bluuebunny/crossref_metadata_2025 huggingface dataset:
Lactate activates the mitochondrial electron transport chain independent of its metabolism
Authors: Cai Xin , Ng Charles C. , Jones Olivia , Fung Tak Shun , Ryu Keunwoo , Li Dayi , Thompson Craig B.
Published: August 2023

Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.
WITHDRAWN:MHC-BDiversity and Signs of Respiratory Illness in Wild, East African Chimpanzees (Pan troglodytes schweinfurthii)
Authors: Phillips SR
Published: August 2023

Withdrawal Statement

bioRxiv has withdrawn this preprint following a formal investigation by the University of New Mexico Office of Research Integrity and Compliance.
Heterogeneous presynaptic receptive fields contribute to directional tuning in starburst amacrine cells
Authors: Gaynes John A. , Budoff Samuel A. , Grybko Michael J. , Poleg-Polsky Alon
Published: August 2023

The processing of visual information by retinal starburst amacrine cells (SACs) involves transforming excitatory input from bipolar cells (BCs) into directional calcium output. While previous studies have suggested that an asymmetry in the kinetic properties of bipolar cells along the soma-dendritic axes of the postsynaptic cell could enhance directional tuning at the level of individual branches, it remains unclear whether biologically relevant presynaptic kinetics contribute to direction selectivity when visual stimulation engages the entire dendritic tree. To address this question, we built multicompartmental models of the bipolar–SAC circuit and trained them to boost directional tuning. We report that despite significant dendritic crosstalk and dissimilar directional preferences along the dendrites that occur during whole-cell stimulation, the rules that guide BC kinetics leading to optimal directional selectivity are similar to the single-dendrite condition. To correlate model predictions to empirical findings, we utilized two-photon glutamate imaging to study the dynamics of bipolar release onto ON- and OFF- starburst dendrites in the murine retina. We reveal diverse presynaptic dynamics in response to motion in both BC populations; algorithms trained on the experimental data suggested that the differences in the temporal release kinetics are likely to correspond to heterogeneous receptive field (RF) properties among the different BC types, including the spatial extent of the center and surround components. In addition, we demonstrate that circuit architecture composed of presynaptic units with experimentally recorded dynamics could enhance directional drive but not to levels that replicate empirical findings, suggesting other DS mechanisms are required to explain SAC function. Our study provides new insights into the complex mechanisms underlying direction selectivity in retinal processing and highlights the potential contribution of presynaptic kinetics to the computation of visual information by starburst amacrine cells.
CONDITIONALLY MUTANT ANIMAL MODEL FOR INVESTIGATING THE INVASIVE TROPHOBLAST CELL LINEAGE
Authors: Iqbal Khursheed , Nixon Brandon , Crnkovich Benjamin , Dominguez Esteban M. , Moreno-Irusta Ayelen , Scott Regan L. , Vu Ha T.H. , Tuteja Geetu , Vivian Jay L. , Soares Michael J.
Published: August 2023

Placental development involves coordinated expansion and differentiation of trophoblast cell lineages possessing specialized functions. Among the differentiated trophoblast cell lineages are invasive trophoblast cells, which exit the placenta and invade into the uterus where they restructure the uterine parenchyma and facilitate remodeling of uterine spiral arteries. The rat exhibits deep intrauterine trophoblast cell invasion, a feature shared with human placentation, and is also amenable to gene manipulation using genome editing techniques. In this investigation, we generated a conditional rat model targeting the invasive trophoblast cell lineage. Prolactin family 7, subfamily b, member 1 (Prl7b1) is uniquely and abundantly expressed in the rat invasive trophoblast cell lineage. Disruption ofPrl7b1did not adversely affect placental development. We demonstrated that thePrl7b1locus could be effectively used to drive the expression of Cre recombinase in invasive trophoblast cells. Our rat model represents a new tool for investigating candidate genes contributing to the regulation of invasive trophoblast cells and their contributions to trophoblast-guided uterine spiral artery remodeling.
Predicting brain activity using Transformers
Authors: Adeli Hossein , Minni Sun , Kriegeskorte Nikolaus
Published: August 2023

The Algonauts challenge [Gifford et al., 2023] called on the community to provide novel solutions for predicting brain activity of humans viewing natural scenes. This report provides an overview and technical details of our submitted solution. We use a general transformer encoder-decoder model to map images to fMRI responses. The encoder model is a vision transformer trained using self-supervised methods (DINOv2). The decoder uses queries corresponding to different brain regions of interests (ROI) in different hemispheres to gather relevant information from the encoder output for predicting neural activity in each ROI. The output tokens from the decoder are then linearly mapped to the fMRI activity. The predictive success (challenge score: 63.5229, rank 2) suggests that features from self-supervised transformers may deserve consideration as models of human visual brain representations and shows the effectiveness of transformer mechanisms (self and cross-attention) to learn the mapping from features to brain responses. Code is available in thisgithub repository.
Multi-day Neuron Tracking in High Density Electrophysiology Recordings using EMD
Authors: Yuan Augustine(Xiaoran) , Colonell Jennifer , Lebedeva Anna , Okun Michael , Charles Adam S. , Harris Timothy D.
Published: August 2023

Accurate tracking of the same neurons across multiple days is crucial for studying changes in neuronal activity during learning and adaptation. Advances in high density extracellular electrophysiology recording probes, such as Neuropixels, provide a promising avenue to accomplish this goal. Identifying the same neurons in multiple recordings is, however, complicated by non-rigid movement of the tissue relative to the recording sites (drift) and loss of signal from some neurons. Here we propose a neuron tracking method that can identify the same cells independent of firing statistics, that are used by most existing methods. Our method is based on between-day non-rigid alignment of spike sorted clusters. We verified the same cell identity in mice using measured visual receptive fields. This method succeeds on datasets separated from one to 47 days, with an 84% average recovery rate.
A reverse-central-dogma pathway underlies ribosome-antibiotic efficacy and accelerates resistance evolution
Authors: Zheng Yayun , Chai Ruochen , Xu Zeqi , He Yihui , Shen Ping , Liu Jintao , Wang Tianmin
Published: August 2023

Withdrawal Statement

This manuscript has been withdrawn by the authors as it was submitted and made public without the full consent of all the authors. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
A single-cell transcriptomic analysis of the mouse hippocampus after voluntary exercise
Authors: Methi Aditi , Islam Md Rezaul , Kaurani Lalit , Sakib M Sadman , Krüger Dennis M. , Burkhardt Susanne , Liebetanz David , Fischer André
Published: August 2023

Exercise has been recognized as a beneficial factor for cognitive health, particularly in relation to the hippocampus, a vital brain region responsible for learning and memory. Previous research has demonstrated that exercise-mediated improvement of learning and memory in humans and rodents correlates with increased adult neurogenesis and processes related to enhanced synaptic plasticity. Nevertheless, the underlying molecular mechanisms are not fully understood. With the aim to further elucidate these mechanisms we provide a comprehensive dataset of the mouse hippocampal transcriptome at the single-cell level after four weeks of voluntary wheel-running. Our analysis provides a number of interesting observations. For example, the results suggest that exercise affects adult neurogenesis by accelerating the maturation of a subpopulation ofPrdm16-expressing neurons. Moreover, we uncover the existence of an intricate crosstalk among multiple vital signaling pathways such as NF-κB, Wnt/β-catenin, Notch, retinoic acid (RA) pathways altered upon exercise in a specific cluster of excitatory neurons within the Cornu Ammonis (CA) region of the hippocampus. In conclusion, our study provides an important resource dataset and sheds further light on the molecular changes induced by exercise in the hippocampus. These findings have implications for developing targeted interventions aimed at optimizing cognitive health and preventing age-related cognitive decline.
Txnip deletions and missense alleles prolong the survival of cones in a retinitis pigmentosa mouse model
Authors: Xue Yunlu , Zhou Yimin , Cepko Constance L.
Published: August 2023

Retinitis pigmentosa (RP) is a prevalent inherited retinal degenerative disease worldwide, affecting 1 in 4,000 people. The disease is characterized by an initial loss of night vision followed by a loss of daylight and color vision. Many of the RP disease genes are expressed in the rod photoreceptors, the cell type that initiates dim light vision. Following loss of rods, the cone photoreceptors, which initiate daylight vision, also are affected and can die leading to total loss of vision. The reasons for loss of cone vision are not entirely clear, but appear to be due to loss of the rods. Previously we showed that overexpressing Txnip, an α-arrestin protein, in mouse models of RP using AAV gene therapy prolonged the survival of RP cones (Xue et al., 2021). At least part of the mechanism for cone survival was a switch in the fuel source, from glucose to lactate. In addition, the mitochondria of cones were both morphologically and functionally improved by delivery of Txnip. We have gone on to test several alleles of Txnip for the ability to prolong cone survival inrd1, a mouse model of RP. In addition, proteins that bind to Txnip and/or have homology to Txnip were tested. Five different deletion alleles of Txnip were expressed in cones or the retinal pigmented epithelium (RPE). Here we show that the C-terminal half of Txnip (149-397aa) is sufficient to remove GLUT1 from the RPE cell surface, and improvedrd1cone survival when expressed specifically in the RPE. Overexpressing Arrdc4, an α-arrestin that shares 60% similar protein sequence to Txnip, reducedrd1cone survival. Reduction of the expression of HSP90AB1, a protein that interacts with Txnip and regulates metabolism, improved the survival ofrd1cones alone and was additive for cone survival when combined with Txnip. However, full length Txnip with a single amino acid change, C247S, as we tested in our original study, remains the most highly efficacious form of the gene for cone rescue. The above observations suggest that only a subset of the hypothesized and known activities of Txnip play a role in promoting RP cone survival, and that the activities of Txnip in the RPE differ from those in cone photoreceptors.
Comparative genomics of two closely related coral species with different spawning seasons reveals genomic regions possibly associated with gametogenesis
Authors: Takahashi-Kariyazono Shiho , Iguchi Akira , Terai Yohey
Published: August 2023

Marine invertebrates release their gametes at an optimal time to produce the next generation. In reef-building scleractinian corals, synchronous spawning is essential for reproductive success. Molecular mechanisms of scleractinian gametogenesis have been studied; however, the mechanism by which coral gametes mature at specific times has yet to be discovered. The present study focused on twoAcroporaspecies with different spawning seasons. In Okinawa, Japan,Acropora digitiferaspawns from May to June, whereasAcroporasp. 1 spawns in August. Comparative genomic analyses revealed that 39 candidate genes are differentiated between the two species, suggesting a possible association with timing of gametogenesis. Among candidate genes, we identified anAcroporasp. 1-specific amino acid change in geneWDR59, one of the components of a mTORC1 activator, GATOR2. Since regulation of gametogenesis by mTORC1 is widely conserved among eukaryotes, the difference in timing of gamete maturation observed in the twoAcroporaspecies may be caused by a substitution in WDR59 that slightly affects timing of mTORC1 activation via GATOR2. In addition, this substitution may lead to reproductive isolation between the two species, due to different spawning periods. Thus, we propose that A. digitifera and Acropora sp. 1 species pair is an effective model for studying coral speciation and understanding the molecular mechanisms that control coral spawning timing.

Significance statement (required)

For successful coral reproduction, conspecific corals must spawn synchronously. Gamete production initiates coral spawning. Regulation of gamete maturation by a protein complex, mTORC1, is widely conserved among organisms, but little is known about it in cnidarians. In this study, we analyzed genomes of two closely relatedAcroporaspecies with different spawning months, May/June and August. Our analyses revealed that 39 genes are genetically differentiated between the two species. One of these is a component of mTORC1 activator, suggesting that this gene may be associated with the difference in spawning times of these two species.
Mechanical forces and ligand-binding modulatePseudomonas aeruginosaPilY1 mechanosensitive protein
Authors: Cao-Garcia Francisco J. , Walker Jane E. , Board Stephanie , Alonso-Caballero Alvaro
Published: August 2023

Bacteria initiate colonization and biofilm formation in response to mechanical cues caused by surface proximity. The protein PilY1 has been proposed as a key actor mediating mechanosensing. PilY1 is a calcium and integrin-binding protein with additional roles in host adhesion and functional regulation of the type IV pili (T4P), the appendages involved in twitching motility, and various aspects of the surface-associated life of bacteria. Due to its extracellular location and involvement in several surface processes, PilY1 is exposed to mechanical forces that could modulate its different roles. Herein, we explore the effect of mechanical forces and ligand binding on the conformational dynamics of the PilY1 C-terminal domain. Our single-molecule approach demonstrates that PilY1 acts as a ligand-modulated force sensor. At high forces, PilY1 unfolding occurs through a hierarchical sequence of intermediates. When calcium is bound to its cognate site linked to T4P regulation, there is a long-range mechanical stabilization affecting several PilY1 domains, which ensures the structural integrity of the protein. In the low-force regime, the integrin-binding domain of PilY1 exhibits calcium-tuned force sensitivity and conformational dynamics akin to those of mechanosensor proteins. Integrin binding to this domain occurs under force, inducing a shortening of its unfolded extension. Our findings suggest that the roles of the PilY1 C-terminal domain are force and ligand-modulated, which could entail a mechanical-based compartmentalization of its functions.
Determining the structure of protein-bound ceramides, essential lipids for skin barrier function
Authors: Ohno Yusuke , Nakamura Tetsuya , Iwasaki Takafumi , Katsuyama Akira , Ichikawa Satoshi , Kihara Akio
Published: August 2023

Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the β-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE-ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides.

Highlights

Epoxy-enone (EE) ceramides form stable conjugates with Cys.

Cys binds to the β-carbon of the enone via a Michael addition reaction.

Cys-bound EE ceramides exist in the mouse epidermis.

Cys-bound EE ceramides are the predominant protein-bound ceramides.

In Brief

Protein-bound ceramides are specialized ceramides essential for skin permeability barrier function. In the present study, we determined the exact structure of protein-bound ceramides as Cys-bound epoxy-enone ceramides where the β-carbon of the epoxy-enone ceramides is attached by Cys residues of corneocyte surface proteins via a Michael addition reaction.
Comparing full variation profile analysis with the conventional consensus method in SARS-CoV-2 phylogeny
Authors: Nóra Fiam Regina , István Csabai , Norbert Solymosi
Published: August 2023

This study proposes a novel approach to studying SARS-CoV-2 virus mutations through sequencing data comparison. Traditional consensus-based methods, which focus on the most common nucleotide at each position, might overlook or obscure the presence of low-frequency variants. Our method, in contrast, retains all sequenced nucleotides at each position, forming a genomic matrix. Utilizing simulated short reads from genomes with specified mutations, we contrasted our genomic matrix approach with the consensus sequence method. Our matrix methodology accurately reflected the known mutations and true compositions, demonstrating its efficacy in understanding the sample variability and their interconnections. Further tests using real data from GISAID and NCBI-SRA confirmed its reliability and robustness. As we see, the genomic matrix approach offers a more accurate representation of the viral genomic diversity, thereby providing superior insights into virus evolution and epidemiology. Future application recommendations are provided based on our observed results.
Parallel proteomics and phosphoproteomics defines starvation signal specific processes in cell quiescence
Authors: Sun Siyu , Tranchina Daniel , Gresham David
Published: August 2023

Cells arrest growth and enter a quiescent state upon nutrient deprivation. However, the molecular processes by which cells respond to different starvation signals to regulate exit from the cell division cycle and initiation of quiescence remains poorly understood. To study the role of protein expression and signaling in quiescence we combined temporal profiling of the proteome and phosphoproteome using stable isotope labeling with amino acids in cell culture (SILAC) inSaccharomyces cerevisiae(budding yeast). We find that carbon and phosphorus starvation signals activate quiescence through largely distinct remodeling of the proteome and phosphoproteome. However, increased expression of mitochondrial proteins is associated with quiescence establishment in response to both starvation signals. Deletion of the putative quiescence regulatorRIM15, which encodes a serine-threonine kinase, results in reduced survival of cells starved for phosphorus and nitrogen, but not carbon. However, we identified common protein phosphorylation roles for RIM15 in quiescence that are enriched for RNA metabolism and translation. We also find evidence for RIM15-mediated phosphorylation of some targets, including IGO1, prior to starvation consistent with a functional role for RIM15 in proliferative cells. Finally, our results reveal widespread catabolism of amino acids in response to nitrogen starvation, indicating widespread amino acid recycling via salvage pathways in conditions lacking environmental nitrogen. Our study defines an expanded quiescent proteome and phosphoproteome in yeast, and highlights the multiple coordinated molecular processes at the level of protein expression and phosphorylation that are required for quiescence.
A graded neonatal mouse model of necrotizing enterocolitis demonstrates that mild enterocolitis is sufficient to activate microglia and increase cerebral cytokine expression
Authors: Sha Cuilee , Van Brunt Trevor , Kudria Jacob , Schmidt Donna , Yurovsky Alisa , Bandovic Jela , Giarrizzo Michael , Lin Joyce , Tsirka Styliani-Anna , Bialkowska Agnieszka B , Wollmuth Lonnie , Speer Esther , Hsieh Helen
Published: August 2023

Background

Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal process that afflicts approximately 10% of preterm infants born in the United States each year, with a mortality rate of 30%. NEC severity is graded using Bell’s classification system, from stage I mild NEC to stage III severe NEC. Over half of NEC survivors present with neurodevelopmental impairment during adolescence, a long-term complication that is poorly understood but can occur even after mild NEC. Although multiple animal models exist, none allow the experimenter to control nor represent the gradient of symptom severities seen in NEC patients. We bridge this knowledge gap by developing a graded murine model of NEC and studying its relationship with neuroinflammation across a range of NEC severities.

Methods

Postnatal day 3 (P3) C57BL/6 mice were fed a formula containing different concentrations (0% control, 0.25%, 1%, 2%, and 3%) of dextran sodium sulfate (DSS). P3 mice were fed every 3 hours for 72-hours. We collected data on weight gain and behavior (activity, response, body color) during feeding. At the end of the experiment, we collected tissues (intestine, liver, plasma, brain) for immunohistochemistry, immunofluorescence, and cytokine and chemokine analysis.

Results

Throughout NEC induction, mice fed higher concentrations of DSS died sooner, lost weight faster, and became sick or lethargic earlier. Intestinal characteristics (dilation, color, friability) were worse in mice fed with higher DSS concentrations. Histology revealed small intestinal disarray among mice fed all DSS concentrations, while higher DSS concentrations resulted in reduced small intestinal cellular proliferation and increased hepatic and systemic inflammation. In the brain, IL-2, G-CSF, and CXCL1 concentrations increased with higher DSS concentrations. Although the number of neurons and microglia in the CA1 hippocampal region did not differ, microglial branching was significantly reduced in DSS-fed mice.

Conclusion

We characterize a novel graded model of NEC that recapitulates the full range of NEC severities. We show that mild NEC is sufficient to initiate neuroinflammation and microglia activation. This model will facilitate studies on the neurodevelopmental effects of NEC.
Ranking and serial thinking: A geometric solution
Authors: Di Antonio Gabriele , Raglio Sofia , Mattia Maurizio
Published: August 2023

A general mathematical description of the way the brain encodes ordinal knowledge of sequences is still lacking. Coherently with the well-established idea of mixed selectivity in high-dimensional state spaces, we conjectured the existence of a linear solution for serial learning tasks. In this theoretical framework, the neural representation of the items in a sequence are read out as ordered projections along a suited “geometric” mental line learnedviaclassical conditioning (delta rule learning). We show that the derived model explains all the behavioral effects observed in humans and other animal species performing the transitive inference task in presence of noisy sensory information and stochastic neural activity. This result is generalized to the case of recurrent neural networks performing motor decision, where the same geometric mental line is learned showing a tight correlation with the motor plan of the responses. Network activity is then eventually modulated according to the symbolic distance of presented item pairs, as observed in associative cortices of nonhuman primates. Serial ordering is thus predicted to emerge as a linear mapping between sensory input and behavioral output, highlighting a possible pivotal role of motor-related associative cortices in the transitive inference task.
Correlative single-cell hard X-ray tomography and X-ray fluorescence imaging
Authors: Lin Zihan , Zhang Xiao , Nandi Purbasha , Lin Yuewei , Wang Liguo , Chu Yong , Paape Timothy , Yang Yang , Xiao Xianghui , Liu Qun
Published: August 2023

X-ray tomography (XRT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XRT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XRT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.
Interface-guided phenotyping of coding variants in the transcription factor RUNX1 with SEUSS
Authors: Ozturk Kivilcim , Panwala Rebecca , Sheen Jeanna , Ford Kyle , Payne Nathan , Zhang Dong-Er , Hutter Stephan , Haferlach Torsten , Ideker Trey , Mali Prashant , Carter Hannah
Published: August 2023

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions.
The essential genome ofXanthomonas citri
Authors: Wang Xiaolan , Wu Manying , Ge Yifei , Lv Weiwei , Liu Chaoying , Ding Xiaojun , Zhang Yu , Wang Jihua , Zhang Yunzeng , Li Lei , Zhou Xiaofeng
Published: August 2023

Citrus canker, caused by the bacteriumXanthomonas citrisubsp.citri, is a devastating disease with significant economic implications for the citrus industry worldwide. Understanding the molecular basis ofXanthomonascell cycle and identifying therapeutic targets is crucial for effective disease management. In this study, we employed hyper-saturated transposon mutagenesis combined with high-throughput sequencing to determine the essential features of theXanthomonas citrigenome at ∼7-bp resolution. Our analysis revealed 525 essential genes, 181 high fitness cost genes, 7 small non-coding RNAs, 25 transfer RNAs, 4 ribosomal RNAs, and the origin of replication. Notably, the use of a newly designed Tn5 transposon with an outward pointinglacpromoter significantly reduced false positives caused by polar effects associated with conventional transposons. Functional enrichment analysis showed that essential genes were significantly enriched in processes related to ribosome biogenesis, energy production and conversion, and membrane metabolism. Interestingly, the distribution of essential genes inX. citrishowed similarities to that of the model organismE. coli, suggesting a conserved mode of genome organization that influences transposon accessibility. Our comprehensive analysis provides valuable target genes for potential therapeutic interventions against citrus canker and other related plant diseases.
Seed or soil: tracing back the plant mycobiota primary sources
Authors: Laurent-Webb Liam , Maurice Kenji , Perez-Lamarque Benoît , Bourceret Amélia , Ducousso Marc , Selosse Marc-André
Published: August 2023

Plants host diverse communities of fungi (collectively called the mycobiota) which play crucial roles in their development. The assembly processes of the mycobiota, however, remain poorly understood, in particular, whether it is transmitted by parents through the seeds (vertical transmission) or recruited in the environment (horizontal transmission). Here we attempt to quantify the relative contributions of horizontal and vertical transmission in the mycobiota assembly of a desert shrub,Haloxylon salicornicum,by comparing the mycobiota ofin situbulk soil and seeds to that of (i)in situadult individuals and (ii)in vitro-germinated seedlings in soil collectedin situ, either autoclaved or not. We show that the mycobiota is partially transmitted through the seeds to seedlings. In contrast, root mycobiota of adults are highly similar to that of bulk soil, whereas adult leaf mycobiota remain similar to that of seeds. Thus, the mycobiota is transmitted both horizontally and vertically depending on the plant tissue. Despite discrepancies betweenin situandin vitroapproaches, our result may also suggest a compositional turnover in plant mycobiota during plant development. Understanding the respective contribution of these transmission paths to the plant mycobiota is fundamental to deciphering potential coevolutionary processes between plants and fungi.
hGRAD – a versatile ʹone-fits-allʹ system for the acute depletion of RNA binding proteins in nuclear condensates
Authors: Arnold Benjamin , Riegger Ricarda J. , Okuda Ellen Kazumi , Slišcović Irena , Keller Mario , McNicoll François , Zarnack Kathi , Müller-McNicoll Michaela
Published: August 2023

Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and cross- regulation. They are highly abundant and often localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects.

Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a ʹone-fits-allʹ plasmid, and integration into any cell line that expresses endogenously GFP-tagged proteins allows an inducible, rapid and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2 and NONO are completely cleared from nuclear speckles and paraspeckles within two hours. hGRAD works in various cell types, is more efficient than other methods and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered highly dynamic transient transcript changes, compensatory mechanisms and that SRSF5 promotes transcript stability.
Rapid and automated design of two-component protein nanomaterials using ProteinMPNN
Authors: de Haas Robbert J. , Brunette Natalie , Goodson Alex , Dauparas Justas , Yi Sue Y. , Yang Erin C. , Dowling Quinton , Nguyen Hannah , Kang Alex , Bera Asim K. , Sankaran Banumathi , de Vries Renko , Baker David , King Neil P.
Published: August 2023

The design of novel protein-protein interfaces using physics-based design methods such as Rosetta requires substantial computational resources and manual refinement by expert structural biologists. A new generation of deep learning methods promises to simplify protein-protein interface design and enable its application to a wide variety of problems by researchers from various scientific disciplines. Here we test the ability of a deep learning method for protein sequence design, ProteinMPNN, to design two-component tetrahedral protein nanomaterials and benchmark its performance against Rosetta. ProteinMPNN had a similar success rate to Rosetta, yielding 13 new experimentally confirmed assemblies, but required orders of magnitude less computation and no manual refinement. The interfaces designed by ProteinMPNN were substantially more polar than those designed by Rosetta, which facilitatedin vitroassembly of the designed nanomaterials from independently purified components. Crystal structures of several of the assemblies confirmed the accuracy of the design method at high resolution. Our results showcase the potential of deep learning-based methods to unlock the widespread application of designed protein-protein interfaces and self-assembling protein nanomaterials in biotechnology.
Time-resolved X-ray solution scattering unveils the sequence of events leading to human Hb heme capture byStaphylococcus aureusIsdB
Authors: Bei Omar De , Marchetti Marialaura , Guglielmo Stefano , Gianquinto Eleonora , Spyrakis Francesca , Campanini Barbara , Bettati Stefano , Levantino Matteo , Ronda Luca
Published: August 2023

Infections caused byStaphylococcus aureusdepend on its ability to acquire nutrients. One essential nutrient is iron, which is obtained from the heme of the human host hemoglobin (Hb) through a protein machinery called Iron-regulated Surface Determinant (Isd). IsdB is the protein in charge of heme extraction from Hb, which is the first step of the chain of events leading to iron transfer to the bacterium cell interior. In order to elucidate the molecular events leading from the formation of the initial IsdB:Hb complex to heme extraction, we have performed a time-resolved X-ray solution scattering (TR-XSS) investigation combined with a rapid mixing triggering approach. We succeeded in defining the stoichiometry of IsdB:Hb binding and in describing the kinetics of the subsequent structural changes. The presented approach is potentially applicable to unveil the complex kinetic pathways generated by protein-protein interaction in different biological systems.
Molecular mechanism of bacteriophage tail contraction-structure of an S-layer-penetrating bacteriophage
Authors: Wilson Jason S. , Fortier Louis-Charles , Fagan Robert P. , Bullough Per A.
Published: August 2023

Viruses that infect bacteria (bacteriophages or phages) attach to the host cell envelope, inject their genetic material into the host cytosol and either persist as prophage or hijack the host machinery to produce progeny virions. Attachment is mediated through phage receptor binding proteins that are specific for different host cell surface molecules. A subset of phage, the myoviruses, possess contractile tails, the outer sheath of which contracts upon receptor binding, driving an inner tail tube through the cell envelope and delivering the phage genome into the host cytosol. The molecular details of phage tail contraction and mode of cell envelope penetration have remained poorly understood and were completely unknown for any phage infecting bacteria enveloped by a proteinaceous S-layer. Here we reveal the extended and contracted atomic structures of an intact contractile-tail phage that binds to and penetrates the protective S-layer of the Gram positive human pathogenClostridioides difficile. Surprisingly, we find no evidence of the intrinsic enzymatic domains that other phages exploit in cell wall penetration, suggesting that sufficient energy is released upon tail contraction to penetrate the S-layer and the thick cell wall without enzymatic activity. However, it is also notable that the tail sheath subunits move less than those studied in related contractile injection systems such as the model phage T4. Instead, the unusually long tail length and flexibility upon contraction likely contribute towards the required free energy release for envelope penetration. Our results show that the principles of phage contraction and infection as determined in the model system of T4 are not universal. We anticipate that our structures will form a strong foundation to engineerC. difficilephages as therapeutics, and highlight important adaptations made in order to infect S-layer containing pathogens.
Air pollution modifies key colonization factors of the beneficial bee gut symbiontSnodgrassella alviand disrupts the bumblebee (Bombus terrestris) gut microbiome
Authors: Sampson Hannah R. , Allcock Natalie , Mallon Eamonn B. , Ketley Julian M. , Morrissey Julie A.
Published: August 2023

Air pollution is the world’s largest environmental health risk. Particulate pollutants, a major component of air pollution, are detrimental to human health and a significant risk to wildlife and ecosystems globally. Black carbon, a by-product of fossil fuel and biomass burning, is a key constituent of air pollution with levels continuing to increase worldwide. Here we describe the effects of black carbon on the beneficial gut microbiome of an important global insect pollinator, the buff-tailed bumblebee (Bombus terrestris). Our data shows that exposure to black carbon particulates alters the biofilm structure, gene expression and initial adhesion of beneficial bee gut coloniser,Snodgrassella alvi in vitro.Additionally, our results show that black carbon disrupts adultBombus terrestrisgut microbiome composition, a vital component to bee health. Exposure to black carbon increased bees’ viable gut bacteria and significantly altered the abundance of beneficial core bacteriaGilliamellaandBombilactobacillusin the microbiome. These findings demonstrate that exposure to black carbon air pollution has direct, measurable effects on bees’ beneficial gut commensal bacteria and microbiome. Together these data highlight that particulate pollutants are an underexplored risk for the health of insect pollinators.
YfmR is a translation factor that prevents ribosome stalling and cell death in the absence of EF-P
Authors: Hong Hye-Rim , Prince Cassidy R. , Tetreault Daniel D. , Wu Letian , Feaga Heather A.
Published: August 2023

Protein synthesis is performed by the ribosome and a host of highly conserved elongation factors. Elongation factor P (EF-P) prevents ribosome stalling at difficult-to-translate sequences, particularly polyproline tracts. In bacteria, phenotypes associated withefpdeletion range from modest to lethal, suggesting that some species encode an additional translation factor that has similar function to EF-P. Here we identify YfmR as a translation factor that is essential in the absence of EF-P inB. subtilis. YfmR is an ABCF ATPase that is closely related to both Uup and EttA, ABCFs that bind the ribosomal E-site and are conserved in more than 50% of bacterial genomes. We show that YfmR associates with actively translating ribosomes and that depleting YfmR from Δefpcells causes severe ribosome stalling at a polyproline tractin vivo. YfmR depletion from Δefpcells was lethal, and caused reduced levels of actively translating ribosomes. Our results therefore identify YfmR as an important translation factor that is essential inB. subtilisin the absence of EF-P.

Significance

Translation is one of the most ancient and energetically demanding processes that occurs in the cell. Ribosomes constitute more than 60% of cellular mass in actively growing cells, and ribosomes are a major target of antimicrobials and chemotherapeutics. Here, we identify YfmR as a translation factor that is essential in the absence of EF-P. YfmR is a member of the ABCF family of ATPases whose role in translation is only beginning to be understood. Given the broad distribution of ABCFs from bacteria to fungi, we expect our results to have implications for understanding translation elongation in diverse organisms.
Nonstructural protein 1 of swine arterivirus PRRSV downregulates promyelocytic leukemia nuclear bodies and promotes viral replication
Authors: Su Chia-Ming , Han Mingyuan , Everett Roger , Yoo Dongwan
Published: August 2023

Porcine reproductive and respiratory syndrome virus (PRRSV) inhibits the type I interferon (IFN) production and signaling pathways during infection, and the nonstructural protein 1 (nsp1) has been identified as a potent IFN antagonist. Promyelocytic leukemia (PML) protein is a major scaffold protein organizing the PML nuclear bodies (NBs) of the cell and plays a diverse role in maintaining the cellular homeostasis including antiviral response among many other processes. The present study reveals a significant reduction of PML NBs in cells during infection of PRRSV, implicating the negative regulation of PML gene expression by PRRSV. Subsequently, the nsp1β protein was identified as the viral regulator for PML expression. The overexpression of PML isoforms restricted viral replication, while the gene silencing of endogenous PML promoted viral replication. The downregulation of PML expression by PRRSV was post-translational via the ubiquitination-proteasome pathway. Of six isoforms, PML-II and PML-IV exhibited the most potent suppressive activity against viral replication. PRRSV nsp1β bound to PML directly, and this interaction was mediated through the small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) on nsp1β. Further studies revealed that double mutations in SIM1 and SIM4 abolished the binding of nsp1β to PML and prevented the PML degradation. The PML downregulation by nsp1 was common in other arteriviruses including equine arteritis virus, murine lactate dehydrogenase elevating virus, and simian hemorrhagic fever virus. Our study unveils the evolutionary conservation of the viral immune evasion strategy employed by arteriviruses, which promotes their replication by targeting PML for downregulation.

IMPORTANCE OF THE STUDY

Porcine reproductive and respiratory syndrome (PRRS) is an economically significant disease in the swine industry worldwide. One of the immunological hallmarks in virus-infected animals is the suppression of type I interferon response during an early-stage infection, leading to the consequence of adaptive immunity and viral persistence. In the present study, we report that the nsp1-beta protein of PRRS virus degrades the promyelocytic leukemia (PML) protein and downregulates PML nuclear body (NB) formation. The PML downregulation by PRRS virus results in enhanced viral replication. The PML downregulation by nsp1 is common in other arteriviruses, unveiling the basic understanding of cell-virus interactions and immune evasion strategies for arteriviruses.
OsNAC2 maintains the homeostasis of immune responses to bacterial blight through the OsEREBP1 in rice
Authors: Zhong Qun , Yu Jiangtao , Wu Yiding , Yao Xuefeng , Meng Xiangzong , Ming Feng
Published: August 2023

Rice bacterial blight, caused byXanthomonas oryzaepv.Oryzae(Xoo), threatens plant growth and yield. However, the molecular mechanisms underlying rice immunity againstXooremain elusive. Here, we demonstrated down-regulation of a NAC (NAM-ATAF-CUC) transcription factorOsNAC2enhanced resistance to bacterial blight disease in rice. Consistently, salicylic acid (SA) biosynthesis was greatly attenuated when OsNAC2 overexpressed. Furthermore, study demonstrated that OsNAC2 can down-regulated the expression of SA synthesis genes, thus mediating SA signal transmission. Simultaneously, OsNAC2 interacted with OsEREBP1 (AP2/ERF family) in the nucleus, may be bloking the inhibition of OsNAC2 toOsICS1,OsPAL3and so on. Furthermore, OsEREBP1 interacted with OsXb22a in the cytoplasm to exert its positive regulatory effects to bacterial blight. However,OsNAC2-overexpression kept OsEREBP1 in the nucleus and be rapidly degraded in pathogen infection, which adversely affects the interaction of OsEREBP1-OsXb22a. Our results determined that OsNAC2 inhibits the SA signaling and stably interacts with OsEREBP1 to maintain disease resistance. This OsNAC2-OsEREBP1-based homeostatic mechanism provides new insights into rice disease resistance, and it may be useful for improving the disease resistance of important crops through breeding.
A Bright, Photostable Dye that Enables Multicolor, Time Lapse, and Super-Resolution Imaging of Acidic Organelles
Authors: Lesiak Lauren , Dadina Neville , Zheng Shuai , Schelvis Marianne , Schepartz Alanna
Published: August 2023

Lysosomes have long been known for their acidic lumen and efficient degradation of cellular byproducts. In recent years it has become clear that their function is far more sophisticated, involving multiple cell signaling pathways and interactions with other organelles. Unfortunately, their acidic interior, fast dynamics, and small size makes lysosomes difficult to image with fluorescence microscopy. Here we report a far-red small molecule, HMSiR680-Me, that fluoresces only under acidic conditions, causing selective labeling of acidic organelles in live cells. HMSiR680-Me can be used alongside other far-red dyes in multicolor imaging experiments and is superior to existing lysosome probes in terms of photostability and maintaining cell health and lyso-some motility. We demonstrate that HMSiR680-Me is compatible with overnight time lapse experiments, as well as time lapse super-resolution microscopy with a fast frame rate for at least 1000 frames. HMSiR680-Me can also be used alongside silicon rhodamine dyes in a multiplexed super-resolution microscopy experiment to visualize interactions between the inner mitochondrial membrane and lysosomes with only a single excitation laser and simultaneous depletion. We envision this dye permitting more detailed study of the role of lysosomes in dynamic cellular processes and disease.