bluuebunny/crossref_metadata_2025 huggingface dataset:
Environmental drivers of the decline of the fen orchid, Liparis loeselii
Authors: Andersen Dagmar Kappel , Ejrnæs Rasmus , Minter Martine , Riis Tenna , Vinther Erik , Bruun Hans Henrik
Published: February 2024

Background

Liparis loeseliiis a rare and declining orchid species restricted to rich fens in the Northern Hemisphere. Habitat destruction, eutrophication, drainage and scrub encroachment have been suggested as reasons for the decline. However, which factors are most important is not well understood.

Aims

Based on vegetation and environmental properties from extant, potential and historicalL. loeseliisites, we i) developed habitat suitability models from either Ellenberg Indicator Values or field-measured environmental properties, and ii) identified the primary reasons for the observed decline ofL. loeselii.

Results

Nutrient status was the most important predictor forL. loeseliioccurrence, followed by hydrology proxies (Ellenberg reaction and Ellenberg moisture). Vegetation height and Ellenberg light were of minor importance.

Conclusion

Effect partitioning based on sites, from whichL. loeseliihas gone locally extinct, pinpointed eutrophication and drainage to be the most likely primary drivers of the species’ demise. Phosphorus limitation induced by discharge of Calcium-rich groundwater seems to be crucial forL. loeseliito sustain populations in landscapes dominated by intensive agricultural.
The Apurinic/Apyrimidinic Endodeoxyribonuclease 1 is an RNA G-quadruplex binding protein and regulates miR-92b expression in cancer cells
Authors: Bellina Alessia , Malfatti Matilde Clarissa , Salgado Gilmar , Fleming Aaron M. , Antoniali Giulia , Gualandi Nicolò , La Manna Sara , Marasco Daniela , Dassi Erik , Burrows Cynthia J. , Tell Gianluca
Published: February 2024

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as a DNA repair enzyme, unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized- or abasic-miRNAs during oxidative stress conditions. Furthermore, in recent years several non-canonical pathways of miRNA biogenesis have been described, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. In this study, we show that several miRNA precursors, dysregulated upon APE1-depletion, contain an rG4 motif and that their corresponding target genes are upregulated after APE1-depletion. We also show, both byin vitroassays and by using a HeLa cell model, that APE1 can bind and regulate the folding of an rG4 structure contained in pre-miR92b, with a mechanism strictly dependent on critical lysine residues present in the N-terminal disordered region. Furthermore, APE1 depletion in HeLa cells alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Lastly, bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings for cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver cancer, as suggested by their involvement in several cancer-related pathways.

Significance Statement

We highlight an undescribed non-canonical role of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) in the context of RNA G-quadruplexes (rG4), specifically in the alternative pathway of miRNA maturation of guanosine-rich miRNA precursors. Specifically, APE1 binds these structures and modulates their folding, mainly through its N-terminal region and some residues in its catalytic domain. Moreover, we showed an interesting new role of APE1 in regulating the shuttling and accumulation of miR-92b between the nuclear and cytosolic compartments, opening new perspectives on how APE1 may exercise its role in the miRNA maturation pathway and function. Moreover, APE1-depleted dysregulated miRNAs with rG4 motifs in their precursors have significant prognostic value in lung, cervical, and liver tumors, suggesting potential targets for cancer therapy.
Escape from X inactivation is directly modulated by levels of Xist non-coding RNA
Authors: Hauth Antonia , Panten Jasper , Kneuss Emma , Picard Christel , Servant Nicolas , Rall Isabell , Pérez-Rico Yuvia A. , Clerquin Lena , Servaas Nila , Villacorta Laura , Jung Ferris , Luong Christy , Chang Howard Y. , Zaugg Judith B. , Stegle Oliver , Odom Duncan T. , Loda Agnese , Heard Edith
Published: February 2024

In placental females, one copy of the two X chromosomes is largely silenced during a narrow developmental time window, in a process mediated by the non-coding RNA Xist1. Here, we demonstrate that Xist can initiate X-chromosome inactivation (XCI) well beyond early embryogenesis. By modifying its endogenous level, we show that Xist has the capacity to actively silence genes that escape XCI both in neuronal progenitor cells (NPCs) andin vivo, in mouse embryos. We also show that Xist plays a direct role in eliminating TAD-like structures associated with clusters of escapee genes on the inactive X chromosome, and that this is dependent on Xist’s XCI initiation partner, SPEN2. We further demonstrate that Xist’s function in suppressing gene expression of escapees and topological domain formation is reversible for up to seven days post-induction, but that sustained Xist up-regulation leads to progressively irreversible silencing and CpG island DNA methylation of facultative escapees. Thus, the distinctive transcriptional and regulatory topologies of the silenced X chromosome is actively, directly - and reversibly - controlled by Xist RNA throughout life.
Septin-associated PIPKIγ splice variants drive centralspindlin association with the midbody via PI(4,5)P2
Authors: Russo Giulia , Hümpfer Nadja , Jaensch Nina , Restel Steffen , Schmied Christopher , Heyd Florian , Lehmann Martin , Haucke Volker , Ewers Helge , Krauss Michael
Published: February 2024

Mammalian cytokinesis critically depends on the phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] which serves as docking site for crucial components of the cytokinetic machinery at the plasma membrane. PI(4,5)P2supports several stages of cytokinesis, including actomyosin ring assembly and constriction, membrane tethering of spindle microtubules, and midbody organization. How these various activities of PI(4,5)P2and the underlying mechanisms of local PI(4,5)P2synthesis are orchestrated in space and time has remained elusive. Here, we identify a pivotal role of septin-binding splice variants of PIPKIγ that couple nanoscale PI(4,5)P2synthesis at the ingressing cleavage furrow to late midbody formation. Depletion of PIPKIγ isoforms causes multinucleation and perturbs anillin and septin deposition at the intercellular bridge and at the midbody. These defects are rescued by wild-type kinase, but not by septin binding-deficient or catalytically inactive PIPKIγ variants. We further show that both, septins and PIPKIγ form a complex with centralspindlin, and thereby facilitate the recruitment of centralspindlin to the midbody. Taken together, our findings establish septin-associated PIPKIγ isoforms as key regulators of midbody organization that act through generating a local pool of PI4,5P2required for centralspindlin recruitment and maintenance at the midbody, and for septin association with microtubules.
Human MX1 orchestrates the cytoplasmic sequestration of neo-synthesized influenza A virus vRNPs
Authors: McKellar Joe , García de Gracia Francisco , Aubé Corentin , Chaves Valadão Ana Luiza , Tauziet Marine , Arnaud-Arnould Mary , Rebendenne Antoine , Neyret Aymeric , Labaronne Emmanuel , Ricci Emiliano , Delaval Bénédicte , Gaudin Raphaël , Naffakh Nadia , Gallois-Montbrun Sarah , Moncorgé Olivier , Goujon Caroline
Published: February 2024

Interferon-inducible Myxovirus resistance 1 (MX1) proteins are well-known to restrict influenza A virus (IAV) at early stages during viral replication, impairing the viral transcription/replication process. Herein, we show that this early restriction was only partial against human IAVs, whereas a strong inhibition of viral production was observed. Indeed, relatively high levels of IAV mRNAs and proteins were observed in the presence of human (Hs) and mouse (Mm) MX1 proteins but additional inhibition processes occurred at later stages of IAV life cycle. Hence, MmMx1 induced an abnormal nuclear accumulation of the viral nucleoprotein (NP) at late time points post-infection. This block was also observed, albeit to a much lower extent, with HsMX1. In most HsMX1-expressing cells, vRNPs could be exported from the nucleus to the cytoplasm however a potent defect in subsequent vRNP cytoplasmic trafficking was observed. Indeed, vRNPs were found sequestrated together with cellular co-factors YBX1 and Rab11a in large clusters in the vicinity of the microtubule organization center (MTOC). Live imaging experiments revealed that the transient association of HsMX1 with Rab11a-associated vRNPs favoured their dynein-dependant retrograde transport along microtubules towards the MTOC. Importantly, dynein inhibition prevented the vRNP sequestration and significantly rescued infectious viral production in the presence of HsMX1, showing a significant contribution of these abnormal vRNP clusters in HsMX1 antiviral activity. In conclusion, this study provides the first evidence of IAV vRNPs being re-routed and accumulated away from the plasma membrane, through the coordinated action of HsMX1 restriction factor, dynein and the microtubule network.
Genotypic variation in blueberry flower morphology and nectar reward content affects pollinator attraction in a diverse breeding population
Authors: Cromie Juliana , Ternest John J. , Komatz Andrew P. , Adunola Paul M. , Azevedo Camila , Mallinger Rachel E. , Muñoz Patricio R.
Published: February 2024

Pollination is crucial to obtain optimal blueberry yield and fruit quality. Despite substantial investments in seasonal beekeeping services, blueberry producers consistently report suboptimal pollinator visitation and fruit set in some cultivars. Flower morphology and floral rewards are among the key factors that have shown to contribute to pollinator attraction, however little is known about their relative importance for improving yield in the context of plant breeding. Clarifying the relationships between flower morphology, nectar reward content, pollinator recruitment, and pollination outcomes, as well as their genetic components, can inform breeding priorities for enhancing yield. In the present study, we measured ten flower and nectar traits and indices of successful pollination, including fruit set, seed count, and fruit weight in 38 southern highbush blueberry genotypes. Additionally, we assessed pollinator visitation frequency and foraging behavior over two growing seasons. Several statistical models were tested to optimize the prediction of pollinator visitation and pollination success, including partial least squares, BayesB, ridge-regression, and random forest. Random forest models obtained high predictive abilities for pollinator visitation frequency, with values of 0.54, 0.52, and 0.66 for honey bee, bumble bee, and total pollinator visits, respectively. The BayesB model provided the most consistent prediction of fruit set, fruit weight, and seed set, with predictive abilities of 0.07, -0.08, and 0.42, respectively. Variable importance analysis revealed that genotypic differences in nectar volume had the greatest impact on honey bee and bumble bee recruitment, although preferences for flower morphological traits varied depending on the foraging task. Flower density was a major driving factor attracting nectar-foraging honey bees and bumble bees, while pollen-foraging bumble bees were most influenced by flower accessibility, specifically corolla length and the length-to-width ratio. Corolla length and the length-to-width ratio were also identified as the main predictors of fruit set, fruit weight, and seed count, suggesting that bumble bees and their foraging preferences may play a pivotal role in fruit production. Moderate to high narrow-sense heritability values (ranging from 0.30 to 0.77) were obtained for all floral traits, indicating that selective breeding efforts may enhance cultivar attractiveness to pollinators.
Rational protein engineering to enhance MHC-independent T cell receptors
Authors: Chang Ju-Fang , Landmann Jack H. , Chang Tien-Ching , Tenzin Yangdon , Warrington John M. , Selli Mehmet Emrah , Ritchey Julie , Hsu Yu-Sung , Slade Michael , Gupta Deepesh Kumar , DiPersio John F. , Singh Nathan
Published: February 2024

Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity against cancer, however remain limited in their scope and long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the endogenous T cell response, we designed hybrid MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant domains. While functional, we observed stark differences in miTCR-driven T cell function that were dependent on receptor orientation. Using predictive structural modeling, we observed significant biochemical conflicts at the hybrid variable-constant domain interface. To overcome this, we performed iterative sequence modifications and structural modeling to design a panel of miTCR variants predicted to have improved interface stability. Functional screening nominated a variant with superior efficacy to all other miTCRs as well as a standard CAR against high burdens of leukemia.

Statement of Significance

Improving the durability of engineered T cell immunotherapies is critical to enhancing efficacy. We used structure-informed design to evolve MHC-independent T cell receptors that drive improved tumor control. This work underscores the central role of synthetic receptor structure on T cell function and provides a framework for improved receptor engineering.
Elucidating TolC Protein Dynamics: Structural Shifts Facilitate Efflux Mediated β-lactam Resistance
Authors: Kantarcioglu Isik , Gaszek Ilona K. , Guclu Tandac F. , Yildiz M. Sadik , Atilgan Ali Rana , Toprak Erdal , Atilgan Canan
Published: February 2024

Efflux-mediated β-lactam resistance represents a significant public health challenge, limiting the efficacy of various β-lactam antibiotics against numerous clinically relevant pathogenic bacteria. Structural and functional analyses have revealed that the efflux protein TolC in several Gram-negative bacteria serves as a conduit for antibiotics, bacteriocins, and phages, affecting bacterial susceptibility and virulence. In this study, we conducted a comprehensive examination of the efflux of β-lactam drugs mediated by TolC, employing extensive experimental and computational analyses. Our computational investigations into the molecular dynamics of drug-free TolC revealed critical unidirectional movements of the trimeric TolC and identified residues significantly involved in TolC opening. To corroborate these findings, we performed a whole-gene-saturation mutagenesis assay, systematically mutating each residue of TolC to 19 other amino acids and measuring the fitness effects of these mutations under β-lactam-induced selection. The β-lactams oxacillin, piperacillin, and carbenicillin were selected for this study because they are effluxed by the AcrAB-TolC complex with varying efficiencies. This approach clarified the similarities and differences in the efflux processes of the three β-lactam antibiotics through the trimeric TolC. Further analysis of TolC’s efflux mechanism for these β-lactam antibiotics via steered molecular dynamics simulations revealed the existence of general and drug-specific mechanisms employed by TolC. We identified key positions at the periplasmic entry of TolC whose altered dynamics influence long-range efflux motions as allosteric modulators. Our findings provide valuable insights into the structural dynamics of TolC, establishing a foundation for understanding the key mechanisms behind multidrug resistance and principles for designing new antibiotics and antibiotic derivatives capable of circumventing the bacterial efflux mechanism.
Nova-ST: Nano-Patterned Ultra-Dense platform for spatial transcriptomics
Authors: Poovathingal Suresh , Davie Kristofer , Vandepoel Roel , Poulvellarie Nicholas , Verfaillie Annelien , Corthout Nikky , Aerts Stein
Published: February 2024

Spatial transcriptomics workflows using barcoded capture arrays are commonly used for resolving gene expression in tissues. However, existing techniques are either limited by capture array density or are cost prohibitive for large scale atlasing. We present Nova-ST, a dense nano-patterned spatial transcriptomics technique derived from randomly barcoded Illumina sequencing flow cells. Nova-ST enables customized, low cost, flexible, and high-resolution spatial profiling of large tissue sections. Benchmarking on mouse brain sections demonstrates significantly higher sensitivity compared to existing methods, at reduced cost.

Motivation

Spatial transcriptomics analysis is becoming exceedingly important in biomedical and clinical research. Several platforms for spatial transcriptomics are currently available, but most of these technologies are commercialized making them inflexible and cost prohibitive. The motivation for this work was to establish an open source, flexible and sensitive sequencing-based spatial transcriptomics platform that can provide a considerable cost advantage for performing large scale spatial profiling. We provide thorough and in-depth guidance and resources both for the experimental and computational components of the workflow, to facilitate easy implementation of Nova-ST.
Androgens modulate the immune profile in a mouse model of polycystic ovary syndrome
Authors: Torstensson Sara , Ascani Angelo , Risal Sanjiv , Lu Haojiang , Zhao Allan , Espinosa Alexander , Lindgren Eva , Johansson Maria H. , Eriksson Gustaw , Barakat Maya , Karlsson Mikael C.I. , Svensson Camilla , Benrick Anna , Stener-Victorin Elisabet
Published: February 2024

Polycystic ovary syndrome (PCOS) is associated with a low-grade inflammation, but it is unknown how hyperandrogenism, the hallmark of PCOS, affects the immune system. Using a well-established PCOS-like mouse model, we demonstrate that androgen exposure affects immune cell populations in reproductive, metabolic, and immunological tissues differently in a site-specific manner. Co-treatment with flutamide, an androgen receptor antagonist, prevents most of these alterations, demonstrating that these effects are mediated through androgen receptor activation. Dihydrotestosterone (DHT)-exposed mice display a drastically reduced eosinophil population in uterus compared to controls, coupled with lower levels of eotaxin (CCL11), suggesting a reduced recruitment from blood. Decreased frequencies of eosinophils were also seen in visceral adipose tissue (VAT). A higher expression level of CD69, a marker of activation or tissue residency, was consistently found on natural killer (NK) cells in all analyzed tissues. However, a higher frequency of NK cells and elevated levels of IFN-γ and TNF-α were only seen in uteri of androgen-exposed mice, while NK cell frequencies were unaffected in all other analyzed compartments. Distinct alterations of macrophages in ovaries, uterus and VAT were also found in DHT-exposed mice and could potentially be linked to PCOS-like traits of the model. Indeed, androgen-exposed mice were insulin resistant and displayed an aberrant immune profile in VAT, albeit unaltered fat mass. Collectively, we demonstrate that hyperandrogenism causes tissue-specific alterations of immune cells in reproductive organs and VAT, which could have considerable implications on tissue function and contribute to the reduced fertility and metabolic comorbidities associated with PCOS.
Comparison of gnotobiotic communities reveals unexpected amino acid metabolism by the pre-weaning microbiome
Authors: Lubin Jean-Bernard , Silverman Michael A. , Planet Paul J.
Published: February 2024

The intestinal microbiome during infancy and childhood has distinct compositions and metabolic functions to that of adults. We recently published a gnotobiotic mouse model of the pre-weaning microbiome (PedsCom), which retains a pre-weaning configuration during the transition from a milk-based diet to solid foods and leads to a stunted immune system and susceptibility to enteric infection. Here we compared the phylogenetic and metabolic relationship of the PedsCom consortium to the adult-derived gnotobiotic communities, Altered Schaedler Flora and Oligo-MM12. We find that PedsCom contains several unique functions relative to adult-derived mouse consortia. In particular, amino acid degradation metabolic modules are more prevalent among PedsCom isolates, which is in line with the ready availability of these nutrients in milk. Indeed, metabolomic analysis showed significantly lower levels of free amino acids in the intestinal contents of adult PedsCom colonized mice versus Oligo-MM12 controls. Thus, enhanced amino acid metabolism is a prominent feature of the pre-weaning microbiome that may facilitate design of early life microbiome interventions.
Activity of DNA polymerase κ across the genome in human fibroblasts
Authors: Torres Mariela C. , Sun Dongxiao , Spratt Thomas E.
Published: February 2024

DNA polymerase κ (Pol κ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replicating repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Pol κ utilizing a Pol κ - specific substrate,N2-(4-ethynylbenzyl)-2′-deoxyguanosine (EBndG). After shearing of the DNA into 200-500bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)- catalyzed alkyne–azide cycloaddition reaction, and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method polymerase κ sequencing (polK-seq). Here we present the human genome maps for pol κ activity in an undamaged cell line. We found that pol κ activity was enhanced in euchromatin regions, the promoter of genes, and in DNA that is replicated early in S-phase.
Drug-Target Interactions Prediction at Scale: the Komet Algorithm with the LCIdb Dataset
Authors: Guichaoua Gwenn , Pinel Philippe , Hoffmann Brice , Azencott Chloé-Agathe , Stoven Véronique
Published: February 2024

Drug-target interactions (DTIs) prediction algorithms are used are various stages of the drug discovery process. In this context, specific problems such as de-orphanization of a new therapeutic target, or target identification of a drug candidate arising from phenotypic screens require large-scale predictions across the protein and molecule spaces. DTI prediction heavily relies on supervised learning algorithms that use known DTIs to learn associations between molecule and protein features, allowing for the prediction of new interactions based on learned patterns. The algorithms must be broadly applicable to enable reliable predictions, even in regions of the protein or molecule spaces where data may be scarce. In this paper, we address two key challenges to fulfil these goals: building large, high-quality training datasets and designing prediction methods that can scale, in order to be trained on such large datasets. First, we introduce LCIdb, a curated, large-sized dataset of DTIs, offering extensive coverage of both the molecule and druggable protein spaces. Notably, LCIdb contains a much higher number of molecules than publicly available benchmarks, expanding coverage of the molecule space. Second, we propose Komet (Kronecker Optimized METhod), a DTI prediction pipeline designed for scalability without compromising performance. Komet leverages a three-step framework, incorporating efficient computation choices tailored for large datasets and involving the Nyström approximation. Specifically, Komet employs a Kronecker interaction module for (molecule, protein) pairs, which efficiently captures determinants in DTIs, and whose structure allows for reduced computational complexity and quasi-Newton optimization, ensuring that the model can handle large training sets, without compromising on performance. Our method is implemented in open-source software, leveraging GPU parallel computation for efficiency. We demonstrate the interest of our pipeline on various datasets, showing that Komet displays superior scalability and prediction performance compared to state-of-the-art deep learning approaches. Additionally, we illustrate the generalization properties of Komet by showing its performance on an external dataset, and on the publicly availableℒℌbenchmark designed for scaffold hopping problems. Komet is available open source athttps://komet.readthedocs.ioand all datasets, including LCIdb, can be found athttps://zenodo.org/records/10731712.
Protein aggregation is a consequence of the dormancy-inducing membrane toxin TisB inEscherichia coli
Authors: Leinberger Florian H. , Cassidy Liam , Edelmann Daniel , Schmid Nicole E. , Blumenkamp Patrick , Schmidt Sebastian , Natriashvili Ana , Ulbrich Maximilian H. , Tholey Andreas , Koch Hans-Georg , Berghoff Bork A.
Published: February 2024

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present an optimized plasmid expression system for toxin genes based on an artificial 5’ untranslated region. We applied the system to induce expression of the toxin genetisBfrom the chromosomal type I toxin- antitoxin systemtisB/istR-1inEscherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA- sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genesibpB,spyandcpxP. The importance of chaperone genes suggested that TisB- producing cells are prone to protein aggregation, which was validated by anin vivofluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin inE. coliwild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells.

Importance

Protein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including the exposure to antibiotics. InEscherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which in turn promotes a deeper state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.
TNFR1-mediated senescence and lack of TNFR2-signaling limit human intervertebral disc cell repair in back pain conditions
Authors: Gansau Jennifer , Grossi Elena , Rodriguez Levon , Wang Minghui , Laudier Damien M. , Chaudhary Saad , Hecht Andrew C. , Fu Wenyu , Sebra Robert , Liu Chuanju , Iatridis James C.
Published: February 2024

Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of TNFα-receptors (TNFRs) in contributing to poor healing in painful IVDD. We first isolated IVDD tissue of back pain subjects and determined the complex pro-inflammatory mixture contained many chemokines for recruiting inflammatory cells. Single-cell RNA-sequencing of human IVDD tissues revealed these pro- inflammatory cytokines were dominantly expressed by a small macrophage-population. Human annulus fibrosus (hAF) cells treated with IVDD-conditioned media (CM) underwent senescence with greatly reduced metabolic rates and limited inflammatory responses. TNFR1 inhibition partially restored hAF cell metabolism sufficiently to enable a robust chemokine and cytokine response to CM. We showed that the pro-reparative TNFR2 was very limited on hIVD cell membranes so that TNFR2 inhibition with blocking antibodies or activation using Atsttrin had no effect on hAF cells with CM challenge. However, TNFR2 was expressed in high levels on macrophages identified in scRNA-seq analyses, suggesting their role in repair responses. Results therefore point to therapeutic strategies for painful IVDD involving immunomodulation of TNFR1 signaling in IVD cells to enhance metabolism and enable a more robust inflammatory response including recruitment or delivery of TNFR2 expressing immune cells to enhance IVD repair.

SUMMARY STATEMENT

TNFR1 signaling drives cells towards senesce and muted inflammatory response in painful intervertebral disc degeneration, while limited TNFR2 signaling may limit disc cell repair responses.
Kinetochores grip microtubules with directionally asymmetric strength
Authors: Larson Joshua D. , Heitkamp Natalie A. , Murray Lucas E. , Popchock Andrew R. , Biggins Sue , Asbury Charles L.
Published: February 2024

For accurate mitosis, all chromosomes must achieve ‘bi-orientation’, with replicated sister chromatids coupled via kinetochores to the plus ends of opposing microtubules. However, kinetochores first bind the sides of microtubules and subsequently find plus ends by directed transport or when side-attached microtubules shorten and bring their ends to the kinetochores. Mitotic accuracy depends on the selective release of erroneous attachments and proposed mechanisms have focused mainly on plus-end attachments. Whether erroneous side-attachments are distinguished from correct side-attachments is unknown. Here we show that side-attached kinetochores are very sensitive to microtubule polarity, gripping six-fold more strongly when pulled toward plus versus minus ends. This directionally asymmetric grip correlates with changes in the axial arrangement of subcomplexes within the kinetochores, suggesting that internal architecture dictates attachment strength. We propose that the kinetochore’s directional grip promotes accuracy specifically during early mitosis, by stabilizing correct attachments even before both sisters have found plus ends.
Effects of childhood hearing loss on the subcortical and cortical representation of speech
Authors: Calcus Axelle , Rosen Stuart
Published: February 2024

Little is known about the effects of childhood mild-to-moderate sensorineural hearing loss (MM HL) on the function of the auditory pathway. We aimed to examine the effect of childhood MM HL and the benefit of frequency-specific amplification on both subcortical and cortical auditory processing, and to relate it to speech-perceptual abilities. We recorded subcortical and cortical responses to speech syllables in nineteen children with congenital MM HL (unamplified and amplified), and sixteen children with typical hearing (unamplified sounds only). Speech perception was measured behaviourally. Congenital HL led to smaller subcortical and cortical responses to unamplified speech sounds. There was a significant benefit of amplification on subcortical and early, but not late, cortical responses, with some effects differing across age. No relationship was found between the neural and behavioural measures. Childhood MM HL affects both subcortical and cortical processing of speech. Amplification mostly benefits subcortical processing of speech in younger children. Childhood HL leads to functional changes in the processing of sounds, with amplification differentially affecting subcortical and cortical levels of the auditory pathway.
Drivers of Vessel Progenitor Fate Define Intermediate Mesoderm Dimensions by Inhibiting Kidney Progenitor Specification
Authors: Perens Elliot A. , Yelon Deborah
Published: February 2024

Proper organ formation depends on the precise delineation of organ territories containing defined numbers of progenitor cells. Kidney progenitors reside in bilateral stripes of posterior mesoderm that are referred to as the intermediate mesoderm (IM). Previously, we showed that the transcription factors Hand2 and Osr1 act to strike a balance between the specification of the kidney progenitors in the IM and the vessel progenitors in the laterally adjacent territory. Recently, the transcription factor Npas4l – an early and essential driver of vessel and blood progenitor formation – was shown to inhibit kidney development. Here we demonstrate how kidney progenitor specification is coordinated byhand2,osr1, andnpas4l. We find thatnpas4lis necessary to inhibit IM formation. Consistent with the expression ofnpas4lflanking the medial and lateral sides of the IM, our findings suggest roles fornpas4lin defining the IM boundaries at each of these borders. At the lateral IM border,hand2promotes andosr1inhibits the formation ofnpas4l-expressing lateral vessel progenitors, andhand2requiresnpas4lto inhibit IM formation and to promote vessel formation. Meanwhile,npas4lappears to have an additional role in suppressing IM fate at the medial border:npas4lloss-of-function enhanceshand2mutant IM defects and results in excess IM generated outside of the lateralhand2-expressing territory. Together, our findings reveal that establishment of the medial and lateral boundaries of the IM requires inhibition of kidney progenitor specification by the neighboring drivers of vessel progenitor fate.
Hypoxia disrupts circadian rhythms in astrocytes and causes synapse engulfment defects
Authors: Li Li , Choi Jong Bin , Shin Chi Hong , Htun Saw , Mestan Sherry , Voss Anna , Shadrach Jennifer L. , Puno Alyssa , Nagar Dhriti , Ramirez Nephy , Rojo Daniela , Lee Samuel H. , Gibson Erin M. , Kaltschmidt Julia A. , Sloan Steven A. , Chung Won-Suk , Pasca Anca M.
Published: February 2024

Astrocytes are emerging as key regulators of neuronal synaptic network maturation and function, through control of synaptic pruning. This is important, because individuals with ASD have excess glutamatergic synapses in the cortex, but the biological mechanisms underlying this phenotype remain unclear.

Here, we used human cortical organoids (hCO) derived from induced pluripotent stem cells (hiPSCs), to examine the effect of hypoxia on synapse engulfment in human astrocytes at postnatal-equivalent stages of development. We identified that hypoxia significantly inhibits the synaptosome phagocytosis, and that this phenotype is mediated through disruptions in the astrocytic circadian rhythm molecular pathway and subsequent decreased expression of MEGF10. Lastly, we demonstrated that circadian clock disruptions are sufficient to induce these observed phenotypes even in the absence of hypoxia, both in hCOs and within the mouse hippocampusin vivo.

Our study uncovers a novel mechanistic link between hypoxia, circadian rhythms disruptions, and synapse pruning by astrocytes, and provides insight into the pathophysiology of ASD, and other neuropsychiatric diseases. Separately, the demonstration of the presence of circadian rhythms in hCOs opens an unprecedented opportunity to dissect the role of circadian clocks in normal brain development and how it contributes to specific diseases of environmental or genetic origin.
Discovery of novel compounds and target mechanisms using a high throughput, multiparametric phenotypic screen in a human neuronal model of Tuberous Sclerosis
Authors: Williams Luis A. , Ryan Steven J. , Joshi Vaibhav , Lewarch Caitlin , Elder Amy , McManus Owen , Godard Patrice , Sridhar Srinidhi , Jacques Jane , Grooms Jennifer , Fink James J. , Zhang Hongkang , Gillard Michel , Pegurier Cécile , Sabnis Yogesh , André Véronique , Steward Lucinda , Dedeurwaerdere Stefanie , Devinsky Orrin , Wolff Christian , Dempsey Graham T.
Published: February 2024

Tuberous sclerosis complex (TSC) is a rare genetic disorder caused by mutations in the mTOR pathway genesTSC1orTSC2. TSC can affect multiple organs including the brain, and most patients (75-90%) present with seizures during early childhood and intractable epilepsy throughout life. mTOR inhibitors, part of the current standard of care, lack the optimal characteristics to fully address patient phenotypes. Here, we report on the application of our all-optical electrophysiology platform for phenotypic screening in a human neuronal model of TSC. We used CRISPR/Cas9-isogenicTSC2−/−iPS cell lines to identify disease-associated changes to neuronal morphology, transcript expression and neuronal excitability. We established a robust multiparametric electrophysiological phenotype which we then validated in TSC patient-derived neurons. We used this phenotype to conduct a screen of ∼30,000 small molecule compounds in human iPS cell-derived neurons and identified chemical scaffolds that rescued the functional TSC disease parameters. Confirmed hits may act via different mechanisms than direct mTOR pathway inhibition. This strategy provides molecular starting points for therapeutic development in TSC and a framework for phenotype discovery and drug screening in other neurological disorders.
WITHDRAWN: Investigating the ability of music to induce calm in young adults
Authors: Shepherd Daniel , Pedersen Mangor , Vashista Geet , Kercher Amy , Hautus Michael J.
Published: February 2024

Withdrawal Statement

The authors have withdrawn their manuscript owing to reviewer feedback. A revised version of this work will be uploaded with significant changes. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.
Direction-aware functional class scoring enrichment analysis of Infinium DNA methylation data
Authors: Ziemann Mark , Abeysooriya Mandhri , Bora Anusuiya , Lamon Séverine , Kasu Mary Sravya , Norris Mitchell W. , Wong Yen Ting , Craig Jeffrey M.
Published: February 2024

Infinium Methylation BeadChip arrays remain one of the most popular platforms for epigenome-wide association studies, but tools for downstream pathway analysis have their limitations. Functional class scoring (FCS) is a group of pathway enrichment techniques that involve the ranking of genes and evaluation of their collective regulation in biological systems, but the implementations described for Infinium methylation array data do not retain direction information, which is important for mechanistic understanding of genomic regulation. Here, we evaluate several candidate FCS methods that retain directional information. According to simulation results, the best-performing method involves the mean aggregation of probe limma t-statistics by gene followed by a rank-ANOVA enrichment test using the mitch package. This method, which we call “LAM”, outperformed an existing over-representation analysis method in simulations, and showed higher sensitivity and robustness in an analysis of real lung tumour-normal paired datasets. Using matched RNA-seq data we examine the relationship of methylation differences at promoters and gene bodies with RNA expression at the level of pathways in lung cancer. To demonstrate the utility of our approach, we apply it to three other contexts where public data were available. Firstly, we examine differential pathway methylation associated with chronological age. Secondly, we investigate pathway methylation differences in infants conceived with in vitro fertilisation. Lastly, we analyse differential pathway methylation in 19 disease states, identifying hundreds of novel associations. These results show LAM is a powerful method for the detection of differential pathway methylation as compared to existing methods. A reproducible vignette is provided to illustrate how to implement this method.
Identification and characterization of a bacterial periplasmic solute binding protein that binds L-amino acid amides
Authors: Smith Oliver B. , Frkic Rebecca L. , Rahman Marina G. , Jackson Colin J. , Kaczmarski Joe A.
Published: February 2024

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP fromMesorhizobiumsp. A09 (MeAmi_SBP) and show thatMeAmi_SBP bindsl-amino acid amides but not the correspondingl-amino acids. An X-ray crystal structure ofMeAmi_SBP bound tol-serinamide highlights the residues that impart distinct specificity forl-amino acid amides and reveals a structural Ca2+binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+binding are conserved amongst the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake and metabolism ofl-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose thatMeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions withl-amino acid amides and bacterial nitrogen utilization.
Power-to-Vitamins: Producing Folate (Vitamin B9) from Renewable Electric Power and CO2with a Microbial Protein System
Authors: Schmitz Lisa Marie , Kreitli Nicolai , Obermaier Lisa , Weber Nadine , Rychlik Michael , Angenent Largus T.
Published: February 2024

Meeting a surging demand for superior micronutrient-rich protein sources and finding production practices that are less detrimental to the climate will be critical challenges of the 21stcentury. New technologies are needed to decouple food production from land use. Our group previously proposed a two-stage Power-to-Protein technology to produce microbial protein from renewable electric power and CO2. Two stages were operatedin series:(1)Clostridium ljungdahliiin Stage A to utilize H2 to reduce CO2 into acetate; and(2)Saccharomyces cerevisiaein Stage B to utilize O2 and produce microbial protein from acetate. Renewable energy would power water electrolysis to produce H2 and O2. A disadvantage ofC. ljungdahliiin Stage A is the need to continuously feed vitamins to sustain growth and acid production. Changing to the more robust thermophilic acetogenThermoanaerobacter kivuiavoids providing any vitamins. Additionally,S. cerevisiaeproduces folate when grown with acetate as a sole carbon source under aerobic conditions. A total folate concentration of 6.7 mg per 100 g biomass with an average biomass concentration of 3 g L-1in Stage B is achieved. The developed Power-to-Vitamin system enables folate production from renewable power and CO2 with zero or negative net-carbon emissions.
End-user feedback of rapid diagnostics in rural Kenya
Authors: Beard Jeffrey W. , Pariser Daphne N. , Monticelli Stephanie , Miller Benjamin L.
Published: February 2024

Underserved communities in low-resource countries are disproportionately impacted by communicable diseases when compared to those in developed countries. These communities have limited access to life saving diagnostic laboratory tests making it difficult to treat communicable diseases like SARS-CoV-2 and Human Immunodeficiency Virus (HIV). Rapid diagnostic tests, like the COVID-19 antigen (Ag) test, play a crucial role in underserved communities by enabling fast and inexpensive diagnosis in low-resource settings. Unfortunately, these rapid test platforms often lack the accuracy and precision of their laboratory-based analogs, resulting in a need for improved rapid diagnostics. The World Health Organization’s (WHO) ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end-users) criteria are often referenced in the development of diagnostic tests. In this work, we aim to provide guidance to the “user-friendly” component of ASSURED through end-user surveys taken in rural Kenya. In these surveys, we examine the user-friendliness of two of the most commonly used rapid diagnostic tests, the COVID-19 Ag test and pregnancy test, by assessing participants’ familiarity with the tests, their opinion of test appearance, and the perceived complexity of the operator’s workflow. We also examine community acceptance and desire for a self-test for the highly stigmatized HIV. We intend these results to help guide developers of future rapid diagnostic tests intended for low-resource communities.
Demonstrating the Beneficial Effect of Low Protein Diet in Primary Sclerosing Cholangitis through a Randomized Clinical Trial and Multi-omics Data Analysis
Authors: Yin Xiaole , Sasson Gila , Sun Zheng , Ke Shanlin , Babazadeh Demsina , Mahmood Shaikh Danish , Andrews Macie , Hurwitz Shelley , Chikowore Tinashe , Paul Maia , Javier Nadine , Dave Malav , Austin Alexandra , Gray Linda , Steinberg Francene , Souza Elaine , Bowlus Christopher , Liu Yang-Yu , Korzenik Joshua
Published: February 2024

Primary sclerosing cholangitis (PSC), a progressive cholestatic hepatobiliary disease characterized by inflammation and fibrosis of the bile ducts, has a pathophysiology that is not understood. No effective therapies exist. The only treatment option for PSC is liver transplant. We undertook a pilot randomized trial of diet to investigate the pathophysiology of the disease, the role of diet and to advance potential therapy. We enrolled 20 patients with PSC and randomly assigned them to a Low Protein/low sulfur Diet (LPD, n=10) or the Specific Carbohydrate Diet (SCD, n=10) for 8 weeks. Results showed that low protein intake benefits PSC patients, whereas higher protein levels exacerbate the condition. We further identified gut bacterial markers useful for distinguishing LPD responders (mostly PSC with concomitant ulcerative colitis) from non-responders. Additionally, by integrating multi-omics data, we propose that this diet modifies the intestinal sulfur cycle reducing hydrogen sulfide (H2S) production. Our findings provide an understanding of the beneficial effect of LPD as well as insights into a possible key driver of inflammation in PSC.