with a homodimer of the transposition protein TniQ and leverages the sequence specificity of Cascade to direct the integration activity of the heteromeric transposase tnsA/B, resulting in site-specific transposition of Tn6677. We desire to uncover the molecular details behind R Loop formation of 'Cascade-TniQ.' Due to the lack of a complete model of Cascade-TniQ available at atom-level resolution, we first build a complete model using AlphaFold V2.1. We then simulate this model via classical molecular dynamics and umbrella sampling to study an important regulatory component within Cascade-TniQ, known as the Cas8 'bundle.' Particularly, we show that this alpha helical bundle experiences a free energy barrier to its large-scale translatory motions and relative free energies of its states primarily dependent on a loop within a Cas7 subunit in Cascade-TniQ. Further, we comment on additional structural and dynamical regulatory points of Cascade-TniQ during R Loop formation, such as Cascade-TniQ backbone rigidity, and the potential role TniQ plays in regulating bundle dynamics. In summary, our outcomes provide the first all-atom dynamic representation of one of the largest CRISPR systems, with information that can contribute to understanding the mechanism of nucleic acid binding and, eventually, to transposase recruitment itself. Such information may prove informative to advance genome engineering efforts.
PubMed: 38948825
DOI: 10.1101/2024.06.21.600075 -
BioRxiv : the Preprint Server For... Jun 2024The role of dynamics in enzymatic function is a highly debated topic. Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been...
The role of dynamics in enzymatic function is a highly debated topic. Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been studied, is a model system in this debate. Myriad previous works have identified networks of residues in positions near to and remote from the active site that are involved in dynamics and others that are important for catalysis. For example, specific mutations on the Met20 loop in DHFR (N23PP/S148A) are known to disrupt millisecond-timescale motions and reduce catalytic activity. However, how and if networks of dynamically coupled residues influence the evolution of DHFR is still an unanswered question. In this study, we first identify, by statistical coupling analysis and molecular dynamic simulations, a network of coevolving residues, which possess increased correlated motions. We then go on to show that allosteric communication in this network is selectively knocked down in N23PP/S148A mutant DHFR. Finally, we identify two sites in the human DHFR sector which may accommodate the Met20 loop double proline mutation while preserving dynamics. These findings strongly implicate protein dynamics as a driving force for evolution.
PubMed: 38948820
DOI: 10.1101/2024.06.18.599103 -
BioRxiv : the Preprint Server For... Jun 2024Platelet Derived Growth Factor Receptor Beta (Pdgfrβ) suppresses the formation of cold temperature-induced beige adipocytes in aged mammals. We aimed to determine if...
OBJECTIVE
Platelet Derived Growth Factor Receptor Beta (Pdgfrβ) suppresses the formation of cold temperature-induced beige adipocytes in aged mammals. We aimed to determine if deleting Pdgfrβ in aged mice could rejuvenate metabolically active beige adipocytes by activating group 2 innate lymphoid cells (ILC2), and whether this effect could counteract diet-induced obesity-associated beige fat decline.
METHODS
We employed Pdgfrβ gain-of-function and loss-of-function mouse models targeting beige adipocyte progenitor cells (APCs). Our approach included cold exposure, metabolic cage analysis, and age and diet-induced obesity models to examine beige fat development and metabolic function under varied Pdgfrβ activity.
RESULTS
Acute cold exposure alone enhanced metabolic benefits in aged mice, irrespective of beige fat generation. However, Pdgfrβ deletion in aged mice reestablished the formation of metabolically functional beige adipocytes, enhancing metabolism. Conversely, constitutive Pdgfrβ activation in young mice stymied beige fat development. Mechanistically, Pdgfrβ deletion upregulated IL-33, promoting ILC2 recruitment and activation, whereas Pdgfrβ activation reduced IL-33 levels and suppressed ILC2 activity. Notably, diet-induced obesity markedly increased Pdgfrβ expression and Stat1 signaling, which inhibited IL-33 induction and ILC2 activation. Genetic deletion of Pdgfrβ restored beige fat formation in obese mice, improving whole-body metabolism.
CONCLUSION
This study reveals that cold temperature exposure alone can trigger metabolic activation in aged mammals. However, reversing Pdgfrβ signaling in aged and obese mice not only restores beige fat formation but also renews metabolic function and enhances the immunological environment of white adipose tissue (WAT). These findings highlight Pdgfrβ as a crucial target for therapeutic strategies aimed at combating age- and obesity-related metabolic decline.
PubMed: 38948810
DOI: 10.1101/2024.06.17.599436