bluuebunny/crossref_metadata_2025 huggingface dataset:
Uncovering nitroxoline activity spectrum, mode of action and resistance across Gram-negative bacteria
Authors: Cacace Elisabetta , Tietgen Manuela , Steinhauer Meike , Mateus André , Schultze Tilman G. , Eckermann Marina , Galardini Marco , Varik Vallo , Koumoutsi Alexandra , Parzeller Jordan J. , Corona Federico , Orakov Askarbek , Knopp Michael , Brauer-Nikonow Amber , Bork Peer , Romao Celia V. , Zimmermann Michael , Cloetens Peter , Savitski Mikhail M. , Typas Athanasios , Göttig Stephan
Published: June 2024

Nitroxoline is a bacteriostatic quinoline antibiotic, considered a metal chelator inhibiting the activity of RNA-polymerase1. Its clinical indications are limited to uncomplicated urinary tract infections (UTIs), with a clinical susceptibility breakpoint only available forEscherichia coli2. By testing > 1,000 clinical isolates, here we demonstrate a much broader activity spectrum and species-specific bactericidal activity, including multidrug-resistant Gram-negative bacteria for which therapeutic options are limited due to resistance. By combining systematic genetic and proteomic approaches with direct measurement of intracellular metals, we dissect nitroxoline perturbation of metal homeostasis and unveil additional effects on bacterial physiology. We show that nitroxoline affects outer membrane integrity, synergizing with large-scaffold antibiotics and resensitizing colistin-resistant Enterobacteriaceaein vitroandin vivo. We further characterise resistance mechanisms acrossE. coli,Acinetobacter baumanniiandKlebsiella pneumoniae, recapitulating knownE. coliresistance determinants and uncovering novel and conserved mechanisms across species, demonstrating their common effect on nitroxoline efflux.
Wound closure after brain injury relies on force generation by microglia in zebrafish
Authors: El-Daher Francois , Drake Louisa K. , Enos Stephen J. , Wehner Daniel , Westphal Markus , Porter Nicola J. , Becker Catherina G. , Becker Thomas
Published: June 2024

Wound closure after a brain injury is critical for tissue restoration but this process is still not well characterised at the tissue level. We use live observation of wound closure in larval zebrafish after inflicting a stab wound to the brain. We demonstrate that the wound closes in the first 24 hours after injury by global tissue contraction. Microglia accumulation at the point of tissue convergence precedes wound closure and computational modelling of this process indicates that physical traction by microglia could lead to wound closure. Indeed, genetically or pharmacologically depleting microglia leads to defective tissue repair. Live observations indicate centripetal deformation of astrocytic processes contacted by migrating microglia. Severing such contacts leads to retraction of cellular processes, indicating tension. Weakening tension by disrupting the F-actin stabilising gene lcp1 in microglial cells, leads to failure of wound closure. Therefore, we propose a previously unidentified mechanism of brain repair in which microglia has an essential role in contracting spared tissue. Understanding the mechanical role of microglia will support advances in traumatic brain injury therapies

Graphical Abstract
Sampling bias corrections for accurate neural measures of redundant, unique, and synergistic information
Authors: Koçillari Loren , Lorenz Gabriel Matías , Engel Nicola Marie , Celotto Marco , Curreli Sebastiano , Blanco Malerba Simone , Engel Andreas K. , Fellin Tommaso , Panzeri Stefano
Published: June 2024

Shannon Information theory has long been a tool of choice to measure empirically how populations of neurons in the brain encode information about cognitive variables. Recently, Partial Information Decomposition (PID) has emerged as principled way to break down this information into components identifying not only the unique information carried by each neuron, but also whether relationships between neurons generate synergistic or redundant information. While it has been long recognized that Shannon information measures on neural activity suffer from a (mostly upward) limited sampling estimation bias, this issue has largely been ignored in the burgeoning field of PID analysis of neural activity. We used simulations to investigate the limited sampling bias of PID computed from discrete probabilities (suited to describe neural spiking activity). We found that PID suffers from a large bias that is uneven across components, with synergy by far the most biased. Using approximate analytical expansions, we found that the bias of synergy increases quadratically with the number of discrete responses of each neuron, whereas the bias of unique and redundant information increase only linearly or sub-linearly. Based on the understanding of the PID bias properties, we developed simple yet effective procedures that correct for the bias effectively, and that improve greatly the PID estimation with respect to current state-of-the-art procedures. We apply these PID bias correction procedures to datasets of 53117 pairs neurons in auditory cortex, posterior parietal cortex and hippocampus of mice performing cognitive tasks, deriving precise estimates and bounds of how synergy and redundancy vary across these brain regions.
Cadmium Disrupts Blood-Testis Barrier (BTB) via An Oxidative Stress-Dependent Autophagy in Prepubertal Rats
Authors: Man Yonghong , Du Bingxue , Huang Jiaolong , Sun Yu , Liu Yunhao , Zhang Ling
Published: June 2024

Blood testis barrier (BTB) is an important target of cadmium (Cd) toxicology, but the mechanism underlying the Cd-induced impairment of BTB function remains fully elucidated. The mammalian target of rapamycin (mTOR) complex 2 (mTOR2) regulates BTB function via its effects on cellular junctions and the cytoskeleton of Sertoli cells. In this study, we investigated whether mTOR2 was involved in the effects of Cd exposure on BTB integrity. A Cd exposure modelin vivowas established in prepubertal male rats using a single intraperitoneal injection of Cadmium Chloride (CdCl2). A Cd exposure model of Sertoli cell was established using a CdCl2-treated TM4 cell line. The Acute cadmium exposure decreased the activity of mTOR2 signaling and adhesin proteins which is linked to the induction of oxidative stress-induced autophagy. In the presence of CdCl2, mTOR, the catalytic subunit of the mTOR2 complex, exhibits a reduction in levels of phosphorylation, accompanied by decreased adhesin proteins and Rictor, the key component of the mTOR2 complex. CdCl2treatment also drives a process of oxidative stress-induced autophagy, evidenced by alterations in cellular markers for oxidative stress and autophagy. Pharmaceutical inhibition of oxidative stress and/or autophagy alleviates the alternations in mTOR2 signaling and adhesin proteins upon CdCl2treatment in TM4, a Sertoli cell line. This work is the first to examine the effects of cadmium exposure on rictor/mTOR2 signaling pathways. Our results suggest that Cadmium might exert testicular toxicology via the perturbation in mTOR2 signaling, which can be associated with the cellular stress-related protolysis in Sertoli cells.
Potency, dissociation kinetics and reversibility of fentanyls and nitazenes by naloxone at the μ opioid receptor
Authors: Alhosan Norah , Cavallo Damiana , Santiago Marina , Kelly Eamonn , Henderson Graeme
Published: June 2024

Background and Purpose

Fentanyls and nitazenes are μ opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we compared the potency, dissociation kinetics and antagonism by naloxone at the μ receptor of several fentanyl and nitazene analogues and compared them to morphine and DAMGO.

Experimental Approach

In vitroassays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing μ receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment.

Key Results

The rate of agonist dissociation from the μ receptor varied, with morphine, DAMGO, alfentanil and fentanyl dissociating rapidly whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow rate of dissociation was not due to G protein receptor kinase-mediated arrestin recruitment as its rate of dissociation was not affected by inhibition of GRKs with Compound 101. Thein vitrorelative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed inin vivoexperiments.

Conclusions and Implications

With fentanyls and nitazenes, that slowly dissociate from the μ opioid receptor, antagonism by naloxone is pseudo competitive. In overdoses involving fentanyls and nitazenes higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose.

What is already known

“Fentanyls” and “nitazenes” are potent agonists at the μ opioid receptor.

What does this study add

Some fentanyls and nitazenes dissociate slowly and are less sensitive to naloxone antagonism

What is the clinical significance

More naloxone may be required to reverse overdoses involving fentanyls and nitazenes
Reducing competition betweenmsdand genomic DNA significantly improved the editing efficiency of the retron editing system
Authors: Ni Yuyang , Wang Yifei , Shi Xinyu , Ruan Qingmin , Na Tian , He Jin , Wang Xun
Published: June 2024

A retron is a distinct system encoding reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). The ability of msDNA to serve as a homologous recombination donor for gene editing has attracted great interest. However, the mechanism by which msDNA expression affects editing efficiency remains unclear. In this study, we show that an increase inmsdnumber increased msDNA yield but was not necessarily accompanied by an increase in editing efficiency. Mechanistic studies indicate thatmsdand genomic regions competed for msDNA during recombination. As the number ofmsdincreased, the amount of msDNA allocated to the genomic targets decreased, resulting in a decrease in editing efficiency. Finally, we reducedmsdediting by expressing msDNA corresponding to the plasmid replication leading strand sequence, thus constructing a retron-based gene editing system that achieved 100% editing efficiency in the shortest time reported to date. The above results reveal a completely different features between retron-based gene editing system and oligonucleotide-mediated gene editing system and will provide theoretical guidance for the design and application of the retron system.
Chromosome-level genome assembly of the sacoglossan sea slugElysia timida(Risso, 1818)
Authors: Männer Lisa , Schell Tilman , Spies Julia , Galià-Camps Carles , Baranski Damian , Ben Hamadou Alexander , Gerheim Charlotte , Neveling Kornelia , Helfrich Eric J. N. , Greve Carola
Published: June 2024

Background

Sequencing and annotating genomes of non-model organisms helps to understand genome architecture, the genetic processes underlying species traits, and how these genes have evolved in closely-related taxa, among many other biological processes. However, many metazoan groups, such as the extremely diverse molluscs, are still underrepresented in the number of sequenced and annotated genomes. Although sequencing techniques have recently improved in quality and quantity, molluscs are still neglected due to difficulties in applying standardized protocols for obtaining genomic data.

Results

In this study, we present the chromosome-level genome assembly and annotation of the marine sacoglossan speciesElysia timida, known for its ability to store the chloroplasts of its food algae. In particular, by optimizing the Long-read and chromosome conformation capture library preparations, the genome assembly was performed using PacBio HiFi and Arima HiC data. The scaffold and contig N50s, at 41.8 Mb and 1.92 Mb, respectively, are 100-fold and 4-fold higher compared to other published sacoglossan genome assemblies. Structural annotation resulted in 19,904 protein-coding genes, which are more contiguous and complete compared to publicly available annotations of Sacoglossa. We detected genes encoding polyketide synthases inE. timida, indicating that polypropionates are produced. HPLC-MS/MS analysis confirmed the presence of a large number of polypropionates, including known and yet uncharacterised compounds.

Conclusions

We can show that our methodological approach helps to obtain a high-quality genome assembly even for a “difficult-to-sequence” organism, which may facilitate genome sequencing in molluscs. This will enable a better understanding of complex biological processes in molluscs, such as functional kleptoplasty in Sacoglossa, by significantly improving the quality of genome assemblies and annotations.
AARS2 ameliorates myocardial ischemiaviafine-tuning PKM2-mediated metabolism
Authors: Zhang Zongwang , Zheng Lixia , Chen Yang , Chen Yuanyuan , Hou Junjie , Xiao Chenglu , Zhu Xiaojun , Zhao Shi-Min , Xiong Jing-Wei
Published: June 2024

AARS2, an alanyl-tRNA synthase, is essential for protein translation, but its function in mouse hearts is not fully addressed. Here, we found that cardiomyocyte-specific deletion of mouse AARS2 exhibited evident cardiomyopathy with impaired cardiac function, notable cardiac fibrosis and cardiomyocyte apoptosis. Cardiomyocyte-specific AARS2 overexpression in mice improved cardiac function and reduced cardiac fibrosis after myocardial infarction (MI), without affecting cardiomyocyte proliferation and coronary angiogenesis. Mechanistically, AARS2 overexpression suppressed cardiomyocyte apoptosis and mitochondrial reactive oxide species production, and changed cellular metabolism from oxidative phosphorylation toward glycolysis in cardiomyocytes, thus leading to cardiomyocyte survival from ischemia and hypoxia stress. Ribo-Seq revealed that AARS2 overexpression increased pyruvate kinase M2 (PKM2) protein translation and the ratio of PKM2 dimers to tetramers that promote glycolysis. Additionally, PKM2 activator TEPP-46 reversed cardiomyocyte apoptosis and cardiac fibrosis caused by AARS2 deficiency. Thus, this study demonstrates that AARS2 plays an essential role in protecting cardiomyocytes from ischemic pressureviafine-tuning PKM2-mediated energy metabolism, and presents a novel cardiac protective AARS2-PKM2 signaling during the pathogenesis of MI.
The genetic architecture of floral trait divergence between hummingbird- and self-pollinated monkeyflower (Mimulus) species
Authors: Chen Hongfei , Berg Colette S. , Samuli Matthew , Sotola V. Alex , Sweigart Andrea L. , Yuan Yao-Wu , Fishman Lila
Published: June 2024

Pollination syndromes are a key component of flowering plant diversification, prompting questions about the architecture of single traits and genetic coordination among traits. Here, we investigate the genetics of extreme floral divergence between naturally hybridizing monkeyflowersMimulus parishii(self-pollinated) andM. cardinalis(hummingbird-pollinated).

We mapped quantitative trait loci (QTLs) for 18 pigment, pollinator reward/handling, and dimensional traits in parallel sets of F2hybrids plus recombinant inbred lines and generated nearly isogenic lines (NILs) for two dimensional traits, pistil length and corolla size.

Our multi-population approach revealed a highly polygenic basis (n = 190 QTLs total) for pollination syndrome divergence, capturing minor QTLs even for pigment traits with leading major loci. There was significant QTL overlap within pigment and dimensional categories. Nectar volume QTLs clustered with those for floral dimensions, suggesting a partially shared module. The NILs refined two pistil length QTLs, only one of which has tightly correlated effects on other dimensional traits.

An overall polygenic architecture of floral divergence is partially coordinated by genetic modules formed by linkage (pigments) and likely pleiotropy (dimensions plus nectar). This work illuminates pollinator syndrome evolution in a model radiation and generates a robust framework for molecular and ecological genomics.
Water-Glycan Interactions Drive the SARS-CoV-2 Spike Dynamics: Insights into Glycan-Gate Control and Camouflage Mechanisms
Authors: Blazhynska Marharyta , Lagardère Louis , Liu Chengwen , Adjoua Olivier , Ren Pengyu , Piquemal Jean-Philip
Published: June 2024

To develop therapeutic strategies against COVID-19, we introduce a high-resolution all-atom polarizable model capturing many-body effects of protein, glycans, solvent, and membrane components in SARS-CoV-2 spike protein open and closed states. Employingμs-long molecular dynamics simulations powered by high-performance cloud-computing and unsupervised density-driven adaptive sampling, we investigated the differences in bulk-solvent-glycan and protein-solvent-glycan interfaces between these states. We unraveled a sophisticated solvent-glycan polarization interaction network involving the N165/N343 residues that provide structural support for the open state and identified key water molecules that could potentially be targeted to destabilize this configuration. In the closed state, the reduced solvent polarization diminishes the overall N165/N343 dipoles, yet internal interactions and a reorganized sugar coat stabilize this state. Despite variations, our glycan-solvent accessibility analysis reveals the glycan shield capability to conserve constant interactions with the solvent, effectively camouflaging the virus from immune detection in both states. The presented insights advance our comprehension of viral pathogenesis at an atomic level, offering potential to combat COVID-19.
Extremely charged coral protein AGARP regulates calcium carbonate growth through liquid phase separation
Authors: Klepka Barbara P. , Michaś Agnieszka , Wojciechowski Tomasz , Niedźwiecka Anna
Published: June 2024

Biomineralizationviathe non-classical crystallization pathway is postulated to involve a transient liquid phase of calcium carbonate formed in the presence of polymers. In the context of coral biocalcification, these polymers may include coral acid-rich proteins (CARPs), secreted into the skeletal organic matrix. However, direct evidence for the existence of this liquid phase with proteins is lacking. Here we report the properties of the intrinsically disordered aspartic and glutamic acid-rich protein (AGARP), which is the first CARP cloned from one of the most studied scleractinian coral of the Great Barrier Reef,Acropora millepora, a model species for biomineralization and ecological studies. We show that AGARP can significantly influence early stages of CaCO3nucleation and crystal growth through liquid-liquid phase separation. Consequently, we introduce the concept of a biologically relevant crystallization precursor, a liquid protein-calcium condensate composed of CARP molecules and Ca2+ions, that forms as a result of liquid phase separation in a viscous, crowded environment. Our work bridges the gap between the liquid phase separation and biomineralization research.
Human left ventricle circRNA-miRNA-mRNA network analyses reveals a novel proangiogenic role for circNPHP1 under ischemic conditions
Authors: Anwar Maryam , Sarkar Moumita , Ford Kerrie , Angelini Gianni D , Punjabi Prakash , Laftah Abas , Chamorro-Jorganes Aránzazu , Ji Jiahui , Srivastava Prashant K , Petretto Enrico , Emanueli Costanza
Published: June 2024

Background

Ischemic heart disease (IHD) is characterized by insufficient myocardial blood flow in the left ventricle and aggravated by diabetes mellitus. Endothelial resilience and reparative angiogenesis are tightly controlled processes. Gene expression is regulated by multimodal interactions between RNA species. Circular RNAs (circRNAs) can sponge microRNAs (miRNAs) to reduce the repressive effects of miRNAs on its messenger RNA (mRNAs) targets.

Methods

Left ventricle whole RNA-sequencing (circRNAs, mRNAs) and small RNA- sequencing (miRNAs) datasets were obtained from 3 patient groups: IHD with/out T2DM and controls (N=11 to 12/group) as part of a prospective observational cardiac surgery study.

The interactions between differentially expressed (DE) circRNAs, miRNA and mRNAs were identified with a customized bioinformatics pipeline. The emerging networks were screened using endothelial-specific RNA-sequencing datasets from GEO resulting in EC-rich networks. CircRNAs from these networks were subsequently screened (RT-PCR) in endothelial cells (ECs) exposed to disease-mimicking conditions vs control. Afterwards, circRNA pulldown allowed to interrogate the circRNA-miRNAs interactome in ECs. EC biology assays using loss-of-function and gain-of-function approaches corroborated the study.

Results

We identified novel circRNA-miRNA-mRNA interactions in the human diseased heart. CircNPHP1, which is upregulated in IHD with/out Type-2 diabetes mellitus (T2DM), sponges miR-221-3p to de-repress VEGF-A and BCL2, increasing the angiogenic capacity of ECs under disease and disease-mimicking conditions.

Conclusions

The interactions between individual members of different RNA species are affected by IHD. The therapeutic value of circNPHP1/miR-221-3p axis could be further investigated.

Visual Abstract

Highlights

We found a novel circRNA-miRNA-mRNA network in IHD and Type 2 diabetes.

CircNPHP1 regulates angiogenesis and proliferation in the cardiac ECs exposed to conditions mimicking IHD and Type 2 diabetes.

We elucidated a novel pro-angiogenic subnetwork commanded by circNPHP1/miR-221-3p/BCL2/VEGFA.

We identified circNPHP1 as a potential new target for therapeutic angiogenesis.
Distinct computational mechanisms of uncertainty processing explain opposing exploratory behaviors in anxiety and apathy
Authors: Yan Xinyuan , Ebitz R. Becket , Grissom Nicola , Darrow David P. , Herman Alexander B.
Published: June 2024

Decision-making in uncertain environments often leads to varied outcomes. Understanding how individuals interpret the causes of unexpected feedback is crucial for adaptive behavior and mental well-being. Uncertainty can be broadly categorized into two components: volatility and stochasticity. Volatility is about how quickly conditions change, impacting results. Stochasticity, on the other hand, refers to outcomes affected by random chance or “luck”. Understanding these factors enables individuals to have more effective environmental analysis and strategy implementation (explore or exploit) for future decisions. This study investigates how anxiety and apathy, two prevalent affective states, influence the perceptions of uncertainty and exploratory behavior. Participants (N = 1001) completed a restless three-armed bandit task that was analyzed using latent state models. Anxious individuals perceived uncertainty as more volatile, leading toincreasedexploration and learning rates, especially after reward omission. Conversely, apathetic individuals viewed uncertainty as more stochastic, resulting indecreasedexploration and learning rates. The perceived volatility-to-stochasticity ratio mediated the anxiety-exploration relationship post-adverse outcomes. Dimensionality reduction showed exploration and uncertainty estimation to be distinct but related latent factors shaping a manifold of adaptive behavior that is modulated by anxiety and apathy. These findings reveal distinct computational mechanisms for how anxiety and apathy influence decision-making, providing a framework for understanding cognitive and affective processes in neuropsychiatric disorders.
Phage detection by a bacterial NLR-related protein is mediated by DnaJ
Authors: Conte Amy N. , Ruchel Madison E. , Ridgeway Samantha M. , Kibby Emily M. , Nagy Toni A. , Whiteley Aaron T.
Published: June 2024

Bacteria encode a wide range of antiphage systems and a subset of these proteins are homologous to components of the human innate immune system. Mammalian nucleotide-binding and leucine-rich repeat containing proteins (NLRs) and bacterial NLR-related proteins use a central NACHT domain to link detection of infection with initiation of an antimicrobial response. Bacterial NACHT proteins provide defense against both DNA and RNA phages. Here we determine the mechanism of RNA phage detection by the bacterial NLR-related protein bNACHT25 inE. coli. bNACHT25 was specifically activated byEmesvirusssRNA phages and analysis of MS2 phage escaper mutants that evaded detection revealed a critical role for Coat Protein (CP). A genetic assay confirmed CP was sufficient to activate bNACHT25 but the two proteins did not directly interact. Instead, we found bNACHT25 requires the host chaperone DnaJ to detect CP. Our data suggest that bNACHT25 detects a wide range of phages by guarding a host cell process rather than binding a specific phage-derived molecule.
Metabolic imprint of an intracellular pathogen drives histone hypermethylation and tunes the host transcriptional response to infection
Authors: Charendoff Chloé I. , Louchez Félix V. , Wu Yongzheng , Dolat Lee , Velasco Guillaume , Perrinet Stéphanie , Torres Adrian Gabriel , Blanchet Laure , Duchateau Magalie , Gianetto Quentin Giai , Matondo Mariette , Maestro Laurence Del , Ait-Si-Ali Slimane , Bonhomme Frédéric , Millot Gaël A. , Ribas de Pouplana Lluís , Martinez Elisabeth D. , Valdivia Raphael H. , Subtil Agathe
Published: June 2024

Chlamydia trachomatis, an intracellular bacterium, highjacks metabolites from the host cell. We provide evidence of global hypermethylation of the host proteome, including histones, during the late stages of infection and that histone hypermethylation is the result of metabolic imbalance favoring the activity of lysine methyl transferases over demethylases. We find that histones hypermethylated at residues H3K4 and H3K9 are distributed throughout the chromatin. Inhibition of bacterial growth, or supplementation of the culture medium with iron or with dimethyl-ketoglutarate (DMKG) reduced histone hypermethylation. DMKG supplementation modified the transcription of about one third of the infection-responsive genes, including genes involved in the innate response to infection. Transfer RNA (tRNA) levels decreased late in infection and DMKG supplementation prevented this phenomenon. Finally, we uncovered a robust, histone demethylase dependent shut-down of the innate response in the mouse genital tract shortly after the acute phase of infection. Overall, our data show that the metabolic pressure exerted by a pathogen with an intracellular lifestyle drives an epigenetic imprint that tunes the transcriptional response of its host.
A high-throughput search for intracellular factors that affect RNA folding identifiesE. coliproteins PepA and YagL as RNA chaperones that promote RNA remodeling
Authors: Rojano-Nisimura Alejandra Matsuri , Miller Lucas G. , Anantharaman Aparna , Middleton Aaron T. , Kibret Elroi , Jung Sung H. , Russell Rick , Contreras Lydia M.
Published: June 2024

General RNA chaperones are RNA-binding proteins (RBPs) that interact transiently and non-specifically with RNA substrates and assist in their folding into native state. In bacteria, these chaperones impact both coding and non-coding RNAs and are particularly important for large, structured RNAs which are prone to becoming kinetically trapped in misfolded states. Currently, due to the limited number of well-characterized examples and the lack of a consensus structural or sequence motif, it is difficult to identify general RNA chaperones in bacteria. Here, we adapted a previously publishedin vivoRNA regional accessibility probing assay to screen genome wide for intracellular factors inE. coliaffecting RNA folding, among which we aimed to uncover novel RNA chaperones. Through this method, we identified and validated eight proteins whose deletion gives changes in regional accessibility within the exogenously expressedTetrahymenagroup I intron ribozyme. Further, we purified and measuredin vitroproperties of two of these proteins, YagL and PepA, which were especially attractive as general chaperone candidates. We showed that both proteins bind RNA and that YagL accelerates native refolding of the ribozyme from a long-lived misfolded state. Further dissection of YagL showed that a putative helix-turn-helix (HTH) domain is responsible for most of its RNA binding activity but only the full protein shows chaperone activity. Altogether, this work expands the current repertoire of known general RNA chaperones in bacteria.
Phosphatases control the duration and range of cAMP/PKA microdomains
Authors: Conca Filippo , Bayburtlu Doruk Kaan , Vismara Mauro , Surdo Nicoletta C. , Tavoni Alessandra , Nogara Leonardo , Sarra Adamo , Ciciliot Stefano , Di Benedetto Giulietta , Iannucci Liliana F. , Lefkimmiatis Konstantinos
Published: June 2024

The spatiotemporal interplay between the second messenger cyclic AMP (cAMP) and its main effector, Protein Kinase A (PKA) is crucial for the pleotropic nature of this cascade. To maintain a high degree of specificity, the cAMP/PKA axis is organized into functional multiprotein complexes, called microdomains, precisely distributed within the cell. While the subcellular allocation of PKA is guaranteed by a family of tethers called A-Kinase-anchoring Proteins (AKAPs), the mechanisms underlying the efficient confinement of a microdomain’s functional effects are not fully understood. Here we used FRET-based sensors to detect cAMP levels and PKA-dependent phosphorylation within specific subcellular compartments and found that, while free cAMP is responsible for the activation of local PKA enzymes, the dephosphorylating actions of phosphatases dictate the duration of the microdomain’s effects. To test the range of action of PKA microdomains we used rigid aminoacidic nanorulers to distance our FRET sensors from their original location for 10 or 30 nm. Interestingly, we established that cAMP levels do not affect the spatial range of the microdomain while on the contrary, phosphatase activity acts as the main functional boundary for phosphorylated PKA targets. Our findings contribute to the design of a picture where two microdomain-forming events have distinct roles. Cyclic AMP elevations trigger the initial activation of subcellular PKA moieties, while the temporal and spatial extent of the PKA’s actions is regulated by phosphatases.
Conserved residues of the immunosuppressive domain of MLV are essential for regulating the fusion-critical SU-TM disulfide bond
Authors: Hogan Victoria A. , Harmon Julia , Cid-Rosas Miguel , Hall Laura R. , Johnson Welkin E.
Published: June 2024

The ENV protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type ENVs are found in retroviruses and related endogenous retroviruses (ERV) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX6CC motif. The extraordinary conservation of the ISD and its invariant association with the CX6CC suggests a fundamental contribution to Env function. To investigate function of the ISD, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the mutants, while TM/CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion and entry. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild type MLV Env. These results reveal a role for residues at MLV ISD positions 2, 3, 4, 7, and 10 in regulating a late step in fusion, and suggest that the ISD is part of a larger domain, encompassing both the ISD and CX6CC motifs, that is critical for formation and regulation of the metastable, intersubunit disulfide bond.

IMPORTANCE

The gamma-type Env is an extremely prevalent viral fusogen, extensively found within retroviruses and endogenous loci across vertebrate species and are further found in filoviruses such as Ebola virus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of IAV and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of fusion with the host membrane. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs.
A mouse model of TB-associated lung fibrosis reveals persistent inflammatory macrophage populations during treatment
Authors: Boucau Julie , Naidoo Threnesan , Liu Yuming , Dasgupta Shatavisha , Jain Neha , Castillo Jennie Ruelas , Jacobson Nicholas E. , Nargan Kievershen , Cimini Beth A. , Eliceiri Kevin W. , Steyn Adrie J.C. , Barczak Amy K.
Published: June 2024

Post-TB lung disease (PTLD) causes a significant burden of global disease. Fibrosis is a central component of many clinical features of PTLD. To date, we have a limited understanding of the mechanisms of TB-associated fibrosis and how these mechanisms are similar to or dissimilar from other fibrotic lung pathologies. We have adapted a mouse model of TB infection to facilitate the mechanistic study of TB-associated lung fibrosis. We find that the morphologies of fibrosis that develop in the mouse model are similar to the morphologies of fibrosis observed in human tissue samples. Using Second Harmonic Generation (SHG) microscopy, we are able to quantify a major component of fibrosis, fibrillar collagen, over time and with treatment. Inflammatory macrophage subpopulations persist during treatment; matrix remodeling enzymes and inflammatory gene signatures remain elevated. Our mouse model suggests that there is a therapeutic window during which adjunctive therapies could change matrix remodeling or inflammatory drivers of tissue pathology to improve functional outcomes after treatment for TB infection.
Emergence of Slc11 clade MCbgut: a parsimonious hypothesis for the dawn of Lactobacillales in the gut of early vertebrates
Authors: Cellier M. FM
Published: June 2024

The Lactobacillales (LB) stand apart among bacterial orders, using manganese (Mn) instead of iron to support their growth and swiftly ferment complex foods while acidifying their environment. The present work investigates whether a shift in the use of Mn could mark the origin of LB. Transmembrane carriers of the ubiquitous Slc11 family play key roles in LB physiology by catalyzing proton-dependent Mn import. In prior studies, the Slc11 clade found in LB (MntH Cb, MCb) showed both remarkable structural plasticity and highly efficient Mn uptake, and another Slc11 clade, MCg1, demonstrated divergent evolution coinciding with emergence of bacterial genera (e.g.,Bordetella,Achromobacter). Herein, Slc11 clade MCb is subdivided in sister groups: MCbieand MCbgut. MCbiederives directly from Slc11 clade MCa, pointing an intermediate stage in the evolution of MCbgut. MCbiepredominates in marine Bacillaceae, is more conserved than MCbgut, lacks the structural plasticity that typify MCbgutcarriers, and responds differently to identical mutagenesis. Exchanging MCbie/MCbgutamino acid residues at sites that distinguish these clades showed conformation-dependent effects with both MCbieand MCbguttemplates and the 3D location of the targeted sites in the carrier structure together suggest the mechanism to open the inner gate, and release Mn into the cytoplasm, differs between MCbieand MCbgut. Building on the established phylogeny forEnterococcusrevealed that a pair of genes encoding MCbgutwas present in the common ancestor of LB, as MCbgu1and MCbgu2templates exhibit distinct structural dynamics properties. These data are discussed examining whether MCb+LB could emerge in the upper gut of early vertebrates (ca. 540 mya), through genome contraction and evolution toward Mn-centrism, as they specialized as gastric aids favoring stomach establishment in jawed vertebrates through bi-directional communication with host nervous, endocrine and immune systems.
High-affinity PQQ import is widespread in Gram-negative bacteria
Authors: Munder Fabian , Voutsinos Marcos , Hantke Klaus , Venugopal Hari , Grinter Rhys
Published: June 2024

PQQ is a soluble redox cofactor used by diverse bacteria to oxidise fuel compounds as a source of electrons for the respiratory chain. Many Gram-negative bacteria that encode PQQ-dependent enzymes do not possess the biosynthetic machinery for its production and instead obtain it from the environment. To achieve this the bacteriumEscherichia coliuses the TonB-dependent transporter PqqU as a high-affinity PQQ importer, allowing it to use PQQ at an external concentration as low as 1 nM. Here, we show that PqqU achieves this by binding PQQ with a very high affinity. Using cryo-electron microscopy we determine the structure of the PqqU-PQQ complex at a resolution of 1.99 Å, revealing that the extracellular loops of PqqU undergo significant conformational changes upon PQQ binding, which captures the cofactor in an internal cavity. This cavity likely facilitates an airlock-style gating mechanism that prevents non-specific import through PqqU. Using structural modelling we show that the change in PqqU structure upon PQQ binding precludes the binding of bacteriophage, which targets it as a cell surface receptor. Guided by the PqqU-PQQ complex structure we use phenotypic analysis to identify the amino acids essential for PQQ import and leverage this information to map the presence of PqqU across Gram-negative bacteria. This reveals that PqqU is encoded by Gram-negative bacteria from at least 22 phyla from diverse habitats, including those found in aquatic, soil, host-associated, and extreme environments. This indicates that PQQ is a ubiquitous nutrient in many environments, and an important cofactor for bacteria that adopt diverse lifestyles and metabolic strategies.

Significance Statement

Many enzymes form complexes with molecules called cofactors to perform their function. PQQ is a cofactor used by bacterial enzymes that provide energy by breaking down food molecules. While some bacteria make their own PQQ, other bacteria use the transport protein PqqU to bind PQQ from the environment and import it into their cells. We show that PqqU binds PQQ very tightly, allowing bacteria to acquire it at very low concentrations. Using cryo-electron microscopy we image the PqqU-PQQ complex on an atomic level, revealing how PQQ is bound so tightly. Using this the information to analyse microbial genomes, we show that PQQ scavenging is employed by diverse bacteria, implying that PQQ is an important common good of diverse microbiomes.
Multiplexed optical barcoding and sequencing for spatial omics
Authors: Venkatramani Aditya , Ciftci Didar , Cohen Limor , Li Christopher , Pham Khanh , Zhuang Xiaowei
Published: June 2024

Spatial omics has brought a fundamental change in the way that we study cell and tissue biology in health and disease. Among various spatial omics methods, genome-scale imaging allows transcriptomic, 3D-genomic, and epigenomic profiling of individual cells with high spatial (subcellular) resolution but often requires a preselection of targeted genes or genomic loci. On the other hand, spatially dependent barcoding of molecules followed by sequencing provides untargeted, genome-wide profiling but typically lacks single-cell resolution. Here, we report a spatial omics method that could potentially combine the power of the two approaches by optically controlled spatial barcoding followed by sequencing. Specifically, we utilize patterned light to encode the locations of cells in tissues using oligonucleotide-based barcodes and then identify the barcoded molecular content, such as mRNAs, by sequencing. This optical barcoding method is designed with multiplexing and error-correction capacity and achieved by a light-directed ligation chemistry that attaches distinct nucleic-acid sequences to the reverse transcribed cDNA products at different locations. As a proof of principle for this method, we demonstrated high-efficiency in situ light-directed ligation, spatially dependent barcoding with multiplexed light-controlled ligations, and high-accuracy detection of spatially barcoded mRNAs in cells.
How many vector charmoniumlike states lie in the mass range 4.2–4.35 GeV?
Authors: von Detten Leon , Baru Vadim , Hanhart Christoph , Wang Qian , Winney Daniel , Zhao Qiang
Published: June 2024

In recent years many vector charmonium(like) states were reported by different electron-positron collider experiments above 4.2 GeV. However, so far, there not only exists sizable tension in the parameters of those states, but there is also no consensus on the number of the vector states in this energy range. To some extend, this might be caused by the fact that the experimental data were typically analyzed in single channel analyses employing overlapping Breit-Wigner functions, in particular ignoring the effect of opening thresholds. In this study, we focus on the mass range between 4.2 GeV and 4.35 GeV, conducting a comprehensive analysis of eight different final states in e+e− annihilation. Our findings demonstrate that, within this mass range, a single vector charmoniumlike state, exhibiting properties consistent with a D1D¯ molecular structure and characterized by a pole location spoleY(4230)=(4227±4−i2(50−2+8))  MeV, can effectively describe all the collected data. This is made possible by allowing for an interference with the well-established vector charmonium ψ(4160) along with the inclusion of the D1D¯ threshold effect. Moreover, in contrast to experimental analyses, our study reveals that the highly asymmetric total cross sections for e+e−→J/ψππ and e+e−→J/ψKK¯ around 4230 MeV stem from the same physics, rooted in the approximate SU(3) flavor symmetry of QCD.

Published by the American Physical Society
2024
Primordial black holes and wormholes from domain wall networks
Authors: Gouttenoire Yann , Vitagliano Edoardo
Published: June 2024

Domain walls (DWs) are topological defects originating from phase transitions in the early Universe. In the presence of an energy imbalance between distinct vacua, enclosed DW cavities shrink until the entire network disappears. By studying the dynamics of thin-shell bubbles in general relativity, we demonstrate that closed DWs with sizes exceeding the cosmic horizon tend to annihilate later than the average. This delayed annihilation allows for the formation of large overdensities, which, upon entering the Hubble horizon, eventually collapse to form primordial black holes (PBHs). We rely on 3D percolation theory to calculate the number density of these late-annihilating DWs, enabling us to infer the abundance of PBHs. A key insight from our study is that DW networks with the potential to emit observable gravitational waves are also likely to yield detectable PBHs. Additionally, we study the production of wormholes connected to baby universes and conclude on the possibility to generate a multiverse.

Published by the American Physical Society
2024
Sustainability and development of EWE communities in Ghana through indigenous knowledge management practices
Authors: Dei De-Graft Johnson
Published: June 2024

Purpose

Losing indigenous knowledge is to lose the future and impoverish the societies because indigenous knowledge is considered one of the cornerstones and survival of communities, societies and economies. This study, thus, aims to explore and assess the contextual enablers of indigenous knowledge and their role in developing and sustaining the Ewe communities in Ghana.

Design/methodology/approach

The qualitative research design, which enabled the researcher to engage the participants in an interview process and observation, was used in this study. It was augmented with a quantitative design using structured questions. Data analysis was guided by the basic principles of grounded theory. The coding system was aided by NVivo to analyze the qualitative data, while the quantitative data was analyzed using the SPSS. Descriptive analysis and graphs were deployed in the presentation of the findings.

Findings

The study discovered that the communities are in possession of several types of indigenous knowledge, ranging from tacit to explicit knowledge, which are embedded in their cultural and traditional systems and unique to every community; the culture of trust among the citizens and motivation to create and share knowledge in communities was high. Same time, family heads, farmer groups, libraries and museums played key roles in the preservation and management of indigenous knowledge in the communities. On the other hand, information officers and institutions like churches and police posts played insignificant roles in the management and preservation of knowledge in the communities just as the absence of a culture of willingness by the community members to create and share knowledge. Additionally, there was an absence of technological infrastructure, platforms, databases and policies to aid the management and preservation of knowledge in the communities. The study concludes that indigenous knowledge plays a vital role in the development and sustainability of communities in Ghana.

Practical implications

Community leaders and political leaders need to invest in systems and structures that will promote the management and preservation of indigenous knowledge for the development of the communities and the state as a whole.

Originality/value

The study demonstrates its originality in terms of scope, setting, population and empirical evidence by focusing on the role of indigenous knowledge in the sustainability and development of Ewe communities in Ghana.