bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigation ofAspergillus fumigatussmall RNA biogenesis uncovers evidence of double-stranded RNA-dependent growth arrest
Authors: Kelani Abdulrahman A. , Pan Xiaoqing , Prabakar Swatika , Schrettenbrunner Lukas , Blango Matthew G. 
Published: May 2024

ABSTRACTAspergillus fumigatusis a ubiquitous filamentous fungus and dangerous human pathogen that produces a limited pool of small RNAs, consisting in large part of tRNA-derived RNAs (tDRs). Here, we improve our understanding of the small RNAs produced in conidia and mycelium ofA. fumigatususing small RNA-sequencing of strains lacking RNA interference (RNAi) machinery and a cutting-edge tDR-sequencing approach. We find little evidence of small RNAs dependent on the canonical RNAi machinery under laboratory growth conditions, but reveal tDRs to be differentially abundant across fungal morphotypes, with specific fragments proving dominant in each assessed condition (e.g., Asp(GTC)-5’tRH in conidia; His(GTG)-5’tRH in mycelium). Consistent with the literature, we observed distinct patterns of tDRs from nuclear- and mitochondria-derived tDRs, which was confirmed for wild-type fungus with tDR-seq. By inducing canonical RNAi with overexpression of an inverted-repeat transgene, we determinedA. fumigatusto produce predominantly 20-nt, 5’ uridine-containing small RNAs from the transgene, a population reliant on Argonaute and Dicer-like proteins. Surprisingly, we found that overexpression of this double-stranded RNA (dsRNA) limited growth of both wild-type and RNAi-deficient strains, with a strain lacking the twoA. fumigatusRNA-dependent RNA polymerase orthologs particularly vulnerable. Dicer-like- and Argonaute-double knockout strains with few detectable transgene-derived small RNAs were also susceptible to growth inhibition, suggesting excessive dsRNA limits growth inA. fumigatus. Ultimately, we have provided an improved description of small RNA biogenesis inA. fumigatusand uncovered an intriguing link between dsRNA metabolism and filamentous growth.IMPORTANCEThe mechanisms of small RNA biogenesis in fungi are diverse and multifactorial. Here, using a panel of RNA interference gene deletion strains and multiple small RNA sequencing approaches, we reveal limited input of the canonical RNAi machinery on the small RNA landscape of the human fungal pathogen,Aspergillus fumigatusunder normal growth conditions. We identify specific patterns of tRNA-derived RNAs unique to fungal morphotypes, which may hold promise for future diagnostic efforts. We then probed the mechanism of double-stranded RNA processing by the organism after artificial induction, revealing a potential link between proper double-stranded RNA processing and fungal growth arrest, improving our understanding of the evolution of double-stranded RNA regulation in a ubiquitous mold.
Viral microRNA regulation of Akt is necessary for reactivation of Human Cytomegalovirus from latency in CD34+hematopoietic progenitor cells and humanized mice
Authors: Diggins Nicole L. , Pham Andrew H. , Mitchell Jennifer , Parkins Christopher J. , Slind Luke , Turner Rebekah , Caposio Patrizia , Nelson Jay A. , Hancock Meaghan H. 
Published: May 2024

ABSTRACTHuman cytomegalovirus (HCMV) actively manipulates cellular signaling pathways to benefit viral replication. Phosphatidyl-inositol 3-kinase (PI3K)/Akt signaling is an important negative regulator of HCMV replication, and during lytic infection the virus utilizes pUL38 to limit Akt phosphorylation and activation. During latency, PI3K/Akt signaling also limits virus replication, but how this is overcome at the time of reactivation is unknown. Virally encoded microRNAs (miRNAs) are a key component of the virus arsenal used to alter signaling during latency and reactivation. In the present study we show that three HCMV miRNAs (miR-UL36, miR-UL112 and miR-UL148D) downregulate Akt expression and attenuate downstream signaling, resulting in the activation of FOXO3a and enhanced internal promoter-driven IE transcription. A virus lacking expression of all three miRNAs is unable to reactivate from latency both in CD34+hematopoietic progenitor cells and in a humanized mouse model of HCMV infection, however downregulating Akt restores the ability of the mutant virus to replicate. These findings highlight the negative role Akt signaling plays in HCMV replication in lytic and latent infection and how the virus has evolved miRNA-mediated countermeasures to promote successful reactivation.AUTHOR SUMMARYHuman cytomegalovirus (HCMV) infection results in lifelong persistence of the virus through the establishment of latency, and viral reactivation is a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is established in CD34+hematopoietic progenitor cells (HPCs) where the virus manipulates cell signaling pathways to maintain the viral genome and remain poised to reinitiate gene expression under the appropriate conditions, although the molecular mechanisms surrounding these processes are poorly understood. HCMV encodes microRNAs (miRNAs) that modulate expression of hundreds of cellular and viral genes and play important roles in regulating signaling in HPCs. In this study, we show that HCMV miR-UL36, miR-UL112, and miR-UL148D coordinately inhibit Akt expression, activation, and downstream signaling through nonconventional mechanisms. A mutant lacking these miRNAs is unable to reactivate from latency, yet complementing Akt regulation restores the ability of the mutant virus to reactivate, pointing to an important role for miRNA-mediated inhibition of Akt to promote HCMV reactivation.
Acanthamoeba castellaniias a model for unveilingCampylobacter jejunihost-pathogen dynamics
Authors: Nasher Fauzy , Lehri Burhan , Stabler Richard , Wren Brendan W. 
Published: May 2024

AbstractThe persistence of the major enteric pathogenCampylobacter jejuniin the natural environment, despite being microaerophilic, remains unsolved. Its survival in the natural atmospheric environment likely stems from several factors, including interactions with amoebae.C. jejunitransiently interacts with Acanthamoebae and this is thought to provide protection against unfavourable atmospheric conditions and subsequently prime the bacteria for interactions with warm-blooded hosts. Acanthamoebae play vital roles in microbial ecosystems by preying on bacterial species, some of which are clinically important. We analysed the whole transcriptome ofA. castellaniiinfected withC. jejuni11168H. Our findings provide evidence that infection ofA. castellaniiwithC. jejunitriggers distinct and reproducible cellular responses. Upregulated genes were associated with protein synthesis, DNA damage and repair, gluconeogenic pathways, and protein folding and targeting, while downregulated genes were involved in calcium ion transport, osmotic stress response, energy reserve metabolic processes, and protein hydroxylation. From this data we characterized Cj0979c, named hereC. jejuniendonuclease (CjeN), which induces DNA damage inA. castellanii. High-resolution microscopy revealed an unexpected association betweenC. jejuniand host mitochondria, while infected cells show elevated cytosolic calcium levels and metabolic changes favouring “Warburg-like” metabolism. The increased lactate production was subsequently depleted, suggesting that this host metabolic by-product may supportC. jejunisurvival. These findings identify an unexpected interaction between amoebae and a microaerophilic bacterium and provides a useful model for further research on host-pathogen interactions.
Gene drive-based population suppression in the malaria vectorAnopheles stephensi
Authors: Xu Xuejiao , Chen Jingheng , Wang You , Liu Yiran , Zhang Yongjie , Yang Jie , Yang Xiaozhen , He Zhengbo , Champer Jackson 
Published: May 2024

AbstractGene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we constructed a homing suppression drive in the major urban malaria vectorAnopheles stephensitargeting the female-specific exon ofdoublesex, incorporating two gRNAs and ananos-Cas9 promoter to reduce functional resistance and improve female heterozygote fitness. Our result showed that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes showed only moderate drive conversion, indicating that thenospromoter has lower activity inA. stephensithan inAnopheles gambiae. By amplicon sequencing, we detected a very low level of resistance allele formation. Combination of the homing suppression and avasa-Cas9 line demonstrated a boost in the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission.
The HCoV-HKU1 N-terminal domain binds a wide range of 9-O-acetylated sialic acids presented on different glycan cores
Authors: Tomris Ilhan , Kimpel Anne , Liang Ruonan , van der Woude Roosmarijn , Boons Geert-Jan , Li Zeshi , de Vries Robert P. 
Published: May 2024

AbstractCoronaviruses recognize a wide array of protein and glycan receptors using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal (S1-NTD) and C-terminal (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possess an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated α2-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GNTI-cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high-mannose-containing NTD proteins revealed a much broader receptor binding profile compared to its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated α2-3 linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding towards 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids,N-orO-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding. (218)
ProtMamba: a homology-aware but alignment-free protein state space model
Authors: Sgarbossa Damiano , Malbranke Cyril , Bitbol Anne-Florence 
Published: May 2024

AbstractProtein design has important implications for drug discovery, personalized medicine, and biotechnology. Models based on multiple sequence alignments efficiently capture the evolutionary information in homologous protein sequences, but multiple sequence alignment construction is imperfect. We present ProtMamba, a homology-aware but alignment-free protein language model based on the Mamba architecture. In contrast with attention-based models, ProtMamba efficiently handles very long context, comprising hundreds of protein sequences. We train ProtMamba on a large dataset of concatenated homologous sequences, using two GPUs. We combine autoregressive modeling and masked language modeling through a fill-in-the-middle training objective. This makes the model adapted to various protein design applications. We demonstrate ProtMamba’s usefulness for the generation of novel sequences and for fitness prediction. ProtMamba reaches competitive performance with other protein language models despite its smaller size, which sheds light on the importance of long-context conditioning.
Progression ofampCamplification during de novo amoxicillin resistance development inE. coli
Authors: Nong Luyuan , Jonker Martijs , de Leeuw Wim , Wortel Meike T. , ter Kuile Benno 
Published: May 2024

ABSTRACTBeta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance inE. coli. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression ofampCamplification andampCpromoter mutations in the evolution of resistance by stepwise increasing amoxicillin concentration.AmpCpromoter mutations occur by day two, while the amplification by a factor of approximately eight occurs after more than six days of amoxicillin exposure. The combination of amplification and promoter mutations increaseampCmRNA level by an average factor of 200 after 22 days. An IS1 insertion was identified in the amplification junction, suggesting the amplification is facilitated by mobile genetic elements transposition. In order to identify the essential genes forampCamplification, the chromosomal mutations of strains with induced amoxicillin resistance were compared a similarly evolved resistant ΔampCknockout. The evolved ΔampCcontained several resistance mutations that were absent in the WT, which accumulated more mutations in stress response genes. The amoxicillin evolved ΔampCdoes not show amplification of the fragment around the originalampCposition but exhibits a large duplication or triplication at another position, suggesting selection of genes to amplify is essential for resistance adaption.IMPORTANCEAmoxicillin is one of the most used antimicrobial against bacterial infections. DNA fragments containingampCare amplified upon prolonged and stepwise increasing exposure to amoxicillin. TheseampCamplification fragments have been identified in extended-spectrum beta-lactamases (ESBLs) plasmids, which are considered the main cause of beta- lactam resistance. Understanding the progression ofampCamplification enables amoxicillin resistance prevention. In this study, we show the time course of two important factors forampCtranscription enhancement,ampCamplification andampCpromoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification and that the sigma factor 70 regulatesampCoverexpression.
Cyclic di-AMP drives developmental cycle progression inChlamydia trachomatis
Authors: Lee Junghoon , Ouellette Scot P. 
Published: May 2024

AbstractThe obligate intracellular bacteriumChlamydiaalternates between two functional forms during its developmental cycle: elementary body (EB) and reticulate body (RB). However, the molecular mechanisms governing the transitions between these forms are unknown. Here, we present evidence cyclic di-AMP (c-di-AMP) is a key factor in triggering the transition from RB to EB (i.e., secondary differentiation) in the chlamydial developmental cycle. By overexpressing or knocking down expression of c-di-AMP synthase genes, we made strains producing different levels of c-di-AMP, which we linked to changes in secondary differentiation status. Increases in c-di-AMP resulted in an earlier increase in transcription of EB-associated genes, and this was further manifested in earlier production of EBs. In contrast, when c-di-AMP levels were decreased, secondary differentiation was delayed. Based on these data, we conclude there is a threshold level of c-di-AMP needed to trigger secondary differentiation inChlamydia. This is the first study to identify a mechanism by which secondary differentiation is initiated inChlamydiaand reveals a critical role for the second messenger signaling molecule c-di-AMP in this process.
Influenza A Virus NS1 Limits Recognition of Double-Stranded Transposable Elements by Cytosolic RNA Sensors
Authors: Lork Marie , Childs Liam , Lieber Gauthier , König Renate , Hale Benjamin G. 
Published: May 2024

AbstractInfluenza A virus (IAV) infection triggers de-repression of host transposable elements (TEs), which have the potential to form double-stranded (ds)RNAs and stimulate innate antiviral immunity. However, as wild-type IAV is generally a poor inducer of innate immunity, it remains unclear whether de-repressed TEs actually form dsRNAs recognizable by host cytosolic RNA sensors, or whether IAV might antagonize such sensing. Here, we performed strand-specific total RNA-Seq on nuclear and cytosolic fractions from cells infected with wild-type IAV or a recombinant IAV lacking NS1, a viral dsRNA-binding protein. Both infections led to global increases in host TE RNAs with bioinformatic and experimental evidence for double-strandedness. However, only NS1-deficient IAV infection led to significant amounts of TE-dsRNAs translocating to the cytosol, and co-precipitations identified that wild-type NS1 associates with TE-dsRNAs. Furthermore, a functional screen indicated that TE-dsRNAs can be engaged by various host cytosolic RNA sensors, including RIG-I, MDA5, ZBP1, and PKR. Our data reveal the double-stranded nature of infection-triggered host TEs and suggest an NS1-mediated sequestration mechanism to limit their cytosolic abundance and broad activation of diverse sensors.
Intercellular epigenomic signaling during B cell maturation
Authors: Wai Yim Kevin Ho , Hrout Alaa Al , Chahwan Richard 
Published: May 2024

ABSTRACTB cell maturation is crucial for effective adaptive immunity. It requires a complex signaling network to mediate antibody diversification through mutagenesis. B cells also rely on queues from other cells within the germinal center. Recently, a novel class of intercellular signals mediated by extracellular vesicles (EVs) has emerged. Studies have shown B cell EV-mediated signaling is involved in immune response regulation and tumorigenesis. However, the mechanistic role of B cell EVs is not yet established. We herein study the biological properties and physiological function of B cell EVs during B cell maturation. We use emerging technologies to profile B cell EVs surface marker signatures at the single particle level, molecular cargo, and physiological roles in B cell maturation. EV ncRNA cargo, characterized by RNA-seq, identified an EV-mediated novel non-coding RNA regulatory network for B cell maturation. A previously uncharacterized micro-RNA (miR-5099) in combination with a set of long non-coding RNA carried within B cell EVs is shown to be important for antibody diversification. The physiological role of EVs in B cell maturation is investigated using EV blockade assays and complementation studies using diverse EV sources further confirmed the physiological role and mode of action of EVs in B cell maturation.
The pioneer transcription factor ELF2 remodels the nucleosome near transcription start sites
Authors: Xiao Tianyi , Crowe-McAuliffe Caillan , Ochmann Moritz , Du Yepeng , Hou Tianyun , Chen Yinan , Zhu Fangjie , Seyednasrollah Fatemeh , Osmala Maria , Vanharanta Sakari , Morgunova Ekaterina , Cramer Patrick , Rogerson Connor , Dienemann Christian , Taipale Jussi 
Published: May 2024

AbstractPioneer transcription factors are DNA-binding proteins that can bind to nucleosomes in closed chromatin regions, exposing enhancers and promoters of genes for transcription. The action of these factors underpin stem cell pluripotency, cell reprogramming and differentiation. ELF2 is an ETS family pioneer factor that has a strong preference for oriented binding on nucleosomes at a composite head-to-tail dimeric sequence motif with a 2 bp spacing between the GGAA core elements. In this study, we investigated the interaction between ELF2 and a nucleosome using single-particle cryo-electron microscopy (cryo-EM). The ELF2-nucleosome structure shows two ELF2 proteins bound to the nucleosome cooperatively at superhelical location +4. The recognition ɑ4 helices of both ELF2 monomers dock into the major groove at the core GGAA motifs and unwrap approximately four helical turns of the DNA from the surface of the nucleosome. The unwrapping almost completely exposes one histone H2A:H2B dimer, which dissociates in a subset of particles to form an ELF2-bound hexasome. ChIP-seq combined with genome-wide motif mapping indicates that all unmethylated high-affinity ELF2 double motifs are occupied, indicating that ELF2 is able to use its binding energy to function as a pioneer factor bothin vitroandin vivo. ELF2 binding is highly enriched downstream of transcription start sites (TSS) of highly expressed genes, with the motifs oriented in such a way that the TSS becomes accessible upon ELF2 binding. This mechanism is essential for cells, as mutation of ELF2 motifs result in decreased cell proliferation. Taken together, our results indicate that ELF2 can use its binding energy to open chromatin in an oriented fashion, facilitating nucleosome positioning and transcription initiation at transcription start sites.
Nuclear eNOS interacts with andS-nitrosates ADAR1 to modulate type I interferon signaling and endothelial function
Authors: Zhou Xiaozhu , Kuenne Carsten , Günther Stefan , Wittig Ilka , Güven Beyza , Boutguetait Doha , Lagos Fredy Delgado , Sachs Nadja , Maegdefessel Lars , Münch Christian , Offermanns Stefan , Fleming Ingrid , Siragusa Mauro 
Published: May 2024

AbstractBackgroundNitric oxide (NO) generated by the endothelial NO synthase (eNOS) regulates vascular tone and endothelial homeostasis to counteract vascular inflammation. Most eNOS is localized at the cell membrane or in the Golgi apparatus, but the enzyme is also present in the endothelial cell nucleus. Here we assessed the relevance of nuclear eNOS and NO signaling for endothelial cell function.MethodseNOS gain- and loss-of-function approaches were combined with confocal microscopy, biochemical, histological and multi-omics analyses. The pathophysiological relevance of the findings was assessed in murine models of atherogenesis (ApoE-/-and partial carotid ligation) as well as in samples from patients with atherosclerosis.ResultseNOS was present in the nucleus of unstimulated human and murine endothelial cells (in vitroandex vivo) and stimulation with vascular endothelial growth factor (VEGF) enhanced its nuclear localization. Co-immunoprecipitation studies coupled with proteomics revealed the association of nuclear eNOS with 81 proteins involved in RNA binding and processing. Among the latter was double-stranded RNA-specific adenosine deaminase (ADAR1), an enzyme involved in editing double-stranded RNA (dsRNA) via the deamination of adenosine (A) to inosine (I). ADAR1 wasS-nitrosated in human endothelial cells, and the knockdown of eNOS resulted in altered ADAR1-mediated A-to-I editing and an increase in dsRNA. Mitochondrial antiviral signaling protein (MAVS) was the transcript most affected by eNOS depletion, and endothelial cells lacking eNOS accumulated dsRNA as well as MAVS protein aggregates. These changes resulted in activation of the type I interferon (IFN) signaling pathway and a marked downregulation of cell cycle-related genes. ADAR1 depletion elicited similar effects on the activation of type I IFN signaling. As a result, growth factor-stimulated cell proliferation was abrogated and basal as well as stimulated cell death were increased. Endothelial dysfunction in mice as well as in subjects with atherosclerosis was accompanied by the accumulation of dsRNA and the activation of the type I IFN signaling. Preserving NO bioavailabilityin vivoin an eNOS gain-of-function mouse model (Tyr657Phe eNOS) prevented these effects.ConclusionsOur findings have uncovered a novel mechanism linking nuclear eNOS-derived NO with the activity of ADAR1 to maintain vascular homeostasis. Reduced NO bioavailability results in the previously unrecognized activation of a type I IFN response in the endothelium, which contributes to atherogenesis.