bluuebunny/crossref_metadata_2025_split huggingface dataset:
Accounting for fast vs slow exchange in single molecule FRET experiments reveals hidden conformational states
Authors: Miller Justin J. , Mallimadugula Upasana L. , Zimmerman Maxwell I. , Stuchell-Brereton Melissa D. , Soranno Andrea , Bowman Gregory R. 
Published: June 2024

AbstractProteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles remains challenging, limiting our understanding of the relationships between sequence, structure, and function. Combining single molecule Förster resonance energy transfer (smFRET) experiments with molecular dynamics simulations could provide experimentally grounded, all-atom models of a protein’s structural ensemble. However, agreement between the two techniques is often insufficient to achieve this goal. Here, we explore whether accounting for important experimental details like averaging across structures sampled during a given smFRET measurement is responsible for this apparent discrepancy. We present an approach to account for this time-averaging by leveraging the kinetic information available from Markov state models of a protein’s dynamics. This allows us to accurately assess which timescales are averaged during an experiment. We find this approach significantly improves agreement between simulations and experiments in proteins with varying degrees of dynamics, including the well-ordered protein T4 lysozyme, the partially disordered protein apolipoprotein E (ApoE), and a disordered amyloid protein (Aβ40). We find evidence for hidden states that are not apparent in smFRET experiments because of time averaging with other structures, akin to states in fast exchange in NMR, and evaluate different force fields. Finally, we show how remaining discrepancies between computations and experiments can be used to guide additional simulations and build structural models for states that were previously unaccounted for. We expect our approach will enable combining simulations and experiments to understand the link between sequence, structure, and function in many settings.
The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale
Authors: Wayment-Steele Hannah K. , Otten Renee , Pitsawong Warintra , Ojoawo Adedolapo M. , Glaser Andrew , Calderone Logan A. , Kern Dorothee 
Published: June 2024

AbstractHow can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB fromRhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the “Ground” and “Fold-switched” (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is thecis-transisomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a “partially disordered” (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB’s landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the “Enigma” state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this “Enigma” state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.Significance StatementOne can conceptualize KaiB as an on-off switch to regulate circadian rhythms in bacteria, where the “On state” is the Fold-switched state that binds KaiC and other proteins, and the “Off state” is the Ground state. Our work exemplifies how evolution tuned the kinetics of interconversion to align with the hour-long timescale of its biological function. The Ground state is dramatically destabilized at cold temperatures, and the system contains an alternate “off” conformation that exchanges with the primary “off” conformation at faster timescales than the rate-limiting step. Our findings demonstrate a simple principle for evolving a protein switch: one part of a protein domain remains stably folded to serve as a scaffold for the rest of the protein to re-fold.
Reduced Non-Specific Binding of Super-Resolution DNA-PAINT Markers by Shielded DNA-PAINT Labeling Protocols
Authors: Lučinskaitė Evelina , Bokhobza Alexandre F. E. , Stannard Andrew , Meletiou Anna , Estell Chris , West Steven , Di Michele Lorenzo , Soeller Christian , Clowsley Alexander H. 
Published: June 2024

AbstractThe DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events ∼5 fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific background.
Artificial intelligence as a ploy to delve into the intricate relationship between genetics and mitochondria in MASLD patients
Authors: Longo Miriam , Paolini Erika , Meroni Marica , Ripolone Michela , Napoli Laura , Gentile Francesco , Cespiati Annalisa , Maggioni Marco , Alisi Anna , Miele Luca , Soardo Giorgio , Moggio Maurizio , Fracanzani Anna Ludovica , Dongiovanni Paola 
Published: June 2024

AbstractBackground and AimsMitochondrial (mt-) dysfunction is a hallmark of progressive MASLD. MtDNA copy number (mtDNA-CN) and cell-free circulating mtDNA (ccf-mtDNA), which reflect mt-mass and mt-dysfunction, respectively, are gaining attention as non-invasive disease biomarkers. We previously demonstrated thatPNPLA3/MBOAT7/TM6SF2deficiency in HepG2 cells increased mt-mass, mtDNA-CN and ccf-mtDNA. This study furtherly explored mt-biogenesis, function and mt-biomarkers in biopsied MASLD patients from a Discovery (n=28) and a Validation (n=824) cohort, stratified by the number of risk variants (NRV=3). We took advantage of artificial intelligence (AI) to develop new risk scores, predicting MASLD evolution by integrating anthropometric and genetic data (Age, BMI, NRV) with mt-biomarkers.MethodsHepatic mt-morphology and dynamics were assessed by TEM, IHC and gene expression. mtDNA-CN and ccf-mtDNA were measured in PBMCs and serum samples. GPT-4 was employed as AI tool to support the construction of novel risk scores for MASLD progressive forms (MASH, fibrosis and HCC).ResultsIn the Discovery cohort, NRV=3 patients showed the highest mt-mass and significant mt-morphological changes (i.e. membranes rupture). An elevated PGC-1α, OPA1, DRP1 and PINK1, markers of mt-biogenesis, fusion and fission were found in these patients, supporting an enhanced mt-dynamics. However, PRKN protein levels were reduced, suggesting a premature block of mitophagy. In the Validation cohort,PGC-1αmRNA levels and mtDNA-CN were significantly higher in NRV=3 compared to patients with 1,2 or no variants. Circulating mtDNA-CN and ccf-mtDNA were augmented in NRV=3 patients and correlated with genetics and MASLD severity at multivariate analysis, supporting that both may independently modulate mt-dynamics and activity. By exploiting rsGPT-4 we then optimized the combination of non-invasive variables to get prediction models named Mitochondrial, Anthropometric, and Genetic Integration with Computational intelligence (“MAGIC-“) for assessing MASH, fibrosis, and HCC, respectively. The MAGIC-MASH and MAGIC-Fib models showed AUCs of 73% and 76% in detecting MASH and fibrosis >1. Of note, MAGIC-HCC achieved an AUC of 86% (95% CI: 0.823-0.885), with 78.6% sensitivity and 81.5% specificity thus resulting the best score for the desired outcome.ConclusionsmtDNA-CN and ccf-mtDNA may have pathological and prognostic significance in MASLD patients, especially in those genetically-predisposed.
Phosphorothioate DNA modification by BREX Type 4 systems in the human gut microbiome
Authors: Yuan Yifeng , DeMott Michael S. , Byrne Shane R. , Flores Katia , Poyet Mathilde , Groussin Mathieu ,   Global Microbiome Conservancy, Berdy Brittany , Comstock Laurie , Alm Eric J. , Dedon Peter C. 
Published: June 2024

AbstractAmong dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen bydndandsspgene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possesseddndorsspgenes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis uncovered several putative new PT synthesis systems, including Type 4 Bacteriophage Exclusion (BREX)brxgenes, which were genetically validated inBacteroides salyersiae.Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessingdnd,ssp, andbrxgenes revealed 8 PT dinucleotide settings confirmed in 6 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.One-sentence SummaryApplication of informatic, mass spectrometric, and sequencing-based mapping tools to human gut bacteria revealed new phosphorothioate epigenetic systems widespread in the gut microbiome.
Single-nucleus transcriptomic analysis reveals the regulatory circuitry of myofiber XBP1 during regenerative myogenesis
Authors: Joshi Aniket S. , Castillo Micah B. , Tomaz da Silva Meiricris , Vuong Anh Tuan , Gunaratne Preethi H. , Darabi Radbod , Liu Yu , Kumar Ashok 
Published: June 2024

SUMMARYEndoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) are activated in skeletal muscle in multiple conditions. However, the role of the UPR in the regulation of muscle regeneration remains less understood. We demonstrate that gene expression of various markers of the UPR is induced in both myogenic and non-myogenic cells in regenerating muscle. Genetic ablation of XBP1, a downstream target of the IRE1α arm of the UPR, in myofibers attenuates muscle regeneration in adult mice. Single nucleus RNA sequencing (snRNA-seq) analysis showed that deletion of XBP1 in myofibers perturbs proteolytic systems and mitochondrial function in myogenic cells. Trajectory analysis of snRNA-seq dataset showed that XBP1 regulates the abundance of satellite cells and the formation of new myofibers in regenerating muscle. In addition, ablation of XBP1 disrupts the composition of non-myogenic cells in injured muscle microenvironment. Collectively, our study suggests that myofiber XBP1 regulates muscle regeneration through both cell-autonomous and -non-autonomous mechanisms.HIGHLIGHTSThe UPR is activated in different cell types during muscle regeneration.Targeted deletion of XBP1 impairs muscle regeneration in adult miceMyofiber XBP1 regulates satellite cell dynamics during regenerative myogenesisMyofiber XBP1 regulates abundance of non-myogenic cells in regenerating muscle
Insights into the Contribution of Type VI Secretion Towards AHPND Pathogenesis
Authors: Cavanagh Damian , Martinez Karina , Froelich Brett 
Published: June 2024

AbstractThe Type VI Secretion System (T6SS) is a bacterial organelle resembling a poison-tipped spear that can be deployed by a wide range of gram-negative bacteria to intoxicate prokaryotic and eukaryotic prey. In this capacity, Type VI Secretion Systems (T6SSs) play various roles in facilitating interbacterial competition and augmenting virulence towards animal and plant hosts. Prior reports have elucidated that many strains ofVibrio parahaemolyticusthat cause Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp harbor two functional T6SSs (T6SS1 and T6SS2), leading to speculations about the contribution of these systems to disease progression. In the present study, we demonstrate the antibacterial functionality of T6SS1 and T6SS2 in a representative VPAHPNDstrain, and provide direct evidence that T6SS1 augments the lethality of this isolate against whiteleg shrimp (Litopenaeus vannamei) postlarvae under conditions relevant to commercial aquaculture. Our findings provide early evidence that Type VI Secretion (T6S) plays a role in facilitating AHPND pathogenesis, and invites additional lines of inquiry aimed at uncovering the mechanisms responsible for this contribution.Author SummaryType VI Secretion Systems (T6SSs) have drawn interest as important determinants of virulence and fitness in the bacterial taxa that utilize them. Included among these species are members of the genusVibrio, many of which have clinical and commercial relevance as pathogens or aquaculture pests.Vibrio parahaemolyticusisolates that harbor thepirA/Btoxin genes encoded on pVA1-like plasmids have been identified as a causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND), an illness which results in mass mortality events in shrimp aquaculture systems. It has previously been noted that, aside from the PirA/B binary toxin, many VPAHPNDstrains harbor two T6SSs (T6SS1 and T6SS2), which they may rely on to outcompete and displace the native shrimp microbiome during colonization. In this study, we have directly assessed the effects of inactivating T6SS1 and T6SS2 on the lethality of a representative VPAHPNDisolate againstL. vannameipostlarvae (PL) using a simple infection assay. Our findings indicate that T6SS1 putatively contributes to the pathogenicity of VPAHPNDagainstL. vannameiPL under warm, marine-like conditions. These initial results help to provide insight into the factors which enable AHPND pathogenesis, and may inform future efforts to develop countermeasures against this disease.
Bhlhe40 limits proliferation and prevents excessive angiogenesis in mouse embryoid bodies under hypoxia
Authors: Acosta-Iborra Barbara , Gil-Acero Ana Isabel , Sanz-Gómez Marta , Berrouayel Yosra , Puente-Santamaría Laura , Alieva Maria , del Peso Luis , Jiménez Benilde 
Published: June 2024

AbstractKnowledge of the molecular mechanisms that underlie the regulation of the major adaptive responses to an unbalanced oxygen tension is central to understanding tissue homeostasis and disease. Hypoxia-inducible transcription factors (HIFs) coordinate changes in the transcriptome that control these adaptive responses. Here, we focused on the functional role of the transcriptional repressor basic-helix-loop-helix family member e40 (Bhlhe40), which we previously identified in a meta-analysis as one of the most consistently up-regulated genes in response to hypoxia across various cell types. We investigated the role of Bhlhe40 in controlling proliferation and angiogenesis using a gene editing strategy in mouse embryonic stem cells (mESCs) that we differentiated in embryoid bodies (EBs). We observed that hypoxia-induced Bhlhe40 expression was compatible with the rapid proliferation of pluripotent mESCs under low oxygen tension. However, in EBs, hypoxia triggered a Bhlhe40-dependent cell cycle arrest in most progenitor cells and endothelial cells within vascular structures. Furthermore, Bhlhe40 knockout increased the basal vascularization of the EBs in normoxia and exacerbated the hypoxia-induced vascularization, supporting a novel role for Bhlhe40 as a negative regulator of blood vessel formation. Our findings implicate Bhlhe40 in mediating key functional adaptive responses to hypoxia, such as proliferation arrest and angiogenesis.
Exploring memory-related network via dorsal hippocampus suppression
Authors: Han Xu , Cramer Samuel R. , Chan Dennis C.Y. , Zhang Nanyin 
Published: June 2024

AbstractMemory is a complex brain process that requires coordinated activities in a large-scale brain network. However, the relationship between coordinated brain network activities and memory-related behavior is not well understood. In this study, we investigated this issue by suppressing the activity in the dorsal hippocampus (dHP) using chemogenetics and measuring the corresponding changes in brain-wide resting-state functional connectivity (RSFC) and memory behavior in awake rats. We identified an extended brain network contributing to the performance in a spatial-memory related task. Our results were cross-validated using two different chemogenetic actuators, clozapine (CLZ) and clozapine-N-oxide (CNO). This study provides a brain network interpretation of memory performance, indicating that memory is associated with coordinated brain-wide neural activities.Significance StatementSuccessful memory processes require coordinated activity in a large-scale brain network, extending beyond a few key, well-known brain regions like the hippocampus. However, the specific brain regions involved and how they orchestrate their activity that is pertinent to memory processing remain unclear. Our study, using a chemogenetics-rsfMRI- behavior approach in awake rats, elucidates a comprehensive framework of the extended memory-associated network. This knowledge offers a broader interpretation of memory processes, enhancing our understanding of the neural mechanisms behind memory function, particularly from a network perspective.
APOE Christchurch enhances a disease-associated microglial response to plaque but suppresses response to tau pathology
Authors: Tran Kristine M. , Kwang Nellie , Gomez-Arboledas Angela , Kawauchi Shimako , Mar Cassandra , Chao Donna , Da Cunha Celia , Wang Shuling , Collins Sherilyn , Walker Amber , Shi Kai-Xuan , Alcantara Joshua A. , Neumann Jonathan , Tenner Andrea J. , LaFerla Frank M. , Hohsfield Lindsay A. , Swarup Vivek , MacGregor Grant R. , Green Kim N. 
Published: June 2024

ABSTRACTBackgroundApolipoprotein E ε4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer’s disease (LOAD). A recent case report identified a rare variant in APOE, APOE3-R136S (Christchurch), proposed to confer resistance to autosomal dominant Alzheimer’s Disease (AD). However, it remains unclear whether and how this variant exerts its protective effects.MethodsWe introduced the R136S variant into mouseApoe(ApoeCh) and investigated its effect on the development of AD-related pathology using the 5xFAD model of amyloidosis and the PS19 model of tauopathy. We used immunohistochemical and biochemical analysis along with single-cell spatial transcriptomics and proteomics to explore the impact of theApoeChvariant on AD pathological development and the brain’s response to plaques and tau.ResultsIn 5xFAD mice,ApoeChenhances a Disease-Associated Microglia (DAM) phenotype in microglia surrounding plaques, and reduces plaque load, dystrophic neurites, and plasma neurofilament light chain. By contrast, in PS19 mice,ApoeChsuppresses the microglial and astrocytic responses to tau-laden neurons and does not reduce tau accumulation or phosphorylation, but partially rescues tau-induced synaptic and myelin loss. We compared how microglia responses differ between the two mouse models to elucidate the distinct DAM signatures induced byApoeCh. We identified upregulation of antigen presentation-related genes in the DAM response in a PS19 compared to a 5xFAD background, suggesting a differential response to amyloid versus tau pathology that is modulated by the presence ofApoeCh.ConclusionsThese findings highlight the ability of theApoeChvariant to modulate microglial responses based on the type of pathology, enhancing DAM reactivity in amyloid models and dampening neuroinflammation to promote protection in tau models. This suggests that the Christchurch variant’s protective effects likely involve multiple mechanisms, including changes in receptor binding and microglial programming.
Machine learning elucidates electrophysiological properties predictive of multi- and single-firing human and mouse dorsal root ganglia neurons
Authors: Zurek Nesia A. , Thiyagarajan Sherwin , Ehsanian Reza , Goins Aleyah E. , Goyal Sachin , Shilling Mark , Lambert Christophe G. , Westlund Karin N. , Alles Sascha R.A. 
Published: June 2024

AbstractHuman and mouse dorsal root ganglia (hDRG and mDRG) neurons are important tools in understanding the molecular and electrophysiological mechanisms that underlie nociception and drive pain behaviors. One of the simplest differences in firing phenotypes is that neurons are single-firing (exhibit only one action potential) or multi-firing (exhibit 2 or more action potentials). To determine if single- and multi-firing hDRG exhibit differences in intrinsic properties, firing phenotypes, and AP waveform properties, and if these properties could be used to predict multi-firing, we measured 22 electrophysiological properties by whole-cell patch-clamp electrophysiology of 94 hDRG neurons from 6 male and 4 female donors. We then analyzed the data using several machine learning models to determine if these properties could be used to predict multi-firing. We used 1000 iterations of Monte Carlo Cross Validation to split the data into different train and test sets and tested the Logistic Regression, k-Nearest Neighbors, Random Forest, Supported Vector Classification, and XGBoost machine learning models. All models tested had a greater than 80% accuracy on average, with Supported Vector Classification and XGBoost performing the best. We found that several properties correlated with multi-firing hDRG neurons and together could be used to predict multi-firing neurons in hDRG including a long decay time, a low rheobase, and long first spike latency. We also found that the hDRG models were able to predict multi-firing with 90% accuracy in mDRG. Targeting the neuronal properties that lead to multi-firing could elucidate better targets for treatment of chronic pain.