bluuebunny/crossref_metadata_2025 huggingface dataset:
Episomal virus maintenance enables bacterial population recovery from infection and virus-bacterial coexistence
Authors: Sanchez-Martinez Rodrigo , Arani Akash , Krupovic Mart , Weitz Joshua S. , Santos Fernando , Anton Josefa
Published: July 2024

Hypersaline environments harbor the highest concentrations of virus-like particles (VLPs) reported for aquatic ecosystems. The substantial densities of both microbial populations and VLPs challenge traditional explanations of top-down control exerted by viruses. At close to saturation salinities, prokaryotic populations are dominated byArchaeaand the bacterial cladeSalinibacter. In this work we examine the episomal maintenance of a virus within aSalinibacter ruberhost. We found that infected cultures ofSal. ruberM1 developed a population-level resistance and underwent systematic and reproducible recovery post infection that was counter-intuitively dependent on the multiplicity of infection (MOI), where higher MOI led to better host outcomes. Furthermore, we developed a nonlinear population dynamics model that successfully reproduced the qualitative features of the recovery. This suggests that the maintenance of the virus episomally, often referred to as pseudolysogeny, and lysis inhibition allow for host-virus co-existence under high MOI infections. Our results emphasize the ecological importance of exploring a spectrum of viral infection strategies beyond the conventional binary of lysis or lysogeny.
A survey of ADP-ribosyltransferase families in the pathogenicLegionella
Authors: Krysińska Marianna , Gradowski Marcin , Baranowski Bartosz , Pawłowski Krzysztof , Dudkiewicz Małgorzata
Published: July 2024

Background

ADP-ribosyltransferases (ARTs) are a superfamily of enzymes implicated in various cellular processes, including pathogenic mechanisms. TheLegionellagenus, known for causing Legionnaires’ disease, possesses diverse ART-like effectors. This study explores the proteomes of 41Legionellaspecies to bioinformatically identify and characterise novel ART-like families, providing insights into their potential roles in pathogenesis and host interactions.

Methods

We conducted a comprehensive bioinformatic survey of 41Legionellaspecies to identify proteins with significant sequence or structural similarity to known ARTs. Sensitive sequence searches were performed to detect candidate ART-like families. Subsequent validation, including structure prediction of such families, was achieved using artificial intelligence-driven tools, such as AlphaFold. Comparative analyses were performed to assess sequence and structural similarities between the novel ART-like families and known ARTs.

Results

Our analysis identified 63 proteins with convincing similarity to ARTs, organised into 39 ART-like families, including 26 novel families. Key findings include:

DUF2971 family: exhibits sequence similarity to DarT toxins and other DNA-acting ARTs.

DUF4291 family: the largest newly identified family shows structural and sequence similarity to the diphtheria toxin, suggesting the ability to modify proteins.

Most members of the novel ART families are predicted effectors. Although experimental validation of the predicted ART effector functions is necessary, the novel ART-like families identified present promising targets for understandingLegionellapathogenicity and developing therapeutic strategies. We publish a complete catalogue of our results in the astARTe database:http://bioinfo.sggw.edu.pl/astarte/.
Water availability determines plant regeneration fates
Authors: Kareem Abdul , van Wüllen Anna K. , Zhang Ai , Walckiers Gabriel , Fasth Ellen , Melnyk Charles W.
Published: July 2024

Wounding and hormones serve as diverse triggers for regeneration in animals and plants but how organisms determine regeneration outcomes remains largely unknown. Here, we demonstrated that wounded Arabidopsis tissues regenerate two distinct fates, wound-induced callus or de novo root formation, that are driven by antagonizing molecular pathways related to cambium and root development. We discovered that local water availability dictated these regeneration outcomes in Arabidopsis and tomato, with high water triggering root fate and low water initiating callus fate. Moreover, distinct spatial distributions of auxin response maxima were critical for fate progression and water availability regulated these auxin maxima through the hormones ethylene and jasmonic acid. We propose that water availability determines environmental control of regeneration plasticity with applied potential for improving regeneration in agriculture.
In silicoPK predictions in Drug Discovery: Benchmarking of Strategies to Integrate Machine Learning with Empiric and Mechanistic PK modelling
Authors: Walter Moritz , Aljayyoussi Ghaith , Gerner Bettina , Rapp Hermann , Tautermann Christofer S. , Balazki Pavel , Skalic Miha , Borghardt Jens M. , Humbeck Lina
Published: July 2024

A successful drug needs to combine several properties including high potency and good pharmacokinetic (PK) properties to sustain efficacious plasma concentration over time. To estimate required doses for preclinical animal efficacy models or for the clinics,in vivoPK studies need to be conducted. While the prediction of ADME properties of compounds using Machine Learning (ML) models based on chemical structures is well established in drug discovery, the prediction of complete plasma concentration-time profiles has only recently gained attention. In this study, we systematically compare various approaches that integrate ML models with mechanistic PK models to predict PK profiles in rats after i.v. administration prior to synthesis. More specifically, we compare a standard noncompartmental analysis (NCA) based approach (prediction of CL and Vss), a pure ML approach (non-mechanistic PK description), a compartmental modeling approach, and a physiologically based pharmacokinetic (PBPK) approach. Our study based on internal preclinical data shows that the latter three approaches yield PK profile predictions of comparable accuracy (evaluated as geometric mean fold errors for each profile) across a large test set (>1000 small molecules). In summary, we demonstrate the improved ability to prioritize drug candidates with desirable PK properties prior to synthesis with ML predictions.
Circulating mucosal-like IgA responses associate with severity of Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome
Authors: Cabrera Luz E. , Buckner Cienna , Martinez Veronica , Mäki Sanna , Vapalahti Olli , Vaheri Antti , Hepojoki Jussi , Tietäväinen Johanna , Mäkelä Satu , Mustonen Jukka , Strandin Tomas
Published: July 2024

Old World Orthohantaviruses cause hemorhagic fever with renal syndrome (HFRS) characterized by increased vascular permeability and acute kidney injury (AKI). Despite the systemic nature of the disease, the virus enters humans through inhalation and therefore initially encounters the immunoglobulin class A (IgA) dominated mucosal immune system. Herein, we characterized systemic IgA responses and their potential relationship to the mucosal immune activation by examining blood samples obtained from patients hospitalized due to acute Puumala orthohantavirus infection. Our findings reveal increased frequencies of IgA-expressing circulating mucosal-associated B1 cells and plasmablasts, as well as elevated levels of polyreactive, polymeric, virus-specific and secretory IgA in the acute stage of the disease. Importantly, the levels of circulating virus-specific and secretory IgA associated with the severity of AKI. Furthermore, circulating polymeric IgA displayed enhanced effector functions by forming stable complexes with the IgA receptor CD89 and induced pro-inflammatory neutrophil responses. These results suggest that, while an efficient mucosal immune response is likely to be crucial for infection clearance, an excessive mucosal immune activation may contribute to HFRS disease progression.
Senescent cell clearance ameliorates temporal lobe epilepsy and associated spatial memory deficits in mice
Authors: Khan Tahiyana , McFall David J. , Hussain Abbas I. , Frayser Logan A. , Casilli Timothy P. , Steck Meaghan C. , Sanchez-Brualla Irene , Kuehn Noah M. , Cho Michelle , Barnes Jacqueline A. , Harris Brent T. , Vicini Stefano , Forcelli Patrick A.
Published: July 2024

Current therapies for the epilepsies only treat the symptoms, but do not prevent epileptogenesis (the process in which epilepsy develops). Many cellular responses during epileptogenesis are also common hallmarks ofcellular senescence, which halts proliferation of damaged cells. Clearing senescent cells (SCs) restores function in several age-associated and neurodegenerative disease models. It is unknown whether SC accumulation contributes to epileptogenesis and associated cognitive impairments. To address this question, we used a mouse model of temporal lobe epilepsy (TLE) and characterized the senescence phenotype throughout epileptogenesis. SCs accumulated 2 weeks after SE and were predominantly microglia. We ablated SCs and reduced (and in some cases prevented) the emergence of spontaneous seizures and normalized cognitive function in mice. Suggesting that this is a translationally-relevant target we also found SC accumulation in resected hippocampi from patients with TLE. These findings indicate that SC ablation after an epileptogenic insult is a potential anti-epileptogenic therapy.
The reducing end of cell wall oligosaccharides is critical for DAMP activity inArabidopsis thalianaand can be exploited by oligosaccharide oxidases in the scavenging of phenolic radicals
Authors: Giovannoni Moira , Scortica Anna , Scafati Valentina , Piccirilli Emilia , Sorio Daniela , Benedetti Manuel , Mattei Benedetta
Published: July 2024

The enzymatic hydrolysis of cell wall polysaccharides results in the production of oligosaccharides with nature of damage-associated molecular patterns (DAMPs) that are perceived by plants as danger signals.In vitro, the oxidation of oligogalacturonides and cellodextrins by plant FAD-dependent oligosaccharide-oxidases (OSOXs) suppresses their elicitor activity, suggesting a protective role of OSOXs against a prolonged activation of defense responses potentially deleterious for plant health. However, OSOXs are also produced by phytopathogens and saprotrophs, complicating the understanding of their role in plant-microbe interactions.

The oxidation catalyzed by specific fungal OSOXs also converts the elicitor-active cello-tetraose and xylo-tetraose into elicitor-inactive forms, indicating that the oxidation state of cell wall oligosaccharides is crucial for their DAMP function, irrespective of whether the OSOX originates from fungi or plants.

Certain OSOXs can transfer the electrons from the reducing end of these oligosaccharides to phenolic radicals instead of molecular O2, highlighting an unexpected sub-functionalization of these enzymes.

The activity of OSOXs may be crucial for a thorough understanding of cell wall metabolism since these enzymes can redirect the reducing power from sugars to phenolic components of the plant cell wall, an insight with relevant implications for both plant physiology and green technologies.
Understanding the Transfer and Persistence of Antimicrobial Resistance in Aquaculture Using a Model Teleost Gut System
Authors: Barcan Alexandru Stefan , Humble Joseph , Kasaragod Sandeep , Sajib Mohammad Saiful Islam , Barcan Rares Andrei , McGinnity Philip , Welch Timothy J. , Robertson Brendan , Vamanu Emanuel , Bacigalupo Antonella , Llewellyn Martin Stephen , Samsing Francisca
Published: July 2024

The development, progression, and dissemination of antimicrobial resistance (AMR) is determined by interlinked human, animal, and environmental drivers, posing severe risks to human health. Conjugative plasmid transfer drives the rapid dissemination of AMR among bacteria. Besides antibiotic judicious use and implementation of antibiotic stewardship programs, mitigating antibiotic resistance spread requires an understanding of the dynamics of AMR transfer among microbial communities, as well as the role of various microbial taxa as potential reservoirs that promote long term AMR persistence. Here, we employed Hi-C, a high-throughput, culture-free technique, combined with qPCR, to monitor carriage and transfer of a multidrug-resistant plasmid within an Atlantic salmon in vitro gut model during florfenicol treatment, a benzenesulfonyl antibiotic widely deployed in fin-fish aquaculture. Microbial communities from the pyloric ceaca of three healthy adult farmed salmon were inoculated into three bioreactors developed for the SalmoSim gut system. The model system was then inoculated with anEscherichia colistrain ATCC 25922 carrying plasmid pM07-1 and treated with florfenicol at a concentration of 150 mg/L fish feed media for five days prior to a washout/recovery phase. Hi-C and metagenomic sequencing identified numerous transfer events, including to gram-negative and gram-positive taxa and, crucially, continuing transfer and persistence of the plasmid once florfenicol treatment had been withdrawn. Our findings highlight the role of commensal teleost gut flora as a reservoir for AMR, and our system provides a model to study how different treatment regimes and interventions may be deployed to mitigate AMR persistence.
Insights into Therapeutic Discovery Through the Kelch Domain Structure of Keap1 at Ambient Temperature
Authors: Yilmaz Merve , Sever Belgin , Kutlu Yigit , Gul Mehmet , Okuducu Ceren , Tavli Serra , Otsuka Masami , Fujita Mikako , Haliloglu Turkan , Ciftci Halilibrahim , DeMirci Hasan
Published: July 2024

TheKelch-like-ECH associated protein 1(Keap1) is a part of the E3-ubiquitin ligase complex that binds toNuclear factor erythroid 2-related factor 2(Nrf2) protein and facilitates its degradation by the eukaryotic 26S proteasome. The Kelch domain of Keap1 includes six repeated structural signature motifs, approximately 45–55 amino acid residues in length. Each Kelch repeat contains highly conserved residues and is known to form one blade of beta-propeller structure. Here, we report the dimeric Kelch domain of Keap1 determined at 3.0 Å resolution at the Turkish Light Source‘Turkish DeLight’ at ambient temperature. Our structure provides new structural dynamics information of the dimeric Keap1 Kelch domain at ambient temperature. It displays potential conformational changes of Keap1 residues, particularly at the Dimethyl fumarate (DMF) and Nrf2 binding regions, due to observed temperature shifts. Supported by the Gaussian Network Model (GNM) analysis, the dynamics of the Kelch domain revealed that the allosteric behavior of DMF binding residues is fully established in the ambient temperature structure. We also performed complementary molecular docking studies using our ambient temperature structure for numerous compounds acting as electrophilic irreversible indirect or non-covalent direct inhibitors of the Keap1/Nrf2 complex. Our data suggest that our previously reported novel compound, a hybrid ofL-carnosine andL-histidyl hydrazide (CNN), revealed the most favorable scoring functions and prominent interactions with critical Keap1 residues. Collectively, ourin silicoandin crystalloresults suggest a new potential lead compound for Keap1 inhibition. Additionally, understanding the dimeric form of the Keap1 Kelch domain and conformational changes around the DMF and Nrf2 binding sites at ambient temperature is critical for understanding Keap1-Nrf2 interaction dynamics.
Method and timing of rhizobacteria inoculation to plant roots alters success and strength of aphid suppression
Authors: Blenkinsopp Sophie , Henderson Milo , Zytynska Sharon E
Published: July 2024

Insect pests cause substantial yield losses globally, necessitating novel pest control approaches beyond chemical pesticides. Rhizobacteria, beneficial root-associated bacteria, present a promising alternative by enhancing plant growth and defences against pests. This study explores the use of sodium alginate encapsulation for precise inoculation of two rhizobacteria,Acidovorax radicisandBacillus subtilis, to suppress aphid populations on barley (Hordeum vulgare). We optimized a method using 4mm diameter wet-matrix alginate beads for controlled bacterial delivery directly to plant roots. Two experiments evaluated the impact of inoculation methods and timing on aphid suppression. Results indicated that rhizobacteria inoculation via alginate beads at root emergence significantly reduced aphid reproductive output, outperforming seed soaking methods. This suggests that more consistent bacterial establishment and prolonged release from alginate beads enhances plant defence priming. Additionally, alginate bead inoculation demonstrated effective long-term bacterial viability following storage at 4° C for eight months, supporting potential field application. Our findings highlight the potential of alginate bead-encapsulated rhizobacteria for aphid suppression on barley, but emphasizing the need for precise inoculation timing and placement. This approach offers a robust method for empirical research and practical agricultural application, paving the way for sustainable pest management strategies. Future work should focus on optimizing formulations and understanding plant-microbe interactions to enhance field efficacy.
CHANGES IN MUSCLE QUALITY FOLLOWING SHORT-TERM RESISTANCE TRAINING IN OLDER ADULTS: A COMPARISON OF ECHO INTENSITY AND TEXTURE ANALYSIS
Authors: Knowles Kevan S. , Pagan Jason I. , Beausejour Jonathan P. , Mongold Scott J. , Anderson Abigail W. , Stout Jeffrey R. , Stock Matt S.
Published: July 2024

Background

Skeletal muscle echo intensity (EI) is associated with functional outcomes in older adults, but resistance training interventions have shown mixed results. Texture analysis has been proposed as a novel approach for assessing muscle quality, as it captures spatial relationships between pixels.

Purpose

To examine changes in first-order (EI) and second-order (texture) features of muscle quality following lower-body resistance training in older adults.

Methods

Twelve older adults (2 males, 10 females; mean ± SD age = 70 ± 5 years) completed 6 weeks of progressive resistance training, consisting of twice-weekly sessions at 85% of estimated 1RM. Pre- and post-intervention assessments included ultrasound imaging of the rectus femoris (RF) and vastus lateralis (VL), 5-repetition maximum (5RM) leg extension strength, and maximal voluntary isometric contraction (MVIC) force. Ultrasound images were analyzed for EI and texture features using gray-level co-occurrence matrix (GLCM) analysis.

Results

Large improvements were observed in 5RM leg extension strength (p< 0.001,d= 2.09), MVIC force (p= 0.006,d= 0.969), and RF EI (uncorrected:p= 0.003,d= 0.727; corrected:p= 0.012,d= 0.864). No significant changes were observed in muscle size, VL EI, or texture features for either muscle.

Conclusions

Short-term resistance training improved strength and RF muscle quality as measured by EI. However, texture analysis features were not sensitive to changes following training. These findings suggest that traditional EI measures may be more appropriate than texture analysis for tracking changes in muscle quality following resistance training in older adults.

NEW & NOTEWORTHY

We studied muscle quality changes after 6 weeks of resistance training in older adults, comparing traditional EI to texture analysis. Despite significant strength gains, only RF EI improved, with minimal changes in muscle size, VL EI, or texture parameters. Surprisingly, texture analysis was less sensitive than traditional EI in detecting muscle quality changes. Our results suggest that conventional EI measures may be more suitable for evaluating short-term adaptations to resistance training in older adults.
Muscarinic receptor activation preferentially inhibits rebound in vulnerable dopaminergic neurons
Authors: Beaver Megan L. , Evans Rebekah C.
Published: July 2024

Dopaminergic subpopulations of the substantia nigrapars compacta(SNc) differentially degenerate in Parkinson’s disease and are characterized by unique electrophysiological properties. The vulnerable population expresses a T-type calcium channel-mediated afterdepolarization (ADP) and shows rebound activity upon release from inhibition, whereas the resilient population does not have an ADP and is slower to fire after hyperpolarization. This rebound activity can trigger dopamine release in the striatum, an important component of basal ganglia function. Using whole-cell patch clamp electrophysiology onex vivoslices from adult mice of both sexes, we find that muscarinic activation with the non-selective muscarinic agonist Oxotremorine inhibits rebound activity more strongly in vulnerable vs resilient SNc neurons. Here, we show that this effect depends on the direct activation of muscarinic receptors on the SNc dopaminergic neurons. Through a series of pharmacological and transgenic knock-out experiments, we tested whether the muscarinic inhibition of rebound was mediated through the canonical rebound-related ion channels: T-type calcium channels, hyperpolarization-activated cation channels (HCN), and A-type potassium channels. We find that muscarinic receptor activation inhibits HCN-mediated current (Ih) in vulnerable SNc neurons, but that Ihactivity is not necessary for the muscarinic inhibition of rebound activity. Similarly, we find that Oxotremorine inhibits rebound activity independently of T-type calcium channels and A-type potassium channels. Together these findings reveal new principles governing acetylcholine and dopamine interactions, showing that muscarinic receptors directly affect SNc rebound activity in the midbrain at the somatodendritic level and differentially modify information processing in distinct SNc subpopulations.

Significance Statement

Dopaminergic neurons in the substantia nigrapars compacta(SNc) can be divided into functional subpopulations with distinct basal ganglia connectivity and different degeneration patterns in Parkinson’s disease. We show that the vulnerable and resilient subpopulations of SNc dopaminergic neurons are differentially modulated by muscarinic receptor activation. Specifically, muscarinic receptor activation inhibits rebound activity more strongly in the vulnerable SNc neurons than in the resilient. We find that this inhibition occurs through a non-canonical rebound-related pathway and is not mediated through the channels best known for modulating rebound in midbrain dopaminergic neurons. These findings are important because they reveal novel acetylcholine-dopamine interactions that occur in the midbrain and affect information processing in distinct basal ganglia circuits.
ALPHA: A High Throughput System for Quantifying Growth In Aquatic Plants
Authors: Robinson Kassidy A. , Augoustides Victoria , Madenyika Tanaka , Sartor Ryan C.
Published: July 2024

The need for more sustainable agricultural systems is becoming increasingly apparent. The global demand for agricultural products — food, feed, fuel and fiber — will continue to increase as the global population continues to grow. This challenge is compounded by climate change. Not only does a changing climate make it difficult to maintain stable yields but current agricultural systems are a major source of greenhouse gas emissions and continue to drive the problem further. Therefore, future agricultural systems must not only increase production but also significantly decrease negative environmental impacts. One approach to addressing this is to begin breeding and cultivating new plant species that have fundamental sustainability advantages over our existing crops. The Lemnaceae, a.k.a duckweeds, are one such species that have potential to increase output and reduce the negative environmental impacts of agricultural production. Herein we describe the Automated Lab-scale PHenotyping Apparatus, ALPHA, for high-throughput phenotyping of Lemnaceae. ALPHA is being used for selective breeding of one species,Lemna gibba, toward the goal of creating a new crop for use in sustainable agricultural systems. ALPHA can be used on many small aquatic plant species to assess growth rates in different environmental conditions. A proof of principle use case is demonstrated where ALPHA is used to determine saltwater tolerance of 6 different varieties ofL. gibba.
A replicable and modular benchmark for long-read transcript quantification methods
Authors: Zare Jousheghani Zahra , Singh Noor Pratap , Patro Rob
Published: July 2024

We provide a replicable benchmark for long-read transcript quantification, and evaluate the performance of some recently-introduced tools on several synthetic long-read RNA-seq datasets. This benchmark is designed to allow the results to be easily replicated by other researchers, and the structure of the underlyingSnakemakeworkflow is modular to make the addition of new tools or new data sets relatively easy. In analyzing previously assessed simulations, we find discrepancies with recently-published results. We also demonstrate that the robustness of certain approaches hinge critically on the quality and “cleanness” of the simulated data.

Availability

The Snakemake scripts for the benchmark are available athttps://github.com/COMBINE-lab/lr_quant_benchmarks, the data used as input for the benchmarks (reference sequences, annotations, and simulated reads) are available athttps://doi.org/10.5281/zenodo.13130623.
Immunodominant extracellular loops ofTreponema pallidumFadL outer membrane proteins elicit antibodies with opsonic and growth-inhibitory activities
Authors: Delgado Kristina N. , Caimano Melissa J. , Orbe Isabel C. , Vicente Crystal F. , La Vake Carson J. , Grassmann André A. , Moody M. Anthony , Radolf Justin D. , Hawley Kelly L.
Published: July 2024

The global resurgence of syphilis has created a potent stimulus for vaccine development. To identify potentially protective antibodies (Abs) againstTreponema pallidum(TPA), we usedPyrococcus furiosusthioredoxin (PfTrx) to display extracellular loops (ECLs) from threeTPAouter membrane protein families (outer membrane factors for efflux pumps, eight-stranded β-barrels, and FadLs) to assess their reactivity with immune rabbit serum (IRS). Five ECLs from the FadL orthologs TP0856, TP0858 and TP0865 were immunodominant. Rabbits and mice immunized with these fivePfTrx constructs produced ECL-specific Abs that promoted opsonophagocytosis ofTPAby rabbit peritoneal and murine bone marrow-derived macrophages at levels comparable to IRS and mouse syphilitic serum. ECL-specific rabbit and mouse Abs also impaired viability, motility, and cellular attachment of spirochetes duringin vitrocultivation. The results support the use of ECL-based vaccines and suggest that ECL-specific Abs promote spirochete clearance via Fc receptor-independent as well as Fc receptor-dependent mechanisms.

Author Summary

The resurgence of syphilis emphasizes the critical need for vaccine development againstTreponema pallidum(TPA). Research utilizing immune rabbit serum (IRS) suggests that an effective syphilis vaccine should induce “functional” antibodies (Abs) capable of enhancing the opsonophagocytosis of treponemes by activated macrophages. Structural models ofTPAouter membrane proteins (OMPs), specifically the extracellular loops (ECLs), guided the identification of potential vaccine candidates. Antigenic analysis with IRS of individual ECLs from threeTPAOMP families scaffolded ontoPyrococcus furiosusthioredoxin (PfTrx) revealed five FadL antigenic ECLs. Immunization with immunodominant ECL antigens elicited robust ECL-specific Abs, demonstrating functional opsonic activity in the opsonophagocytosis assays. Furthermore, these Abs effectively inhibited the growth inhibition ofin vitro-cultivatedTPAin both rabbit and mouse models. Our findings underscore the value of antigenic analysis in identifying promisingTPAOMP ECL vaccine targets and highlight the multifaceted protective capacity of ECL Abs againstTPA. This approach also extends to identifying potential OMP vaccinogens in other bacterial pathogens, offering valuable insights for broader vaccine development strategies.
Spreading depolarizations exhaust neuronal ATP in a model of cerebral ischemia
Authors: Schoknecht Karl , Baeza-Lehnert Felipe , Hirrlinger Johannes , Dreier Jens P. , Eilers Jens
Published: July 2024

Spreading depolarizations (SDs) have been identified in various brain pathologies. SDs increase the cerebral energy demand and, concomitantly, oxygen consumption, which indicates enhanced synthesis of adenosine triphosphate (ATP) by oxidative phosphorylation. Therefore, SDs are considered particularly detrimental during reduced supply of oxygen and glucose. However, measurements of intracellular neuronal ATP ([ATP]i), ultimately reporting the balance of ATP synthesis and consumption during SDs, have not yet been conducted.

In this study, we investigated neuronal ATP homeostasis during SDs using 2-photon imaging in acute brain slices from adult mice, expressing the ATP sensor ATeam1.03YEMKin neurons. SDs were induced by application of potassium chloride or by oxygen and glucose deprivation (OGD) and were detected by recording the local field potential, extracellular potassium, as well as the intrinsic optical signal.

We found that, in the presence of oxygen and glucose, SDs were accompanied by a substantial but transient drop in neuronal [ATP]i. OGD, which prior to SD was accompanied by a slight reduction in [ATP]ionly, led to an even larger, terminal drop in [ATP]iduring SDs. Subsequently, we investigated whether neurons could still regenerate ATP if oxygen and glucose were promptly resupplied following SD detection. The data show that ATP depletion was essentially reversible in most cells.

Our findings indicate that SDs are accompanied by a substantial increase in ATP consumption beyond production. This, under conditions that mimic reduced blood supply, leads to a breakdown of [ATP]i. Therefore, our findings support therapeutic strategies targeting SDs after cerebral ischemia.

Significance statement

Spreading depolarizations occur in many brain pathologies, and are suggested to contribute to brain damage through a mismatch in energy supply and demand. However, measurements directly demonstrating an imbalance between ATP production and consumption, particularly in individual neurons, have not been reported. Here we show that spreading depolarizations lead to a transient decrease in intracellular neuronal ATP even in presence of ample glucose and oxygen. When the supply of oxygen and glucose was interrupted, ATP declined gradually until spreading depolarization led to the exhaustion of neuronal ATP. This process would be terminal without the renewed supply of oxygen and glucose. Therefore, therapies targeting spreading depolarizations could preserve neuronal ATP or delay its loss in brain pathologies.
HIV-1 Vif disrupts phosphatase feedback regulation at the kinetochore, leading to a pronounced pseudo-metaphase arrest
Authors: Ghone Dhaval , Evans Edward L. , Bandini Madison , Stephenson Kaelyn G. , Sherer Nathan M. , Suzuki Aussie
Published: July 2024

Virion Infectivity Factor (Vif) of the Human Immunodeficiency Virus type 1 (HIV-1) targets and degrades cellular APOBEC3 proteins, key regulators of intrinsic and innate antiretroviral immune responses, thereby facilitating HIV-1 infection. While Vif’s role in degrading APOBEC3G is well-studied, Vif is also known to cause cell cycle arrest, but the detailed nature of Vif’s effects on the cell cycle has yet to be delineated. In this study, we employed high-temporal single-cell live imaging and super-resolution microscopy to monitor individual cells during Vif-induced cell cycle arrest. Our findings reveal that Vif does not affect the G2/M boundary as previously thought. Instead, Vif triggers a unique and robust pseudo-metaphase arrest, distinct from the mild prometaphase arrest induced by Vpr. During this arrest, chromosomes align properly and form the metaphase plate, but later lose alignment, resulting in polar chromosomes. Notably, Vif, unlike Vpr, significantly reduces the levels of both Protein Phosphatase 1 (PP1) and 2A (PP2A) at kinetochores, which regulate chromosome-microtubule interactions. These results unveil a novel role for Vif in kinetochore regulation that governs the spatial organization of chromosomes during mitosis.
Capillary connections between sensory circumventricular organs and adjacent parenchyma enable local volume transmission
Authors: Yao Yifan , Chen Yannan , Tomer Raju , Silver Rae
Published: July 2024

Among contributors to diffusible signaling are portal systems which join two capillary beds through connecting veins (Dorland 2020). Portal systems allow diffusible signals to be transported in high concentrations directly from one capillary bed to the other without dilution in the systemic circulation. Two portal systems have been identified in the brain. The first was discovered almost a century ago and connects the median eminence to the anterior pituitary gland (Popa & Fielding 1930). The second was discovered a few years ago, and links the suprachiasmatic nucleus to the organum vasculosum of the lamina terminalis, a sensory circumventricular organ (CVO) (Yao et al. 2021). Sensory CVOs bear neuronal receptors for sensing signals in the fluid milieu (McKinley et al. 2003). They line the surface of brain ventricles and bear fenestrated capillaries, thereby lacking blood brain barriers. It is not known whether the other sensory CVOs, namely the subfornical organ (SFO), and area postrema (AP) form portal neurovascular connections with nearby parenchymal tissue. This has been difficult to establish as the structures lie at the midline and protrude into the ventricular space. To preserve the integrity of the vasculature of CVOs and their adjacent neuropil, we combined iDISCO clearing and light-sheet microscopy to acquire volumetric images of blood vessels. The results indicate that there is a portal pathway linking the capillary vessels of the SFO and the posterior septal nuclei, namely the septofimbrial nucleus and the triangular nucleus of the septum. Unlike the latter arrangement, the AP and the nucleus of the solitary tract share their capillary beds. Taken together, the results reveal that all three sensory circumventricular organs bear specialized capillary connections to adjacent neuropil, providing a direct route for diffusible signals to travel from their source to their targets.
CIB2 function is distinct from Whirlin in the development of cochlear stereocilia staircase pattern
Authors: Giese Arnaud P. J. , Parker Andrew , Rehman Sakina , Brown Steve D. M. , Riazuddin Saima , Vander Kooi Craig W. , Bowl Michael R. , Ahmed Zubair M.
Published: July 2024

Variations in genes coding for calcium and integrin binding protein 2 (CIB2) and whirlin cause deafness both in humans and mice. We previously reported that CIB2 binds to whirlin, and is essential for normal staircase architecture of auditory hair cells stereocilia. Here, we refine the interacting domains between these proteins and provide evidence that both proteins have distinct role in the development and organization of stereocilia bundles required for auditory transduction. Using a series of CIB2 and whirlin deletion constructs and nanoscale pulldown (NanoSPD) assays, we localized the regions of CIB2 that are critical for interaction with whirlin. AlphaFold 2 multimer, independently identified the same interacting regions between CIB2 and whirlin proteins, providing a detailed structural model of the interaction between the CIB2 EF2 domain and whirlin HHD2 domain. Next, we investigated genetic interaction between murineCib2andWhrnusing genetic approaches. Hearing in mice double heterozygous for functionally null alleles (Cib2KO/+;Whrnwi/+) was similar to age-matched wild type mice, indicating that partial deficiency for bothCib2andWhrndoes not impair hearing. Double homozygous mutant mice (Cib2KO/KO;Whrnwi/wi) had profound hearing loss and cochlear stereocilia exhibited a predominant phenotype seen in singleWhrnwi/wimutants. Furthermore, over-expression ofWhrninCib2KO/KOmice did not rescue the stereocilia morphology. These data suggest that, CIB2 is multifunctional, with key independent functions in development and/or maintenance of stereocilia staircase pattern in auditory hair cells.
Modular small RNA drives the emergence of virulence traits and environmental trade-offs inVibrio cholerae
Authors: Balasubramanian Deepak , Crist Cole , Almagro-Moreno Salvador
Published: July 2024

The sole gain of laterally acquired virulence genes does not fully explain the transition of environmental strains into human pathogens. To date, the specific molecular drivers and fitness trade-offs that enable some strains within a population to undergo this process remain enigmatic. Here, we describe a small RNA (sRNA) with a unique modular structure that shapes the evolution of toxigenicVibrio cholerae, the agent of cholera. The sRNA comprises of a highly variable 5’ module located within theompUORF and a conserved 3’ one downstream from the gene. This atypical location confers a distinct bimodular structure to the OmpU-encoded sRNA (OueS), generating allelic variants that differentially contribute to the emergence of virulence potential in some strains and associated fitness trade-offs between human infection and environmental survival. Unlike environmental counterparts, the OueS allele from toxigenic strains controls phenotypes essential during host colonization: a) stringently inhibits biofilm formation via a novel sRNA-sRNA interaction by suppressing the iron-responsive sRNA RyhB, and b) confers resistance against intestinal bacteriophages by activating the recently discovered CBASS phage defense system. Toxigenic OueS is also required for successful intestinal colonization and, as the first known example of its kind, acts as a functional surrogate of the master virulence regulator ToxR, controlling over 84% of its regulome. On the other hand, isogenic strains encoding environmental alleles of OueS exhibit higher competitive fitness than those harboring toxigenic ones during colonization of natural reservoirs such as crustaceans and phytoplankton. Our findings provide critical insights into the evolution of toxigenicV. choleraeand reveal specific molecular mechanisms and fitness costs associated with the emergence of pathogenic traits in bacteria.
Enhancer AAVs for targeting spinal motor neurons and descending motor pathways in rodents and macaque
Authors: Kussick Emily , Johansen Nelson , Taskin Naz , Wynalda Brooke , Martinez Refugio , Groce Erin L. , Reding Melissa , Liang Elizabeth , Shulga Lyudmila , Huang Cindy , Casper Tamara , Clark Michael , Ho Windy , Gao Yuan , van Velthoven Cindy T.J. , Sobieski Cassandra , Ferrer Rebecca , Berg Melissa R. , Curtis Britni C. , English Chris , Day Jesse C. , Fortuna Michal , Donadio Nicholas , Newman Dakota , Yao Shenqin , Chakka Anish Bhaswanth , Goldy Jeff , Torkelson Amy , Guzman Junitta B. , Chakrabarty Rushil , Nguy Beagen , Guilford Nathan , Pham Trangthanh H. , Wright Vonn , Ronellenfitch Kara , Gudsnuk Kathryn , Thyagarajan Bargavi , Smith Kimberly A. , Dee Nick , Zeng Hongkui , Yao Zizhen , Tasic Bosiljka , Levi Boaz P. , Hodge Rebecca , Bakken Trygve E. , Lein Ed S. , Ting Jonathan T. , Daigle Tanya L.
Published: July 2024

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, which function to integrate information from the brain and control movement through direct innervation of effector muscles, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. The most promising candidate enhancers were then extensively characterized using imaging and molecular techniques and further tested in rat and macaque to show conservation of LMN labeling. Additionally, we combined enhancer elements into a single vector to achieve simultaneous labeling of upper motor neurons (UMNs) and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.
Enzymatic synthesis of isotopically labeled hydrogen peroxide for mass spectrometry-based applications
Authors: Hoare Margaret , Tan Ruiyue , Militi Isabella , Welle Kevin A. , Swovick Kyle , Hryhorenko Jennifer R. , Ghaemmaghami Sina
Published: July 2024

Methionine oxidation is involved in multiple biological processes including protein misfolding and enzyme regulation. However, it is often challenging to measure levels of methionine oxidation by mass spectrometry, in part due to the prevalence of artifactual oxidation that occurs during sample preparation and ionization steps of typical proteomic workflows. Isotopically labeled hydrogen peroxide (H218O2) can be used to block unoxidized methionines and enable accurate measurement ofin vivolevels of methionine oxidation. However, H218O2is an expensive reagent that can be difficult to obtain from commercial sources. Here, we report a method for synthesizing H218O2in house. Glucose oxidase catalyzes the oxidation of β-D-glucose and produces hydrogen peroxide in the process. We took advantage of this reaction to enzymatically synthesize H218O2from18O2and assessed its concentration, purity, and utility in measuring methionine oxidation levels by mass spectrometry.
Subjective salience ratings are a reliable proxy for physiological measures of arousal
Authors: Hadjis Georgia E , Atlas Lauren Y , Mouseli Pedram , Sexton Christine A. , McAndrews Mary Pat , Moayedi Massieh
Published: July 2024

Pain is an inherently salient multidimensional experience that signals potential bodily threats and promotes nocifensive behaviours. Any stimulus can be salient depending on its features and context. This poses a challenge in delineating pain-specific processes in the brain, rather than salience-driven activity. It is thus essential to salience match control (innocuous) stimuli and noxious stimuli, to remove salience effects, when aiming to delineate pain-specific mechanisms. Previous studies have salience-matched either through subjective salience ratings or the skin conductance response (SCR). The construct of salience is not intuitive, and thus matching through self-report poses challenges. SCR is used as a proxy measure that captures physiological arousal, which overcomes the nebulous construct of salience. However, SCR cannot be used to salience-match in real-time (i.e., during an experiment) and assumes an association between salience and physiological arousal elicited by painful and non-painful stimuli, but this has not been explicitly tested. To determine whether salience and physiological arousal are associated, thirty-five healthy adults experienced 30 heat pain and 30 non-painful electric stimuli of varying intensities. Stimuli were subjectively matched for salience and SCR was measured to each presentation. A linear mixed model found no differences in SCR between salience-matched heat and electric stimuli. A mediation analysis showed that salience fully mediated the relationship between stimulus intensity and SCR (proportion mediated=83%). In conclusion, salience and physiological arousal are associated, and subjective salience ratings are a suitable for salience-matching pain with non-painful stimuli. Future work can thus use subjective salience ratings to delineate pain-specific processes.
Mechanism of neurodegeneration mediated by clonal inflammatory microglia
Authors: Vicario Rocio , Fragkogianni Stamatina , Pokrovskii Maria , Mayer Carina , Lopez-Rodrigo Estibaliz , Hu Yang , Ogishi Masato , Alberdi Araitz , Baako Ann , Ay Oyku , Plu Isabelle , Sazdovitch Véronique , Heritier Sebastien , Cohen-Aubart Fleur , Shor Natalia , Miyara Makoto , Nguyen-Khac Florence , Viale Agnes , Idbaih Ahmed , Amoura Zahir , Rosenblum Marc K. , Zhang Haochen , Karnoub Elias-Ramzey , Sashittal Palash , Jakatdar Akhil , Iacobuzio-Donahue Christine A. , Abdel-Wahab Omar , Tabar Viviane , Socci Nicholas D. , Elemento Olivier , Diamond Eli L , Boisson Bertrand , Casanova Jean-Laurent , Seilhean Danielle , Haroche Julien , Donadieu Jean , Geissmann Frederic
Published: July 2024

Langerhans cell Histiocytosis (LCH) and Erdheim-Chester disease (ECD) are clonal myeloid disorders, associated with MAP-Kinase activating mutations and an increased risk of neurodegeneration. Surprisingly, we found pervasive PU.1+microglia mutant clones across the brain of LCH and ECD patients with and without neurological symptoms, associated with microgliosis, reactive astrocytosis, and neuronal loss. The disease predominated in the grey nuclei of the rhombencephalon, a topography attributable to a local proliferative advantage of mutant microglia. Presence of clinical symptoms was associated with a longer evolution of the disease and a larger size of PU.1+clones (p= 0.0003). Genetic lineage tracing of PU.1+clones suggest a resident macrophage lineage or a bone marrow precursor origin depending on patients. Finally, a CSF1R-inhibitor depleted mutant microglia and limited neuronal loss in mice suggesting an alternative to MAPK inhibitors. These studies characterize a progressive neurodegenerative disease, caused by clonal proliferation of inflammatory microglia (CPIM), with a decade(s)-long preclinical stage of incipient disease that represent a therapeutic window for prevention of neuronal death.
Aggregation Dynamics of a 150 kDa Aβ42 Oligomer: Insights from Cryo Electron Microscopy and Multimodal Analysis
Authors: Kamalaldinezabadi S. Shirin , Watzlawik Jens O. , Rosenberry Terrone L. , Paravastu Anant K. , Stagg Scott M.
Published: July 2024

I.

Protein misfolding is a widespread phenomenon that can result in the formation of protein aggregates, which are markers of various disease states, including Alzheimer’s disease (AD). In AD, amyloid beta (Aβ) peptides, particularly Aβ40 and Aβ42, are key players in the disease’s progression, as they aggregate to form amyloid plaques and contribute to neuronal toxicity. Recent research has shifted attention from solely Aβ fibrils to also include Aβ protofibrils and oligomers as potentially critical pathogenic agents. Particularly, oligomers demonstrate greater toxicity compared to other Aβ specie. Hence, there is an increased interest in studying the correlation between toxicity and their structure and aggregation pathway. The present study investigates the aggregation of a 150 kDa Aβ42 oligomer that does not lead to fibril formation over time. Using negative stain transmission electron microscopy (TEM), size exclusion chromatography (SEC), dynamic light scattering (DLS), and cryo-electron microscopy (cryo-EM), we demonstrate that 150 kDa Aβ42 oligomers form higher-order string-like assemblies over time. The strings are unique from the classical Aβ fibril structures. The significance of our work lies in elucidating molecular behavior of a novel non-fibrillar form of Aβ42 aggregate.
The structural basis for the selective antagonism of soluble TNF-alpha by variable new antigen receptors
Authors: Ubah Obinna C. , Lake Eric W. , Priyanka Stella , Shi Ke , Moeller Nicholas H. , Porter Andrew J. , Aihara Hideki , LeBeau Aaron M. , Barelle Caroline J.
Published: July 2024

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF)-α is synthesized as transmembrane TNF-α (tmTNF-α) where proteolytic processing releases soluble TNF-α (sTNF-α). tmTNF-α can act as either a ligand by activating TNF receptors, or a receptor that transmits outside-to-inside signals (reverse signalling) after binding to native receptors. All TNF-α therapies bind tmTNF-α and induce reverse signalling which can result in immunosuppression leading to infection. We present crystal structures of two anti-TNF-α Variable New Antigen Receptors (VNAR) in complex with sTNF-αviatwo distinct epitopes. The VNAR-D1 recognized an epitope that selectively engaged sTNF-α while VNAR-C4 bound an epitope that overlapped with other biologic therapies. In activated CD4+T cells, our VNARs did not bind tmTNF-α in contrast to commercially available therapies that demonstrated induction of reverse signalling. Our findings suggest that neutralisation through a unique mechanism may lead to anti-TNF-α agents with an improved safety profile that will benefit high-risk patients.