bluuebunny/crossref_metadata_2025 huggingface dataset:
Rolling with the F-words Life Wheel: reflexive thematic analysis of a coaching-based, holistic approach to pediatric occupational therapy
Authors: Sehajpal Khushi , McCrostie Claire , Charles Lucy , Hamill Arul , Terei Pio , Hamill James
Published: March 2023

Background and purpose

The F-words Life Wheel approaches child development by hybridizing a holistic model in the F-words for Child Development, and a coaching model in Occupational Performance Coaching, along with a life-flow approach in the Kawa model. The effect of the F-words Life Wheel has not been previously studied. The purpose of this paper is to report parents’ experiences with the F-words Life Wheel.

Methods

This was a qualitative study based on interviews with parents of children with developmental needs and experts in child development. Interviews were conducted in person and transcribed verbatim. The researchers used reflexive thematic analysis within a critical realist paradigm.

Results

A total of 13 interviews were conducted, 11 with parents of children with developmental needs and two with child development experts. Interview transcripts totaled 42,763 words from which we developed 45 codes and three themes. The themes were 1) overwhelming, 2) power rebalance, and 3) connectedness. The overwhelming theme addresses how life with developmental needs is challenging, engaging with the health and disability system is difficult, and the focus on deficits can lead to a sense of being overwhelmed. The power rebalance theme addresses the transition from professionals calling the shots to giving agency to the child and family. Holistic goal setting empowers parents and children to direct and prioritize therapy, and helps shift from a deficit-focused to a “can-do” attitude. The connectedness theme addresses the linkages between psychological health, physical health, the extended family, and the planet as a whole.

Conclusions

The F-words Life Wheel approach appears to be empowering and motivating for children and families. Further research is needed to explore how holistic models of therapy such as the F-words Life Wheel can promote family-centered care and connectedness on a wider scale.
L1 retrotransposons drive human neuronal transcriptome complexity and functional diversification
Authors: Garza Raquel , Atacho Diahann , Adami Anita , Gerdes Patricia , Vinod Meghna , Hsieh PingHsun , Karlsson Ofelia , Horvath Vivien , Johansson Pia A. , Pandiloski Ninoslav , Matas Jon , Quaegebeur Annelies , Kouli Antonina , Sharma Yogita , Jönsson Marie E , Monni Emanuela , Englund Elisabet , Eichler Evan E. , Hammell Molly , Barker Roger A. , Kokaia Zaal , Douse Christopher H. , Jakobsson Johan
Published: March 2023

The genetic mechanisms underlying the expansion in size and complexity of the human brain remains poorly understood. L1 retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution is largely unknown. Using multi-omic profiling we here demonstrate that L1-promoters are dynamically active in the developing and adult human brain. L1s generate hundreds of developmentally regulated and cell-type specific transcripts, many which are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived lncRNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPRi-silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.
Uncovering strain- and age-dependent differences in innate immune response to SARS-CoV-2 infection in nasal epithelia using 10X single-cell sequencing
Authors: Chang Jessie J.-Y. , Grimley Samantha , Tran Bang M. , Deliyannis Georgia , Tumpach Carolin , Steinig Eike , Wong Sharon L. , Waters Shafagh A. , Stinear Timothy P. , Pitt Miranda E. , Purcell Damian , Vincan Elizabeth , Coin Lachlan J. M.
Published: March 2023

Assessing the impact of SARS-CoV-2 variants on the host is crucial with continuous emergence of new variants. We employed single-cell sequencing to investigate host transcriptomic response to ancestral and Alpha-strain SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from adults and adolescents. Strong innate immune responses were observed across lowly-infected and bystander cell-types, and heightened in Alpha-infection. Contrastingly, the innate immune response of highly-infected cells was like mock-control cells. Alpha highly-infected cells showed increased expression of protein refolding genes compared with ancestral-strain-infected adolescent cells. Oxidative phosphorylation- and translation-related genes were down-regulated in bystander cells versus infected and mock-control cells, suggesting that the down-regulation is protective and up-regulation supports viral activity. Infected adult cells revealed up-regulation of these pathways compared with infected adolescents, implying enhanced pro-viral states in infected adults. Overall, this highlights the complexity of cell-type-, age- and viral-strain-dependent host epithelial responses to SARS-CoV-2 and the value of air-liquid-interface cultures.
Generally-healthy individuals with aberrant bowel movement frequencies show enrichment for microbially-derived blood metabolites associated with reduced kidney function
Authors: Johnson-Martínez Johannes P. , Diener Christian , Levine Anne E. , Wilmanski Tomasz , Suskind David L. , Ralevski Alexandra , Hadlock Jennifer , Magis Andrew T. , Hood Leroy , Rappaport Noa , Gibbons Sean M.
Published: March 2023

Bowel movement frequency (BMF) has been linked to changes in the composition of the human gut microbiome and to many chronic conditions, like metabolic disorders, neurodegenerative diseases, chronic kidney disease (CKD), and other intestinal pathologies like irritable bowel syndrome and inflammatory bowel disease. Lower BMF (constipation) can lead to compromised intestinal barrier integrity and a switch from saccharolytic to proteolytic fermentation within the microbiota, giving rise to microbially-derived toxins that may make their way into circulation and cause damage to organ systems. However, the connections between BMF, gut microbial metabolism, and the early-stage development and progression of chronic disease remain underexplored. Here, we examined the phenotypic impact of BMF variation in a cohort of generally-healthy, community dwelling adults with detailed clinical, lifestyle, and multi-omic data. We showed significant differences in microbially-derived blood plasma metabolites, gut bacterial genera, clinical chemistries, and lifestyle factors across BMF groups that have been linked to inflammation, cardiometabolic health, liver function, and CKD severity and progression. We found that the higher plasma levels of 3-indoxyl sulfate (3-IS), a microbially-derived metabolite associated with constipation, was in turn negatively associated with estimated glomerular filtration rate (eGFR), a measure of kidney function. Causal mediation analysis revealed that the effect of BMF on eGFR was significantly mediated by 3-IS. Finally, we identify self-reported diet, lifestyle, and psychological factors associated with BMF variation, which indicate several common-sense strategies for mitigating constipation and diarrhea. Overall, we suggest that aberrant BMF is an underappreciated risk factor in the development of chronic diseases, even in otherwise healthy populations.
Light-Guided Rabies Virus Tracing for Neural Circuit Analysis
Authors: Zhang Shuyang , Ma Yunhan , Ngamkanjanarat Wasu , Takahashi Sola , Gibbs Daniel , Coleman Todd , Doan Sapphire , Kyriakakis Phillip
Published: March 2023

Neuronal tracing methods are essential tools to understand the fundamental architecture of neural circuits and their connection to the overall functional behavior of the brain. Viral vectors used to map these transsynaptic connections are capable of cell-type-specific and directional-specific labeling of the neuronal connections. Herein, we describe a novel approach to guide the transsynaptic spreading of the Rabies Virus (RV) retrograde tracer using light. We built a Baculovirus (BV) as a helper virus to deliver all the functional components necessary and sufficient for a nontoxic RV to spread from neuron to neuron, with a light-actuated gene switch to control the RV polymerase, theL gene. This design should allow for precisely controlled polysynaptic viral tracing with minimal viral toxicity. To use this system in a highly scalable and automated manner, we built optoelectronics for controlling this systemin vitrowith a large field of view using an off-the-shelf CMOS sensor, OLED display panel, and microcontrollers. We describe the assembly of these genetic circuits using the uLoop DNA assembly method and a library of genetic parts designed for the uLoop system. Combining these tools provides a framework for increasing the capabilities of nontoxic tracing through multiple synapses and increasing the throughput of neural tracing using viruses.
Deficiency in neprilysin causes laminar impairments in the visual cortex
Authors: Bae Haneui , Hensch Takao K.
Published: March 2023

1

Neprilysin (Nep) is a membrane-bound zinc-dependent endopeptidase that cleaves a wide variety of small peptides in the body. In the brain, it has gained fame as an important endogenous degrader of amyloid beta peptide, responsible for the pathophysiology of Alzheimer’s disease. Nep is expressed specifically by parvalbumin (PV)-expressing inhibitory neurons in the adult cortex, the maturation of which regulates critical period plasticity. Given that PV neuron soma and proximal dendrites are primary sites of perineuronal net (PNN) formation, we investigated the role that Nep plays in PNN and PV neuron maturation and cortical development in the mouse visual cortex, using mice whose Nep expression is constitutively knocked out (Nep KO mice). Nep expression is high in young wildtype mice (P10) and is downregulated rapidly throughout postnatal development. It is especially prominent in layer 5 where it is highly expressed by inhibitory neurons and also expressed at low levels by many excitatory neurons. Contrary to our hypothesis, Nep KO mice did not show an alteration in the relative density or gross morphology of PNNs. Instead, Nep KO mice showed reduced maximal activation of L5 after white matter stimulation and decreased number of inhibitory neurons in L4. These laminar defects may lead to impaired development of optomotor acuity in Nep KO mice.
O-GlcNAc Signaling Increases Neuron Regeneration Through One-Carbon Metabolism inCaenorhabditis elegans
Authors: Yadav Dilip Kumar , Chang Andrew C , Gabel Christopher V
Published: March 2023

Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked β-N-acetylglucosamine (O-GlcNAc), a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhancein vivoneuron regeneration. Here we define the specific metabolic pathway by which mutation of the O-GlcNAc transferase (ogt-1)increases regenerative outgrowth. Performingin vivolaser axotomy and measuring subsequent regeneration of individual neurons inC. elegans, we find that theogt-1mutation increases regeneration by diverting the metabolic flux of enhanced glycolysis towards one carbon metabolism (OCM) and the downstream transsulfuration metabolic pathway (TSP). These effects are abrogated by genetic and/or pharmacological disruption of OCM or the serine synthesis pathway (SSP) that links OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA-sequencing that reveals dramatic transcriptional changes, by theogt-1mutation, in the genes involved in glycolysis, OCM, TSP and ATP metabolism. Strikingly, the beneficial effects of theogt-1mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron inogt-1animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.Abstarct Figure.

Metabolic pathways involved in the enhanced neuronal regeneration inogt-1animals:

The green highlighted pathway illustrates the metabolic rewiring inogt-1mutant animals supporting enhanced axonal regeneration of injured neuronsin vivo.
A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity
Authors: Ahmed Wesam S , Geethakumari Anupriya M , Sultana Asfia , Fatima Asma , Philip Angelin M , Uddin S. M. Nasir , Biswas Kabir H
Published: March 2023

NanoLuc (NLuc) luciferase has found extensive application in designing a range of biological assays including gene expression analysis, protein-protein interaction and protein conformational changes due to its enhanced brightness and small size. However, questions related to its mechanism of interaction with the substrate, furimazine, as well as bioluminescence activity remains elusive. Here, we combined molecular dynamics (MD) simulation and mutational analysis to show that the R162A mutation results in a decreased but stable bioluminescence activity of NLuc in vitro. Specifically, we performed multiple, all-atom, explicit solvent MD simulations of the apo and furimazine-docked (holo) NLuc structures revealing differential dynamics of the protein in the absence and presence of the ligand. Further, analysis of trajectories for hydrogen bonds (H-bonds) formed between NLuc and furimazine revealed substantial H-bond interaction between R162 and Q32 residues. Mutation of the two residues in NLuc revealed a decreased but stable activity of the R162A, but not Q32A, mutant NLuc in in vitro assays performed with furimazine. In addition to highlighting the role of the R162 residue in NLuc activity, we believe that the mutant NLuc will find wide application in designing in vitro assays requiring extended monitoring of NLuc bioluminescence activity.
You can move, but you can't hide: identification of mobile genetic elements with geNomad
Authors: Camargo Antonio Pedro , Roux Simon , Schulz Frederik , Babinski Michal , Xu Yan , Hu Bin , Chain Patrick S. G. , Nayfach Stephen , Kyrpides Nikos C.
Published: March 2023

Identifying and characterizing mobile genetic elements (MGEs) in sequencing data is essential for understanding their diversity, ecology, biotechnological applications, and impact on public health. Here, we introduce geNomad, a classification and annotation framework that combines information from gene content and a deep neural network to identify sequences of plasmids and viruses. geNomad uses a large dataset of marker proteins to provide functional gene annotation and taxonomic assignment of viral genomes. Using a conditional random field model, geNomad also detects proviruses integrated into host genomes with high precision. In benchmarks that included diverse MGE and chromosome sequences, geNomad significantly outperformed other tools in all evaluated clades of plasmids and viruses. Leveraging geNomad's speed and scalability, we were able to process public metagenomes and metatranscriptomes, leading to the discovery of millions of new viruses and plasmids that are available through the IMG/VR and IMG/PR databases. We anticipate that geNomad will enable further advancements in MGE research, and it is available at https://portal.nersc.gov/genomad.
Functional profiling of theToxoplasmagenome during acute mouse infection
Authors: Giuliano Christopher J. , Wei Kenneth J. , Harling Faye M. , Waldman Benjamin S. , Farringer Madeline A. , Boydston Elizabeth A. , Lan Tammy C. T. , Thomas Raina W. , Herneisen Alice L. , Sanderlin Allen G. , Coppens Isabelle , Dvorin Jeffrey D. , Lourido Sebastian
Published: March 2023

Within a host, pathogens encounter a diverse and changing landscape of cell types, nutrients, and immune responses. Examining host-pathogen interactions in animal models can therefore reveal aspects of infection absent from cell culture. We use CRISPR-based screens to functionally profile the entire genome of the model apicomplexan parasiteToxoplasma gondiiduring mouse infection. Barcoded gRNAs were used to track mutant parasite lineages, enabling detection of bottlenecks and mapping of population structures. We uncovered over 300 genes that modulate parasite fitness in mice with previously unknown roles in infection. These candidates span multiple axes of host-parasite interaction, including determinants of tropism, host organelle remodeling, and metabolic rewiring. We mechanistically characterized three novel candidates, including GTP cyclohydrolase I, against which a small-molecule inhibitor could be repurposed as an antiparasitic compound. This compound exhibited antiparasitic activity againstT. gondiiandPlasmodium falciparum,the most lethal agent of malaria. Taken together, we present the first complete survey of an apicomplexan genome during infection of an animal host, and point to novel interfaces of host-parasite interaction that may offer new avenues for treatment.
Accelerated Dynamic Time Warping on GPU for Selective Nanopore Sequencing
Authors: Sadasivan Harisankar , Stiffler Daniel , Tirumala Ajay , Israeli Johnny , Narayanasamy Satish
Published: March 2023

The design and supply of RT-PCR primers for accurate virus testing is a complex process. The MinION is a revolutionary portable nanopore DNA sequencer that may be used to sequence the whole genome of a target virus in a biological sample. Human samples have more than 99% of non-target host DNA and Read Until is a protocol that enables the MinION to selectively eject reads in real-time. However, the MinION does not have any in-built compute power to select non-target reads. SquiggleFilter is a prior work that identified the accuracy and throughput challenges in performing Read Until using the state-of-the-art solution and proposed a hardware-accelerated subsequence Dynamic Time Warping (sDTW) based programmable filter on an ASIC. However, SquiggleFilter does not work for genomes larger than 100Kb. We optimize SquiggleFilter’s sDTW algorithm onto the more commonly available GPUs.DTWaxbetter uses tensor core pipes, 2X-SIMD FP16 computations and efficient data handling strategies using offline pre-processing, coalesced global memory loads, warp shuffles and shared memory buffering among other optimizations.DTWaxenables Read Until and yields 1.92X sequencing speedup and 3.64X compute speedup: costup over a sequencing workflow that does not use Read Until.
Different lanthanide elements induce strong gene expression changes in a lanthanide-accumulating methylotroph
Authors: Gorniak Linda , Bechwar Julia , Westermann Martin , Steiniger Frank , Wegner Carl-Eric
Published: March 2023

Lanthanides are the most recently described life metals and are central to methylotrophy in diverse taxa. We recently characterized a novel, lanthanide-dependent, and lanthanide-accumulating methylotroph, Beijerinckiaceae bacterium RH AL1, that utilizes lighter lanthanides (La, Ce, Nd) for methanol oxidation. We show that lanthanum concentration and different lanthanide (Ln) elements strongly affect gene expression and intracellular lanthanide accumulation. Differential gene expression analysis based on incubations with either La (50 nM or 1 µM), Nd (1 µM), or a lanthanide cocktail ([La, Ce, Nd, Dy, Ho, Er, Yb], equimolarly pooled, 1 µM), revealed that up to 41% of the encoded genes were differentially expressed. The effects of lanthanum concentration and Ln elements were not limited to lanthanide-dependent methanol oxidation but reached into many aspects of metabolism. We observed that lanthanides control the flagellar and chemotactic machinery and that they affect polyhydroxyalkanoate (PHA) biosynthesis. Secretion and various uptake systems, and carbohydrate metabolism were highly responsive. The most differentially expressed genes encode various unknown or hypothetical proteins, but alsolanM, coding for the well-characterized lanthanide-binding protein lanmodulin, and a glucose dehydrogenase gene linked to the conversion of β-D-glucose to gluconolactone, a known metal chelator. Electron microscopy, together with RNAseq, suggested different and potentially selective mechanisms for the uptake and accumulation of individual Ln elements. Mechanisms for discriminating lanthanides and links between lanthanides and various aspects of metabolism underline a broader functional role for lanthanides, possibly by functioning as calcium complements or antagonists.

Importance

Since its discovery, lanthanide-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding lanthanide recycling and circular economy. The physiological role of lanthanides is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) lanthanides affect metabolism have rarely been done. Our research shows that strain RH AL1 distinguishes different lanthanide elements and that the effect of lanthanides reaches into many aspects of physiology, for instance, motility and polyhydroxyalkanoate metabolism. Numerous differentially expressed genes coding for unknown or hypothetical proteins might hide so far unknown lanthanide-binding proteins. Our findings regarding lanthanide accumulation suggest different mechanisms for dealing with individual lanthanide elements and provide insights relating to intracellular lanthanide homeostasis. Understanding comprehensively how microbes distinguish and handle different lanthanide elements is key for turning knowledge into application regarding lanthanide-centered biometallurgy.
Potential of Negative Ion Mode Proteomics: MS1-Only Approach
Authors: Penanes Pelayo A. , Gorshkov Vladimir , Ivanov Mark V. , Gorshkov Mikhail V. , Kjeldsen Frank
Published: March 2023

Current proteomics approaches rely almost exclusively on using positive ionization mode, which results in inefficient ionization of many acidic peptides. With an equal quantity of acidic and basic proteins and, correspondingly, the similar number for their derived peptides in case of the human proteome, this inefficient ionization poses both a substantial challenge and a potential. In this work, we study the efficiency of protein identification in the bottom-up proteomic analysis performed in negative ionization mode, using the recently introduced MS1-only ultra-fast data acquisition method DirectMS1. This method is based on accurate peptide mass measurements and predicted retention times. Our method achieves the highest rate of protein identifications in negative ion mode to date, with over 1,000 proteins identified in a human cell line at a 1% false discovery rate using a single-shot 10-min separation gradient, which is comparable with hours-long MS/MS-based analyses. Evaluating the proteins as a function of pI indicated preferable identification of the acidic part of the proteome. Optimization of separation and mass spectrometric experimental conditions facilitated the performance of the method with the best results in terms of spray stability and signal abundance obtained using mobile buffers at 2.5 mM imidazole and 3% isopropanol. The work also highlighted the complementarity of data acquired in positive and negative modes: Combining the results for all replicates for both polarities, the number of identified proteins increased up to 1,774. Finally, we performed analysis of the method’s efficiency when different proteases are used for protein digestion. Among the four studied proteases (LysC, GluC, AspN, and trypsin), we found that trypsin and LysC performed best in terms of protein identification yield. Thus, digestion procedures used for positive mode proteomics can be efficiently utilized for analysis in negative ion mode.
Enhanced Behavioral Performance through Interareal Gamma and Beta Synchronization
Authors: Parto-Dezfouli Mohsen , Vezoli Julien , Bosman Conrado Arturo , Fries Pascal
Published: March 2023

Cognitive functioning requires coordination between brain areas. Between visual areas, feedforward gamma synchronization improves behavioral performance. Here, we investigate whether similar principles hold across brain regions and frequency bands, using simultaneous local field potential recordings from 15 areas during performance of a selective attention task. Short behavioral reaction times (RTs), an index of efficient interareal communication, occurred when occipital areas V1, V2, V4, DP showed gamma synchronization, and fronto-central areas S1, 5, F1, F2, F4 showed beta synchronization. For both area clusters and corresponding frequency bands, deviations from the typically observed phase relations increased RTs. Across clusters and frequency bands, good phase relations occurred in a correlated manner specifically when they processed the behaviorally relevant stimulus. Furthermore, the fronto- central cluster exerted a beta-band influence onto the occipital cluster whose strength predicted short RTs. These results suggest that local gamma and beta synchronization and their inter-regional coordination jointly improve behavioral performance.
The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells
Authors: Koch Jana , Xin Qilin , Obr Martin , Schäfer Alicia , Rolfs Nina , Anagho Holda , Kudulyte Aiste , Woltereck Lea , Kummer Susann , Campos Joaquin , Uckeley Zina M. , Bell-Sakyi Lesley , Kräusslich Hans-Georg , Schur Florian KM , Acuna Claudio , Lozach Pierre-Yves
Published: March 2023

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific but inefficient, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.
Neural activity induces strongly coupled electro-chemo-mechanical interactions and fluid flow in astrocyte networks and extracellular space – a computational study
Authors: Sætra Marte J. , Ellingsrud Ada J. , Rognes Marie E.
Published: March 2023

The complex interplay between chemical, electrical, and mechanical factors is fundamental to the function and homeostasis of the brain, but the effect of electrochemical gradients on brain interstitial fluid flow, solute transport, and clearance remains poorly quantified. Here, via in-silico experiments based on biophysical modeling, we estimate water movement across astrocyte cell membranes, within astrocyte networks, and within the extracellular space (ECS) induced by neuronal activity, and quantify the relative role of different forces (osmotic, hydrostatic, and electrical) on transport and fluid flow under such conditions. Our results demonstrate how neuronal activity in the form of extracellular ionic input fluxes may induce complex and strongly-coupled chemical-electrical-mechanical interactions in astrocytes and ECS. Furthermore, we observe that the fluid dynamics are crucially coupled to the spatial organization of the intracellular network, with convective and electrical drift dominating ionic diffusion in astrocyte syncytia.

Author Summary

Over the last decades, the neuroscience community has paid increased attention to the astrocytes – star-shaped brain cells providing structural and functional support for neurons. Astrocyte networks are likely to be a crucial pathway for fluid flow through brain tissue, which is essential for the brain’s volume homeostasis and waste clearance. However, numerous questions related to the role of osmotic pressures and astrocytic membrane properties remain unanswered. There are also substantial gaps in our understanding of the driving forces underlying fluid flow through brain tissue. Answering these questions requires a better understanding of the interplay between electrical, chemical, and mechanical forces in brain tissue. Due to the complex nature of this interplay and experimental limitations, computational modeling can be a critical tool. Here, we present a high fidelity computational model of an astrocyte network and the extracellular space. The model predicts the evolution in time and distribution in space of intra- and extracellular volumes, ion concentrations, electrical potentials, and hydrostatic pressures following neural activity. Our findings show that neural activity induces strongly coupled chemical-mechanical-electrical interactions in the tissue and suggest that chemical gradients inside astrocyte syncytia strengthen fluid flow at the microscale.
Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast
Authors: Xu Yong-jie , Bhadra Sankhadip , Mahdi Alaa Taha A. , Dev Kamal , Yurtsever Ilknur , Nakamura Toru M.
Published: March 2023

Replication protein A (RPA) is a heterotrimeric complex and the major single-strand DNA (ssDNA) binding protein in eukaryotes. It plays important roles in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling. Because RPA is essential for cell survival, understanding its checkpoint signaling function in cells has been challenging. Several RPA mutants have been reported previously in fission yeast. None of them, however, has a defined checkpoint defect. A separation-of-function mutant of RPA, if identified, would provide significant insights into the checkpoint initiation mechanisms. We have explored this possibility and carried out an extensive genetic screening for Rpa1/Ssb1, the large subunit of RPA in fission yeast, looking for mutants with defects in checkpoint signaling. This screen has identified twenty-five primary mutants that are sensitive to genotoxins. Among these mutants, two have been confirmed partially defective in checkpoint signaling primarily at the replication fork, not the DNA damage site. The remaining mutants are likely defective in other functions such as DNA repair or telomere maintenance. Our screened mutants, therefore, provide a valuable tool for future dissection of the multiple functions of RPA in fission yeast.

AUTHOR SUMMARY

Originally discovered as a protein required for replication of simian virus SV40 DNA, replication protein A is now known to function in DNA replication, repair, recombination, telomere maintenance, and checkpoint signaling in all eukaryotes. The protein is a complex of three subunits and the two larger ones are essential for cell growth. This essential function however complicates the studies in living cells, and for this reason, its checkpoint function remains to be fully understood. We have carried out an genetic screening of the largest subunit of this protein in fission yeast, aiming to find a non-lethal mutant that lacks the checkpoint function. This extensive screen has uncovered two mutants with a partial defect in checkpoint signaling when DNA replication is arrested. Surprisingly, although the two mutants also have a defect in DNA repair, their checkpoint signaling remains largely functional in the presence of DNA damage. We have also uncovered twenty-three mutants with defects in DNA repair or telomere maintenance, but not checkpoint signaling. Therefore, the non-lethal mutants uncovered by this study provide a valuable tool for dissecting the multiple functions of this biologically important protein in fission yeast.
The EDC4-XRN1 axis controls P-body dynamics to link mRNA decapping with decay
Authors: Brothers William R. , Ali Farah , Kajjo Sam , Fabian Marc R.
Published: March 2023

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors associate with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5’-3’ exoribonuclease XRN1 interact with enhancer of mRNA decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein (RNP) granules that are often enriched with mRNAs targeted for decay, such as microRNA (miRNA)-targeted mRNAs, yet paradoxically are not required for mRNA decay to occur. In this study, we show that disrupting the interaction between XRN1 and EDC4 or altering their stoichiometry leads to an inhibition of mRNA decapping, with miRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are directly responsible for preventing mRNA decapping under these conditions. Finally, we demonstrate that P-bodies act to support cell viability and prevent stress granule formation under conditions when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5’-3’ decay in human cells.

HIGHLIGHTS

XRN1-EDC4 interaction couples mRNA decapping with mRNA decay.

Disrupting XRN1-EDC4 contact generates larger P-bodies that, in turn, inhibit decapping.

P-bodies support cellular fitness in the absence of XRN1.
Thiol reductive stress activates the hypoxia response pathway
Authors: Ravi  , Kumar Ajay , Bhattacharyya Shalmoli , Singh Jogender
Published: March 2023

Owing to their capability to disrupt the oxidative protein folding environment in the endoplasmic reticulum (ER), thiol antioxidants such as dithiothreitol (DTT) are used as ER-specific stressors. We recently showed that thiol antioxidants modulate the methionine-homocysteine cycle by upregulating an S-adenosylmethionine-dependent methyltransferase,rips-1, inCaenorhabditis elegans. However, the changes in cellular physiology induced by thiol stress that modulate the methionine-homocysteine cycle remain uncharacterized. Here, using forward genetic screens inC. elegans, we discover that thiol stress enhancesrips-1expression via the hypoxia response pathway. We demonstrate that thiol stress activates the hypoxia response pathway. The activation of the hypoxia response pathway by thiol stress is conserved in human cells. The hypoxia response pathway enhances thiol toxicity viarips-1expression and confers protection against thiol toxicity viarips-1-independent mechanisms. Finally, we show that DTT might activate the hypoxia response pathway by producing hydrogen sulfide. Our studies reveal an intriguing interaction between thiol-mediated reductive stress and the hypoxia response pathway and challenge the current model that thiol antioxidant DTT disrupts only the ER milieu in the cell.
Genus-Wide Genomic Characterization ofMacrococcus: Insights into Evolution, Population Structure, and Functional Potential
Authors: Carroll Laura M. , Pierneef Rian , Mafuna Thendo , Magwedere Kudakwashe , Matle Itumeleng
Published: March 2023

Macrococcusspecies have been isolated from a range of mammals and mammal-derived food products. While they are largely considered to be animal commensals,Macrococcusspp. can be opportunistic pathogens in both veterinary and human clinical settings. This study aimed to provide insight into the evolution, population structure, and functional potential of theMacrococcusgenus, with an emphasis on antimicrobial resistance (AMR) and virulence potential. All high-quality, publicly availableMacrococcusgenomes (n= 104, accessed 27 August 2022), plus six South African genomes sequenced here (two strains from bovine clinical mastitis cases and four strains from beef products), underwent taxonomic assignment (using four different approaches), AMR determinant detection (via AMRFinderPlus), and virulence factor detection (using DIAMOND and the core Virulence Factor Database). Overall, the 110Macrococcusgenomes were of animal commensal, veterinary clinical, food-associated (including food spoilage), and environmental origins; five genomes (4.5%) originated from human clinical cases. Notably, none of the taxonomic assignment methods produced identical results, highlighting the potential forMacrococcusspecies misidentifications. The most common predicted antimicrobial classes associated with AMR determinants identified acrossMacrococcusincluded macrolides, beta-lactams, and aminoglycosides (n= 81, 61, and 44 of 110 genomes; 73.6, 55.5, and 40.0%, respectively). Genes showing homology toStaphylococcus aureusexoenzyme aureolysin were detected across multiple species (using 90% coverage,n= 40 and 77 genomes harboring aureolysin-like genes at 60% and 40% amino acid [AA] identity, respectively).Staphylococcus aureusPanton-Valentine leucocidin toxin-associatedlukF-PVandlukS-PVhomologs were identified in eightM. canisgenomes (≥40% AA identity, >85% coverage). Using a method that delineates populations using recent gene flow (PopCOGenT), two species (M. caseolyticusandM. armenti) were composed of multiple within-species populations. Notably,M. armentiwas partitioned into two populations, which differed in functional potential (e.g., one harbored beta-lactamase family, type II toxin-antitoxin system, and stress response proteins, while the other possessed a Type VII secretion system; PopCOGenTP< 0.05). Overall, this study leverages all publicly availableMacrococcusgenomes in addition to newly sequenced genomes from South Africa to identify genomic elements associated with AMR or virulence potential, which can be queried in future experiments.
FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase
Authors: Wasserman Jason S , Faezov Bulat , Patel Kishan R , Kurimchak Alison N , Palacio Seren M , Fowle Holly , McEwan Brennan C , Xu Qifang , Zhao Ziran , Cressey Lauren , Johnson Neil , Duncan James S , Kettenbach Arminja N , Dunbrack Roland L , Graña Xavier
Published: March 2023

The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM [RK]-V-x-x-[VI]-Rin a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowleet al. eLife2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A. This conserved SLiM is necessary for FAM122A binding to B55αin vitroand in cells. Computational structure prediction withAlphaFold2predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the ‘SLiM’ in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents binding of substrates inin vitrocompetition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a ‘SLiM’-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.
Structural and functional properties of the transporter SLC26A6 reveal mechanism of coupled anion exchange
Authors: Tippett David N. , Breen Colum , Butler Stephen J. , Sawicka Marta , Dutzler Raimund
Published: March 2023

Members of the SLC26 family constitute a conserved class of anion transport proteins, which encompasses uncoupled transporters with channel-like properties, coupled exchangers and motor proteins. Among the ten functional paralogs in humans, several participate in the secretion of bicarbonate in exchange with chloride and thus play an important role in maintaining pH homeostasis. Previously, we have elucidated the structure of murine SLC26A9 and defined its function as an uncoupled chloride transporter (Walter, Sawicka, & Dutzler, 2019). Here we have determined the structure of the closely related human transporter SLC26A6 and characterized it as a coupled exchanger of chloride with bicarbonate and presumably also oxalate. The structure defines an inward-facing conformation of the protein that generally resembles known structures of SLC26A9. The altered anion selectivity between both paralogs is a consequence of a remodeled ion binding site located in the center of a mobile unit of the membrane-inserted domain, which also accounts for differences in the coupling mechanism.
Identification of a carbohydrate-recognition motif of purinergic receptors
Authors: Zhao Lifen , Wei Fangyu , He Xinheng , Jiang Hualiang , Wen Liuqing , Cheng Xi
Published: March 2023

As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane helices (TM) 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36, KDKE) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.
Comparative genomics analysis of three conserved plasmid families in the Western Hemisphere soft tick-borne relapsing fever borreliae provides insight into variation in genome structure and antigenic variation systems
Authors: Kneubehl Alexander R. , Lopez Job E.
Published: March 2023

Borreliaspirochetes, causative agents of Lyme disease and relapsing fever (RF), have a uniquely complex genome consisting of a linear chromosome and circular and linear plasmids. The plasmids harbor genes important for the vector-host life cycle of these tick-borne bacteria. The role of Lyme disease causingBorreliaplasmids is more refined compared to RF spirochetes because of limited plasmid-resolved genomes for RF spirochetes. We recently addressed this limitation and found that three linear plasmid families (F6, F27, and F28) were syntenic across all species. Given this conservation, we further investigated the three plasmid families. The F6 family, also known as the megaplasmid, contained regions of repetitive DNA. The F27 was the smallest, encoding genes with unknown function. The F28 family encoded the expression locus for antigenic variation in all species exceptBorrelia hermsiiandBorrelia anserina.Taken together, this work provides a foundation for future investigations to identify essential plasmid-localized genes that drive the vector-host life cycle of RFBorrelia.

IMPORTANCE

Borreliaspp. spirochetes are arthropod-borne bacteria found globally and infect humans and other vertebrates. RF borreliae are understudied and misdiagnosed pathogens because of the vague clinical presentation of disease and the elusive feeding behavior of argasid ticks. Consequently, genomics resources for RF spirochetes have been limited. Analyses ofBorreliaplasmids have been challenging because they are often highly fragmented and unassembled. By utilizing Oxford Nanopore Technologies, we recently generated plasmid-resolved genomes for sevenBorreliaspp. found in the Western Hemisphere. This current study is a more in-depth investigation into the linear plasmids that were conserved and syntenic across species. This analysis determined differences in genome structure and, importantly, in antigenic variation systems between species. This work is an important step in identifying crucial plasmid-borne genetic elements essential for the life cycle of RF spirochetes.
Glycans modulate lipid binding in Lili-Mip lipocalin protein
Authors: SureshKumar Harini , Appadurai Rajeswari , Srivastava Anand
Published: March 2023

The unique viviparous Pacific Beetle cockroaches provide nutrition to their embryo by secreting milk proteins Lili-Mip, which is a lipid-binding glycoprotein that crystallizes in vivo. The resolved in vivo crystal structure of variably glycosylated Lili-Mip shows a classical Lipocalin fold with an eight-stranded antiparallel beta-barrel enclosing a fatty acid. The availability of physiologically unaltered glycoprotein structure makes Lili-Mip a very attractive model system to investigate the role of glycans on protein structure, dynamics, and function. Towards that end, we have employed all-atom molecular dynamics simulations on various glycosylated stages of a bound and free Lili-Mip protein and characterized the impact of glycans and the bound lipid on the dynamics of this glycoconjugate. Our work provides important molecular-level mechanistic insights into the role of glycans in the nutrient storage function of the Lili-Mip protein. Our analyses show that the glycans locally stabilize spatially proximal residues and regulate the low amplitude opening motions of the residues at the entrance of the binding pocket. Glycans, which are located at the portal end of the barrel, also restrict the distal barrel depth and allosterically modulate the lipid dynamics in the barrel. A simple but effective distance-based network analysis of the protein also reveals the role of glycans in the subtle rewiring of residues crucial for determining the barrel depth and lipid orientation.