bluuebunny/crossref_metadata_2025 huggingface dataset:
Design, synthesis, and characterization of [18F]mG2P026 as a high contrast PET imaging ligand for metabotropic glutamate receptor 2
Authors: Yuan Gengyang , Dhaynaut Maeva , Guehl Nicolas J. , Afshar Sepideh , Huynh Dalena , Moon Sung-Hyun , Iyengar Suhasini , Kang Hye Jin , Ondrechen Mary Jo , Fakhri Georges El , Normandin Marc D. , Brownell Anna-Liisa
Published: July 2021

An array of triazolopyridines based on JNJ-46356479 (6) were synthesized as potential PET imaging ligands for metabotropic glutamate receptor 2 (mGluR2) in the brain. The selected candidates 8-11 featured an enhanced positive allosteric modulator (PAM) activity (37-fold max.) and an apparent mGluR2 agonist activity (25-fold max.) compared to compound 6. Radiolabeling of compounds 8 and 9 (also named mG2P026) was achieved via the Cu(I)-mediated radiofluorination in the automated TRACERLabTM FXF-N platform. Both [18F]8 and [18F]9 were obtained with satisfactory radiochemical yields (> 5%, non-decay corrected), high molar activity (> 180 GBq/μmol), and excellent chemical and radiochemical purities (> 98%). Preliminary characterization of [18F]8 and [18F]9 in rats confirmed their excellent brain permeability with [18F]9 showing better brain heterogeneity and favorable binding kinetics. Pretreatment with different classes of PAMs enhanced the radioactivity uptake for both [18F]8 and [18F]9 at the regions of interest by 20.3-40.9% and 16.7-81.6%, respectively, due to their pharmacological effects. Further evaluation of [18F]9 in a nonhuman primate confirmed its superior brain heterogeneity in mapping mGlu2 receptors and its higher specific binding than [18F]6. Pretreatment with 0.5 mg/kg BINA led (2) to an enhanced brain uptake of [18F]9 by 3% in high tracer uptake regions that was consistent with the rat studies. Therefore, [18F]9 has the potential to be translated for human studies.
Unrevealing water and carbon relations during and after heat and hot drought stress in Pinus sylvestris
Authors: Rehschuh Romy , Ruehr Nadine K.
Published: July 2021

Forests are increasingly affected by heatwaves, often co-occurring with drought, with consequences for water and carbon (C) cycling. However, our ability to project the resilience of trees to an intensification of hot droughts remains limited. Here, we used single tree cuvettes (n=18) allowing us to investigate transpiration (E), net assimilation (Anet), root respiration (Rroot) and stem diameter change in Scots pine seedlings during gradually intensifying heat or drought-heat stress (max. 42°C), and post-stress. Further, we assessed indicators of stress impacts and recovery capacities.

Under heat stress, well-watered seedlings prevented overheating of leaves effectively via increased E, while under drought-heat leaf temperatures increased to 46°C. However, leaf electrolyte leakage was negligible, but F’v/F’m declined alongside Anet moderately in heat but strongly in drought-heat seedlings, in which respiration exceeded C uptake. Further, the decrease of needle water potential (ψNeedle) to −2.7 MPa and relative needle water content (RWCNeedle) under drought-heat reflected a decline of leaf hydraulic conductance (KLeaf) by 90% and stem hydraulic conductivity (KS) by 25%. Alongside, we observed pronounced stem diameter shrinkage.

Heat stress alone resulted in low functional impairment and all measured parameters recovered fast. In contrast, larger impacts following combined heat and drought led to the incomplete recovery of KLeaf and KS. Despite Anet tended to be reduced albeit F’v/F’m had recovered, the seedlings’ net C balance reached control values 2 d after stress release and stem growth rates exceeded control rates in the 2nd week post-stress. This indicates that a new equilibrium of C uptake and release was maintained at the tree level, slowly supporting regaining of stress-induced losses.

In summary, we highlight that under moderate heatwaves with low functional impairment, recovery is fast in Scots pine, while in combination with drought hydraulic and thermal stress are intensified, resulting in functional damage and delayed recovery processes. The incomplete recovery of hydraulic conductance indicates limited water transport capacities that could become critical under repeated heat events.
TACSTD2 upregulation is an early reaction to lung infection
Authors: Lenárt Sára , Lenárt Peter , Knopfová Lucia , Kotasová Hana , Pelková Vendula , Sedláková Veronika , Čan Vladimír , Šmarda Jan , Souček Karel , Hampl Aleš , Beneš Petr
Published: July 2021

TACSTD2encodes a transmembrane glycoprotein Trop2 commonly overexpressed in carcinomas. While the Trop2 protein was discovered already in 1981 and first antibody-drug conjugate targeting Trop2 were recently approved cancer therapy, the physiological role of Trop2 is still not fully understood. In this article, we show that TACSTD2/Trop2 expression is evolutionarily conserved in lungs of various vertebrates. By analysis of publicly available transcriptomic data we demonstrate thatTACSTD2level consistently increases in lungs infected with miscellaneous pathogens. Single cell and subpopulation based transcriptomic data revealed that the major source ofTACSTD2transcript are lung epithelial cells and their progenitors and thatTACSTD2is induced directly in lung epithelial cells following infection. This increase may represent a mechanism to maintain/restore epithelial barrier function and contribute to regeneration process in infected/damaged lungs.
The Role of Action Potential Waveform in Failure of Excitation Contraction Coupling
Authors: Wang Xueyong , Nawaz Murad , Burke Steve R.A. , Bannister Roger , Foy Brent D. , Voss Andrew A. , Rich Mark M.
Published: July 2021

Excitation contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, ICU acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a range of several mV of resting potential. While some studies have hypothesized the sudden failure of ECC is due to all-or-none failure of excitation, other studies suggest failure of excitation is graded. Intracellular recordings of action potentials (APs) in individual fibers during depolarization revealed that APs do not fail in an all-or-none manner. Simultaneous imaging of Ca2+transients during depolarization revealed failure over a narrow range of resting potentials. An AP property that closely correlated with the sudden failure of the Ca2+transient was the integral of AP voltage with respect to time. We hypothesize the close correlation is due to the combined dependence on time and voltage of Ca2+release from the sarcoplasmic reticulum. The quantitative relationships established between resting potential, APs and Ca2+transients provide the foundation for future studies of depolarization-induced failure of ECC in diseases such as periodic paralysis.
Extent, impact, and mitigation of batch effects in tumor biomarker studies using tissue microarrays
Authors: Stopsack Konrad H. , Tyekucheva Svitlana , Wang Molin , Gerke Travis A. , Vaselkiv J. Bailey , Penney Kathryn L. , Kantoff Philip W. , Finn Stephen P. , Fiorentino Michelangelo , Loda Massimo , Lotan Tamara L. , Parmigiani Giovanni , Mucci Lorelei A.
Published: July 2021

Tissue microarrays (TMAs) have been used in thousands of cancer biomarker studies. To what extent batch effects, measurement error in biomarker levels between slides, affects TMA-based studies has not been assessed systematically. We evaluated 20 protein biomarkers on 14 TMAs with prospectively collected tumor tissue from 1,448 primary prostate cancers. In half of the biomarkers, more than 10% of biomarker variance was attributable to between-TMA differences (range, 1–48%). We implemented different methods to mitigate batch effects (R package batchtma), tested in plasmode simulation. Biomarker levels were more similar between mitigation approaches compared to uncorrected values. For some biomarkers, associations with clinical features changed substantially after addressing batch effects. Batch effects and resulting bias are not an error of an individual study but an inherent feature of TMA-based protein biomarker studies. They always need to be considered during study design and addressed analytically in studies using more than one TMA.
Mapping of the fibrinogen-binding site on the staphylocoagulase C-terminal repeat region
Authors: Maddur Ashoka A. , Voehler Markus , Panizzi Peter , Meiler Jens , Bock Paul E. , Verhamme Ingrid M.
Published: July 2021

The N-terminus ofS. aureusstaphylocoagulase (SC) triggers activation of host prothrombin (ProT), and the SC·ProT* complex cleaves host fibrinogen (Fbg) to form fibrin (Fbn) deposits, a hallmark of SC-positive endocarditis. The C-terminal domain of the prototypical Newman D2 Tager 104 SC contains 1 pseudo-repeat (PR) and 7 repeats (R1→R7) that bind Fbg/Fbn Fragment D (Frag D). This work defines affinities and stoichiometries of Frag D binding to single- and multi-repeat C-terminal constructs, using fluorescence equilibrium binding, NMR titration, Ala scanning, and native PAGE. Constructs containing PR and each single repeat bound Frag D withKD~50 - 130 nM and a 1:1 stoichiometry, indicating a conserved binding site shared between PR and each repeat. NMR titration of PR-R7 with Frag D revealed that residues 22-49, bridging PR and R7, constituted the minimal peptide (MP) required for binding, corroborated by Ala scanning, and binding of labeled MP to Frag D. MP alignment with the PR-repeat and inter-repeat junctions identified conserved residues critical for binding. Labeled PR-(R1→R7) bound Frag D withKD~7 - 32 nM and stoichiometry of 1:5; and PR-R1R2R3, PR-R1R6R7, PR-R3R4R7, and PR-R3R6R7 competed with PR-(R1→R7) for Frag D binding, with a 1:3 stoichiometry andKD~7 - 42 nM. These findings are consistent with binding at the PR-R junctions with modest inter-repeat sequence variability. Circular dichroism of PR-R7 and PR-(R1→R7) suggested a largely disordered structure and conformational flexibility, allowing binding of multiple fibrin(ogen) molecules. This property facilitates pathogen localization on host fibrin networks.
Proper control of R-loop homeostasis is required for maintenance of gene expression and neuronal function during aging
Authors: Jauregui-Lozano Juan , Escobedo Spencer , Easton Alyssa , Lanman Nadia A. , Weake Vikki M. , Hall Hana
Published: July 2021

Age-related loss of cellular function and increased cell death are characteristic hallmarks of aging. While defects in gene expression and RNA metabolism have been linked with age-associated human neuropathies, it is not clear how the changes that occur in aging neurons contribute to loss of gene expression homeostasis. R-loops are RNA-DNA hybrids that typically form co-transcriptionally via annealing of the nascent RNA to the template DNA strand, displacing the non-template DNA strand. Dysregulation of R-loop homeostasis has been associated with both transcriptional impairment and genome instability. Importantly, a growing body of evidence links R-loop accumulation with cellular dysfunction, increased cell death and chronic disease onset. Here, we characterized the R-loop landscape in agingDrosophila melanogasterphotoreceptor neurons and showed that bulk R-loop levels increased with age. Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R-loops over gene bodies during aging, which correlated with decreased expression of long and highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, overexpression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging. Together, our studies highlight the functional link between dysregulation of R-loop homeostasis, gene expression and visual function during aging.
PET imaging studies to investigate functional expression of mGluR2 using [11C]mG2P001
Authors: Yuan Gengyang , Dhaynaut Maeva , Guehl Nicolas J. , Neelamegam Ramesh , Moon Sung-Hyun , Qu Xiying , Poutiainen Pekka , Afshar Sepideh , Fakhri Georges El , Brownell Anna-Liisa , Normandin Marc D.
Published: July 2021

Metabotropic glutamate receptor 2 (mGluR2) has been extensively studied for the treatment of various neurological and psychiatric disorders. Understanding of the mGluR2 function is pivotal in supporting the drug discovery targeting mGluR2. Herein, the positive allosteric modulation of mGluR2 was investigated via the in vivo positron emission tomography (PET) imaging using 2-((4-(2-[11C]methoxy-4-(trifluoromethyl)phenyl)piperidin-1-yl)methyl)-1-methyl-1H-imidazo[4,5-b]pyridine ([11C]mG2P001).Distinct from the orthosteric compounds, pretreatment with the unlabeled mG2P001, a potent mGluR2 positive allosteric modulator (PAM), resulted in a significant increase instead of decrease of the [11C]mG2P001 accumulation in rat brain detected by PET imaging. Subsequent in vitro studies with [3H]mG2P001 revealed the cooperative binding mechanism of mG2P001 with glutamate and its pharmacological effect that contributed to the enhanced binding of [3H]mG2P001 in transfected CHO cells expressing mGluR2. The in vivo PET imaging and quantitative analysis of [11C]mG2P001 in non-human primates (NHPs) further validated the characteristics of [11C]mG2P001 as an imaging ligand for mGluR2. Self-blocking studies in primates enhanced accumulation of [11C]mG2P001 dose- and delivery-dependently. Altogether, these studies show that [11C]mG2P001 is a sensitive biomarker for mGluR2 expression and the binding is affected by the tissue glutamate concentration.
All-optical interrogation of neural circuits in behaving mice
Authors: Russell Lloyd E. , Dalgleish Henry W. P. , Nutbrown Rebecca , Gauld Oliver M. , Herrmann Dustin , Fişek Mehmet , Packer Adam M. , Häusser Michael
Published: July 2021

Recent advances combining two-photon calcium imaging and two-photon optogenetics with digital holography now allow us to read and write neural activity in vivo at cellular resolution with millisecond temporal precision. Such “all-optical” techniques enable experimenters to probe the impact of functionally defined neurons on neural circuit function and behavioural output with new levels of precision. This protocol describes the experimental strategy and workflow for successful completion of typical all-optical interrogation experiments in awake, behaving head-fixed mice. We describe modular procedures for the setup and calibration of an all-optical system, the preparation of an indicator and opsin-expressing and task-performing animal, the characterization of functional and photostimulation responses and the design and implementation of an all-optical experiment. We discuss optimizations for efficiently selecting and targeting neuronal ensembles for photostimulation sequences, as well as generating photostimulation response maps from the imaging data that can be used to examine the impact of photostimulation on the local circuit. We demonstrate the utility of this strategy using all-optical experiments in three different brain areas – barrel cortex, visual cortex and hippocampus – using different experimental setups. This approach can in principle be adapted to any brain area for all-optical interrogation experiments to probe functional connectivity in neural circuits and for investigating the relationship between neural circuit activity and behaviour.
Prenatal alcohol exposure disrupts hippocampal sharp-wave ripple-associated spike dynamics
Authors: Harvey Ryan E. , Berkowitz Laura E. , Savage Daniel D. , Clark Benjamin J.
Published: July 2021

Prenatal alcohol exposure (PAE) is among the most common developmental insults to the nervous system and is characterized by memory disruption. There is a pressing need to identify physiological alterations that help explain this memory impairment. Hippocampal sharp-wave ripples (SPW-Rs) are a compelling candidate for this purpose as they are the electrophysiological signatures of memory consolidation. We report that rats exposed to moderate prenatal alcohol display abnormalities restricted to SPW-R episodes that manifest as decreased recruitment of CA1 pyramidal cells and interneurons to SPW-R events, altered excitation during SPW-Rs, and decreased cell assembly activation rate. These differences observed at the single neuron and the population level may limit the ability of memory trace reactivation during SPW-Rs through the disruption of the intrinsic structure of cell sequences. Together, our results suggest that alterations in hippocampal SPW-R spike dynamics may underlie alcohol exposure-related memory deficits.
Capturing hidden regulation based on noise change of gene expression level from single cell RNA-seq in yeast
Authors: Itoh Thoma , Makino Takashi
Published: July 2021

Recent progress in high throughput single cell RNA-seq (scRNA-seq) has activated the development of data-driven inferring methods of gene regulatory networks. Most network estimations assume that perturbations produce downstream effects. However, the effects of gene perturbations are sometimes compensated by a gene with redundant functionality (functional compensation). In order to avoid functional compensation, previous studies constructed double gene deletions, but its vast nature of gene combinations was not suitable for comprehensive network estimation. We hypothesized that functional compensation may emerge as a noise change without mean change (noise-only change) due to varying physical properties and strong compensation effects. Here, we show compensated interactions, which are not detected by mean change, are captured by noise-only change quantified from scRNA-seq. We investigated whether noise-only change genes caused by a single deletion of STP1 and STP2, which have strong functional compensation, are enriched in redundantly regulated genes. As a result, noise-only change genes are enriched in their redundantly regulated genes. Furthermore, novel downstream genes detected from noise change are enriched in “transport”, which is related to known downstream genes. Herein, we suggest the noise difference comparison has the potential to be applied as a new strategy for network estimation that capture even compensated interaction.
Epigenetic regulation of frog innate immunity: Discovery of frog microRNAs associated with antiviral responses and ranavirus infection using a Xenopus laevis skin epithelial-like cell line
Authors: Todd Lauren A. , Bui-Marinos Maxwell P. , Katzenback Barbara A.
Published: July 2021

Epigenetic regulators such as microRNAs are emerging as conserved regulators of innate antiviral immunity in vertebrates, yet their roles in amphibian antiviral responses remain uncharacterized. We profiled changes in microRNA expressions in the Xenopus laevis skin epithelial-like cell line Xela DS2 in response to poly(I:C) – an analogue of double-stranded viral RNA and inducer of type I interferons – or frog virus 3 (FV3), an immunoevasive virus associated with amphibian mortality events. We sequenced small RNA libraries generated from untreated, poly(I:C)-treated, and FV3-infected cells. We detected 136 known X. laevis microRNAs and discovered 133 novel X. laevis microRNAs. Sixty-five microRNAs were differentially expressed in response to poly(I:C), many of which were predicted to target regulators of antiviral pathways such as cGAS-STING, RIG-I/MDA-5, TLR signaling, and type I interferon signaling, as well as products of these pathways (NF-κB-induced and interferon-stimulated genes). In contrast, only 49 microRNAs were altered by FV3 infection, fewer of which were predicted to interact with antiviral pathways. Interestingly, poly(I:C) treatment or FV3 infection downregulated transcripts encoding factors of the host microRNA biogenesis pathway. Our study is the first to suggest that host microRNAs regulate innate antiviral immunity in frogs, and sheds light on microRNA-mediated mechanisms of immunoevasion by FV3.
Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme
Authors: Lewis Morag A. , Ingham Neil J. , Chen Jing , Pearson Selina , Di Domenico Francesca , Rekhi Sohinder , Allen Rochelle , Drake Matthew , Willaert Annelore , Rook Victoria , Pass Johanna , Keane Thomas , Adams David , Tucker Abigail S. , White Jacqueline K. , Steel Karen P.
Published: July 2021

Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.
Genomic Modules and Intramodular Network Convergency of Susceptibility and Resilience in Multimodeled Stress in Male Mice
Authors: Marrocco Jordan , Caradonna Salvatore G. , Zhang Tie-Yuan , O’Toole Nicholas , Shen Mo-Jun , Khalil Huzefa , Einhorn Nathan R. , Wen Xianglan , Parent Carine , Lee Francis S. , Akil Huda , McEwen Bruce S. , Meaney Michael J.
Published: July 2021

The multifactorial etiology of stress-related disorders is a challenge in developing synchronized medical standards for treatment and diagnosis. It is largely unknown whether there exists molecular convergence in preclinical models of stress generated using disparate construct validity. Using RNA-sequencing (RNA-seq), we investigated the genomic signatures in the ventral hippocampus, which mostly regulates affective behavior, in mouse models that recapitulate the hallmarks of anxiety and depression. Chronic oral corticosterone (CORT), a model that causes a blunted endocrine response to stress, induced anxiety- and depression-like behavior in wildtype mice and mice heterozygous for the gene coding for brain-derived neurotrophic factor (BDNF) Val66Met, a variant associated with genetic susceptibility to stress. In a separate set of mice, chronic social defeat stress led to a susceptible or a resilient population, whose proportion was dependent on housing conditions, standard housing or enriched environment. A rank-rank-hypergeometric (RRHO) analysis of the RNA-seq data set across models demonstrated that in mice treated with CORT and susceptible mice raised in standard housing differentially expressed genes (DEGs) converged toward gene networks involved in similar biological functions. Weighted gene co-expression analysis generated 54 unique modules of interconnected gene hubs, two of which included a combination of all experimental groups and were significantly enriched in DEGs, whose function was consistent with that predicted in the RRHO GO analysis. This multimodel approach showed transcriptional synchrony between models of stress with hormonal, environmental or genetic construct validity shedding light on common genomic drivers that embody the multifaceted nature of stress-related disorders.
Pathogens, Virulence and Resistance Genes Surveillance with Metagenomics Can Pre-empt Dissemination and Escalation of Untreatable Infections: A Systematic Review and Meta-analyses
Authors: Sekyere John Osei , Faife Sara Lino
Published: July 2021

Background

The dissemination of pathogens carrying genetic elements such as antimicrobial resistance genes (ARGs), mobile-genetic elements (MGEs), virulome and methylome have a negative impact on food and environment safety, water quality and animal and human health. The applications of metagenomics to monitor and identify the prevalence/endemicity and emergence of these pathogenic agents from different sources were examined.

Methods

Articles published in English language up to October 2020 were searched for on PubMed. Qualitative and quantitative data extracted from the included articles were translated into charts and maps. GraphPad Prism 9.0.2 was used to undertake statistical analysis using descriptive and column statistics, Chi-square, ANOVA, Wilcoxon’s signed-rank, and one-sample t-test.

Results

In all, 143 articles from 39 countries from Europe, America, Asia, and Africa were quantitatively analysed. Metagenomes from sewage/wastewater, surface water samples (ocean, sea, river lake, stream and tap water), WWTP, effluents and sludge samples contained pathogenic bacteria (Aeromonas, Acinetobacter, Pseudomonas, Streptococcus, Bacteroides,Escherichia coli,Salmonella enterica,Klebsiella pneumoniaeandAcinetobacter baumannii), viruses (Adenovirus, Enterovirus, Hepatovirus, Mamastrovirus and Rotavirus) and parasites (Acanthamoeba, Giardia, Entamoeba, Blastocystis and Naegleria). Integrons, plasmids, transposons, insertion sequences, prophages and integrative and conjugative elements were identified. ARGs mediating resistance to important antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and tetracycline, and virulence factors such as secretion system, adherence, antiphagocytosis, capsule, invasion, iron uptake, hemolysin, and flagella.

Conclusion

Clinically important pathogens, ARGs, and MGEs were identified in diverse clinical, environmental, and animal sources through metagenomics, which can be used to determine the prevalence and emergence of known and unknown pathogens and ARGs.

Importance/significance

Global metagenomic analyses of drinking water, effluents, influents, un/-treated sewage, WWTPs, sludge, rivers, lakes, soil, sediments, biosolid, air and plants. showed the global distribution of diverse clinically important ARGs on mobile genetic elements, antimicrobial-resistant bacteria (ARB) and pathogens, metal resistance genes, and virulence genes in almost all environments. These depict the importance of shot-gun metagenomics as a surveillance tool for AMR and infectious disease control to safeguard water & food quality as well as public health from water- and food-borne outbreaks of pathogenic and ARB infections. More concerning was the identification of ARGs to last-resort antibiotics i.e., carbapenems, colistin, & tigecycline.
Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice
Authors: Belouzard Sandrine , Machelart Arnaud , Sencio Valentin , Vausselin Thibaut , Hoffmann Eik , Deboosere Nathalie , Rouillé Yves , Desmarets Lowiese , Séron Karin , Danneels Adeline , Robil Cyril , Belloy Loïc , Moreau Camille , Piveteau Catherine , Biela Alexandre , Vandeputte Alexandre , Heumel Séverine , Deruyter Lucie , Dumont Julie , Leroux Florence , Engelmann Ilka , Alidjinou Enagnon Kazali , Hober Didier , Brodin Priscille , Beghyn Terence , Trottein François , Déprez Benoît , Dubuisson Jean
Published: July 2021

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Antivirals targeting SARS-CoV-2 are sorely needed. In this study, we screened a library of drug compounds and identified clofoctol as an antiviral against SARS-CoV-2. We further demonstrated that, in vivo, this compound reduces inflammatory gene expression and lowers pulmonary pathology.
TAILORED DC INDUCE PROTECTIVE HIV-1 SPECIFIC POLYFUNCTIONAL CD8+ T CELLS IN THE LYMPHOID TISSUE FROM HUMANIZED BLT MICE
Authors: Calvet-Mirabent Marta , Claiborne Daniel T. , Deruaz Maud , Tanno Serah , Serra Carla , Delgado-Arévalo Cristina , Sánchez-Cerrillo Ildefonso , de los Santos Ignacio , Sanz Jesús , García-Fraile Lucio , Sánchez-Madrid Francisco , Alfranca Arantzazu , Muñoz-Fernández María Ángeles , Allen Todd M. , Buzón Maria J. , Balazs Alejandro , Vrbanac Vladimir , Martín-Gayo Enrique
Published: July 2021

Effective function of CD8+ T cells and enhanced innate activation of dendritic cells (DC) in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of TBK1-primed DC inducing protective CD8+ T cell responses in lymphoid tissue and peripheral blood and their association with reduced HIV-1 disease progression in vivo in the humanized bone marrow, liver and thymus (hBLT) mouse model. A higher proportion of hBLT-mice vaccinated with TBK1-primed DC exhibited less severe CD4+ T cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to secondary lymphoid organs and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, TBK1-primed DC might be an useful tool for subsequent vaccine studies.

Author summary

Emulating protective immunological characteristics from individuals capable of spontaneously controlling HIV-1 infection might be useful for the development of a protective vaccine. Enhanced function of dendritic cells (DC) in these HIV-1 controllers depends on the activation of TANK-binding Kinase 1 (TBK1) and might associate with protective T cells. Our study evaluated the ability of DCs trained through TBK1 activation inducing protective adaptive immune responses against HIV-1 and reducing disease progression in vivo, using a humanized mouse model. Our data indicate that mice vaccinated with tailored DC exhibit delayed disease progression, increased induction of protective CD8+ T lymphocyte subsets in the lymphoid tissue and blood upon antigen recognition. Therefore, trained-DC might be an useful tool for future HIV-1 vaccine designs.
The SARS-CoV-2 host cell membrane fusion protein TMPRSS2 is a tumor suppressor and its downregulation correlates with increased antitumor immunity and immunotherapy response in lung adenocarcinoma
Authors: Liu Zhixian , Zhang Zhilan , Feng Qiushi , Wang Xiaosheng
Published: July 2021

Background

TMPRSS2 is a host cell membrane fusion protein for SARS-CoV-2 invading human host cells. It also has an association with cancer, particularly prostate cancer. However, its association with lung cancer remains insufficiently explored. Thus, an in-depth investigation into the association between TMPRSS2 and lung cancer is significant, considering that lung cancer is the leading cause of cancer death and that the lungs are the primary organ SARS-CoV-2 attacks.

Methods

Using five lung adenocarcinoma (LUAD) genomics datasets, we explored associations between TMPRSS2 expression and immune signatures, cancer-associated pathways, tumor progression phenotypes, and clinical prognosis in LUAD by the bioinformatics approach. Furthermore, we validated the findings from the bioinformatics analysis by performing in vitro experiments with the human LUAD cell line A549 and in vivo experiments with mouse tumor models. We also validated our findings in LUAD patients from Jiangsu Cancer Hospital, China.

Results

TMPRSS2 expression levels were negatively correlated with the enrichment levels of CD8+ T and NK cells and immune cytolytic activity in LUAD, which represent antitumor immune signatures. Meanwhile, TMPRSS2 expression levels were negatively correlated with the enrichment levels of CD4+ regulatory T cells and myeloid-derived suppressor cells and PD-L1 expression levels in LUAD, which represent antitumor immunosuppressive signatures. However, TMPRSS2 expression levels showed a significant positive correlation with the ratios of immune-stimulatory/immune-inhibitory signatures (CD8+ T cells/PD-L1) in LUAD. It indicated that TMPRSS2 levels had a stronger negative correlation with immune-inhibitory signatures than with immune-stimulatory signatures. TMPRSS2 downregulation correlated with elevated activities of many oncogenic pathways in LUAD, including cell cycle, mismatch repair, p53, and extracellular matrix (ECM) signaling. TMPRSS2 downregulation correlated with increased proliferation, stemness, genomic instability, tumor advancement, and worse survival in LUAD. In vitro and in vivo experiments validated the association of TMPRSS2 deficiency with increased tumor cell proliferation and invasion and antitumor immunity in LUAD. Moreover, in vivo experiments demonstrated that TMPRSS2-knockdown tumors were more sensitive to BMS-1, an inhibitor of PD-1/PD-L1.

Conclusions

TMPRSS2 is a tumor suppressor, while its downregulation is a positive biomarker of immunotherapy in LUAD. Our data provide a connection between lung cancer and pneumonia caused by SARS-CoV-2 infection.
Chemogenetic activation of midline thalamic nuclei fails to ameliorate memory deficits in two mouse models of Alzheimer’s disease
Authors: Kohli Shivali , Andrianova Lilya , Margetts-Smith Gabriella , Brady Erica S , Craig Michael T
Published: July 2021

One of the main features of Alzheimer’s disease is the progressive loss of memory, likely due to pathological changes within brain regions such as the hippocampus and entorhinal cortex. These structures are embedded within the extended memory circuit, an interconnected set of brain regions that are essential for episodic memory. The anterior thalamic nuclei (ATN) and thalamic nucleus reuniens (NRe) are both extensively and reciprocally connected with these important memory regions, so we sought to test the hypothesis that chemogenetically-enhancing neurotransmission through NRe and ATN would ameliorate memory deficits in two mechanistically-distinct mouse models of Alzheimer’s disease. Using the hAPP-J20 mouse model of amyloidopathy and the Tg4510 mouse model of tauopathy, we carried out stereotaxic injections of viral vectors to transduce hM3Dq (Gq)_mCherry into NRe or the anterio-dorsal/anterio-ventral nuclei of ATN, using mCherry as a control. At nine months (hAPP-J20) or six months (Tg4510) of age, mice underwent a behaviour battery of open field (OF), novel object recognition (NOR) and radial arm maze (RAM), with DREADD agonist 21 administered 30min prior to each behaviour test. Tissue was collected post-behaviour to confirm injection site and virus expression. Both Tg4510 and hAPP-J20 mice show marked hyperactivity in the OF, significant deficits in recognition memory, and a significant impairment in spatial reference and spatial working memory. Unexpectedly, chemogenetic activation of ATN or NRe did not significantly improve spatial memory impairments or reduce the observed hyperactivity, although NRe activation did modestly rescue recognition memory in J20 mice. This may be due to compensation elsewhere within the memory circuit, or that the pathological changes are too far advanced for behaviour reversal.
Acquisition of alveolar fate and differentiation competence by human fetal lung epithelial progenitor cells
Authors: Lim Kyungtae , Tang Walfred , Sun Dawei , He Peng , Teichmann Sarah A. , Marioni John C. , Meyer Kerstin B. , Rawlins Emma L.
Published: July 2021

Variation in lung alveolar development is strongly linked to disease susceptibility. However, the cellular and molecular mechanisms underlying alveolar development are difficult to study in humans. Using primary human fetal lungs we have characterized a tip progenitor cell population with alveolar fate potential. These data allowed us to benchmark a self-organising organoid system which captures key aspects of lung lineage commitment and can be efficiently differentiated to alveolar type 2 cell fate. Our data show that Wnt and FGF signalling, and the downstream transcription factors NKX2.1 and TFAP2C, promote human alveolar or airway fate respectively. Moreover, we have functionally validated cell-cell interactions in human lung alveolar patterning. We show that Wnt signalling from differentiating fibroblasts promotes alveolar type 2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. Our organoid system recapitulates key aspects of human lung development allowing mechanistic experiments to determine the underpinning molecular regulation.
The histone chaperone FACT facilitates heterochromatin spreading through regulation of histone turnover and H3K9 methylation states
Authors: Murawska Magdalena , Greenstein R. A. , Schauer Tamas , Olsen Karl C.F. , Ng Henry , Ladurner Andreas G. , Al-Sady Bassem , Braun Sigurd
Published: July 2021

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT has been implicated in heterochromatin silencing, however, the mechanisms by which FACT engages in this process remain opaque. Here, we pin-pointed its function to the heterochromatin spreading process. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1 via nucleosome stabilization. Together, our study identifies FACT as a histone chaperone that specifically promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of epigenetic marks.
Horizontal Transfer of Histone H3 by Mammalian Cells
Authors: Sulkowski Parker L. , Nicholson Hilary E. , Li Matthew Z. , Perrimon Norbert , Liu James Zhe , Kaelin William G.
Published: July 2021

The authors have withdrawn their manuscript because many of the experiments described in this paper have not been reproducible, or at least are not robust, in the hands of other members of the Kaelin Laboratory who were not initially involved in this work. While we do see apparent secretion of histone H3 under some conditions, it is usually accompanied by secretion of histone H4. In this regard, the Halo tagged-histone H3 and Halo-tagged H4 constructs used for the single molecule imaging studies we reported, which seemingly confirmed specific secretion and transfer of histone H3, were purported to be sequence validated. Upon resequencing these constructs we discovered a non-synonymous mutation in the Halo tag of the H4 construct. We then redid the imaging experiments with the corrected Halo-H4 together with Halo-H3 and, in contrast to our earlier study, unfused Halo. These experiments were difficult to interpret because of the background signal seen with the unfused Halo but did not support specific secretion and transfer of histones (let alone specific secretion and transfer of histone H3).

We have, in some experiments, observed transfer of H3-Cre into reporter cells ex vivo and in vivo, but 1) the transfer is not specific for H3-Cre versus H4-Cre, 2) the transfer efficiency is highly variable, and 3) the transfer efficiency is typically much less than reported in our original paper. We do not yet know whether this lack of reproducibility and robustness reflects technical and biological variables that we do not yet understand and hence were not captured in our experimental protocols.

Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.
Early detection of SARS-CoV-2 in circulating immune cells in a mouse model
Authors: Geng Tingting , Keilich Spencer , Tafas Triantafyllos , Wang Penghua
Published: July 2021

SARS-CoV-2 infects the respiratory tract, lung and then other organs. However, its pathogenesis remains largely unknown. We used RareScope™ Fluorescence Light Sheet Microscopy (FLSM) and fluorescent in situ hybridization of RNA (RNA-FISH) to detect SARS-CoV-2 RNA and dissemination kinetics in mouse blood circulation. By RNA-FISH, we found that SARS-CoV-2 RNA-positive leukocytes, including CD11c cells, appeared as early as one day after infection and continued through day 10 post infection. Our data suggest that SARS-CoV-2-permissive leukocytes contribute to systemic viral dissemination, and RNA-FISH combined with FLSM can be utilized as a sensitive tool for SARS-CoV-2 detection in blood specimens.
A supergene in seaweed flies modulates male traits and female perception
Authors: Enge Swantje , Mérot Claire , Mozūraitis Raimondas , Apšegaitė Violeta , Bernatchez Louis , Martens Gerrit A. , Radžiutė Sandra , Pavia Henrik , Berdan Emma L.
Published: July 2021

Supergenes, tightly linked allelic combinations that underlie complex adaptive phenotypes represent a critical mechanism protecting intra-specific polymorphism [1, 2]. Supergenes represent some of the best examples of balancing selection in nature and there is increasing evidence that disassortative mating, when individuals preferentially mate with dissimilar phenotypes, is a key force stabilizing supergene polymorphisms. Yet, the underlying biological mechanisms and genetic basis of disassortative mating remain poorly known. Here, we examine a possible mechanism of disassortative mating driven by female mate choice in relation to the overdominant Cf-Inv(1) supergene in the seaweed fly Coelopa frigida by investigating chemical communication and its genomic architecture. We show that Cf-Inv(1) strongly affects chemical signaling; cuticular hydrocarbon (CHC) composition differed between genotypes in males but not females across two continents. In tandem, Cf-Inv(1) affected female perception of these compounds; females are able to sense 36 compounds from the male CHC cocktail but show differential perception between genotypes for almost half of them. This indicates that the genetic underpinnings of male traits and female perceptions are tightly linked within Cf-Inv(1) which likely facilitates disassortative mating [3]. A differential expression approach based on candidate genes for CHC biosynthesis and odorant detection revealed differential expression for CHC biosynthesis in males alone but broad changes in odorant receptors across both sexes. Furthermore, odorant genes clustered together within Cf-Inv(1), with some of them differing between arrangements by 8.3% at the protein level, suggesting evolution via tandem duplication then divergence. We propose that the tight linkage between overdominant loci, male traits, and female perception has helped to maintain the Cf-Inv(1) polymorphism across its range in the face of supergene degeneration.
Optogenetic stimulation of medial amygdala GABA neurons with kinetically different channelrhodopsin variants yield opposite behavioral outcomes
Authors: Baleisyte Aiste , Schneggenburger Ralf , Kochubey Olexiy
Published: July 2021

Optogenetic manipulation of genetically-specified neuron populations has become a key tool in circuit neuroscience. The medial amygdala (MeA) receives pheromone information about conspecifics and has crucial functions in social behaviors; interestingly, this amygdalar structure contains a majority of GABAergic projection neurons. A previous study showed that optogenetic activation of MeA GABA neurons with channelrhodopsin-2H134R (ChR2) strongly enhanced inter-male aggression (Hong et al. 2014, Cell). When we attempted to reproduce these findings, accidentally using a faster channelrhodopsin variant (channelrhodopsin-2H134R,E123T or ChETA), we found the opposite results. We therefore systematically compared the behavioral outcome of optogenetic stimulation of MeApd GABA neurons with ChETA versus ChR2, employing two widely used AAV serotypes. This revealed that optogenetic stimulation with ChETA suppressed aggression, whereas optogenetic stimulation with ChR2 increased aggression. Recordings of membrane potential changes following optogenetic stimulation with ChETA versus ChR2 revealed larger plateau depolarizations, smaller action potential amplitudes, and larger local inhibition of neighboring inhibitory neurons with ChR2 as compared to ChETA. Our study shows that channelrhodopsin variants have to be chosen with care for in-vivo optogenetic experiments. Furthermore, the role of MeApd GABA neurons in aggression control should be re- evaluated.