bluuebunny/crossref_metadata_2025 huggingface dataset:
Community-based causal evidence that high habitual caffeine consumption alters distinct polysomnography-derived sleep variables
Authors: Stucky Benjamin , Henckel Leonard , Maathuis Marloes H , Haba-Rubio Jose , Marques-Vidal Pedro , Siclari Francesca , Heinzer Raphael , Landolt Hans-Peter
Published: December 2024

Caffeine is the most widely consumed psychoactive substance in the World and acutely impairs sleep quality when studied in the laboratory. The repercussions on sleep of chronic caffeine intake, which is common in society, are unknown. Because adequate sleep is important for general health, we explored causal effects of habitual caffeine intake on objective and subjective sleep quality with two-sample Mendelian Randomization and matching methods. We analyzed large community-based datasets taken from the UK Biobank (n=485,511) and HypnoLaus (n=1,702) cohorts. We consistently show that intake of four or more caffeinated beverages daily shortens total sleep time compared to three or fewer caffeinated drinks per day. While we found shorter sleep with all models, the estimated reductions varied from 11-229 minutes, likely due to modest instrument strengths. A concurrent increase in sleep intensity indicates at least partial compensation. The findings suggest that high daily caffeine intake causally alters sleep patterns in the general population.
Dopamine regulates the membrane potential and glycine release of AII amacrine cells via D1-like receptor modulation of gap junction coupling
Authors: Strazza Junior Paulo , Wakeham Colin M , von Gersdorff Henrique
Published: December 2024

Dopamine plays a pivotal role in adjusting the flow of information across the retina as luminance changes from night to day. Here we show, under dim photopic conditions, that both dopamine and a D1-like receptor (D1R) agonist hyperpolarized the resting membrane potential (Vm) of AII amacrine cells (AII-ACs). Surprisingly, in the presence of glutamatergic and GABAergic synaptic blockers that isolate glycinergic synapses, D1R agonists are without effect. However, a D1R antagonist depolarized Vmand reduced the input resistance of AII-ACs in wild type mice, but not inCx36−/−mice. Accordingly, D1R antagonists enhanced tonic glycinergic transmission to type-2 OFF-cone bipolar cells (OFF-CBCs). D1Rs thus adjust the Vmand excitability of AII-ACs and, thereby, the level of glycine release to OFF-CBCs by regulating gap junction coupling with ON cone bipolar cells. Our findings provide insights into how the retina may use dopamine to adapt crossover inhibitory microcircuits during changes in luminance.
The applying of immuno-RCA for the high-sensitivity detection of the ABO blood group antibodies on the printed glycoarray
Authors: Kornilova E.E. , Kutukov R.R. , Polyakova S.M. , Nokel A.Yu. , Zavriev S.K. , Ryazantsev D.Yu.
Published: December 2024

Detecting small amounts of analytes, especially antibodies, presents a significant challenge in high-throughput methods. Fortunately, nucleic acid amplification techniques provide a promising solution. We have successfully developed and tested an innovative technology for detecting anti-glycan antibodies, utilizing a printed glycan array combined with a rolling circle amplification reaction based on the ABO blood group antibody model. This breakthrough has dramatically enhanced the sensitivity of our immunoassay, improving it by over an order of magnitude and allowing us to detect concentrations as low as 1 ng/ml. This advancement opens new approaches for research and clinical applications, making previously undetectable analytes accessible for study.
Chromosomal plasticity can drive rapid adaptation in bacteria
Authors: Vega-Cabrera Luis , Skovgaard Ole , Toll-Riera Macarena
Published: December 2024

Understanding the molecular mechanisms underlying rapid adaptation to stress is a fundamental question in evolutionary biology. A common mechanism is copy number changes, which can affect single genes, larger genomic regions, or even entire chromosomes in the case of eukaryotes (aneuploidy). Bacterial species with genomes comprising more than one chromosome are common, yet the role of adaptive copy number changes affecting entire chromosomes is unknown. Here, by using a bacterium with two chromosomes (chrI and chrII) as model system and combining evolution experiments in the presence of severe nutrient stress with whole-genome sequencing, we show that chromosomal copy number changes can drive rapid adaptation. We detected increased copy number of the entire chrII in almost half of the sequenced clones, isolated from twelve independent populations. In most cases, copy number increases were driven by mutations in the replication protein of chrII. A higher chrII copy number is likely to be beneficial because chrII codes for the operon responsible for metabolizing D-gluconic acid, the only carbon source present in the medium. Our results revealed a novel mechanism for rapid adaptation in bacteria. Given the prevalence of bacteria with secondary chromosomes, and the enrichment of secondary chromosomes in genes associated with interaction with the environment, this may be an important mechanism for adapting to novel environments that has been overlooked.
High cumulative viral titers of influenza virus in animals with significant disease fatality rates indicate a potential trade-off between fatality and transmissibility
Authors: Gouveia Ayrton S. , Mata Leonardo G. , Villela Daniel A.M.
Published: December 2024

Evaluating the trade-off hypothesis for the evolution of virulence using empirical data poses significant challenges. The hypothesis suggests that pathogens evolve to maximize transmissibility, but fatality imposes limits as there are diminishing gains in transmissibility. In this study, we analyzed a secondary dataset of influenza virus infections in ferrets (Mustela putorius furo), categorized by Hemagglutinin (HA) and Neuraminidase (NA) subtypes. Subgroups defined by the H7/N9 and H7/N7 combinations exhibited fatality rates of approximately 30% and reached cumulative viral titers close to 7.5 (log10titer/mL). These levels represent intermediate fatality rates, as the H5/N6 and H5/N1 subgroups had higher fatality rates but reached lower cumulative viral titers. Using cumulative viral titer as a proxy for potential secondary transmissions, the analysis suggests that intermediate fatality rates are associated with higher numbers of secondary transmissions. However, there are significant uncertainties in subgroups with lower or no fatalities. Additionally, subgroups without fatalities showed substantial variability in cumulative viral titers.
Comprehensive expression data for two honey bee species,Apis melliferaandApis cerana japonica
Authors: Yokoi Kakeru , Hatakeyama Masatsugu , Kuwazaki Seigo , Maeda Taro , Yoshiyama Mikio , Horigane-Ogihara Mari , Matsuyama Shigeru , Jouraku Akiya , Bono Hidemasa , Kimura Kiyoshi
Published: December 2024

Comprehensive expression datasets were constructed forApis melliferaandApis cerana japonica. Post-oviposition day 6 to day 58 samples ofA. melliferaworkers (larva to adult); day 9, 10, 12, and 13 samples ofA. melliferaqueen (larva to pupa); and day 9 to day 18 samples ofA. cerana japonicaworkers (larva to adult) were prepared, and RNA-Seq data were obtained. ForA. cerana japonica, reference transcript sequence data, predicted amino acid sequence data, and functional annotation data were generated based on the genome sequence and RNA-Seq data. Using the transcript sequence and RNA-Seq data, comprehensive expression data for all transcripts ofA. mellifera and A. cerana japonicawere prepared. Hierarchical clustering analyses and the used sample preparation method ensured that both sets of expression data were reliable for use as comprehensive reference expression datasets. Therefore, these data are applicable for honey bee research or for comparative or evolutionary studies on insect species or social insect species at the genetic and molecular levels.
Classification of Visual Imagery and Imagined Speech EEG based Brain Computer Interfaces using 1D Convolutional Neural Network
Authors: Le Diane
Published: December 2024

Non-invasive brain-computer interfaces (BCI) utilising electroencephalogram (EEG) signals are a current popular, affordable and accessible method for establishing communication paths between the mind and external devices. However, the challenges faced are inter-subject variability, BCI illiteracy and poor machine learning decoding performance. Two emerging intuitive mental paradigms, Visual Imagery (VI) and Imagined Speech (IS) show promise to optimise the development of non-invasive BCIs, which involves the extraction of corresponding neural patterns during the imagined tasks. This study took a comprehensive user-centric approach to build on the current foundation of knowledge on VI and IS EEG-BCIs utilising an adapted 1D-CNN to optimise the classification decoding performance. Twenty healthy participants were assessed for their ability to visualise imagery in their minds and performed the VI and IS mental paradigms in two class conditions “push” and “relax”. It was shown that alpha and beta suppression was observed during the “push” condition of VI compared to the “relax” condition, and those that scored higher in the VVIQ had better VI classification accuracy than those who did not. The adapted 1D-CNN model performed well for classification between the two classes “push” and “relax” at 89.3% and 77.87% performance accuracy for VI and IS, respectively. These findings contribute to the current body of work on VI BCI, that it is a dynamic and plausible option compared to standard BCI paradigms, and VI BCI illiteracy could potentially be controlled by VVIQ. It also demonstrated the potential of the 1D-CNN model in classification of VI and IS EEG-BCIs.
CRISPR-Edited DPSCs, Constitutively Expressing BDNF Enhance Dentin Regeneration in Injured Teeth
Authors: Kim Ji Hyun , Irfan Muhammad , Sreekumar Sreelekshmi , Kim Stephanie , Phimon Atsawasuwan , Chung Seung
Published: December 2024

Dental caries is one of the most common health issues worldwide arising from the complex interactions of bacteria. In response to harmful stimuli, desirable outcome for the tooth is the formation of tertiary dentin, a protective reparative process that generates new hard tissue. This reparative dentinogenesis is associated with significant inflammation, which triggers the recruitment and differentiation of dental pulp stem cells (DPSCs). Previously, we have shown that brain-derived neurotrophic factor (BDNF) and its receptor TrkB, key mediators of neural functions, are activated during the DPSC-mediated dentin regeneration process. In this study, we further define the role of inflammation in this process and apply stem cell engineering to enhance dentin regeneration in injured teeth. Our data show that TrkB expression and activation in DPSCs rapidly increase during odontogenic differentiation, further amplified by inflammatory inducers and mediators such as TNFα, LTA, and LPS. An in vivo dentin formation assessment was conducted using a mouse pulp-capping/caries model, where CRISPR-engineered DPSCs overexpressing BDNF were transplanted into inflamed pulp tissue. This transplantation significantly enhanced dentin regeneration in injured teeth. To further explore potential downstream pathways, we conducted transcriptomic profiling of TNFα-treated DPSCs, both with and without TrkB antagonist CTX-B. The results revealed significant changes in gene expression related to immune response, cytokine signaling, and extracellular matrix interactions. Taken together, our study advances our understanding of the role of BDNF in dental tissue engineering using DPSCs and identifies potential therapeutic avenues for improving dental tissue repair and regeneration strategies.
Universal Study Design for Instrument Changes in Pharmaceutical Release Analytics
Authors: Ries Anne B. , Merkel Maximilian N. , Coßmann Kristina , Paul Marina , Grunwald Robin , Klemmer Daniel , Hübner Franziska , Eggensperger Sabine , Weiß Frederik T.
Published: December 2024

Instrument changes in analytical methods of pharmaceutical quality control are required to maintain release analytics over decades, yet typically pose a challenge.

We designed an efficient instrument comparability study to gain a comprehensive understanding of potential performance differences between instruments and therefore rationalize the risk assessment and decision process for a path forward. The results may either point out whether a full or partial re-validation is necessary or whether a science-based bridging can be pursued based on the data generated in the study. The study design is universally applicable to a substantial range of release analytical methods. In a straightforward setup of two experiments with the new instrument, a statistically meaningful data set is generated for comparison with available historical or validation data of the original instrument.

In a Good Manufacturing Practice (GMP) environment, we realized the study design a first benchmark in imaged capillary isoelectric focusing (icIEF) analytics, comparing the ICE3 and Maurice C instruments. The core-study confirmed equal or better performance of Maurice C in all parameters and serves as a basis for seamless continuation of release measurements on Maurice C.
14-3-3ε inhibits premature centriole disengagement by inhibiting the activity of Plk1 and Separase
Authors: Jaiswal Monika A. , Karn Akshay , Das Aparna , Kumari Anisha , Tiwari Shilu , Dalal Sorab N.
Published: December 2024

The 14-3-3 protein family regulates several pathways in mammalian cells, including centrosome duplication. However, the precise mechanisms by which 14-3-3 paralogs regulate the centrosome cycle remain unclear. To identify the mechanisms by which 14-3-3ε regulates centrosome duplication, we altered two conserved acidic residues in the 14-3-3ε phospho-peptide-binding pocket that regulate complex formation and dissociation with the associated ligands, D127 and E134, to Alanine. Altering these residues to Alanine led to opposing effects on centrosome duplication; the D127A mutant inhibited centrosome duplication, while cells expressing the E134A mutant showed the presence of supernumerary centrosomes. We demonstrate that 14-3-3ε does not inhibit centriole duplication, as reported for 14-3-3γ, but inhibits centriole disengagement. Using a combination of pharmacological and genetic approaches, we demonstrate that 14-3-3ε inhibits the activity of Plk1 and Separase, leading to disengagement defects that ultimately lead to decreased proliferation and cell death. Our work demonstrates that different 14-3-3 paralogs regulate different steps in the centrosome cycle, and disrupting complex formation between 14-3-3ε and Plk1 or Separase could be a novel therapeutic strategy in tumor cells.
Pan-cancerN-glycoproteomic atlas of patient-derived xenografts uncovers FAT2 as a therapeutic target for head and neck cancers
Authors: Govindarajan Meinusha , Mejia-Guerrero Salvador , Chafe Shawn C , Khan Shahbaz , Shi Wei , Waas Matthew , Khoo Amanda , Liu Lydia Y , Ignatchenko Vladimir , Principe Simona , Sepiashvili Lusia , Tatari Nazanin , Venugopal Chitra , Miletic Petar , Topley Maxwell , Grewal Shan , McKenna Dillon , Sandi Maria-Jose , Pham Nhu-An , Casey Alison , Kim Hyeyeon , Karamboulas Christina , Meens Jalna , Bergqvist Peter , Silva Begonia , Chan Patrick , Cerna-Portillo Liza , Chin Jasmine , Rao-Bhatia Abilasha , Tsao Ming-Sound , Khokha Rama , Su Susie , Xu Wei , Goldstein David , Ailles Laurie , Stambolic Vuk , Liu Fei-Fei , Cummins Emma , Samudio Ismael , Singh Sheila K , Kislinger Thomas
Published: December 2024

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central role in cellular signaling. Despite this, their promise remains largely untapped due to the technical challenges inherent to profiling cell surface proteins. Here, we employedN-glycoproteomics to analyze 85 patient-derived xenografts (PDX), constructing Glyco PDXplorer – anin vivopan-cancer atlas of cancer-derived cell surface proteins. We developed a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validated FAT2 as a head and neck squamous cancer (HNSC) enriched surface protein with limited expression in normal tissue. Functional studies revealed that FAT2 is essential for HNSC growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR) T cells targeting FAT2 demonstrated potent anti-tumor activity in HNSC models. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal resource to inform therapeutic target discovery for multiple cancers.

HIGHLIGHTS

Pan-cancer landscape of cancer-derived cell surface proteins detectedin vivo

Development of a multi-omic discovery pipeline to prioritize proteins with optimal expression profiles as immunotherapy targets

Identification and validation of FAT2 as a head and neck squamous cancer enriched surface protein with minimal expression in normal tissues

FAT2 coordinates cell surface organization, adhesion, growth and survival through the integrin-PI3K-AKT pathway

FAT2 CAR T cells demonstrate anti-tumour activity in pre-clinical models
Long-read Transcriptomics of Caviid Gammaherpesvirus 1: Compiling a Comprehensive RNA Atlas
Authors: Torma Gábor , Dörmő Ákos , Fülöp Ádám , Tombácz Dóra , Mizik Máté , Pretory Amanda M. , Lee See-Chi , Toth Zsolt , Boldogkői Zsolt
Published: December 2024

Caviid gammaherpesvirus 1 (CaGHV-1), formerly known as the guinea pig herpes-like virus, is an oncogenic gammaherpesvirus with a sequenced genome but an as-yet uncharacterized transcriptome. Using nanopore long-read RNA sequencing, we annotated the CaGHV-1 genome and constructed a detailed transcriptomic atlas. Our findings reveal diverse viral mRNAs and non-coding RNAs, along with mapped promoter elements for each viral gene. We demonstrated that the CaGHV-1 RTA lytic cycle transcription factor activates its own promoter, similar to KSHV, and that the CaGHV-1 ORF50 promoter responds to RTA proteins from other gammaherpesviruses, highlighting the evolutionary conservation of RTA-mediated transcriptional mechanisms. Additionally, our analysis uncovered extensive transcriptional overlap within the viral genome, suggesting a role in regulating global gene expression. Given its tumorigenic properties, broad host range, and non-human pathogenicity, this work establishes CaGHV-1 as a promising small animal model for investigating human gammaherpesvirus pathogenesis.

IMPORTANCE

The molecular underpinnings of gammaherpesvirus pathogenesis remain poorly understood, partly due to limited animal models. This study provides the first comprehensive transcriptomic atlas of CaGHV-1, highlighting both coding and non-coding RNAs and revealing regulatory elements that drive viral gene expression. Functional studies of the CaGHV-1 RTA transcription factor demonstrated its ability to self-activate and cross-activate promoters from homologous gammaherpesviruses, reflecting conserved mechanisms of transcriptional control. These findings solidify CaGHV-1 as a unique and versatile small animal model, offering new opportunities to investigate gammaherpesvirus replication, transcriptional regulation, and tumorigenesis in a controlled experimental system.
Data-driven synapse classification reveals a logic of glutamate receptor diversity
Authors: Micheva Kristina D. , Simhal Anish K. , Schardt Jenna , Smith Stephen J , Weinberg Richard J. , Owen Scott F.
Published: December 2024

The rich diversity of synapses facilitates the capacity of neural circuits to transmit, process and store information. We used multiplex super-resolution proteometric imaging through array tomography to define features of single synapses in mouse neocortex. We find that glutamatergic synapses cluster into subclasses that parallel the distinct biochemical and functional categories of receptor subunits: GluA1/4, GluA2/3 and GluN1/GluN2B. Two of these subclasses align with physiological expectations based on synaptic plasticity: large AMPAR-rich synapses may represent potentiated synapses, whereas small NMDAR-rich synapses suggest “silent” synapses. The NMDA receptor content of large synapses correlates with spine neck diameter, and thus the potential for coupling to the parent dendrite. Overall, ultrastructural features predict receptor content of synapses better than parent neuron identity does, suggesting synapse subclasses act as fundamental elements of neuronal circuits. No barriers prevent future generalization of this approach to other species, or to study of human disorders and therapeutics.
Search for a genetic cause of variably protease-sensitive prionopathy
Authors: Lian Yuan , Kotobelli Keisi , Hall Stacey , Talkowski Michael E , O’Donnell-Luria Anne , Vallabh Sonia M , Appleby Brian S , Minikel Eric Vallabh
Published: December 2024

Variably protease-sensitive prionopathy (VPSPr) is a rare, atypical subtype of prion disease in which many patients exhibit a family history of dementia. Rare protein-coding variants inPRNP, which are causal for all known forms of genetic prion disease, have been ruled out in all VPSPr cases to date, leading to suspicion that VPSPr could be caused by variants in other genes or by non-coding variation in or nearPRNP. We performed exome sequencing and targeted sequencing ofPRNPnon-coding regions on genomic DNA from autopsy-confirmed VPSPr patients (N=67) in order to search for a possible genetic cause. Our search identified no potentially causal variants for VPSPr. The common polymorphismPRNPM129V was the largest genetic risk factor for VPSPr, with an odds ratio of 7.0. Other variants in and nearPRNPexhibited association to VPSPr risk only in proportion to their linkage disequilibrium with M129V, and upstream expression quantitative trait loci showed no evidence of independent association to VPSPr risk. We cannot rule out the possibility of causal variants hiding in regions or classes of genetic variation that our search did not canvas. Nevertheless, our data support the classification of VPSPr as a sporadic prion disease.

Author Summary

Prion disease is caused by misfolding of the prion protein (PrP), and can be either sporadic genetic, or acquired. Acquired cases arising from infection through dietary or medical routes are exceedingly rare today (<1% of cases). Sporadic cases occur apparently at random, without any major genetic risk factors, and are not passed down to subsequent generations. All cases of genetic prion disease to date have been traced to DNA changes that alter the amino acid sequence of PrP, and children of people with genetic prion disease are at 50/50 risk of inheriting these autosomal dominant DNA changes. Variably protease sensitive prionopathy, or VPSPr, is a rare and unusual subtype of prion disease, in which there are no changes in PrP’s amino acid sequence, and yet, a high proportion of patients appear to have a family history of dementia, although none to date have had family members diagnosed with prion disease specifically. VPSPr is currently categorized as a sporadic prion disease, but it has been speculated that it may have a genetic cause in a different gene or in a region of the PrP gene that does not change the amino acid sequence. Here we performed DNA sequencing on 67 VPSPr cases to search for a genetic cause. We found no DNA changes that could potentially cause VPSPr. While it is difficult to prove the negative — a causal genetic change could still exist in some part of the genome where we did not search — our data support the notion that VPSPr is truly sporadic in nature, and that risk of VPSPr is not transmitted in families.
Integrating patient experiences of healthcare: a critical realist thematic analysis of peri-operative experiences
Authors: Meigh Neil , O’Callaghan Liam , Goldsworthy Adrian , Allan Courtney , Stokes Alison , Mickan Sharon
Published: December 2024

Background

The perioperative experience is central to how patients perceive and navigate their care. While existing research has explored broad themes, the nuanced interplay of emotional, psychological, and clinical dimensions remains underexamined. Building on a recent scoping review, this qualitative study sought to explore perioperative patient experiences in greater depth, investigating both pre-established themes and emergent insights.

Methods

Patients were invited to share their perioperative journeys through semi-structured interviews, offering detailed accounts of their lived experiences. Using a Critical Realist approach, the study examined both observable factors and underlying mechanisms shaping these experiences. By integrating pre-established themes with participants’ narratives, the study constructed a comprehensive framework reflecting the complexity of patient-reported experiences.

Analysis

Four central themes were identified: (1) psychological resilience as a central pillar for patient experience, (2) empowerment and information flow as drivers of patient experience, (3) personalised care as the foundation for patient-centred experience, and (4) integrated care and pain management as central to patient experience. Additionally, social influences and financial barriers emerged as factors shaping the broader context of these experiences, though they were less interconnected with the core themes.

Discussion

This study underscores the multifaceted nature of perioperative patient experiences, where emotional resilience, empowerment, and personalised care are deeply intertwined. These findings highlight the need for health systems and healthcare professionals to address emotional and contextual factors alongside clinical dimensions to improve the overall patient experience.

Conclusion

By prioritising the exploration of patient-reported perioperative experiences, this study provides critical insights into the emotional, psychological, and clinical elements that shape care. The integration of these dimensions offers practical guidance for developing patient-centred approaches that align with individuals’ needs and expectations.
SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis
Authors: Zhang Shuai , Wang Zhiying , Jiao Yutong , Shi Xiaoyu , Ai Yuanbao , Wang Juanping , Liu Sen
Published: December 2024

Upregulation of protein SUMOylation is associated with various diseases, and SUMOylation inhibitors are promising drug candidates. We performed the first virtual screening of SUMO E1 covalent allosteric inhibitors (CAIs) and identified two SUMO E1 CAIs with new scaffolds and covalent warheads. We further demonstrated that these new CAIs perturbed the SUMOylation pathway and protein SUMOylation. Specifically, these CAIs affected the SUMOylation of the methionine adenosyltransferases MAT2A. The inhibition of MAT2A SUMOylation unexpectedly stimulated polyamine synthesis. Lastly, we showed that the combination of SUMO E1 CAIs with a polyamine synthesis inhibitor had synergistic effects in inhibiting T47D cells. Our work demonstrated the first cost-effective virtual screening of SUMO E1 CAIs, found that the downregulation of MAT2A SUMOylation increases polyamine synthesis, and SUMO E1 CAIs can synergize with polyamine synthesis inhibitors. This work would be of great value to the study of SUMOylation, covalent/allosteric drugs, and the polyamine metabolism network.
Inferring gene-pathway associations from consolidated transcriptome datasets: an interactive gene network explorer forTetrahymena thermophila
Authors: Bertagna Michael A. , Bright Lydia J. , Ye Fei , Jiang Yu-Yang , Sarkar Debolina , Pradhan Ajay , Kumar Santosh , Gao Shan , Turkewitz Aaron P. , Tsypin Lev M. Z.
Published: December 2024

Although an established model organism, Tetrahymena thermophilaremains comparatively inaccessible to high throughput screens, and alternative bioinformatic approaches still rely on unconnected datasets and outdated algorithms. Here, we report a new approach to consolidating RNA-seq and microarray data based on a systematic exploration of parameters and computational controls, enabling us to infer functional gene associations from their co-expression patterns. To illustrate the power of this approach, we took advantage of new data regarding a previously studied pathway, the biogenesis of a secretory organelle called the mucocyst. Our untargeted clustering approach recovered over 80% of the genes that were previously verified to play a role in mucocyst biogenesis. Furthermore, we tested four new genes that we predicted to be mucocyst-associated based on their co-expression and found that knocking out each of them results in mucocyst secretion defects. We also found that our approach succeeds in clustering genes associated with several other cellular pathways that we evaluated based on prior literature. We present theTetrahymenaGene Network Explorer (TGNE) as an interactive tool for genetic hypothesis generation and functional annotation in this organism and as a framework for building similar tools for other systems.

Key Points

ur approach integrates nearly 20-year-old microarray and contemporary RNA-seq datasets.

rigorously compare co-expression clustering parametrization by way of computational controls.

Co-expression clustering identifies known and novel functionally associated genes inTetrahymena.

Graphical Abstract
Morphological and Molecular Phylogenetic Characterization of Three New Marine Goniomonad Species
Authors: Phanprasert Yasinee , Kim Sun Young , Kang Nam Seon , Jeong Minseok , Kim Jong Im , Shin Woongghi , Lee Won Je , Kim Eunsoo
Published: December 2024

Goniomonads are commonly found heterotrophic biflagellates in both marine and freshwater environments. Despite the high genetic diversity inferred from 18S rDNA data, many goniomonad species remain undescribed. In this study, we established a total of 21 marine goniomonad culture strains, and from these, describe three new species by using light microscopy, electron microscopy, and 18S rDNA phylogeny. Molecular sequence analyses suggest the presence of several distinct sub-lineages within the marine goniomonad clade. Two of these areGoniomonas ulleungensissp. nov. andG. linguasp. nov., which are similar in size, flagellar length, appendage & orientation and by having a tongue-like protrusion at the anterior. The two species can be differentiated by the periplast plate pattern withG. ulleungensisdisplaying one additional plate on the right side.G. duplexsp. nov. differed from these two species by having two unequal flagella with the longer one trailing posteriorly and having the opposite cell orientation when skidding. Comparative analyses of five marine goniomonad species showed that genetically distinct goniomonad groups can be delineated by morphological data as well, and of several morphological features that are of taxonomic utility, the periplast plate pattern, observable by SEM, is particularly informative in goniomonad taxonomy.
Sexual dimorphism in molecular profiles of resting human skeletal muscle and the response to acute exercise and endurance training
Authors: Dreher Simon , Goj Thomas , von Toerne Christine , Hoene Miriam , Irmler Martin , Ouni Meriem , Jähnert Markus , Beckers Johannes , Hrabe de Angelis Martin , Peter Andreas , Moller Anja , Birkenfeld Andreas L. , Schürmann Annette , Hauck Stefanie M. , Weigert Cora
Published: December 2024

Biological sex has a strong impact on skeletal muscle metabolism and performance. By a comprehensive investigation of epigenetic, transcriptomic and proteomic differences between female and male skeletal muscle of untrained subjects we provide a molecular basis for the sexual dimorphism of glucose and lipid metabolism. The sex-specific multi-OMICs profiles indicate higher degree of glucose turnover and higher abundance of fast-twitch fibers in males and high degree of lipid handling in females. Eight-week endurance training equalized initial differences toward an endurance-trained proteomic profile in both sexes. The untrained muscle of females was more resistant to an acute exercise challenge since stress-responsive transcripts were predominantly upregulated in males.

In myotubes from the same donors, transcriptomic differences were hardly conserved, but could be partially restored by treatment with sex hormones. In conclusion, after only 8 weeks training mitigates deeply rooted sex-specific molecular profiles in skeletal muscle toward a common metabolically beneficial response.
SARS-CoV-2 Nsp14 binds Tollip and activates pro-inflammatory pathways while downregulating interferon-α and interferon-γ receptors
Authors: Thakur Naveen , Chakraborty Poushali , Tufariello JoAnn M. , Basler Christopher F.
Published: December 2024

SARS coronavirus 2 (SARS-CoV-2) non-structural protein 14 (Nsp14) possesses an N-terminal exonuclease (ExoN) domain that provides a proofreading function for the viral RNA-dependent RNA polymerase and a C-terminal N7-methyltransferase (N7-MTase) domain that methylates viral mRNA caps. Nsp14 also modulates host functions. This includes the activation of NF-κB and downregulation of interferon alpha/beta receptor 1 (IFNAR1). Here we demonstrate that Nsp14 exerts broader effects, activating not only NF-κB responses but also ERK, p38 and JNK MAP kinase (MAPK) signaling, promoting cytokine production. Further, Nsp14 downregulates not only IFNAR1 but also IFN-γ receptor 1 (IFNGR1), impairing cellular responses to both IFNα and IFNγ. IFNAR1 and IFNGR1 downregulation is via a lysosomal pathway and also occurs in SARS-CoV-2 infected cells. Analysis of a panel of Nsp14 mutants reveals a consistent pattern. Mutants that disable ExoN function remain active, whereas N7-MTase mutations impair both pro-inflammatory pathway activation and IFN receptor downregulation. Innate immune modulating functions also require the presence of both the ExoN and N7-MTase domains likely reflecting the need for the ExoN domain for N7-MTase activity. We further identify multi-functional host protein Tollip as an Nsp14 interactor. Interaction requires the phosphoinositide-binding C2 domain of Tollip and sequences C-terminal to the C2 domain. Full length Tollip or regions encompassing the Nsp14 interaction domain are sufficient to counteract both Nsp14-mediated and Nsp14-independent activation of NF-κB. Knockdown of Tollip partially reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infected cells, suggesting relevance of Nsp14-Tollip interaction for Nsp14 innate immune evasion functions.

Significance

SARS-CoV-2 protein Nsp14 both activates NF-κB, which promotes virus replication and inflammation, and downregulates IFNAR1, which can render infected cells resistant to the antiviral effects of IFN-α/β. Our study demonstrates that Nsp14 also activates MAPK signaling and downregulates IFNGR1, causing broader impacts than previously recognized. Data from a panel of Nsp14 mutants suggests a common underlying effect of Nsp14 may be responsible for its multiple innate immune activities. We further describe a novel interaction between Nsp14 and Tollip, a selective autophagy receptor. We show that Tollip expression downregulates Nsp14 activation of NF-κB and that Tollip knockdown reverses IFNAR1 and IFNGR1 downregulation in SARS-CoV-2 infection, suggesting that Tollip functions as a regulator of Nsp14 innate immune modulation.
Zinc is a Key Regulator of the Sperm-Specific K+Channel (Slo3) Function
Authors: Andriani Rizki Tsari , Pipatpolkai Tanadet , Miyata Haruhiko , Ikawa Masahito , Okamura Yasushi , Kawai Takafumi
Published: December 2024

The voltage- and pH-gated Slo3 potassium channel is exclusively expressed in mammalian spermatozoa. Its sensitivity to both voltage and alkalization plays a crucial role in sperm fertility, which is tightly coupled to the capacitation process. Here we show that sperm-enriched divalent cation Zn2+undergoes dynamic alteration in spermatozoa during capacitation. We also found that intracellular Zn2+regulates alkalinization-induced hyperpolarization in mice spermatozoa which is mediated by Slo3 channel. Further examination of zinc regulation in mouse Slo3 (mSlo3) revealed that, inXenopusoocyte expression system, intracellular zinc directly inhibits mouse Slo3 currents in dose-dependent manner at micromolar concentrations, with exceptionally slow dissociation. By combining MD simulations and electrophysiology, we also identified amino acid residues contributing to the Zn2+slow dissociation from Slo3 channels. Our studies uncover the importance of intracellular zinc dynamics and its regulatory role in ion channels during sperm capacitation.
Angiotensin peptides enhance SARS-CoV-2 spike protein binding to its host cell receptors
Authors: Oliveira Katelin X. , Suzuki Yuichiro J.
Published: December 2024

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that caused the Coronavirus Disease 2019 (COVID-19) pandemic, has a spike glycoprotein that is involved in recognizing and fusing to host cell receptors, such as angiotensin-converting enzyme 2 (ACE2), neuropilin-1 (NRP1), and AXL tyrosine-protein kinase. Since the major spike protein receptor is ACE2, an enzyme that regulates angiotensin II (1-8), this study tested the hypothesis that angiotensin II (1-8) influences the binding of the spike protein to its receptors. While angiotensin II (1-8) did not influence spike-ACE2 binding, we found that it significantly enhances spike-AXL binding. Our experiments showed that longer lengths of angiotensin, such as angiotensin I (1-10), did not significantly affect spike-AXL binding. In contrast, shorter lengths of angiotensin peptides, in particular, angiotensin IV (3-8), strongly increased spike-AXL binding. Angiotensin IV (3-8) also enhanced spike protein binding to ACE2 and NRP1. The discovery of the enhancing effects of angiotensin peptides on spike-host cell receptor binding may suggest that these peptides could be pharmacological targets to treat COVID-19 and post-acute sequelae of SARS-CoV-2 (PASC), which is also known as long COVID.
Hydrophysiology NMR reveals mechanisms of steady-state water exchange in neural tissue
Authors: Williamson Nathan H. , Ravin Rea , Cai Teddy X. , Rey Julian A. , Basser Peter J.
Published: December 2024

Water molecules exchange incessantly across cell membranes and among different environments within the cell, but it is not known what the dominant transport pathways are and whether they are active or passive. We have developed realtime NMR hydrophysiology methods to study steady-state water exchange and diffusion in viableex vivoneonatal mouse spinal cord samples. We find that water exchange is not active but is linked to tonicity maintained by active transport. Exchange slows following sodium–potassium pump inhibition but recovers to a normal rate after adding extracellular osmolytes. Additional information from apparent water diffusion coefficients (ADC) differentiates normal and inactive samples regardless of tonicity. Data and modeling suggest a multisite exchange mechanism in which tonicity modulates the dominant apparent exchange pathway between fast transmembrane exchange and slow intracellular exchange. The transmembrane pathway has a high activation energy but does not require ions, suggesting that in this pathway exchange likely occurs through the lipid bilayer rather than through channels or cotransporters. These methods may one day be translated to clinical MRI scanners to determine features of the cellular statein vivo. steady state water exchange, homeostasis, diffusion exchange spectroscopy, DEXSY, tissue microstructure, porous media, membrane permeability, low-field single-sided NMR, apparent diffusion coefficient, ADC, gray matter
Development of an enhanced anti-pan-N-formylmethionine-specific antibody
Authors: Kim Dasom , Park Kyu-Sang , Hwang Cheol-Sang
Published: December 2024

Both bacterial and eukaryotic ribosomes can initiate protein synthesis with formylmethionine (fMet), but detecting fMet-bearing peptides and fMet-bearing proteins has been challenging due to the lack of effective anti-pan-fMet antibodies. Previously, we developed a polyclonal anti-fMet antibody using a fMet-Gly-Ser-Gly-Cys pentapeptide that detects those fMet-bearing peptides and fMet-bearing proteins regardless of their sequence context. In this study, we significantly improved the antibody’s specificity and affinity by using a mixture of fMet-Xaa-Cys (fMXC) tripeptides (Xaa, any of the 20 amino acids) as the immunogen. This newly optimized anti-fMet antibody is a powerful, cost-effective tool for detecting fMet-bearing proteins across species. Furthermore, this approach provides a foundation for developing anti-pan-specific antibodies targeting other N-terminal modifications through acylation, alkylation, oxidation, or arginylation, etc.

METHOD SUMMARY

fMet-Gly-Ser-Gly-Cys (fMGSGC), fMet-dPEG4-Cys (fMdPEG4C), and fMet-Xaa-Cys (fMXC; Xaa, any of the 20 amino acids) were used as antigens to generate anti-pan-fMet-specific antibodies (anti-fMet antibodies). The quality of the raised antibodies was evaluated by immunoblotting using lysates fromEscherichia coli(E. coli) DH5α cells and human kidney HK2 cells, as well as by enzyme-linked immunosorbent assay (ELISA) with purified fMet-bearing (fMet-) proteins and their unformylated counterparts.
Cell surface crowding is a tunable biophysical barrier to cell-cell fusion
Authors: Lee Daniel S.W. , Oster Liya F. , Son Sungmin , Fletcher Daniel A.
Published: December 2024

Cell-cell fusion is fundamental to developmental processes such as muscle formation, as well as to viral infections that cause pathological syncytia. An essential step in fusion is close membrane apposition, but cell membranes are crowded with proteins, glycoproteins, and glycolipids, all of which must be cleared before a fusion pore can be nucleated. Here, we find that cell surface crowding drastically reduces fusogenicity in multiple systems, independent of the method for driving fusion. We estimate that cell surface crowding presents an energetic barrier to membrane apposition on the scale of ∼100kBT, greater than that of bare membrane fusion. We show that increasing cell surface crowding reduces fusion efficiency of PEG-mediated and fusogen-mediated cell-cell fusion, as well as synthetic membranes under force. Interestingly, we find that differentiating myoblasts naturally decrease cell surface crowding prior to fusion. Cell surface crowding presents an underappreciated biophysical barrier that may be tuned developmentally and could be targeted externally to control tissue-specific cell-cell fusion.
The DiaGame Study: Multimodal Data Collection in Free-Living Conditions Using Consumer-Level Wearable Devices and Apps in People with Diabetes
Authors: de Vries Ryan , Reinders Edouard F.H. , de Carvalho Danilo Ferreira , Cruts – Van de Meulengraaf Elma M.W.L. , van Gorp Pieter M.E. , Wouters - van Poppel Pleun C.M. , Kaymak Uzay , Hilbers Peter A.J. , van Riel Natal A.W. , Haak Harm R.
Published: December 2024

The everlasting interest in precision medicine has instigated a need for patient-specific multidimensional data collection. By leveraging consumer-level wearable devices and applications we can support this need by collecting multimodal data in uncontrolled free-living conditions. We used a continuous glucose monitor, a smartwatch with in-house developed application, and a smartphone application over a period of 14 days to collect data from 60 participants with diabetes. Data were collected on 77,683 CGM measurements, 4,073 moods, 3,517 meals, 1,394 insulin injections, and 1,364 bouts of physical activity. Participants were able to effectively use the devices and applications, with 45 participants reporting data on all modalities and all participants reporting data on at least two modalities (in addition to the data collected from the smartwatch’s internal sensors). These findings highlight the efficiency and effectiveness of consumer-level wearable devices to collect data from the comfort of the people’s homes. Thereby, facilitating the need for a cost-effective and scalable approach for precision monitoring to the public.
Multianalyte blood-based risk stratification of incidental pancreas lesions
Authors: Marcinak Clayton T. , Stephens Michelle D. , McDonald Bradon R. , Merali Nabeel , Dietmann Elise C. , McGregor Stephanie M. , Ronnekleiv-Kelly Sean M. , Weber Sharon M. , Nkadori Everlyne N. , Stadmeyer Peter G. , Benson Mark E. , Gopal Deepak V. , Zafar Syed Nabeel , Kelly Kaitlyn J. , Pirasteh Ali , Frampton Adam E. , Pfau Patrick R. , Sivakumar Shivan , Minter Rebecca M. , Murtaza Muhammed
Published: December 2024

Purpose

Incidental detection of pancreas lesions (IPLs) is common and creates an opportunity to intercept pancreatic ductal adenocarcinoma (PDAC). However, identifying patients at risk for progression who can benefit from surgical resection remains challenging. The current role of blood tests for risk stratification in patients with IPLs is limited.

Methods

We evaluated the performance of circulating glycoproteins (CA19-9, CEA and CA125), plasma DNA fragmentation analysis, and their combination, in 99 asymptomatic patients with IPLs, for detection of advanced pathology (high-grade dysplasia or invasive carcinoma). Plasma DNA fragmentation was analyzed using a machine learning model, adapted to detect PDAC in 242 patients with cancer and 300 healthy individuals.

Results

During 18.8 months of median follow-up by a multidisciplinary clinical team, 11 of 99 patients with IPLs were diagnosed with advanced pathology. We observed area under the receiver operating characteristic curves (AUROCs) of 0.78, 0.63, 0.54 and 0.74 using CA19-9, CEA, CA125 and plasma DNA fragmentation, respectively. Combined analysis of CA19-9, CA125 and plasma DNA fragmentation showed an AUROC of 0.93, with 91% sensitivity and 53% positive predictive value (PPV) at 90% specificity. In a subset of 25 patients with histologically confirmed diagnoses, AUROC improved to 0.96 and PPV improved to 91%. In one patient with an equivocal initial endoscopy, multi-analyte blood analysis predicted cancer 3 months before diagnosis of stage IA cancer.

Conclusion

Our results demonstrate a multianalyte blood test combining glycoprotein biomarkers with plasma DNA fragmentation could complement current clinical workflows for cancer detection in patients with IPLs.