bluuebunny/crossref_metadata_2025 huggingface dataset:
All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals
Authors: Bergs Amelie C.F. , Liewald Jana F. , Rodriguez-Rozada Silvia , Liu Qiang , Wirt Christin , Bessel Artur , Zeitzschel Nadja , Durmaz Hilal , Nozownik Adrianna , Jospin Maëlle , Vierock Johannes , Bargmann Cornelia I. , Hegemann Peter , Wiegert J. Simon , Gottschalk Alexander
Published: June 2022

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons ofCaenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.
Tactile attention modulates occipital alpha oscillations in early blindness
Authors: Gurtubay-Antolin Ane , Bruña Ricardo , Collignon Olivier , Rodríguez-Fornells Antoni
Published: June 2022

Alpha oscillatory activity is thought to contribute to the cueing of visual attention through the engagement of task-relevant occipital regions. In early blindness, occipital alpha oscillations are systematically reduced, suggesting that occipital alpha depends on visual experience. However, it is still unknown if alpha activity could serve attentional cueing in non-visual modalities in blind people, especially considering previous research that showed the recruitment of the occipital cortex for non-visual processing. To test this idea, we used electroencephalography to answer whether alpha oscillations reflected a differential recruitment of task-relevant regions between expected and unexpected conditions in two (texture and shape discrimination) haptic tasks. As expected, time frequency analyses showed that alpha suppression in parieto-occipital sites was significantly reduced in early blind individuals. The source reconstruction analysis revealed that group differences originated in the middle occipital cortex. In that region, expected trials evoked higher alpha desynchronization than unexpected trials in the EB group only. Our results support the role of alpha rhythms in the recruitment of occipital areas also in early blind participants, and for the first time we show that even if posterior alpha activity is reduced in blindness, it is however sensitive to task-dependent factors. Our findings therefore suggest that occipital alpha activity may be involved in tactile attention in blind individuals, maintaining the function proposed for visual attention in sighted population but switched to the tactile modality. Altogether, our results indicate that attention-dependent modulation of alpha oscillatory activity does not depend on visual experience.

SIGNIFICANCE STATEMENT

Are posterior alpha oscillations and their role in attention dependent on visual experience? Our results show that tactile attention can modulate posterior alpha activity in blind (but not sighted) individuals through the engagement of occipital regions, suggesting that in early blindness, alpha oscillations maintain their proposed role in visual attention but subserve tactile processing. Our findings bring a new understanding to the role that alpha oscillatory activity plays in blindness, contrasting with the view supporting that alpha activity is rather task unspecific.
Operative dimensions in unconstrained connectivity of recurrent neural networks
Authors: Krause Renate , Cook Matthew , Kollmorgen Sepp , Mante Valerio , Indiveri Giacomo
Published: June 2022

Recurrent Neural Networks (RNNs) are commonly used models to study neural computation. However, a comprehensive understanding of how dynamics in RNNs emerge from the underlying connectivity is largely lacking. Previous work derived such an understanding for RNNs fulfilling very specific constraints on their connectivity, but it is unclear whether the resulting insights apply more generally. Here we study how network dynamics are related to network connectivity in RNNs trained without any specific constraints on several tasks previously employed in neuroscience. Despite the apparent high-dimensional connectivity of these RNNs, we show that a low-dimensional, functionally relevant subspace of the weight matrix can be found through the identification ofoperativedimensions, which we define as components of the connectivity whose removal has a large influence on local RNN dynamics. We find that a weight matrix built from only a few operative dimensions is sufficient for the RNNs to operate with the original performance, implying that much of the high-dimensional structure of the trained connectivity is functionally irrelevant. The existence of a low-dimensional, operative subspace in the weight matrix simplifies the challenge of linking connectivity to network dynamics and suggests that independent network functions may be placed in specific, separate subspaces of the weight matrix to avoid catastrophic forgetting in continual learning.
RPG acts as a central determinant for infectosome formation and cellular polarization during intracellular rhizobial infections
Authors: Lace Beatrice , Su Chao , Perez Daniel Invernot , Rodriguez-Franco Marta , Vernié Tatiana , Batzenschlager Morgane , Egli Sabrina , Liu Cheng-Wu , Ott Thomas
Published: June 2022

Host-controlled intracellular accommodation of nitrogen-fixing bacteria is essential for the establishment of a functional Root Nodule Symbiosis (RNS). In many host plants, this occurs via transcellular tubular-structures (infection threads - ITs) that extend across cell layers via polar tip- growth. Comparative phylogenomic studies have identified RPG (RHIZOBIUM-DIRECTED POLAR GROWTH) among the critical genetic determinants for bacterial infection. In Medicago truncatula, RPG is required for effective IT progression within root hairs but the cellular and molecular function of the encoded protein remain elusive. Here, we show that RPG resides in the protein complex formed by the core endosymbiotic components VAPYRIN (VPY) and LUMPY INFECTION (LIN) required for IT polar growth, co- localizes with both VPY and LIN in IT tip- and perinuclear-associated puncta of M. truncatula root hairs undergoing infection and is necessary for VPY recruitment to puncta. Fluorescence Lifetime Imaging Microscopy (FLIM) of phosphoinositide species during bacterial infection revealed that functional RPG is required to sustain strong membrane polarization at the advancing tip of the IT. In addition, loss of RPG functionality alters the cytoskeleton-mediated connectivity between the IT tip and the nucleus and affects polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL). Our results integrate RPG into a core host machinery required to support symbiont accommodation, suggesting that its occurrence in plant host genomes is essential to co-opt a multimeric protein module committed to endosymbiosis to sustain IT-mediated bacterial infection.
Zyxin and non-muscle myosin are required for single fibroblast durotaxis, but Rho-kinase activity and the Arp2/3 complex are dispensable
Authors: Hakeem Reem M. , Subramanian Bhagawat C. , Hockenberry Max A. , King Zayna T. , Butler Mitchell T. , Legant Wesley R. , Bear James E.
Published: June 2022

Durotaxis, migration of cells directed by stiffness gradient, is critical in development and disease. To study the molecular determinants of single cell durotaxis, we developed an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with different slopes, along with uniform stiffness (soft and stiff) regions. We find that fibroblasts rely on non-muscle myosin II (NMII) activity and the LIM-domain protein zyxin for durotaxis on both steep and shallow stiffness gradients. Importantly, unlike haptotaxis, the Arp2/3 complex is dispensable for durotaxis on both stiffness gradients. Lack of Arp2/3 results in a filopodia-based durotactic migration that is equally efficient as that of lamellipodia-based durotactic migration. Finally, we reveal an essential role for the actin-bundler fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis in shallow, but not steep, stiffness gradient. Together, our all-in-one hydrogel system can serve as a platform to identify, discriminate, and characterize stiffness gradient specific molecular mechanisms that cells employ to efficiently durotax.
Spatiotemporal recruitment of inhibition and excitation in the mammalian cortex during electrical stimulation
Authors: Dadarlat Maria C. , Sun Yujiao Jennifer , Stryker Michael P.
Published: June 2022

Electrical stimulation has emerged as a powerful and precise treatment in which to modulate aberrant neural activity patterns common in neural dysfunction and disease; however, the physiological process involved in microstimulation is poorly understood, particularly regarding the contributions of inhibitory neurons to shaping stimulation-evoked activity. To address this issue, we used 2-photon imaging of transgenic mice to measure the widespread responses of inhibitory and excitatory neurons to electrical stimulation through a chronically-implanted cortical microelectrode. We found that increasing stimulation amplitude both raised the fraction of neurons that responded to a stimulus and increased the distance at which inhibitory and excitatory neurons were significantly modulated by stimulation; however, the lateral spread of inhibitory activity preceded that of excitatory activity. By 50 µA, a significantly larger fraction of inhibitory neurons vs. excitatory neurons were modulated by stimulation. Increasing amplitude also shifted the temporal response properties of the population — towards longer-latency excitation close to the electrode tip and strong inhibition of more distant neurons. Animal behavior, specifically their locomotion patterns, strongly correlated with trial-to-trial variability in stimulation-evoked responses. We conclude that changing electrical stimulation amplitude can shift the balance of excitation to inhibition in the brain in a manner that interact with ongoing animal behavior.
Screening the PRISM library against Staphylococcus aureus reveals a sesquiterpene lactone from Liriodendron tulipifera with inhibitory activity
Authors: Kirk Riley D. , Rosario Margaret E. , Oblie Nana , Jouaneh Terra Marie M. , Carro Marina A. , Wu Christine , Leibovitz Elizabeth , Hunter Elizabeth Sage , Literman Robert , Handy Sara M. , Rowley David C. , Bertin Matthew J.
Published: June 2022

Infections caused by the bacterium Staphylococcus aureus continue to pose threats to human health and put a financial burden on the healthcare system. The overuse of antibiotics has contributed to mutations leading to the emergence of methicillin-resistant Staphylococcus aureus, and there is a critical need for the discovery and development of new antibiotics to evade drug resistant bacteria. Medicinal plants have shown promise as sources of new small molecule therapeutics with potential uses against pathogenic infections. The Principal Rhode Island Secondary Metabolite (PRISM) library is a botanical extract library generated from specimens in the URI Heber W. Youngken Jr. Medicinal Garden by upper-division undergraduate students. PRISM extracts were screened for activity against strains of methicillin-susceptible S. aureus (MSSA). An extract generated from the tulip tree (Liriodendron tulipifera) demonstrated growth inhibition against MSSA, and a bioassay-guided approach identified a sesquiterpene lactone, laurenobiolide, as the active constituent. Intriguingly, its isomers tulipinolide and epi-tulipinolide lacked potent activity against MSSA. Laurenobiolide also proved to be more potent against MSSA than the structurally similar sesquiterpene lactones constunolide and dehydrocostus lactone. Laurenobioloide was most abundant in the twig bark of the tulip tree, supporting the historical and cultural usage of twig bark in poultices and teas.

ABSTRACT GRAPHIC
Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2
Authors: Zhang Xizi , Zoltek Madeline , Mozumdar Deepto , Schepartz Alanna
Published: June 2022

Methyl-CpG-binding-protein 2 (MeCP2) is a nuclear protein expressed in all cell types, especially neurons1. Mutations in the MECP2 gene cause Rett syndrome (RTT), an incurable neurological disorder that disproportionately affects young girls2. Strategies to restore MeCP2 expression phenotypically reverse RTT-like symptoms in male and female MeCP2-deficient mice3–5, suggesting that direct nuclear delivery of functional MeCP2 could restore MeCP2 activity. We report that ZF-tMeCP2, a conjugate of MeCP2(Δaa13-71, 313-484)6 and the cell-permeant mini-protein ZF5.37–11, both binds DNA in a methylation-dependent manner and reaches the nucleus of model cell lines intact at concentrations above 700 nM. When delivered to live cells, ZF-tMeCP2 engages the NCoR/SMRT co-repressor complex and selectively represses transcription from methylated promoters. Efficient nuclear delivery of ZF-tMeCP2 relies on a unique endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat-tMeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of protein-derived therapeutics.
Evaluating the breadth of neutralizing antibody responses elicited by infectious bursal disease virus (IBDV) genogroup A1 strains using a novel chicken B-cell rescue system and neutralization assay
Authors: Reddy Vishwanatha R. A. P. , Nazki Salik , Brodrick Andrew J. , Asfor Amin , Urbaniec Joanna , Morris Yasmin , Broadbent Andrew J.
Published: June 2022

Eight infectious bursal disease virus (IBDV) genogroups have been identified based on the sequence of the capsid hypervariable region (HVR) (A1-8), yet many vaccines are based on A1 strains. Given reported vaccine failures, there is a need to evaluate the ability of vaccines to neutralize the different genogroups. To address this, we used a reverse genetics system and the chicken B-cell line DT40 to rescue a panel of chimeric IBDVs and perform neutralization assays. Chimeric viruses had the backbone of a lab-adapted strain (PBG98) and the HVRs from diverse field strains: classical F52-70 (A1), US-variant Del-E (A2), Chinese-variant SHG19 (A2), very-virulent UK661 (A3), M04/09 distinct (A4), Italian ITA-04 (A6), and Australian-variant Vic-01/94 (A8). Rescued viruses showed no substitutions at amino-acid positions 253, 284, or 330, previously associated with cell-culture adaptation. Sera from chickens inoculated with wt (F52-70) or vaccine (228E) A1 strains had the highest mean virus neutralization (VN) titers against the A1 virus (log2 15.41 and 12.66), and the lowest against A2 viruses (log2 7.41-7.91, p=0.0001-0.0274), consistent with A1 viruses being most antigenically distant from A2 strains, which correlated with the extent of differences in the predicted HVR structure. VN titers against the other genogroups ranged from log2 9.32-13.32, and A1 strains were likely more closely antigenically related to genogroups A3 and A4 than A6 and A8. Our data are consistent with field observations, validating our method, which can used to screen future vaccine candidates for breadth of neutralizing antibodies, and evaluate the antigenic relatedness of different genogroups.

Importance

There is a need to evaluate the ability of vaccines to neutralize diverse IBDV genogroups, and to better understand the relationship between HVR sequence, structure, and antigenicity. Here, we used a chicken B cell-line to rescue a panel of chimeric IBDVs with the HVR from seven diverse IBDV field strains, and conduct neutralization assays and protein modelling. We evaluated the ability of serum from vaccinated or infected birds to neutralize the different genogroups. Our novel chicken B-cell rescue system and neutralization assay can be used to screen IBDV vaccine candidates, platforms, and regimens for the breadth of neutralizing antibody responses elicited, evaluate the antigenic relatedness of diverse IBDV strains, and when coupled with structural modelling, elucidate immunodominant and conserved epitopes to strategically design novel IBDV vaccines in the future.
Calcitonin Gene-Related Peptide (CGRP) Receptor Antagonism Reduces Motion Sickness Indicators in Mouse Migraine Models
Authors: Rahman Shafaqat M. , Luebke Anne E.
Published: June 2022

Structured Abstract

Background

Migraine and especially vestibular migraine are associated with enhanced motion sickness and motion-induced nausea. Murine models of migraine have used injections of Calcitonin Gene-Related Peptide (CGRP) or other migraine triggers [i.e., sodium nitroprusside (SNP)] to induce migraine sensitivities to touch and light. Yet, there is limited research on whether these triggers increase motion-induced nausea, and if migraine blockers can reduce these migraine symptoms. We hypothesized that systemic delivery of CGRP or SNP will increase motion sickness susceptibility and motion-induced nausea in mouse models, and that migraine blockers can block these changes induced by systemically delivered CGRP or SNP.

Methods

We investigated two measures of motion sickness assessment [motion sickness index (MSI) scoring and motion-induced thermoregulation] after intraperitoneal injections of either CGRP or SNP in C57B6/J mice. The drugs olcegepant, sumatriptan, and rizatriptan were used to assess the efficacy of migraine blockers.

Results

MSI measures were confounded by CGRP’s effect on gastric distress. However, analysis of tail vasodilations as a surrogate for motion-induced nausea was robust forbothmigraine triggers. Only olcegepant treatment rescued tail vasodilations.

Conclusion

These preclinical findings support the use of small molecule CGRP receptor antagonists for the treatment of motion-induced nausea of migraine, and show that triptan therapeutics are ineffective against motion-induced nausea of migraine.
A lack of distinct cell identities in single-cell measurements: revisiting Waddington’s landscape
Authors: Sparta Breanne , Hamilton Timothy , Hughes Serena , Natesan Gunalan , Deeds Eric J.
Published: June 2022

The prevailing interpretation of Waddington’s landscape is that attractors in gene expression space produce and stabilize distinct cell types. This notion is often applied in single-cell omics data, where groups of cells are clustered for analysis. Here we apply graph theory to characterize the distribution of cells in epigenetic space, using data from various tissues and organisms as well as various single-cell omics technologies. We found that cell types exist in the same regions of epigenetic space, with highly heterogeneous density distributions that are inconsistent with expected densities near an attractor. The lack of attractor structure could not be explained by technical noise, scale variance among genes, nor the subset of genes that were used; nor could it be rescued by any standard set of transformations. These findings pose a challenge for the robust analysis of single-cell data and open the possibility for alternative explanations of canalization during development.
Molecular epidemiology of carbapenemase-producing Acinetobacter spp. from Israel, 2001-2006: earliest report of blaNDM predating the oldest known blaNDM-positive strains
Authors: Grenier Frédéric , Baby Vincent , Allard Sarah , Heynemand Félix , Lévesque Simon , Sullivan Richard , Landecker Hannah L. , Higgins Paul G. , Rodrigue Sébastien , Haraoui Louis-Patrick
Published: June 2022

Background

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a WHO priority 1 critical pathogen. Despite early emergence of elevated CRAb rates in Israel, limited molecular data from this location are available. We searched for carbapenemases among 198 clinical Acinetobacter spp. from Israel between 2001 and 2006.

Methods

Strains from 3 archives underwent whole-genome sequencing (Illumina NovaSeq on all, MinION on a subset) and computational analyses: assembly (Unicycler), annotation (prokka), identification (Kraken, rpoB similarity), search for carbapenemases (ResFinder, BLDB curation).

Findings

A. baumannii (Ab) represented 179 (90·4%) Acinetobacter spp. Eighty-four Ab (46·9%) carried a carbapenemase: 38 (45·2%) blaOXA-72 (blaOXA-24-like); 28 (33·3%) blaOXA-23-like (20 blaOXA-23 and 8 blaOXA-225); 18 (21·5%) blaOXA-58 (16 from 2001-2). Carbapenemase rates increased yearly from 2002 (32%) to 2006 (67%). Eight species of non-baumannii Acinetobacter (NbA) accounted for 19 isolates (9·6%). Two of three A. junii contained blaOXA-58, one of which, Ajun-H1-3, isolated in January 2004, also possessed blaNDM-1. The pNDM-Ajun-H1-3 plasmid matched numerous NDM-positive plasmids reported from 2005 onwards in Acinetobacter spp. as well as Enterobacterales.

Interpretation

We assessed carbapenemase diversity among Acinetobacter spp. in Israel from 2001-2006. Findings in Ab predate observations elsewhere: rapidly rising carbapenemase rates, driven by blaOXA-23-like and blaOXA-24-like genes replacing blaOXA-58. Among NbA, an A. junii isolated in 2004 carried blaNDM-1, making it the earliest NDM-positive isolate reported to date, preceding those from 2005 in India. Further research into blaNDM’s emergence is warranted, in order to shed light on the evolution and spread of this and other antibiotic-resistance genes.

Funding

Centre de recherche Charles-Le Moyne; Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke; Fonds de recherche du Québec – Santé; New Frontiers in Research Fund Grant NFRFE-2019-00444; CIFAR-Azrieli Global Scholars Program.
HIRA loss transforms FH-deficient cells
Authors: Valcarcel-Jimenez Lorea , Rogerson Connor , Yong Cissy , Schmidt Christina , Yang Ming , Harle Victoria , Offord Victoria , Wong Kim , Mora Ariane , Speed Alyson , Caraffini Veronica , Tran Maxine Gia Binh , Maher Eamonn R. , Stewart Grant D. , Vanharanta Sakari , Adams David J. , Frezza Christian
Published: June 2022

Fumarate Hydratase (FH) is a mitochondrial enzyme that catalyses the reversible hydration of fumarate to malate in the TCA cycle. Germline mutations of FH lead to HLRCC, a cancer syndrome characterised by a highly aggressive form of renal cancer(1). Although HLRCC tumours metastasise rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas (2). How Fh1-deficient cells overcome these tumour suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR/Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of HIRA enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss enables the activation of MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.
Exceptional fossil preservation and evolution of the ray-finned fish brain
Authors: Figueroa Rodrigo T. , Goodvin Danielle , Kolmann Matthew A. , Coates Michael I. , Caron Abigail M. , Friedman Matt , Giles Sam
Published: June 2022

Brain anatomy provides key evidence for ray-finned fish relationships1, but two key limitations obscure our understanding of neuroanatomical evolution in this major vertebrate group. First, the deepest branching living lineages are separated from the group’s common ancestor by hundreds of millions of years, with indications that aspects of their brain morphology–like other aspects of their anatomy2,3–are specialised relative to primitive conditions. Second, there are no direct constraints on brain morphology in the earliest ray-finned fishes beyond the coarse picture provided by cranial endocasts: natural or virtual infillings of void spaces within the skull4–8. Here we report brain and cranial nerve soft-tissue preservation in †Coccocephalichthys wildi, a ∼319-million-year-old (Myr) ray-finned fish. This oldest example of a well-preserved vertebrate brain provides a unique window into neural anatomy deep within ray-finned fish phylogeny. †Coccocephalichthys indicates a more complicated pattern of brain evolution than suggested by living species alone, highlighting cladistian apomorphies9 and providing temporal constraints on the origin of traits uniting all extant ray-finned fishes9–11. Our findings, along with a growing set of studies in other animal groups12–16, point to the significance of ancient soft tissue preservation in understanding the deep evolutionary assembly of major anatomical systems outside of the narrow subset of skeletal tissues17–20.
AdipoQ – a simple, open-source software to quantify adipocyte morphology and function in tissues and in vitro
Authors: Sieckmann Katharina , Winnerling Nora , Huebecker Mylene , Leyendecker Philipp , Ribeiro Dalila , Gnad Thorsten , Pfeifer Alexander , Wachten Dagmar , Hansen Jan N.
Published: June 2022

The different adipose tissues can be distinguished according to their function. For example, white adipose tissue (WAT) stores energy in form of lipids, whereas brown adipose tissue (BAT) dissipates energy in the form of heat. These functional differences are represented in the respective adipocyte morphology: whereas white adipocytes contain large, unilocular lipid droplets, brown adipocytes contain smaller, multilocular lipid droplets. However, an automated, image-analysis pipeline to comprehensively analyze adipocytes in vitro in cell culture as well as ex vivo in tissue sections is missing. We here present AdipoQ, an open-source software implemented as ImageJ plugins that allows to analyze adipocytes in tissue sections and in vitro after histological and/or immunofluorescent labelling. AdipoQ is compatible with different imaging modalities and staining methods, allows batch processing of large datasets and simple post-hoc analysis, provides a broad band of parameters, and allows combining multiple fluorescent read-outs. Thereby, AdipoQ is of immediate use not only for basic research but also for clinical diagnosis.
Mucosal host–microbe interactions associate with clinical phenotypes in inflammatory bowel disease
Authors: Hu Shixian , Bourgonje Arno R. , Gacesa Ranko , Jansen Bernadien H. , Björk Johannes R. , Bangma Amber , Hidding Iwan J. , van Dullemen Hendrik M. , Visschedijk Marijn C. , Faber Klaas Nico , Dijkstra Gerard , Harmsen Hermie J. M. , Festen Eleonora A. M. , Vila Arnau Vich , Spekhorst Lieke M. , Weersma Rinse K.
Published: June 2022

Dysregulation of gut mucosal host–microbe interactions is a central feature of inflammatory bowel disease (IBD). To study tissue-specific interactions, we performed transcriptomic (RNA-seq) and microbial (16S-rRNA-seq) profiling of 696 intestinal biopsies derived from 353 patients with IBD and controls. Analysis of transcript-bacteria interactions identified six distinct groups of inflammation-related pathways that were associated with intestinal microbiota, findings we could partially validate in an independent cohort. An increased abundance of Bifidobacterium was associated with higher expression of genes involved in fatty acid metabolism, while Bacteroides was associated with increased metallothionein signaling. In fibrostenotic Crohn’s disease, a transcriptional network dominated by immunoregulatory genes associated with Lachnoclostridium bacteria in non-stenotic tissue. In patients using TNF-α-antagonists, a transcriptional network dominated by fatty acid metabolism genes associated with Ruminococcaceae. Mucosal microbiota composition was associated with enrichment of specific intestinal cell types. Overall, we identify multiple host–microbe interactions that may guide microbiota-directed precision medicine.
UNC-45 has a crucial role in maintaining muscle sarcomeres during aging in Caenorhabditis elegans
Authors: Matheny Courtney J. , Qadota Hiroshi , Kimelman Marion , Bailey Aaron O. , Oberhauser Andres F. , Benian Guy M.
Published: June 2022

Summary/Abstract

As people live longer, age-related diseases, like sarcopenia, will become a greater public health concern. We use the model organism C. elegans to better understand the molecular mechanisms behind muscle maintenance. Muscle function is dependent on having properly organized and functioning thick filaments, which are primarily composed of myosin. The myosin head requires the chaperone UNC-45 to initially fold it after translation and is likely used to re-fold back to functionality after thermal or chemical stress induced unfolding. We observe an early onset of sarcopeania when UNC-45 is perturbed during adulthood. We observe that during adult aging, there is a sequential decline of HSP-90, UNC-45, and then myosin. Myosin and UNC-45 protein decline are independent of steady state mRNA levels. Loss of UNC-45 is correlated with an increase in phosphorylation of the protein. By mass spectrometry, S111 was identified as being phosphorylated and this modification may affect binding to HSP-90. A longevity mutant with delayed onset of sarcopenia also shows a delay in the loss of HSP-90, UNC-45, and myosin. We also see a decrease in UNC-45 protein, but not transcript, in an hsp-90 loss of function mutant, suggesting a role for HSP-90 in stabilizing UNC-45. This leads us to propose the model that during aging, a loss of HSP-90 leads to UNC-45 being post translationally modified, such as phosphorylation, and degraded, which then leads to a loss of myosin, and thus muscle mass and function. A better understanding of how myosin and its chaperone proteins are regulated and affected by aging will lead to better preventative care and treatment of sarcopenia and, possibly, the age-related decline of heart muscle function.

Graphical Abstract

In young adults, under normal conditions the UCS domain of UNC-45 (shown in green) is bound to the myosin head (in red) and the TPR domain (in yellow) is bound to HSP-90 (in purple). Under stress conditions, HSP-90 detaches from the TPR domain, causing a conformational change in UNC-45 that allows the Central domain (in blue) to bind to the myosin neck (in red) resulting in inhibition of the myosin power stroke while the UCS domain protects/re-folds the myosin head. HSP-90 can then rebind the TPR domain, causing the Central domain to release the myosin neck, allowing movement of the myosin motor. However, aged adults experience a loss of HSP-90 and UNC-45 (which has increased post translational modification with aging). The loss of the Myosin chaperones leads to increased aggregation and degradation of Myosin with age.This loss of Myosin at the thick filament results in decline in muscle mass and function, also known as sarcopenia. Note that only the myosin head and neck are shown for simplicity of illustration.
ASAR lncRNAs control DNA replication timing through interactions with multiple hnRNP/RNA binding proteins
Authors: Thayer Mathew J. , Heskett Michael B. , Smith Leslie G. , Spellman Paul T. , Yates Phillip A.
Published: June 2022

ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR lncRNAs remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR lncRNAs, with several hnRNPs as abundant interactors. An ∼7kb domain within the ASAR6-141 lncRNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this ∼7kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes incis. shRNA-mediated depletion of 10 different RNA binding proteins, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, or HNRNPUL1, results in dissociation of ASAR lncRNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and we propose that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.
Anaerobic single particle cryoEM of nitrogenase
Authors: Warmack Rebeccah A. , Rees Douglas C.
Published: June 2022

The enzyme nitrogenase catalyzes the reduction of dinitrogen to ammonia during biological nitrogen fixation through a mechanism involving the ATP dependent interaction of two component proteins adopting multiple conformational states. To date, high resolution structural information has been provided by X-ray crystallography, which restricts the states that can be accessed to those that can be crystallized. Cryo-electron microscopy (cryoEM) presents a new opportunity for structural characterization of nitrogenase solution structures, and may yield new information on the mechanism of nitrogenase by revealing structures of transient or heterogeneous states. In this study, we present single particle cryoEM structures of the MoFe-nitrogenase endogenously isolated from Azotobacter vinelandii. To maintain the fully reduced cluster states of this oxygen sensitive protein, we prepared samples within an anaerobic chamber and employed specialized conditions to minimize partial disordering of the α-subunit at the air-water interface during freezing. Under these conditions, cryoEM structures of the as-isolated MoFe-protein and stabilized MoFe-protein-Fe-protein ADP-AlF4-complex were generally found to closely resemble their corresponding X-ray crystallographic structures. The cryoEM structures did reveal disordering in regions of the MoFe-protein α-subunit reminiscent of that observed previously for the ΔnifB MoFe-protein lacking the FeMo-cofactor, suggesting that this disorder may reflect functionally relevant dynamics, as well as the possibility of asymmetric binding of the Fe-protein to the MoFe-protein in solution. The methods presented here pave the way toward the capture and interrogation of turnover-relevant nitrogenase states by cryoEM.
Autoregulation of a trimeric transporter involves the cytoplasmic domains of both adjacent subunits
Authors: Leone Vanessa , Bradshaw Richard T. , Koshy Caroline , Lee Paul Suhwan , Fenollar-Ferrer Cristina , Heinz Veronika , Ziegler Christine , Forrest Lucy R.
Published: June 2022

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used µs-scale molecular simulations of the BetP trimer to compare reported conformations of the 45 amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport.
Human ERG Oncoprotein Represses Chip/LDB1 LIM-Domain Binding Gene in Drosophila
Authors: Bharti Mahima , Bajpai Anjali , Rautela Umanshi , Manzar Nishat , Ateeq Bushra , Sinha Pradip
Published: June 2022

ERG oncoprotein, a master transcription factor, targets diverse arrays of genes in different cancers. Identifying oncogenically relevant ones from these ERG targets, however, is challenging. Here we show that heterologous ERG disrupts a LIM-homeodomain (LIM-HD) complex, Chip-Tailup, in Drosophila. In the posterior thorax (notum) primordium, ERG-induced upregulation of E(z)/EZH2 trimethylates histones in Chip promoter. A consequent loss of the Chip-Tailup complex releases repression of N-Wg signaling in the notum, inducing de novo wings and, alternatively, carcinogenesis of ERG-expressing notal cells displaying loss of Lgl tumor suppressor. ERG-induced developmental or oncogenic fallouts are abrogated upon gain of Chip, N, or E(z) loss, besides Wg ligand sequestration. ERG-positive prostate cancer (PCa) cells, too, display suppression of mammalian homolog of Drosophila Chip, LIM Domain Binding1, LDB1. Deep homology in gene regulatory networks, like that of Chip-Tup complex, thus help prioritize identification of functionally relevant targets of human oncoproteins in Drosophila.

Highlights

Human ERG suppresses Chip, a LIM-domain binding, LDB gene in Drosophila via E(z)

ERG-mediated Chip loss induces ectopic Wg morphogen signaling in the notum primordium

Chip gain suppresses ERG-induced Wg morphogen and tumor progression in lgl clones

ERG-positive human PCa cell lines show downregulation of a Chip homolog, LDB1

In brief

Mammalian ERG oncoprotein displays a diverse and perplexing range of targets in different cancers. By driving ERG in Drosophila developing appendages, Bharti et al. reveal its repression of a LIM-domain coding gene, Chip/LDB1. ERG-positive prostate cancer cells, too, display Chip/LDB1 repression. Deep homology across phylogeny thus helps uncover oncoprotein targets.
Modeling suicide mortality in US counties using population socioeconomic indicators
Authors: Kandula Sasikiran , Martinez-Alés Gonzalo , Rutherford Caroline , Gimbrone Catherine , Olfson Mark , Gould Madelyn S. , Keyes Katherine M. , Shaman Jeffrey
Published: June 2022

Background

Suicide is one of the leading causes of death in the United States and population risk prediction models can inform the type, location, and timing of public health interventions. Here, we report the development of a prediction model of suicide risk using population characteristics.

Methods

All suicide deaths reported to the Nation Vital Statistics System between 2005-2019 were identified, and age, sex, race, and county-of-residence of the decedents were extracted to calculate baseline risk. County-wise annual measures of socioeconomic predictors of suicide risk — unemployment, weekly wage, poverty prevalence, median household income, and population density — along with two state-wise measures of prevalence of major depressive disorder and firearm ownership were compiled from public sources. Conditional autoregressive (CAR) models, which account for spatiotemporal autocorrelation in response and predictors, were used to estimate county-level risk.

Results

Estimates derived from CAR models were more accurate than from models not adjusted for spatiotemporal autocorrelation. Inclusion of suicide risk/protective covariates further reduced errors. Suicide risk was estimated to increase with each standard deviation increase in firearm ownership (2.8%), prevalence of major depressive episode (1%) and unemployment (2.8%). Conversely, risk was estimated to decrease by 4.3% for each standard deviation increase in both median household income and population density. Increased heterogeneity of risk across counties was also noted.

Conclusions

Area-level characteristics and the CAR model structure can estimate population-level suicide risk and thus inform decisions on resource allocation and focused interventions during outbreaks.
Proteome-wide microarray-based screening of PAR-binding proteins
Authors: Kang Bong Gu , Kang Sung-Ung , Kim Jae Jin , Kwon Ji-Sun , Gagné Jean-Philippe , Lee Seo Yun , Kim Soyeon , Sangwon Karl L. , Ha Shinwon , Jeong Jun Seop , Lee Yun-Il , Zhu Heng , Kim Dongsan , Poirier Guy G. , Kang Ho Chul , Dawson Valina L. , Dawson Ted M.
Published: June 2022

Poly(ADP-ribose) (PAR) plays a crucial role in intracellular signaling and scaffolding through covalent modification or non-covalent binding to target proteins. The non- covalent binding PARylome has not been extensively characterized. Here we performed a PAR-binding screen using a human protein microarray that covers most of the human proteome to characterize the non-covalent binding PARylome. A total of 356 PAR- binding proteins were identified. The PAR-binding PARylome suggests that PAR- binding regulates a variety of biological processes beyond well-characterized DNA damage signaling and DNA repair. Proteins that may be reprogrammed by PAR-binding include signaling molecules, transcription factors, nucleic acid binding proteins, calcium binding proteins, ligases, oxidoreductases, enzymes, transferases, hydrolases, and receptors. The global database of PAR-binding proteins that we established will be a valuable tool for further in-depth analysis of the role of PARylation in a wide range of biological contexts.
Artificial neural network modelling of the neural population code underlying mathematical operations
Authors: Nakai Tomoya , Nishimoto Shinji
Published: June 2022

Mathematical operations have long been regarded as a sparse, symbolic process in neuroimaging studies. In contrast, advances in artificial neural networks (ANN) have enabled extracting distributed representations of mathematical operations. Recent neuroimaging studies have compared distributed representations of the visual, auditory and language domains in ANNs and biological neural networks (BNNs). However, such a relationship has not yet been examined in mathematics. Here we used the fMRI data of a series of mathematical problems with nine different combinations of operators to construct voxel-wise encoding models using both sparse operator and latent ANN features. Representational similarity analysis demonstrated shared representations between ANN and BNN, an effect particularly evident in the intraparietal sulcus. Feature-brain similarity analysis served to reconstruct a sparse representation of mathematical operations based on distributed ANN features. Such reconstruction was more efficient when using features from deeper ANN layers. Moreover, latent ANN features allowed the decoding of novel operators not used during model training from brain activity. The current study provides novel insights into the neural code underlying mathematical thought.
The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm
Authors: Hamdorf Matthias , Imhof Thomas , Bailey-Elkin Ben , Betz Janina , Theobald Sebastian J , Simonis Alexander , Cristanziano Veronica Di , Gieselmann Lutz , Dewald Felix , Lehmann Clara , Augustin Max , Klein Florian , Alcazar Miguel A Alejandre , Rongisch Robert , Fabri Mario , Rybniker Jan , Goebel Heike , Stetefeld Jörg , Brachvogel Bent , Cursiefen Claus , Koch Manuel , Bock Felix
Published: June 2022

The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1β, IL-6, IL-12p70, TNF-α, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases.
Extracellular vesicle formation inCryptococcus deuterogattiiimpacts fungal virulence and requires theNOP16gene
Authors: Castelli Rafael F. , Pereira Alana , Honorato Leandro , Valdez Alessandro , de Oliveira Haroldo C. , Bazioli Jaqueline M. , Garcia Ane W. A. , Freitas Klimeck Tabata D’Maiella , Reis Flavia C. G. , Staats Charley C. , Nimrichter Leonardo , Fill Taícia P. , Rodrigues Marcio L.
Published: June 2022

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known.NOP16is a eukaryotic gene that is required for the activity of benzimidazoles againstCryptococcus deuterogattii. In this study, during the phenotypic characterization ofC. deuterogattiimutants lackingNOP16expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independentnop16Δ mutants revealed that the deletion ofNOP16resulted not only in a reduced number of EVs but also an altered small molecule composition. In aGalleria mellonellamodel of infection, thenop16Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with thenop16Δ cells inG. mellonella. These results reveal a role forNOP16in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.