bluuebunny/crossref_metadata_2025 huggingface dataset:
The RNA-binding selectivity of the RGG/RG motifs of hnRNP U is abolished by elements within the intrinsically disordered region
Authors: Kletzien Otto A. , Wuttke Deborah S. , Batey Robert T.
Published: May 2024

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ~100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U's RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5'-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.
Asesino: a nucleus-forming phage that lacks PhuZ
Authors: Prichard Amy , Sy Annika , Meyer Justin , Villa Elizabeth , Pogliano Joe
Published: May 2024

As nucleus-forming phages become better characterized, understanding their unifying similarities and unique differences will help us understand how they occupy varied niches and infect diverse hosts. All identified nucleus-forming phages fall within the proposed Chimalliviridae family and share a core genome of 68 unique genes including chimallin, the major nuclear shell protein. A well-studied but non-essential protein encoded by many nucleus-forming phages is PhuZ, a tubulin homolog which aids in capsid migration, nucleus rotation, and nucleus positioning. One clade that represents 24% of all currently known chimalliviruses lacks a PhuZ homolog. Here we show thatErwiniaphage Asesino, one member of this PhuZ-less clade, shares a common overall replication mechanism with other characterized nucleus-forming phages despite lacking PhuZ. We show that Asesino replicates via a phage nucleus that encloses phage DNA and partitions proteins in the nuclear compartment and cytoplasm in a manner similar to previously characterized nucleus-forming phages. Consistent with a lack of PhuZ, however, we did not observe active positioning or rotation of the phage nucleus within infected cells. These data show that some nucleus-forming phages have evolved to replicate efficiently without PhuZ, providing an example of a unique variation in the nucleus-based replication pathway.
L-Dopa induced dyskinesias require Cholinergic Interneuron expression of Dopamine 2 receptor
Authors: Uribe-Cano Santiago , Malave Lauren , Kottmann Andreas H.
Published: May 2024

Background

Striatal Cholinergic Interneurons (CIN) are drivers of L-Dopa induced Dyskinesias (LID). However, what signaling pathways elicit aberrant CIN activity remains unclear. CIN express D2 and D5 receptors suggesting repeated activation of these receptors in response to L-Dopa could promote LID. While the role of D5 in this process has recently been probed, little is known about the role of D2.

Method

Mice with CIN-specific D2 ablation (D2CINKO) underwent unilateral 6-OHDA lesion and chronic L-Dopa dosing, throughout which LID severity was quantified. The effect of D2CINKO on histological markers of LID severity and CIN activity were also quantified postmortem.

Results

D2CINKO attenuated LID across L-Dopa doses, reduced expression of histological LID marker p-ERK, and prevented L-Dopa-induced increases in CIN activity marker p-rpS6 in the dorsolateral striatum.

Conclusion

The activation of D2 specifically on CIN is a key driver of LID.
Regulation of brain development by the Minibrain/Rala signaling network
Authors: Brown Melissa , Sciascia Erika , Ning Ken , Adam Wesam , Veraksa Alexey
Published: May 2024

The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer, however the exact mechanism through which it functions is unknown. Here, we have studied the role of theDrosophilaortholog of DYRK1A, Minibrain (Mnb), in brain development. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis ofmnbmutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized early role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in brain development.

Significance statement

The kinase Minibrain(Mnb)/DYRK1A regulates the development of the brain and other tissues across many organisms. Here we show the critical importance of Mnb within the developing neuroepithelium. Advancing our understanding of Mnb function, we identified novel protein interactors of Mnb, Reps and Rlip, which function together with Mnb to regulate growth inDrosophila melanogaster. We also identify and characterize a role for the small GTPase Rala in Mnb-regulated growth and nervous system development. This work reveals an early role of Mnb in brain development and identifies a new Mnb/Reps/Rlip/Rala signaling axis.
A Stochastic Dynamic Operator framework that improves the precision of analysis and prediction relative to the classical spike-triggered average method, extending the toolkit
Authors: Smith Trevor S. , Abolfath-Beygi Maryam , Sanger Terence D. , Giszter Simon F.
Published: May 2024

Here we test the Stochastic Dynamic Operator (SDO) as a new framework for describing physiological signal dynamics relative to spiking or stimulus events. The SDO is a natural extension of existing spike-triggered averaging (STA), or stimulus-triggered averaging, methods currently used in neural analysis. It extends the classic STA to cover state-dependent and probabilistic responses where STA may fail. SDO methods are more sensitive and specific than the STA for identifying state-dependent relationships in simulated data. We have tested SDO analysis for interactions between electrophysiological recordings of spinal interneurons, single motor units, and aggregate muscle electromyograms (EMG) of major muscles in the spinal frog hindlimb. When predicting target signal behavior relative to spiking events, the SDO framework outperformed or matched classical spike-triggered averaging methods. SDO analysis permits more complicated spike-signal relationships to be captured, analyzed, and interpreted visually and intuitively. SDO methods can be applied at different scales of interest where spike-triggered averaging methods are currently employed, and beyond, from single neurons to gross motor behaviors. SDOs may be readily generated and analyzed using the providedSDO Analysis Toolkit. We anticipate this method will be broadly useful for describing dynamical signal behavior and uncovering state-dependent relationships of stochastic signals relative to discrete event times.

SIGNIFICANCE

Here the authors introduce new tools and demonstrate data analysis using a new probabilistic and state-dependent technique, which is an expansion and extension of the classical spike-triggered average, the Stochastic Dynamic Operator. Stochastic Dynamic Operator methods extend application into domains where classical spike triggered averages fail, capture more information on spike correlations, and match or outperform the spike-triggered average when generating predictions of signal amplitude based on spiking events. A data and code package toolkit for utilizing and interpreting Stochastic Dynamic Operator methods is provided together with example analyses. Both the method and the associated toolkit are thus expected to be broadly useful in research domains where the spike triggered average is currently used for analysis, and beyond.
Enterococcal quorum-controlled protease alters phage infection
Authors: Sheriff Emma K. , Salvato Fernanda , Andersen Shelby E. , Chatterjee Anushila , Kleiner Manuel , Duerkop Breck A.
Published: May 2024

Increased prevalence of multidrug resistant bacterial infections has sparked interest in alternative antimicrobials, including bacteriophages (phages). Limited understanding of the phage infection process hampers our ability to utilize phages to their full therapeutic potential. To understand phage infection dynamics we performed proteomics onEnterococcus faecalisinfected with the phage VPE25. We discovered numerous uncharacterized phage proteins are produced during phage infection ofEnterococcus faecalis. Additionally, we identified hundreds of changes in bacterial protein abundances during infection. One such protein, enterococcal gelatinase (GelE), anfsrquorum sensing regulated protease involved in biofilm formation and virulence, was reduced during VPE25 infection. Plaque assays showed that mutation of either thefsrAorgelEresulted in plaques with a “halo” morphology and significantly larger diameters, suggesting decreased protection from phage infection. GelE-associated protection during phage infection is dependent on the murein hydrolase regulator LrgA and antiholin-like protein LrgB, whose expression have been shown to be regulated by GelE. Our work may be leveraged in the development of phage therapies that can modulate the production of GelE thereby altering biofilm formation and decreasingE. faecalisvirulence.
Paired vagus nerve stimulation drives precise remyelination and motor recovery after myelin loss
Authors: Huang Rongchen , Carter Elise R. , Hughes Ethan G. , Welle Cristin G.
Published: May 2024

Myelin loss in the central nervous system can cause permanent motor or cognitive deficits in patients with multiple sclerosis (MS). While current immunotherapy treatments decrease the frequency of demyelinating episodes, they do not promote myelin repair or functional recovery. Vagus nerve stimulation (VNS) is a neuromodulation therapy which enhances neuroplasticity and the recovery of motor function after stroke, but its effects on myelin repair are not known. To determine if VNS influences myelin repair, we applied VNS following a demyelinating injury and measured longitudinal myelin dynamics and functional recovery. We found that VNS promotes remyelination by increasing the generation of myelinating oligodendrocytes. Pairing VNS with a skilled reach task leads to the regeneration of myelin sheaths on previously myelinated axon segments, enhancing the restoration of the original pattern of myelination. Moreover, the magnitude of sheath pattern restoration correlates with long-term motor functional improvement. Together, these results suggest that recovery of the myelin sheath pattern is a key factor in the restoration of motor function following myelin loss and identify paired VNS as a potential remyelination therapy to treat demyelinating diseases.
Homodimerization of CB2cannabinoid receptor triggered by a bivalent ligand enhances cellular signaling
Authors: Navarro Gemma , Gómez-Autet Marc , Morales Paula , Rebassa Joan Biel , Llinas del Torrent Claudia , Jagerovic Nadine , Pardo Leonardo , Franco Rafael
Published: May 2024

G protein-coupled receptors (GPCRs) exist within a landscape of interconvertible conformational states and in dynamic equilibrium between monomers and higher-order oligomers, both influenced by ligand binding. Here, we have shown that a homobivalent ligand formed by equal chromenopyrazole moieties as pharmacophores, connected by 14 methylene units, can modulate the dynamics of the cannabinoid CB2receptor (CB2R) homodimerization by simultaneously binding both protomers of the CB2R-CB2R homodimer. Computational and pharmacological experimentals showed that one of the ligand pharmacophores binds to the orthosteric site of one protomer, and the other pharmacophore to a membrane-oriented pocket between transmembranes 1 and 7 of the partner protomer. This provides unique pharmacological properties, such as increased potency in Gibinding and increased recruitment of β-arrestin. Thus, by modulating dimerization dynamics, it may be possible to fine-tune CB2R activity with potentially improved therapeutic outcomes.

HIGHLIGHTS

A homobivalent ligand of CB2R (PM369) modulates the dynamics of receptor homodimerization

PM369 binds to the orthosteric site of one protomer and to a complementary, membrane-facing, site of the other protomer

PM369 triggers CB2R homodimerization via the TM 1/7 interface that provides unique pharmacological properties

PM369 potentiates signaling, increased potency in Gibinding and increased recruitment of β-arrestin

These results highlight new approaches to control GPCR signaling

GRAPHICAL ABSTRACT
Bacterial Modulation of Intestinal Motility through Macrophage Redistribution
Authors: Ngo Julia S. , Amitabh Piyush , Sokoloff Jonah G. , Trinh Calvin , Wiles Travis J. , Guillemin Karen , Parthasarathy Raghuveer
Published: May 2024

Intestinal microbes, whether resident or transient, influence the physiology of their hosts, altering both the chemical and the physical characteristics of the gut. An example of the latter is the human pathogenVibrio cholerae’sability to induce strong mechanical contractions, discovered in zebrafish. The underlying mechanism has remained unknown, but the phenomenon requires the actin crosslinking domain (ACD) ofVibrio’sType VI Secretion System (T6SS), a multicomponent protein syringe that pierces adjacent cells and delivers toxins. By using a zebrafish-nativeVibrioand imaging-based assays of host intestinal mechanics and immune responses, we find that macrophages mediate the connection between the T6SS ACD and intestinal activity: ACD-dependent tissue damage activates macrophages and recruits them from their unperturbed positions near enteric neurons lining the midgut, spurring strong gut contractions resembling those resulting from genetic depletion of macrophages. In addition to illuminating host-directed actions of the widespread T6SS protein apparatus, our findings highlight how localized bacteria-induced injury can reshape neuro-immune cellular dynamics to impact whole-organ physiology.
SNP assays for DVI: cost, time, and performance information for decision-makers
Authors: Gettings Katherine Butler , Tillmar Andreas , Sturk-Andreaggi Kimberly , Marshall Charla
Published: May 2024

In mass disaster events, forensic DNA laboratories may be called upon to quickly pivot their operations toward identifying bodies and reuniting remains with family members. Ideally, laboratories have considered this possibility in advance and have a plan in place. Compared with traditional short tandem repeat (STR) typing, single nucleotide polymorphisms (SNPs) may be better suited to these disaster victim identification (DVI) scenarios due to their small genomic target size, resulting in an improved success rate in degraded DNA samples. As the landscape of technology has shifted toward DNA sequencing, many forensic laboratories now have benchtop instruments available for massively parallel sequencing (MPS), facilitating this operational pivot from routine forensic STR casework to DVI SNP typing. Herein, we review the commercially available SNP sequencing assays amenable to DVI, we use data simulations to explore the potential for kinship prediction from SNP panels of varying size, and we give an example DVI scenario as context for presenting the matrix of considerations: kinship predictive potential, cost, and throughput of current SNP assay options. This information is intended to assist laboratories in choosing a SNP system for disaster preparedness.

Highlights

3 to 5 bullet points (maximum 100 characters per bullet point, including spaces). Each bullet point should be a full sentence and should outline the key contributions of your manuscript and how it impacts forensic science.

Single nucleotide polymorphisms (SNPs) are useful in disaster victim identification (DVI).

SNP panels amenable to human identification and extended kinship are described.

Simulations demonstrate the potential for kinship prediction from SNP panels of varying size.

Kinship predictive potential, cost, and throughput are presented for an example DVI scenario.

Information is intended to assist laboratories in choosing a SNP system for disaster preparedness.
Conformational switching of Arp5 subunit differentially regulates INO80 chromatin remodeling
Authors: Garcia Jeison , Paul Somnath , Shukla Shagun , Zhong Yuan , Beauchemin Karissa , Bartholomew Blaine
Published: May 2024

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80’s ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.
Complete list of canonical post-transcriptional modifications in theBacillus subtilisribosome and their link to RbgA driven large subunit assembly
Authors: Popova Anna M. , Jain Nikhil , Dong Xiyu , Abdollah-Nia Farshad , Britton Robert A. , Williamson James R.
Published: May 2024

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny.B. subtilisis a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA ofB. subtilis,including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly inB. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, addingB. subtilisto the list of fully annotated species afterE. coliandT. thermophilus,in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.
Decoding Early Psychoses: Unraveling Stable Microstructural Features Associated with Psychopathology Across Independent Cohorts
Authors: Wang Haley R. , Liu Zhen-Qi , Nakua Hajer , Hegarty Catherine E. , Thies Melanie Blair , Patel Pooja K. , Schleifer Charles H. , Boeck Thomas P. , McKinney Rachel A. , Currin Danielle , Leathem Logan , DeRosse Pamela , Bearden Carrie E. , Misic Bratislav , Karlsgodt Katherine H.
Published: May 2024

Background

Early Psychosis patients (EP, within 3 years after psychosis onset) show significant variability, making outcome predictions challenging. Currently, little evidence exists for stable relationships between neural microstructural properties and symptom profiles across EP diagnoses, limiting the development of early interventions.

Methods

A data-driven approach, Partial Least Squares (PLS) correlation, was used across two independent datasets to examine multivariate relationships between white matter (WM) properties and symptomatology, to identify stable and generalizable signatures in EP. The primary cohort included EP patients from the Human Connectome Project-Early Psychosis (n=124). The replication cohort included EP patients from the Feinstein Institute for Medical Research (n=78). Both samples included individuals with schizophrenia, schizoaffective disorder, and psychotic mood disorders.

Results

In both cohorts, a significant latent component (LC) corresponded to a symptom profile combining negative symptoms, primarily diminished expression, with specific somatic symptoms. Both LCs captured comprehensive features of WM disruption, primarily a combination of subcortical and frontal association fibers. Strikingly, the PLS model trained on the primary cohort accurately predicted microstructural features and symptoms in the replication cohort. Findings were not driven by diagnosis, medication, or substance use.

Conclusions

This data-driven transdiagnostic approach revealed a stable and replicable neurobiological signature of microstructural WM alterations in EP, across diagnoses and datasets, showing a strong covariance of these alterations with a unique profile of negative and somatic symptoms. This finding suggests the clinical utility of applying data-driven approaches to reveal symptom domains that share neurobiological underpinnings.
Alternative splicing ofclocktranscript mediates the response of circadian clocks to temperature changes
Authors: Cai Yao D. , Chow Gary K. , Hidalgo Sergio , Liu Xianhui , Jackson Kiya C. , Vasquez Cameron D. , Gao Zita Y. , Lam Vu H. , Tabuloc Christine A. , Zheng Haiyan , Zhao Caifeng , Chiu Joanna C.
Published: May 2024

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. InDrosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) ofperiod(per) andtimeless(tim) genes that encode key transcriptional repressors of clock gene expression. Althoughclock(clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whetherclkexhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed thatclktranscripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternativeclktranscript, hereinafter termedclk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, andin vitroexperiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.
A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting
Authors: Birtwistle Marc R. , Huggins Jonah R. , Zadeh Cameron O. , Sarmah Deepraj , Srikanth Sujata , Jones B. Kelly , Cascio Lauren N. , Dean Delphine
Published: May 2024

Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ~10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ~1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency (cost and time ~8-fold), and capacity for biological characterization relative to established western blots.
Naturalistic acute pain states decoded from neural and facial dynamics
Authors: Huang Yuhao , Gopal Jay , Kakusa Bina , Li Alice , Huang Weichen , Wang Jeffrey B , Persad Amit , Ramayya Ashwin , Parvizi Josef , Buch Vivek P , Keller Corey
Published: May 2024

Pain is a complex experience that remains largely unexplored in naturalistic contexts, hindering our understanding of its neurobehavioral representation in ecologically valid settings. To address this, we employed a multimodal, data-driven approach integrating intracranial electroencephalography, pain self-reports, and facial expression quantification to characterize the neural and behavioral correlates of naturalistic acute pain in twelve epilepsy patients undergoing continuous monitoring with neural and audiovisual recordings. High self-reported pain states were associated with elevated blood pressure, increased pain medication use, and distinct facial muscle activations. Using machine learning, we successfully decoded individual participants' high versus low self-reported pain states from distributed neural activity patterns (mean AUC = 0.70), involving mesolimbic regions, striatum, and temporoparietal cortex. High self-reported pain states exhibited increased low-frequency activity in temporoparietal areas and decreased high-frequency activity in mesolimbic regions (hippocampus, cingulate, and orbitofrontal cortex) compared to low pain states. This neural pain representation remained stable for hours and was modulated by pain onset and relief. Objective facial expression changes also classified self-reported pain states, with results concordant with electrophysiological predictions. Importantly, we identified transient periods of momentary pain as a distinct naturalistic acute pain measure, which could be reliably differentiated from affect-neutral periods using intracranial and facial features, albeit with neural and facial patterns distinct from self-reported pain. These findings reveal reliable neurobehavioral markers of naturalistic acute pain across contexts and timescales, underscoring the potential for developing personalized pain interventions in real-world settings.
Learning to handle flowers increases pollen collection benefits for bees but does not affect pollination success for plants
Authors: Mayberry Maggie M. , Naumer Katherine C. , Novinger Annaliese N. , McCart Dalton M. , Wilkins Rachel V. , Muse Haley , Ashman Tia-Lynn , Russell Avery L.
Published: May 2024

Behavior frequently affects cooperation as well as conflict in plant-pollinator interactions. Pollinators such as bees often modify how they handle flowers with experience and such learning is generally assumed to increase collection of floral food rewards. The complexity of flower morphology also affects how quickly pollinators learn and recall how to access floral rewards from a given flower type. Because learning to handle a flower can increase extraction of food rewards (such as pollen) and often involves the pollinator altering how it interacts with the flower’s reproductive organs, pollinator learning could affect pollination success. Yet how pollinator cognition and flower morphology interact to affect pollination success is unknown. We therefore asked how learning and memory of flower handling by pollen-foraging generalist bumble bees (Bombus impatiens) varied among four morphologically distinct flower types(Phacelia campanularia, Exacum affine,Solanum elaeagnifolium, andErythranthe guttata) and affected pollen collection and pollen deposition on these flowers. We found that bees learned and remembered how to handle some flower types more quickly than others. Learning typically involved development of motor routines unique to each flower type. While bees invariably learned to improve pollen collection, how quickly bees learned and remembered each flower type did not affect pollen collection. Surprisingly, pollen deposition on flowers was not affected by bee foraging experience. Thus, even though learning benefits the bee, it does not alter female (and potentially male) fitness benefits for the plant. We discuss potential reasons for these patterns and consequences for bee behavior and flower evolution.
Eluding anemone nematocysts: are clownfish deprived of N-acetylated sugars in their skin mucus?
Authors: Heim Sara , Teav Tony , Cortesi Fabio , Gallart-Ayala Hector , Ivanisevic Julijana , Salamin Nicolas
Published: May 2024

The clownfish - sea anemone system is a great example of symbiotic mutualism where host 'toxicity' does not impact its symbiont partner, although the underlying protection mechanism remains unclear. The regulation of nematocyst discharge in cnidarians involves N-acetylated sugars like sialic acid, that bind chemoreceptors on the tentacles of sea anemones, leading to the release of stings. It has been suggested that clownfish could be deprived of sialic acid on their skin surface, sparing them from being stung and facilitating mutualism with sea anemones. In this study, we sampled the skin mucus of two anemone symbionts, the clownfish Amphiprion akindynos and the juvenile damselfish Dascyllus trimaculatus, as well as two non-symbiotic adult damselfish Pomacentrus moluccensis and P. pavo. The free and total sialic acid content, including its conjugated form, and three other intermediates of this pathway were quantified using a stable isotope dilution mass spectrometry approach. We found significantly higher levels of sialic acid and its precursor in the non-symbiotic damselfishes. Concentrations of total sialic acid in anemone symbionts ranged between 13 uM and 16 uM, whereas the non-symbiotic damselfishes ranged between 21 uM and 30 uM. The presence of this metabolite and its precursors, as triggers of nematocyst discharge, in anemone symbionts, suggests that this is not the direct mechanism of protection or that the trigger is concentration dependent. This experiment demonstrates that anemone symbionts are not spared by nematocysts because of a lack of N-acetylated sugars, as previously thought.
Visualization of intracellular ATP dynamics in the whole kidney under pathophysiological conditions using the kidney slice culture system
Authors: Yamamoto Shigenori , Yamamoto Shinya , Takahashi Masahiro , Mii Akiko , Okubo Akihiro , Toriu Naoya , Nakagawa Shunsaku , Abe Takaaki , Fukuma Shingo , Imamura Hiromi , Yamamoto Masamichi , Yanagita Motoko
Published: May 2024

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated ATP synthesis pathway, and analyzed intracellular ATP dynamics usingex vivoIR and cisplatin nephropathy models. We demonstrated that proximal tubules (PTs) were strongly dependent on oxidative phosphorylation (OXPHOS), whereas podocytes relied on both OXPHOS and glycolysis using ATP synthesis inhibitors. We also confirmed that theex vivoIR model could recapitulate ATP dynamicsin vivo; ATP recovery in PTs after reperfusion varied depending on ischemia length, whereas ATP in distal tubules (DTs) recovered well even after long ischemia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent delayed cisplatin-induced ATP decrease in PTs. This novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.
Accelerated protein retention expansion microscopy using microwave radiation
Authors: Bullard Meghan R. , Cervantes Juan Carlos Martinez , Quaicoe Norisha B. , Jin Amanda , Adams Danya A. , Lin Jessica M. , Iliadis Elena , Seidler Tess M. , Cervantes-Sandoval Isaac , He Hai-yan
Published: May 2024

Protein retention expansion microscopy (ExM) retains genetically encoded fluorescent proteins or antibody-conjugated fluorescent probes in fixed tissue and isotropically expands the tissue through a swellable polymer network to allow nanoscale (<70 nm) resolution on diffraction-limited confocal microscopes. Despite numerous advantages ExM brings to biological studies, the full protocol is time-consuming and can take multiple days to complete. Here, we adapted the ExM protocol to the vibratome-sectioned brain tissue ofXenopus laevistadpoles and implemented a microwave-assisted protocol to reduce the workflow from days to hours. In addition to the significantly accelerated processing time, our microwave-assisted ExM (M/WExM) protocol maintains the superior resolution and signal-to-noise ratio of the original ExM protocol. Furthermore, theM/WExM protocol yields higher magnitude of expansion, suggesting that in addition to accelerating the process through increased diffusion rate of reagents, microwave radiation may also facilitate the expansion process. To demonstrate the applicability of this method to other specimens and protocols, we adapted the microwave-accelerated protocol to whole mount adult brain tissue ofDrosophila melanogasterfruit flies, and successfully reduced the total processing time of a widely-usedDrosophilaIHC-ExM protocol from 6 days to 2 days. Our results demonstrate that with appropriate adjustment of the microwave parameters (wattage, pulse duration, interval, and number of cycles), this protocol can be readily adapted to different model organisms and tissue types to greatly increase the efficiency of ExM experiments.
Antibiotic treatment induces microbiome dysbiosis and reduction of neuroinflammation following traumatic brain injury in mice
Authors: Flinn Hannah , Marshall Austin , Holcomb Morgan , Cruz Leonardo , Soriano Sirena , Treangen Todd J. , Villapol Sonia
Published: May 2024

The gut microbiome is linked to brain pathology in cases of traumatic brain injury (TBI), yet the specific bacteria that are implicated are not well characterized. To address this gap, in this study, we induced traumatic brain injury (TBI) in male C57BL/6J mice using the controlled cortical impact (CCI) injury model. After 35 days, we administered a broad-spectrum antibiotics (ABX) cocktail (ampicillin, gentamicin, metronidazole, vancomycin) through oral gavage for 2 days to diminish existing microbiota. Subsequently, we inflicted a second TBI on the mice and analyzed the neuropathological outcomes five days later. Longitudinal analysis of the microbiome showed significant shifts in the diversity and abundance of bacterial genera during both acute and chronic inflammation. These changes were particularly dramatic following treatment with ABX and after the second TBI. ABX treatment did not affect the production of short-chain fatty acids (SCFA) but did alter intestinal morphology, characterized by reduced villus width and a lower count of goblet cells, suggesting potential negative impacts on intestinal integrity. Nevertheless, diminishing the intestinal microbiome reduced cortical damage, apoptotic cell density, and microglial/macrophage activation in the cortical and thalamic regions of the brain. Our findings suggest that eliminating colonized gut bacteria via broad-spectrum ABX reduces neuroinflammation and enhances neurological outcomes in TBI despite implications to gut health.
Intradental mechano-nociceptors serve as sentinels that prevent tooth damage
Authors: Ronan Elizabeth A. , Gandhi Akash R. , Constantinescu Brian S. C. , Uchima Koecklin Karin H. , Guenther Mak E. , Nagel Maximilian , Blumberg Kaitlyn J. , Stern Tomer , Li Peng , Emrick Joshua J.
Published: May 2024

Pain is the anticipated output of the trigeminal sensory neurons that innervate the tooth’s vital interior1,2; however, the contribution of intradental neurons to healthy tooth sensation has yet to be defined. Here, we employ in vivo Ca2+imaging to identify and define a population of myelinated high-threshold mechanoreceptors (intradental HTMRs) that detect superficial structural damage of the tooth and initiate jaw opening to protect teeth from damage. Intradental HTMRs remain inactive when direct forces are applied to the intact tooth but become responsive to forces when the structural integrity of the tooth is compromised, and the dentin or pulp is exposed. Their terminals collectively innervate the inner dentin through overlapping receptive fields, allowing them to monitor the superficial structures of the tooth. Indeed, intradental HTMRs detect superficial enamel damage and encode its degree, and their responses persist in the absence of either PIEZO2 or Nav1.83,4. Optogenetic activation of intradental HTMRs triggers a rapid, jaw opening reflex via contraction of the digastric muscle. Taken together, our data indicate that intradental HTMRs serve as sentinels that guard against mechanical threats to the tooth, and their activation results in physical tooth separation to minimize irreversible structural damage. Our work provides a new perspective on the role of intradental neurons as protective rather than exclusively pain-inducing and illustrates additional diversity in the functions of interoreceptors.
Neural mechanisms of resource allocation in working memory
Authors: Li Hsin-Hung , Sprague Thomas C. , Yoo Aspen H. , Ma Wei Ji , Curtis Clayton E.
Published: May 2024

To mitigate capacity limits of working memory, people allocate resources according to an item’s relevance. However, the neural mechanisms supporting such a critical operation remain unknown. Here, we developed computational neuroimaging methods to decode and demix neural responses associated with multiple items in working memory with different priorities. In striate and extrastriate cortex, the gain of neural responses tracked the priority of memoranda. Higher-priority memoranda were decoded with smaller error and lower uncertainty. Moreover, these neural differences predicted behavioral differences in memory prioritization. Remarkably, trialwise variability in the magnitude of delay activity in frontal cortex predicted differences in decoded precision between low and high-priority items in visual cortex. These results suggest a model in which feedback signals broadcast from frontal cortex sculpt the gain of memory representations in visual cortex according to behavioral relevance, thus, identifying a neural mechanism for resource allocation.
Prioritizing working memory resources depends on prefrontal cortex
Authors: Hallenbeck Grace E. , Tardiff Nathan , Sprague Thomas C. , Curtis Clayton E.
Published: May 2024

How the prefrontal cortex contributes to working memory remains controversial, as theories differ in their emphasis on its role in storing memories versus controlling their content. To adjudicate between these competing ideas, we tested how perturbations to the human (both sexes) lateral prefrontal cortex impact the storage and control aspects of working memory during a task that requires human subjects to allocate resources to memory items based on their behavioral priority. Our computational model made a strong prediction that disruption of this control process would counterintuitively improve memory for low-priority items. Remarkably, transcranial magnetic stimulation of retinotopically-defined superior precentral sulcus, but not intraparietal sulcus, unbalanced the prioritization of resources, improving memory for low-priority items as predicted by the model. Therefore, these results provide direct causal support for models in which the prefrontal cortex controls the allocation of resources that support working memory, rather than simply storing the features of memoranda.

SIGNIFICANCE STATEMENT

Although higher-order cognition depends on working memory, the resources that support our memory are severely limited in capacity. To mitigate this limitation, we allocate memory resources according to the behavioral relevance of items. Nonetheless, the neural basis of these abilities remain unclear. Here, we tested the hypothesis that a region in lateral prefrontal cortex controls prioritization in working memory. Indeed, perturbing this region with transcranial magnetic stimulation disrupted the prioritization of working memory resources. Our results provide causal evidence for the hypothesis that prefrontal cortex primarily controls the allocation of memory resources, rather than storing the contents of working memory.
Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting
Authors: Wan Yiming , Helenek Christopher , Coraci Damiano , Balázsi Gábor
Published: May 2024

The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, “collateral damage” by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting fromtranscleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop newMulti-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid(MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.
Active vision in freely moving marmosets using head-mounted eye tracking
Authors: Singh Vikram Pal , Li Jingwen , Dawson Kana , Mitchell Jude F. , Miller Cory T.
Published: May 2024

Our understanding of how vision functions as primates actively navigate the real-world is remarkably sparse. As most data have been limited to chaired and typically head-restrained animals, the synergistic interactions of different motor actions/plans inherent to active sensing –e.g. eyes, head, posture, movement, etc.-on visual perception are largely unknown. To address this considerable gap in knowledge, we developed an innovative wireless head-mounted eye tracking system calledCEREBROfor small mammals, such as marmoset monkeys. Our system performsChair-freeEye-Recording usingBackpack mounted micROcontrollers. Because eye illumination and environment lighting change continuously in natural contexts, we developed a segmentation artificial neural network to perform robust pupil tracking in these conditions. Leveraging this innovative system to investigate active vision, we demonstrate that although freely-moving marmosets exhibit frequent compensatory eye movements equivalent to other primates, including humans, the predictability of the visual behavior (gaze) is higher when animals are freely-moving relative to when they are head-fixed. Moreover, despite increases in eye/head-motion during locomotion, gaze stabilization remains steady because of an increase in VOR gain during locomotion. These results demonstrate the efficient, dynamic visuo-motor mechanisms and related behaviors that enable stable, high-resolution foveal vision in primates as they explore the natural world.

Significance Statement

Vision is arguably the most thoroughly understood of all neural systems in the primate brain. Yet there is little known about how vision functions in real-world contexts in which individuals freely move and explore an environment. This dearth in knowledge is largely due to the lack of technology that can accurately track eye-movements in freely-moving individuals with the speed and resolution needed to quantify primate vision. Here we developed an innovative wireless head-mounted eye-tracking system for marmosets that meets these technical needs and enabled us to quantify facts of primate vision in a manner not previously possible, including a set of discoveries that are likely to transform our understanding of this keystone system.