bluuebunny/crossref_metadata_2025 huggingface dataset:
Spoink, a LTR retrotransposon, invadedD. melanogasterpopulations in the 1990s
Authors: Pianezza Riccardo , Scarpa Almorò , Narayanan Prakash , Signor Sarah , Kofler Robert
Published: November 2023

During the last few centuriesD. melanogasterpopulations were invaded by several transposable elements, the most recent of which was thought to be theP-element between 1950 and 1980. Here we describe a novel TE, which we namedSpoink, that has invadedD. melanogaster. It is a 5216nt LTR retrotransposon of the Ty3/gypsy superfamily. Relying on strains sampled at different times during the last century we show thatSpoinkinvaded worldwideD. melanogasterpopulations after theP-element between 1983 and 1993. This invasion was likely triggered by a horizontal transfer from theD. willistonigroup, much as theP-element.Spoinkis probably silenced by the piRNA pathway in natural populations and about 1/3 of the examined strains have an insertion into a canonical piRNA cluster such as42AB. Given the degree of genetic investigation ofD. melanogasterit is surprising thatSpoinkwas able to invade unnoticed.
Fasten with Pipes
Authors: Katz Lee S. , Den Bakker Henk C.
Published: November 2023

Fasten is a versatile bioinformatics tool suite developed to address gaps in the manipulation of sequence data files in the fastq format. Existing tools often lack support for interleaved fastq files and Unix-style pipe file control. Fasten provides a solution by allowing bioinformaticians to efficiently work with paired-end sequence data, facilitating data transformation, and offering competitive speeds. This suite, implemented in Rust, offers a comprehensive manual, continuous integration, and seamless integration into the command line with Unix pipes. Fasten is poised to become an invaluable resource for routine command-line bioinformatics tasks, enhancing the accessibility and flexibility of data processing in the field.
Extended-depth of field random illumination microscopy, EDF-RIM, provides super-resolved projective imaging
Authors: Mazzella Lorry , Mangeat Thomas , Giroussens Guillaume , Rogez Benoit , Li Hao , Creff Justine , Saadaoui Mehdi , Martins Carla , Bouzignac Ronan , Labouesse Simon , Idier Jérome , Galland Frédéric , Allain Marc , Sentenac Anne , LeGoff Loïc
Published: November 2023

The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images. We have implemented an Extended Depth of Field (EDF) approach in a Random Illumination Microscope (RIM). RIM uses multiple speckled illuminations and variance data processing to double the resolution. It is particularly adapted to the imaging of thick samples as it does not require the knowledge of illumination patterns. We demonstrate highly-resolved projective images of biological tissues and cells. Compared to a sequential scan of the imaged volume with conventional 2D-RIM, EDF-RIM allows an order of magnitude improvement in speed and light dose reduction, with comparable resolution. As the axial information is lost in an EDF modality, we propose a method to retrieve the sample topography for samples that are organized in cell sheets.
4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter
Authors: Rickert Julian D. , Held Marcus O. , Engelhardt Johann , Hell Stefan W.
Published: November 2023

We introduce MINFLUX localization with interferometric illumination through opposing objective lenses for maximizing the attainable precision in 3D-localization of single inelastic scatterers, such as fluorophores. Our 4Pi optical configuration employs three sequentially tilted counter-propagating beam pairs for illumination, each providing a narrow interference minimum of illumination intensity at the focal point. The localization precision is additionally improved by adding the inelastically scattered (fluorescence) photons collected through both objective lenses. Our 4Pi configuration yields the currently highest precision per detected photon among all localization schemes. Tracking gold nanoparticles as non-blinking inelastic scatterers rendered a position uncertainty < 0.4 nm3in volume at a localization frequency of 2.9 kHz. We harnessed the record spatio-temporal precision of our 4Pi MINFLUX approach to examine the diffusion of single fluorophores and fluorescent nanobeads in solutions of sucrose in water, revealing local heterogeneities at the nanoscale. Our results show the applicability of 4Pi MINFLUX to study molecular nano-environments of diffusion and its potential for quantifying rapid movements of molecules in cells and other material composites.
Stimulation of Neurons and Astrocytes via Temporally Interfering Electric Fields
Authors: Ahtiainen Annika , Leydolph Lilly , Tanskanen Jarno M.A. , Hunold Alexander , Haueisen Jens , Hyttinen Jari A.K.
Published: November 2023

A novel non-invasive electrical stimulation method, temporal interference stimulation (TIS), has been proposed to enable spatially steerable stimulation and to selectively activate different brain regions. However, TIS has not been studied with cell cultures that can provide an indepth assessment of its effects on neuronal cells. We successfully established anin vitroTIS setup using rat cortical neurons on microelectrode arrays. Stimulations involved 1) TIS with 653 Hz and 643 Hz, resulting in a 10 Hz frequency envelope, 2) low-frequency stimulation (LFS) at 10 Hz, 3) high-frequency stimulation (HFS) at 653 Hz, and 4) no electrical stimulation (control/sham). HFS and LFS had the least effect on neuronal activity, but TIS elicited neuronal electrophysiological responses, especially 24 hours after stimulation. We also assessed TIS in neuron-astrocyte co-cultures. Interestingly, co-cultures seemed to counteract the TIS effects. Hence, our findings deepen the current understanding of the electrical modulation of neurons during TIS.

Highlights

Temporal interference stimulation (TIS) is a new technique for brain stimulation.

TIS setup was established and combined with microelectrode array (MEA) system.

Rat cortical neurons and astrocytes were stimulated with TISin vitro.

Neurons responded to TIS more prominently without astrocyte support.
Supramolecular Interactions of Teixobactin Analogues in the Crystal State
Authors: Yang Hyunjun , Kreutzer Adam G. , Nowick James S.
Published: November 2023

Teixobactin is a potent peptide antibiotic against Gram-positive bacteria that binds to lipid II and related peptidoglycan precursors and disrupts the cell membrane. This paper presents the X-ray crystallographic structure of theN-methylated teixobactin analogueN-Me-D-Gln4,Lys10-teixobactin (1).N-Methylation at position 4 prevents uncontrolled aggregation and enables the crystallization of the teixobactin analogue. Lysine at position 10 replaces the non-proteinogenic amino acidallo-enduracididine, which is not commercially available. Crystallization from aqueous solution with MgCl2and PEG3350 afforded crystals suitable for X-ray crystallography. The crystallographic phases were solved using SAD phasing on data sets collected at 2.0663 Å. Molecular replacement then enabled the determination of the structure at 1.50 Å resolution using a data set collected at 0.9997Å (PDB 8U78). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel β-sheet dimers in the crystal lattice, with residues 1–3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice. The β-sheet dimers are amphiphilic, with the side chains of the hydrophobic residues on one surface and the side chains of the hydrophilic residues on the other surface. The dimers pack in the lattice through hydrophobic interactions between the hydrophobic surfaces. The crystal structure of teixobactin analogue 1 recapitulates several aspects of the interaction of teixobactin with the cell membrane of Grampositive bacteria, including anion binding, supramolecular assembly through β-sheet formation, and hydrophobic interactions.
Lewy Body Radius Growth: The Hypothesis of the Cube Root of Time Dependency
Authors: Kuznetsov Andrey V.
Published: November 2023

This paper presents a model for the growth of Lewy bodies (LBs), which are pathological hallmarks of Parkinson’s disease (PD). The model simulates the growth of classical LBs, consisting of a core and a halo. The core is assumed to comprise lipid membrane fragments and damaged organelles, while the halo consists of radiating alpha-synuclein (α-syn) fibrils. The Finke-Watzky model is employed to simulate the aggregation of lipid fragments and α-syn monomers. By analytically and numerically exploring the solutions of the governing equations, approximate solutions were derived, which are applicable for large times. The application of these approximate solutions to simulate LB radius growth led to the discovery of the cube root hypothesis, which posits that the LB radius is proportional to the cube root of its growth time. Sensitivity analysis revealed that the LB radius is unaffected by the kinetic rates of nucleation and autocatalytic growth, with growth primarily regulated by the production rates of lipid membrane fragments and α-syn monomers. The model suggests that large LBs relevant to PD can only develop when the machinery responsible for degrading lipid membrane fragments, α-syn monomers, and their aggregates is dysfunctional.
ODL-BCI: Optimal deep learning model for brain-computer interface to classify students confusion via hyperparameter tuning
Authors: Miah Md Ochiuddin , Habiba Umme , Kabir Md Faisal
Published: November 2023

Brain-computer interface (BCI) research has gained increasing attention in educational contexts, offering the potential to monitor and enhance students’ cognitive states. Real-time classification of students’ confusion levels using electroencephalogram (EEG) data presents a significant challenge in this domain. Since real-time EEG data is dynamic and highly dimensional, current approaches have some limitations for predicting mental states based on this data. This paper introduces an optimal deep learning (DL) model for the BCI, ODL-BCI, optimized through hyperparameter tuning techniques to address the limitations of classifying students’ confusion in real time. Leveraging the “confused student EEG brainwave” dataset, we employ Bayesian optimization to fine-tune hyperparameters of the proposed DL model. The model architecture comprises input and output layers, with several hidden layers whose nodes, activation functions, and learning rates are determined utilizing selected hyperparameters. We evaluate and compare the proposed model with some state-of-the-art methods and standard machine learning (ML) classifiers, including Decision Tree, AdaBoost, Bagging, MLP, Näıve Bayes, Random Forest, SVM, and XG Boost, on the EEG confusion dataset. Our experimental results demonstrate the superiority of the optimized DL model, ODL-BCI. It boosts the accuracy between 4% and 9% over the current approaches, outperforming all other classifiers in the process. The ODL-BCI implementation source codes can be accessed by anyone athttps://github.com/MdOchiuddinMiah/ODL-BCI.
Interdependence of cellular and network properties in respiratory rhythmogenesis
Authors: Phillips Ryan S. , Baertsch Nathan A.
Published: November 2023

How breathing is generated by the preBötzinger Complex (preBötC) remains divided between two ideological frameworks, and the persistent sodium current (INaP) lies at the heart of this debate. AlthoughINaPis widely expressed, thepacemaker hypothesisconsiders it essential because it endows a small subset of neurons with intrinsic bursting or “pacemaker” activity. In contrast,burstlet theoryconsidersINaPdispensable because rhythm emerges from “pre-inspiratory” spiking activity driven by feed-forward network interactions. Using computational modeling, we discover that changes in spike shape can dissociateINaPfrom intrinsic bursting. Consistent with many experimental benchmarks, conditional effects on spike shape during simulated changes in oxygenation, development, extracellular potassium, and temperature alter the prevalence of intrinsic bursting and pre-inspiratory spiking without altering the role ofINaP. Our results support a unifying hypothesis whereINaPand excitatory network interactions, but not intrinsic bursting or pre-inspiratory spiking, are critical interdependent features of preBötC rhythmogenesis.

SIGNIFICANCE STATEMENT

Breathing is a vital rhythmic process originating from the preBötzinger complex. Since its discovery in 1991, there has been a spirited debate about whether respiratory rhythm generation emerges as a network property or is driven by a subset of specialized neurons with rhythmic bursting capabilities, endowed by intrinsic currents. Here, using computational modeling, we propose a unifying data-driven model of respiratory rhythm generation which bridges the gap between these competing theories. In this model, both intrinsic cellular properties (a persistent sodium current) and network properties (recurrent excitation), but not intrinsic bursting, are essential and interdependent features of respiratory rhythm generation.
Robust and Accurate Doublet Detection of Single-Cell Sequencing Data via Maximizing Area Under Precision-Recall Curve
Authors: Chen Yanshuo , Wu Xidong , Ni Ke , Hu Haoran , Yue Molin , Chen Wei , Huang Heng
Published: November 2023

Single-cell sequencing has revolutionized our understanding of cellular heterogeneity by offering detailed profiles of individual cells within diverse specimens. However, due to the limitations of sequencing technology, two or more cells may be captured in the same droplet and share the same barcode. These incidents, termed doublets or multiplets, can lead to artifacts in single-cell data analysis. While explicit experimental design can mitigate these issues with the help of auxiliary cell markers, computationally annotating doublets has a broad impact on analyzing the existing public single-cell data and reduces potential experimental costs. Considering that doublets form only a minor fraction of the total dataset, we argue that current doublet detection methods, primarily focused on optimizing classification accuracy, might be inefficient in performing well on the inherently imbalanced data in the area under the precision-recall curve (AUPRC) metric. To address this, we introduce RADO (Robust and Accurate DOublet detection) - an algorithm designed to annotate doublets by maximizing the AUPRC, effectively tackling the imbalance challenge. Benchmarked on 18 public datasets, RADO outperforms other methods in terms of doublet score and achieves similar performance to the current best methods in doublet calling. Furthermore, beyond its application in single-cell RNA-seq data, we demonstrate RADO’s adaptability to single-cell assays for transposase-accessible chromatin sequencing (scATAC-seq) data, where it outperforms other scATAC-seq doublet detection methods. RADO’s open-source implementation is available at:https://github.com/poseidonchan/RADO.
The landscape of the histone-organized chromatin of Bdellovibrionota bacteria
Authors: Marinov Georgi K. , Doughty Benjamin , Kundaje Anshul , Greenleaf William J.
Published: November 2023

Histone proteins have traditionally been thought to be restricted to eukaryotes and most archaea, with eukaryotic nucleosomal histones deriving from their archaeal ancestors. In contrast, bacteria lack histones as a rule. However, histone proteins have recently been identified in a few bacterial clades, most notably the phylum Bdellovibrionota, and these histones have been proposed to exhibit a range of divergent features compared to histones in archaea and eukaryotes. However, no functional genomic studies of the properties of Bdellovibrionota chromatin have been carried out. In this work, we map the landscape of chromatin accessibility, active transcription and three-dimensional genome organization in a member of Bdellovibrionota (aBacteriovoraxstrain). We find that, similar to what is observed in some archaea and in eukaryotes with compact genomes such as yeast,Bacteriovoraxchromatin is characterized by preferential accessibility around promoter regions. Similar to eukaryotes, chromatin accessibility inBacteriovoraxpositively correlates with gene expression. Mapping active transcription through single-strand DNA (ssDNA) profiling revealed that unlike in yeast, but similar to the state of mammalian and fly promoters,Bacteriovoraxpromoters exhibit very strong polymerase pausing. Finally, similar to that of other bacteria without histones, theBacteriovoraxgenome exists in a three-dimensional (3D) configuration organized by the parABS system along the axis defined by replication origin and termination regions. These results provide a foundation for understanding the chromatin biology of the unique Bdellovibrionota bacteria and the functional diversity in chromatin organization across the tree of life.
Increased force and elastic energy storage are not the mechanisms that improve jump performance with accentuated eccentric loading during a constrained vertical jump
Authors: Su Eric Yung-Sheng , Carroll Timothy J. , Farris Dominic J. , Lichtwark Glen
Published: November 2023

Objective

Accentuated eccentric loading (AEL) involves higher load applied during the eccentric phase of a stretch-shortening cycle movement, followed by a sudden removal of load before the concentric phase. Previous studies suggest that AEL enhances human countermovement jump performance, however the mechanism is not fully understood. Here we explore whether isolating additional load during the countermovement is sufficient to increase ground reaction force, and hence elastic energy stored, at the start of the upward movement and whether this leads to increased jump height or power generation.

Methods

We conducted a trunk-constrained vertical jump test on a custom-built device to isolate the effect of additional load while controlling for effects of squat depth, arm swing, and coordination. Twelve healthy, recreationally active adults (7 males, 5 females) performed maximal jumps without AEL, followed by randomised AEL conditions prescribed as a percentage of body mass (10%, 20%, and 30%), before repeating jumps without AEL.Results.No significant changes in vertical ground reaction force at the turning point were observed. High load AEL conditions (20% and 30% body weight) led to slight reductions in jump height, primarily due to decreased hip joint and centre of mass work. AEL conditions did not alter peak or integrated activation levels of the knee extensor muscles.

Conclusion

These findings suggest that increased elastic energy return may not be the primary mechanism behind the potentiating effects of AEL on jump performance, and other factors such as rate of descent, squat depth, or body configuration may contribute to effective AEL.
Principles of Computation by Competitive Protein Dimerization Networks
Authors: Parres-Gold Jacob , Levine Matthew , Emert Benjamin , Stuart Andrew , Elowitz Michael B.
Published: November 2023

Many biological signaling pathways employ proteins that competitively dimerize in diverse combinations. These dimerization networks can perform biochemical computations, in which the concentrations of monomers (inputs) determine the concentrations of dimers (outputs). Despite their prevalence, little is known about the range of input-output computations that dimerization networks can perform (their “expressivity”) and how it depends on network size and connectivity. Using a systematic computational approach, we demonstrate that even small dimerization networks (3-6 monomers) areexpressive, performing diverse multi-input computations. Further, dimerization networks areversatile, performing different computations when their protein components are expressed at different levels, such as in different cell types. Remarkably, individual networks with random interaction affinities, when large enough (≥8 proteins), can perform nearly all (∼90%) potential one-input network computations merely by tuning their monomer expression levels. Thus, even the simple process of competitive dimerization provides a powerful architecture for multi-input, cell-type-specific signal processing.

Graphical Abstract
Berberine impairs spermatogenesis in mice by modulating ornithine metabolism via gut microbiota regulation
Authors: Qu Wei , Xu Yumin , Lei Junhao , Yang Jing , Shi Hanqing , Wang Junli , Yu Xinnai , Chen Jiemin , Wang Binyi , Han Yan , Luo Mengcheng , Liu Rong
Published: November 2023

Berberine (BBR) is used to treat diarrhea clinically, its reproductive toxicity, however, is poorly documented. This study aims to investigate the impact of BBR on the male reproductive system. Gradient doses of BBR were administered orally to experimental mice for consecutive 14 days. The gut microbiota, sperm concentration of cauda epididymis, and serum testosterone levels were measured after the last dose for assessing the effects of BBR. Moreover, the metabolome and transcriptome of the mice and microbiota were also investigated. Intragastric BBR administration resulted in a significant decrease in serum testosterone levels and epididymal sperm concentration in mice, which was attributed to a dramatic decrease of Muribaculaceae abundance in the gut microbiota of mice. Both fecal microbiota transplantation (FMT) and polyethylene glycol (PEG) treatment experiments also demonstrated that Muribaculaceae is necessary for spermatogenesis. Metabolomic analysis revealed that BBR affected the arginine and proline metabolism pathways, of which ornithine levels were downregulated after BBR administration. Intragastric administration ofM.intestinaleand its metabolite ornithine to BBR-treated mice achieved a recovery of sperm concentration and testosterone levels. RNA sequencing of testes showed the genes related to the LDLR-mediated cholesterol-synthesis testosterone pathway were downregulated after BBR administration. The levels of testosterone increased andLdlrgene became more transcriptionally active in TM3 cells cultured in media supplemented with ornithine. This study for the first time revealed an association between BBR-induced gut Muribaculaceae dysbiosis and defects in spermatogenesis via ornithine metabolism, which provided a candidate and strategy for the treatment of infertility caused by a decreased serum testosterone level-induced by gut microbiota dysbiosis.
Post-fertilization transcription initiation in an ancestral LTR retrotransposon drives lineage-specific genomic imprinting ofZDBF2
Authors: Kobayashi Hisato , Igaki Tatsushi , Kumamoto Soichiro , Tanaka Keisuke , Takashima Tomoya , Nagaoka So I. , Suzuki Shunsuke , Hayashi Masaaki , Renfree Marilyn B. , Kawahara Manabu , Saito Shun , Kobayashi Toshihiro , Nagashima Hiroshi , Matsunari Hitomi , Nakano Kazuaki , Uchikura Ayuko , Kiyonari Hiroshi , Kaneko Mari , Imai Hiroo , Nakabayashi Kazuhiko , Lorincz Matthew C. , Kurimoto Kazuki
Published: November 2023

The imprintedZDBF2gene is controlled by oocyte-derived DNA methylation, but its epigenetic regulation is quite different from that of other canonically imprinted genes that are dependent on DNA methylation deposited in the gametes. At theZDBF2locus, maternal DNA methylation in the imprinted differentially methylated region (DMR) does not persist after implantation. Instead, a transient transcript expressed in the early embryo exclusively from the unmethylated paternal allele of the DMR, known asGPR1-ASin humans andLizin mice, contributes to establishing secondary DMRs that maintain paternal expression ofZDBF2in the somatic lineage. While the imprinting ofZDBF2is evident in humans and mice, whether this process is conserved in other mammals has not been addressed. Here, we show that the first exon of humanGPR1-ASoverlaps with that of a long terminal repeat (LTR) belonging to the MER21C subfamily of retrotransposons. Although this LTR family appears and is amplified in Boroeutherians, the magnorder of placental mammals that includes the Euarchontoglires and Laurasiatheria superorders, the MER21C insertion into theGPR1-ASorthologous region occurred specifically in the common ancestor of Euarchontoglires, a clade that includes extant primates, rodents, and rabbits. The first exon of mouseLizdoes not overlap with an annotated LTR in standard repeat annotation; however, promoter activity assay and multiple sequence alignment suggests that it retains a functionally conserved relationship with the MER21C-overlapping first exon ofGPR1-AS. Furthermore, directional RNA sequencing of placental tissues from rabbits and nonhuman primates also revealedGPR1-ASorthologs, with their first exon embedded within the same ancestral LTR. In contrast, allele-specific expression profiling of cow and tammar wallaby, mammals outside the Euarchontoglires group, revealed expression from both alleles in all tissues analyzed. Taken together, these observations suggest that imprinting ofZDBF2in Euarchontoglires had its genesis in the insertion of a MER21C element in their common ancestor. Our previous studies showed that LTRs reactivated in oocytes contribute to lineage-specific imprinting during mammalian evolution. The data presented here suggest that post-fertilization activation of an ancestral LTR-derived sequence can also contribute to the lineage-specific establishment of imprinted genes.
Identification of two novel DnaJ chaperone family proteins as modifiers of Huntingtin aggregates
Authors: Deo Ankita , Ghosh Rishita , Ahire Snehal , Majumdar Amitabha , Bose Tania
Published: November 2023

Huntington’s disease (HD) is a rare neurodegenerative disease. It is caused due to aggregation of Huntingtin (HTT) protein containing Q repeats more than 40. Similar protein aggregation is also a hallmark of several other neurodegenerative diseases related to loss of cognitive function. In search of modifiers of HTT aggregation, we have screened putative chaperone proteins fromDrosophilain both fly and yeast model of HD. DnaJ chaperones were screened by evaluating HTT protein aggregation related phenotypes using growth assays for studying the growth rate of the cells, imaging studies, and gel-based approaches like semi-denaturing detergent agarose gel electrophoresis (SDD-AGE). Our screening led us to categorize several proteins as suppressors and enhancers of the HTT associated phenotypes. Out of the 40 chaperones and co-chaperones, two chaperones that came up strikingly were CG5001 and P58IPK. Protein aggregation was found to be reduced in both S2 cells andDrosophilatransgenic lines with HTT103Q in presence of these class of chaperones. As these DnaJ chaperones have protein sequence similarity across species, these might be used as possible tools to combat the effects of neurodegenerative diseases, as evidenced specifically in Huntington’s disease and Amyotrophic Lateral Sclerosis (ALS).
Prion diseases disrupt the glutamate/glutamine metabolism in skeletal muscle
Authors: Caredio Davide , Koderman Maruša , Frontzek Karl , Sorce Silvia , Nuvolone Mario , Bremer Juliane , Schwarz Petra , Sellitto Stefano , Streichenberger Nathalie , Scheckel Claudia , Aguzzi Adriano
Published: November 2023

In prion diseases, aggregates of misfolded prion protein (PrPSc) accumulate not only in the brain but also in extraneural organs. This raises the question whether prion-specific pathologies arise also extraneurally. Here we sequenced mRNA transcripts in skeletal muscle, spleen and blood of prion-inoculated mice at eight timepoints during disease progression. We detected gene-expression changes in all three organs, with skeletal muscle showing the most consistent alterations. The glutamate-ammonia ligase (GLUL) gene exhibited uniform upregulation in skeletal muscles of mice infected with three distinct scrapie prion strains (RML, ME7, and 22L) and in victims of human sporadic Creutzfeldt-Jakob disease.GLULdysregulation was accompanied by changes in glutamate/glutamine metabolism, leading to reduced glutamate levels in skeletal muscle. None of these changes were observed in skeletal muscle of humans with amyotrophic lateral sclerosis, Alzheimer’s disease, or dementia with Lewy bodies, suggesting that they are specific to prion diseases. These findings reveal an unexpected metabolic dimension of prion infections and point to a potential role for GLUL dysregulation in the glutamate/glutamine metabolism in prion-affected skeletal muscle.
Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration inChlamydomonas reinhardtii
Authors: Penny Gervette M. , Dutcher Susan K.
Published: November 2023

Motile cilia are complex organelles comprised of >800 structural proteins. Variants in 58 of these genes cause primary ciliary dyskinesia (PCD) in humans. We used a second-site non-complementation approach in diploidChlamydomonas reinhardtiistrains to investigate whether reduced dosage of different combinations of motile cilia proteins affects cilia growth / regeneration. Temperature-sensitive mutants in intraflagellar transport genes fail to complement in heterozygous diploids at the restrictive temperature, which may be due to poisonous interactions or a reduction in protein levels. Diploid strains heterozygous for 211 double mutant combinations in 21 genes are phenotypically recessive when unperturbed and do not show SSNC. Consequently, we developed a sensitized screen. When protein synthesis is inhibited, cells utilize the existing cytoplasmic pool of proteins, which is insufficient to build full-length cilia. Six double heterozygous strains in dynein arm preassembly factors (DNAAFs)pf13, pf23, wdr92,andoda8regrow cilia that are shorter than wild-type diploids, which suggests that double heterozygosity limits the pool of assembled dynein arms further. We isolated a new null allele (pf23-4)that shows a more severe loss of ODAs and IDAs in cilia than the previously studiedpf23-1hypomorphic allele. We also identified a new role for PF23 in cytoplasmic modification of IC138, protein of the I1/finner dynein arm. In our SSNC assay, thepf23-4allele also exhibits a more severe phenotype thanpf23-1. Whole cell extract immunoblots show a reduction of PF23 protein to one-half of wild-type levels in both single and double heterozygous genotypes. Thepf13mutant shows SSNC with mutants in two outer dynein arm structural proteins, ODA6, andODA9. We suggest that reduction of multiple dynein preassembly factors limits the pool of assembled dynein arms needed for cilia assembly and regeneration. Our data support PF23 as a scaffolding hub protein involved in dynein arm assembly.

Author Summary

Motile cilia are essential for movement of cells and fluids. In humans, motile cilia defects cause primary ciliary dyskinesia (PCD), a rare disease characterized by recurring respiratory infections, left-right asymmetry defects, ear infections, and infertility. Many genes are involved in motile cilia function and assembly. We useChlamydomonas reinhardtiito study the effects of changes in protein levels in different combinations of proteins needed for motile cilia. Our results show that the proteins that help fold and assemble the dynein motors in the cytoplasm are sensitive to changes in the dosage of these preassembly factors. Reductions in proteins involved in multiple steps of the pathway prevents efficient dynein preassembly and cilia regeneration under stressful cellular conditions.
The scramblases VMP1 and TMEM41b are required for primitive endoderm specification by targeting WNT signaling
Authors: Holzner Markus , Sonicki Tea , Hunn Hugo , Uliana Federico , Jiang Weijun , Gade Vamshidhar R. , Weis Karsten , Wutz Anton , Di Minin Giulio
Published: November 2023

The ER resident proteins VMP1 and TMEM41b share a conserved DedA domain, which confers lipid scramblase activity. Loss of either gene results in embryonic lethality in mice and defects in autophagy and lipid droplet metabolism. We set out to investigate their role in pluripotency and specification. For this purpose, we generated Vmp1 and Tmem41b mutations in mouse embryonic stem cells (ESCs). We observed that ESCs carrying mutations in Vmp1 and Tmem41b show robust self-renewal and an unperturbed pluripotent expression profile but accumulate LC3-positive autophagosomes and lipid droplets consistent with defects in autophagy and lipid metabolism. ESCs carrying combined mutations in Vmp1 and Tmem41b can differentiate into a wide range of embryonic cell types. However, differentiation into primitive endoderm-like cells in culture is impaired, and the establishment of extra- embryonic endoderm stem (XEN) cells is delayed. Mechanistically, we show the deregulation of genes that are associated with WNT signaling. This is further confirmed by cell surface proteome profiling, which identified a significant reduction of the WNT receptor FZD2 at the plasma membrane in Vmp1 and Tmem41b double mutant ESCs. Importantly, we show that transgenic expression of Fzd2 rescues XEN differentiation. Our findings identify the role of the lipid scramblases VMP1 and TMEM41b in WNT signaling during extra-embryonic endoderm development and characterize their distinct and overlapping functions.
Social avoidance of mice in pain in naturalistic conditions
Authors: Moëne Olivia Le , Larsson Max
Published: November 2023

Pain and social behavior are subject to reciprocal modulation. Both humans and rodents experience emotional contagion from afflicted conspecifics, and may act to relieve the afflicted state of these. Little has been done to investigate the motivation of such prosocial behavior in rodents in naturalistic conditions. Here, we analyzed social interactions in mice group-housed in a seminatural environment (SNE). Social buffering reduced nocifensive behavior in formalin-injected mice. These mice were also both socially withdrawn and avoided by other mice. These findings appear counter to those showing empathy in mouse pain models. It is possible that in naturalistic conditions, healthy mice simply avoid individuals in pain and the cost associated with emotional contagion. Interestingly, behaviors involving direct body contact were not different between saline– and formalin-treated mice, and thus may carry a prosocial, altruistic component. These findings unveil new patterns of social modulation by pain in a naturalistic laboratory setting holding high translational value.

Teaser

In a new, ethological assay, mice in pain are socially withdrawn and avoided by others, challenging findings of empathy in rodent models.
Functional characterization of the disease-associatedCCL2rs1024611G-rs13900T haplotype: The role of the RNA-binding protein HuR
Authors: Akhtar Feroz , Ruiz Joselin Hernandez , Liu Ya-Guang , Resendez Roy G. , Feliers Denis , Morales Liza D. , Diaz-Badillo Alvaro , Lehman Donna M. , Arya Rector , Lopez-Alvarenga Juan Carlos , Blangero John , Duggirala Ravindranath , Mummidi Srinivas
Published: November 2023

CC-chemokine ligand 2 (CCL2) is involved in the pathogenesis of several diseases associated with monocyte/macrophage recruitment, such as HIV-associated neurocognitive disorder (HAND), tuberculosis, and atherosclerosis. The rs1024611 (alleles:A>G; G is the risk allele) polymorphism in theCCL2 cis-regulatory region is associated with increased CCL2 expression in vitro and ex vivo, leukocyte mobilization in vivo, and deleterious disease outcomes. However, the molecular basis for the rs1024611-associated differential CCL2 expression remains poorly characterized. It is conceivable that genetic variant(s) in linkage disequilibrium (LD) with rs1024611 could mediate such effects. Previously, we used rs13900 (alleles: C>T) in theCCL23’ untranslated region (3’ UTR) that is in perfect LD with rs1024611 to demonstrate allelic expression imbalance (AEI) ofCCL2in heterozygous individuals. Here we tested the hypothesis that the rs13900 could modulateCCL2expression by altering mRNA turnover and/or translatability. The rs13900 T allele conferred greater stability to theCCL2transcript when compared to the rs13900 C allele. The rs13900 T allele also had increased binding to Human Antigen R (HuR), an RNA-binding protein, in vitro and ex vivo. The rs13900 alleles imparted differential activity to reporter vectors and influenced the translatability of the reporter transcript. We further demonstrated a role for HuR in mediating allele-specific effects on CCL2 expression in overexpression and silencing studies. The presence of the rs1024611G-rs13900T conferred a distinct transcriptomic signature related to inflammation and immunity. Our studies suggest that the differential interactions of HuR with rs13900 could modulate CCL2 expression and explain the interindividual differences in CCL2-mediated disease susceptibility.
Cell type-specific representation of spatial context in the rat prefrontal cortex
Authors: Brünner H , Kim H , Ährlund-Richter S , van Lunteren J , Crestani AP , Meletis K , Carlén M
Published: November 2023

The ability to represent one’s own position in relation to cues, goals, or threats is crucial to successful goal-directed behavior. Using transgenic rats expressing Cre recombinase in parvalbumin (PV) neurons (PV-Cre rats) we demonstrate cell type-specific encoding of spatial and movement variables in the medial prefrontal cortex (mPFC) during reward foraging. Single neurons encoded the conjunction of the animal’s spatial position and the location of the reward, referred to as the spatial context. The spatial context was most prominently represented by the inhibitory PV interneurons. Movement towards the reward was signified by increased local field potential (LFP) oscillations in the gamma band but this LFP signature was not related to the spatial information in the neuronal firing. The results highlight how spatial information is incorporated into cognitive operations in the mPFC. The presented PV-Cre line opens for expanded research approaches in rats.
High-quality long-read genome assemblies reveal evolutionary patterns of transposable elements and DNA methylation in termites
Authors: Qiu Bitao , Elsner Daniel , Korb Judith
Published: November 2023

Termites are social cockroaches that evolved eusociality independently from social Hymenoptera (ants, bees, wasps). Termites’ large genome sizes, generally composed of around 50% of transposable elements, make it challenging to obtain high quality genome assemblies with short-read sequencing technology. Correspondingly, there are only six published termite genome assemblies. Using PacBio HiFi sequencing, we generated high quality genome assemblies using single individuals for three additional termite species (Trinervitermessp.1,Odontotermessp.2,Macrotermes bellicosus) and a highly improved assembly for a fourth species (Cryptotermes secundus). We simultaneously profiled TE abundance and CpG DNA methylation from the same individuals. We show that TE abundance and DNA methylation levels across species reflect their phylogenetic relationships. We identified lineage- and species-specific TE- and DNA-methylation patterns, and revealed a negative correlation between TE abundance and methylation level. These results imply evolutionary arms-races between TE and DNA methylation during termite evolution.
Aberrant splicing in Huntington’s disease via disrupted TDP-43 activity accompanied by altered m6A RNA modification
Authors: Nguyen Thai B. , Miramontes Ricardo , Chillon-Marinas Carlos , Maimon Roy , Vazquez-Sanchez Sonia , Lau Alice L. , McClure Nicolette R. , England Whitney E. , Singha Monika , Stocksdale Jennifer T. , Jang Ki-Hong , Jung Sunhee , McKnight Jharrayne I. , Ho Leanne N. , Faull Richard L.M. , Steffan Joan S. , Reidling Jack C. , Jang Cholsoon , Lee Gina , Cleveland Don W. , Lagier-Tourenne Clotilde , Spitale Robert C. , Thompson Leslie M.
Published: November 2023

Huntington’s disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of theHTTgene encoding huntingtin. Prior reports have established a correlation between CAG expandedHTTand altered gene expression. However, the mechanisms leading to disruption of RNA processing in HD remain unclear. Here, our analysis of the reported HTT protein interactome identifies interactions with known RNA-binding proteins (RBPs). Total, long-read sequencing and targeted RASL-seq of RNAs from cortex and striatum of the HD mouse model R6/2 reveals increased exon skipping which is confirmed in Q150 and Q175 knock-in mice and in HD human brain. We identify the RBP TDP-43 and the N6-methyladenosine (m6A) writer protein methyltransferase 3 (METTL3) to be upstream regulators of exon skipping in HD. Along with this novel mechanistic insight, we observe decreased nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 in HD mice and human brain. In addition, TDP-43 co-localizes with HTT in human HD brain forming novel nuclear aggregate-like bodies distinct from mutant HTT inclusions or previously observed TDP-43 pathologies. Binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a novel mechanism underlying alternative splicing/unannotated exon usage in HD and highlights the critical nature of TDP-43 function across multiple neurodegenerative diseases.
Peptide aggregation-induced immunogenic cell death in a breast cancer spheroid model
Authors: Gunay Gokhan , Maier Katelyn N. , Hamsici Seren , Carvalho Filipa , Timog Tristan A. , Acar Handan
Published: November 2023

Utilizing multicellular aggregates (spheroids) for in vitro cancer research offers a physiologically relevant model that closely mirrors the intricate tumor microenvironment, capturing properties of solid tumors such as cell interactions and drug resistance. In this research, we investigated the Peptide-Aggregation Induced Immunogenic Response (PAIIR), an innovative method employing engineered peptides we designed specifically to induce immunogenic cell death (ICD). We contrasted PAIIR-induced ICD with standard ICD and non-ICD inducer chemotherapeutics within the context of three-dimensional breast cancer tumor spheroids. Our findings reveal that PAIIR outperforms traditional chemotherapeutics in its efficacy to stimulate ICD. This is marked by the release of key damage-associated molecular patterns (DAMPs), which bolster the phagocytic clearance of dying cancer cells by dendritic cells (DCs) and, in turn, activate powerful anti-tumor immune responses. Additionally, we observed that PAIIR results in elevated dendritic cell activation and increased antitumor cytokine presence. This study not only showcases the utility of tumor spheroids for efficient high-throughput screening but also emphasizes PAIIR’s potential as a formidable immunotherapeutic strategy against breast cancer, setting the stage for deeper exploration and potential clinical implementation.
Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function
Authors: Blazeski Adriana , Floryan Marie A. , Fajardo-Ramírez Oscar R. , Meibalan Elamaran , Ortiz-Urbina Jesús , Angelidakis Emmanouil , Shelton Sarah E. , Kamm Roger D. , García-Cardeña Guillermo
Published: November 2023

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell phenotype and vascular structure. While vascular responses to flow are complex and context-dependent, endothelial cell signaling in response to shear stress induced by laminar flows is coordinated by the transcription factor KLF2. The expression of KLF2 in endothelial cells is associated with a quiescent, anti-inflammatory phenotype and has been well characterized in two-dimensional systems, but has not been studied in three-dimensionalin vitrosystems. Here we develop engineered microvascular networks (MVNs) with a KLF2-based endothelial cell sensor within a microfluidic chip, apply continuous flow using an attached microfluidic pump, and study the effects of this flow on vascular structure and function. We found that culture of MVNs exposed to flow for 48 hours that resulted in increased expression of the KLF2-GFP-reporter display larger vessel diameters and decreased vascular branching and resistance. Additionally, vessel diameters after the application of flow were independent of initial MVN morphologies. Finally, we found that MVNs exposed to flow have improved vascular barrier function and decreased platelet adhesion. The MVNs with KLF2-based flow sensors represent a powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.
Improving the accuracy of bulk fitness assays by correcting barcode processing biases
Authors: McGee Ryan Seamus , Kinsler Grant , Petrov Dmitri , Tikhonov Mikhail
Published: November 2023

Measuring the fitnesses of genetic variants is a fundamental objective in evolutionary biology. A standard approach for measuring microbial fitnesses in bulk involves labeling a library of genetic variants with unique sequence barcodes, competing the labeled strains in batch culture, and using deep sequencing to track changes in the barcode abundances over time. However, idiosyncratic properties of barcodes (e.g., GC content) can induce non-uniform amplification or uneven sequencing coverage that cause some barcodes to be over-or under-represented in samples. This systematic bias can result in erroneous read count trajectories and misestimates of fitness. Here we develop a computational method for inferring the effects of processing bias by leveraging the structure of systematic deviations in the data. We illustrate this approach by applying it to fitness assay data collected for a large library of yeast variants, and show that this method estimates and corrects for bias more accurately than standard proxies, such as GC-based corrections. Our method mitigates bias and improves fitness estimates in high-throughput assays with-out introducing additional complexity to the experimental protocols, with potential value in a range of experimental evolution and mutation screening contexts.